16 International Xenopus Conference

Transcription

th
16 International Xenopus Conference
From Fundamental Processes to Modelling
Human Disease
Sunday 28 August - Thursday 1 September,
2016
Orthodox Academy of Crete (OAC), Chania
DELEGATE INFORMATION
Venue Contact
Emanuela Larentzakis
Orthodox Academy of Crete
Kolympari, Kissamos
Postal Code 730 06
Chania - Crete
Telephone: +30 28240 22500 / 22245
Email:
[email protected]
Website:
http://www.oac.gr/en/
The Registration Desk will be open at the following times:
Sunday 28 August
14.00 onwards
Monday 29 August
08.00 onwards
Tuesday 30 August
08.00 onwards
Wednesday 31 August 08.00 onwards
The Registration Desk will be located outside the main meeting room.
Conference Registration
On registering you will receive your name badge. Name badges must be worn for the
duration of the meeting.
Please note that there will be no printed conference materials provided.
Name badges will be coloured accordingly:

Delegates - Green Badge

Exhibitors - Red Badge

Conference Organisers - Purple Badge

Speakers and Session Chairs - Yellow Badge
Arrival at the OAC
Upon your arrival at the OAC, if you have a room booked onsite, please make your way
to the reception in the main entrance where you can check-in and collect your room key.
The reception desk will be open until 23:00.
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Departure from the OAC
If you are staying at the OAC, the check-out time is 12pm. If you require a late check-out
then please speak to a member of staff at the reception desk and they will advise
whether this can be arranged.
For those delegates who have pre-booked the shuttle bus, this will be departing from the
OAC at the designated times. Please ensure that you have your shuttle bus booking
confirmation with you as you will need this in order to board the bus.
If you require a taxi then these can be booked at the OAC Reception Desk located in the
main entrance.
Internet Access
Complimentary wifi is available throughout the venue. If your computer does not connect
automatically then the username is guest and the password is 2016.
Meals and Refreshments
Breakfast (07.30 - 08.30) will be served in the restaurant for those delegates who are
staying at the OAC. Tea/coffee breaks will be served on the terrace. Lunch and evening
meals will be served in the restaurant.
Social Events
Sunday 28 August - Welcome Reception 20:00 onwards
A welcome reception for all delegates will take place on the Terrace on Sunday.
Monday 29 & Tuesday 30 August - Poster Sessions 21.00 onwards
There will be poster sessions taking place at 21.00 hours after dinner on Monday (odd
numbers) and Tuesday (even numbers) with a drinks reception. There will also be a cash
bar available until midnight on all evenings. These sessions will take place in the Foyer
outside of the main meeting room.
Wednesday 31 August - Cretan Evening 20.30 onwards
There will be a Gala Dinner and Prize Giving on the Wednesday. This will be a Cretan
evening with Cretan specialties including dancing with traditional costumes and
musicians.
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Other Programme Events
Monday 29 August - 13.30 - 14.30
Reviewers Workshop Luncheon
Sponsored by Developmental Biology
In this informal workshop we will discuss the joys and sorrows of modern peer-review.
We will cover the key principles and steps to follow when reviewing a manuscript for an
international, high impact journal, but also the key tips for authors on how to prepare the
best response to reviewers' comments. We will then discuss ethical responsibilities in
peer-review and current innovative pilots and developments for reviewers.
The workshop will be presented by Valentina Sasselli, publisher for Cell and
Developmental journals at Elsevier with the participation of the senior editor of
Developmental Biology, Richard Harland.
Early Career Researchers (PhD Students, Post-docs and Junior PIs) are particularly
encouraged to attend this discussion.
A packed lunch will be provided for those who have signed up to the workshop.
Tuesday 30 August - 13.30 - 14.30
Careers Workshop
The careers workshop will consist of round-table discussions with table leaders from
academia and industry.
Lunch will be provided.
The confirmed tables are listed below:
Table 1: Phd Students
Table 2: Postdocs
Table 3: Junior Faculty
Table 4: Non-academic Careers
Table 5: Women in Science
Those who have signed up to attend this workshop will be advised which tables they
have been allocated at the Conference.
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16th International Xenopus - Programme 2016
From Fundamental Processes to Modelling Human Disease
Sunday 28 August – Thursday 1 September, 2016
Sunday 28 August, 2016
14.00 onwards
Registration Opens
18.00 - 20.00
Session 1
Session Chair: Christof Niehrs
18.00 - 18.10
Welcome
18.10 - 18.35
S01 Richard Harland - Molecular and Cell Biology and Center for Integrative Genomics, University of California, Berkeley, CA, USA
Frog genomes
18.35 - 19.00
19.00 - 19.50
20.00 onwards
S02 Masanori Taira - University of Tokyo, Tokyo
The Xenopus laevis genome project reveals the features of subgenomes in the allotetraploid
PL01 Plenary/Prospectives Talk
Mary Lou King - University of Miami Miller School of Medicine, Miami
Maternal Messages to Live By: past, present, future
Welcome Reception
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Monday 29 August, 2016
07.30 - 08.30
Breakfast for those delegates who are staying onsite at the OAC – other guests will take breakfast at their individual hotels
08.00 onwards
Registration Opens
08.30 - 10.30
Session 2
Session Chair: Ali Brivanlou
08.30 - 08.55
S03 Yonglong Chen - South University of Science and Technology of China, Shenzhen, China
Heritable CRISPR/Cas9-mediated targeted integration in Xenopus tropicalis
08.55 - 09.10
O01 James Briggs - Department of Systems Biology, Harvard Medical School, Cambridge, USA
Whole-embryo single-cell analysis of early Xenopus Tropicalis development genomics
09.10 - 09.35
S04 Vincenzo Costanzo - IFOM, FIRC Institute of Molecular Oncology, Italy
Reconstitution of centromeric DNA replication reveals suppression of the ATR dependent checkpoint and formation of DNA loops
09.35 - 09.50
O02 Ai-Sun Tseng – School of Life Sciences, University of Nevada Las Vegas, Las Vegas, USA
Understanding Developmental Eye Repair
09.50 - 10.05
O03 Rachel Stephenson – Department of Molecular, Cellular and Developmental Biology, University of Michigan, MI, USA
Flares of active RhoA locally reinforce cell-cell junctions
10.05 - 10.30
S05 Taejoon Kwon - Ulsan National Institute of Science and Technology, Ulsan
AmphiBase: Comprehensive Genomic Resource of Amphibians And Its Application to Characterize ‘unnamed’ Xenopus Genes
10.30 - 11.00
Refreshment Break and Poster/Exhibition Viewing
11.00 - 13.30
Session 3
Session Chair: Laurent Kodjabachian
11.00 - 11.25
S06 Peter Nemes - George Washington University, Washington DC, USA
Discovery Mass Spectrometry Finds Metabolic Activity Differences between Blastomeres in the Early Xenopus laevis Embryo
11.25 - 11.40
O04 Gary Gorbsky – Cell Cycle and Cancer Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, USA
Isolation of novel cell lines from standard laboratory strains of Xenopus laevis and Xenopus tropicalis embryos
11.40 - 12.05
S07 Darcy Kelley - Columbia University, New York, USA
The evolution of advertisement calls in Xenopus; genomic,developmental and functional insights
12.05 - 12.20
O05 Mike Danilchik – Integrative Biosciences, Oregon Health and Science University, Portland, USA
Exosomal trafficking in the early X. laevis embryo
12.20 - 12.35
S08 Lindsey Marshall - MNHN/CNRS, France
The amphibian Xenopus as a new model to study cardiac regeneration
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12.45 - 13.00
O06 Kris Vleminckx – Department of Biomedical Molecular Biology, Centre for Medical Genetics, Ghent University, Ghent, Belgium
Development of a fast and efficient genetic platform in Xenopus tropicalis for modeling human congenital limb malformations
13.00 - 13.25
S09 Nanette Nascone-Yoder - Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, USA
Budgett’s frog (Lepidobatrachus) embryos reveal mechanisms of left-right asymmetric organ morphogenesis and new left-right asymmetry
genes
13.30 - 14.30
Lunch and Posters/Exhibition Viewing
13.30 - 14.30
Reviewers Workshop Luncheon
Sponsored by Developmental Biology
14.30 - 16.30
Break in conference programme
16.30 - 17.00
17.00 - 20.00
17.00 - 17.25
Session 4
Session Chair: Ken Cho
S10 John Wallingford - Molecular Biosciences, University of Texas, Austin, USA
Emergence of an apical epithelial cell surface in vivo
17.25 - 17.40
O07 Elizabeth Peuchen – Chemistry and Biochemistry, University of Notre Dame, USA
12-time point proteomics of Xenopus laevis allows for broad understanding of proteomic expression file emerging from a mature oocyte to late
neurala stage embryo quantifying more than 6,100 protein profiles
17.40 - 17.55
O08 Magdalena Koziol – Wellcome Trust Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge, UK
Identification of methylated deoxyadenosines in vertebrates reveals diversity in DNA modifications
17.55 - 18.10
O09 Juan Larrain – Cell and Molecular Biology, Universidad Católica, Santiago, Chile
The heterochronic gene Lin28 regulates metamorphosis by inhibiting the thyroid hormone axis
18.10 - 18.20
Comfort Break
18.20 - 19.00
Yoshiki Sasai Memorial Plenary Lecture Chair: Enrique Amaya
PL02 Naoto Ueno - Developmental Biology, National Institute for Basic Biology, Okazaki, Japan
Measurement of force field during the collective cell migration of Xenopus embryonic cells
19.00 - 19.50
Plenary Talk
PL03 Ray Keller - Department of Biology, University of Virginia, Charlottesville, Virginia, USA
Morphogenic Machines of Early Embryogenesis: How embryos put their players on the field
20.00 - 21.00
Dinner
21.00 onwards
Poster viewing (odd numbers) – cash bar
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Tuesday 30 August, 2016
07.30 - 08.30
08.00 onwards
Registration Opens
08.30 - 10.30
08.30 - 08.55
08.55 - 09.10
Session 5
Session Chair: Anne Monsoro-Burq
S11 Carole LaBonne - Northwestern University, Evanston
FGF mediated MAPK and PI3K/Akt Signaling make distinct contributions to the control of pluripotency and the establishment of the Neural
Crest state
O10 Martin Blum – Zoology, University of Hohenheim, Stuttgart, Germany
Cilia and leftward flow determine laterality in conjoined Xenopus twins
09.10 - 09.35
S12 Frank Conlon – Department of Biology and Genetics, McAllister Heart Institute, University of North Carolina, NC, USA
A genetic requirement for the KCP-BMP pathway in vertebrate heart development
09.35 - 09.50
O11 Maximina Yun – Structural and Molecular Biology, University College London, London, UK
Functions of programmed cellular senescence during vertebrate development
09.50 - 10.05
O12 Chenbei Chang – Cell, Developmental and Integrative Biology, University Alabama, Birmingham, USA
Identification of new regulators of embryonic patterning and morphogenesis in Xenopus gastrulae by RNA sequencing
10.05 - 10.30
S13 Andrea Wills - Biochemistry, University of Washington, Seattle, WA, USA
Remodeling the chromatin landscape during Xenopus regeneration
10.30 - 11.00
11.00 - 13.30
Session 6
Session Chair: Tomas Pieler
11.00 - 11.25
S14 Monica Bettencourt Dias – CCR, Instituto Gulbenkian de Ciencia, Oeiras,Portugal
CDK1 Prevents Unscheduled PLK4-STIL Complex Assembly in Centriole Biogenesis
11.25 - 11.40
O13 Bruno Reversade – Laboratory of Human Genetics and Embryology, Institute of Medical Biology (A*STAR), Singapore
Congenital Arhinia: to Have or Not to Have a Nose
11.40 - 12.05
S15 Matt Good - Cell and Developmental Biology, University of Pennsylvania, Philadelphia, PA, USA
Probing the Impacts of Cell Boundary Conditions on Intracellular Signaling
12.05 - 12.20
O14 Haruki Ochi – Institute for Promotion of Medical Science Research, Faculty of Medicine, Yamagata University, Yamagata, Japan
Evolutionary conserved regeneration signal response enhancers for renal regeneration
12.20 - 12.45
S16 Soeren Lienkamp - Renal Division, University Hospital Freiburg, Freiburg, Germany
Transcriptional control of tubule cell identity
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12.45 - 13.00
O15 Hiroki Danno – Advanced Center for Computing and Communication, RIKEN, Wako, Japan
Disassembling regionalization of neuroectoderm by single-cell transcriptome analysis
13.00 - 13.25
S17 Dale Frank - Technion Israel Institute of Technology, Haifa, Israel
New Approaches to the Signaling Pathways Regulating Neural AP Patterning
13.30 - 14.30
13.30 - 14.30
Careers Workshop
14.30 - 16.30
16.30 - 17.00
17.00 - 20.00
Session 7
Session Chair: Jerry Thomsen
17.00 - 17.25
S18 Douglas Houston - University of Iowa, Iowa City, USA
The role of Syntabulin in germ plasm aggregation during PGC formation
17.25 - 17.40
O16 Pamela Mancini – Division of Developmental Biology, Cincinnati Children's Hospital Research Foundation , Cincinnati, OH, USA
The molecular and cellular bases of tracheo-esophageal birth defects
17.40 - 18.05
S19 Sarah Woolner – Faculty of Life Sciences, University of Manchester, Manchester, UK
Cell division and the mechanical tissue environment
18.05 - 18.20
O17 Ira Daar – Laboratory of Cell and Developmental Signaling, National Cancer Institute, Frederick, MD, USA
The newly identified ephrinB2 binding partner, TBC1d24, plays a role in neural crest cell migration.
18.20 - 18.45
S20 Enrique Amaya - University of Manchester, Manchester, UK
A role for reactive oxygen species during appendage regeneration and early embryonic development
18.45 - 19.00
Comfort Break
19.00 - 19.50
Plenary Lecture
PL04 Isabelle Vernos - CRG, Barcelona
Kindly Sponsored by Cytoskeleton
The mechanism of bipolar spindle assembly: Using the Xenopus egg extract system to unravel the chromosome RanGTP
dependent microtubule assembly pathway and its integration with the centrosomal pathway in animal cells
20.00 - 21.00
Dinner
21.00 onwards
Poster viewing (even numbers) – cash bar
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Wednesday 31 August, 2016
07.30 - 08.30
08.00 onwards
Registration Opens
08.30 - 10.30
Session 8
Session Chair: Paul Krieg
08.30 - 08.55
S21 Todd Stukenberg - University of Virginia, School of Medicine, Virginia, USA
The Ska complex is recruited to kinetochores by Ndc80 proteins clustered along microtubule protofilaments
08.55 - 09.10
O18 Takayoshi Yamamoto – Department of Biological Sciences, Graduate School of Science, University of Tokyo, Japan
Distribution and signaling of Wnt and BMP ligands are modulated by secreted antagonists and heparosan/heparan sulfate proteoglycans in
Xenopus embryos
09.10 - 09.35
S22 Nancy Papalopulu - Faculty of Life Sciences, University of Manchester, Manchester, UK
A new view of cell state transitions and their timing, based on the dynamics of gene expression
09.35 - 09.50
O19 Eamon Dubaissi – Faculty of Biology, Medicine and Health, University of Manchester, UK
Xenopus epidermal mucin forms a barrier to infection and is important for osmoregulation
09.50 - 10.05
O20 Amanda Butler - Department of Cell Biology, University of Miami Miller School of Medicine, Miami, USA
Defining the Primordial Germ Cell Transcriptome: A High Throughput Analysis
10.05 - 10.30
S23 Aga Gambus - Institute for Cancer and Genomic Sciences, University of Birmingham, Birmingham, UK
Replisome disasembly mechanism at the termination of DNA replication forks
10.30 - 11.00
11.00 - 13.30
Session 9
Session Chair: Anna Philpott
11.00 - 11.25
S24 Betsy Pownall – Department of Biology, University of York, York, UK
Modulation of canonical and non-canonical Wnt signalling by Sulf1
11.25 - 11.40
11.40 - 11.55
11.55 - 12.20
O21 John Griffin – Craniofacial Development and Stem Cell Biology, King's College London UK, Pediatrics, Yale University School of
Medicine, New Haven, USA
A Novel GTPase System Regulates β-Catenin Nuclear Transport in Development and Disease
O22 Daria Korotkova – Laboratory of Molecular Bases of Embryogenesis, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian
Academy of Moscow
Answer, the gene lost during evolution in higher vertebrates regulates regeneration and early forebrain development in Xenopus laevis
S25 Stefan Hoppler – Institute of Medical Sciences University of Aberdeen, Aberdeen, UK
Tissue- and stage-specific Wnt target gene expression is controlled subsequent to beta-catenin recruitment to cis-regulatory modules
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12.20 - 12.35
12.35 - 12.50
12.50 - 13.15
O23 Vikram Khedgikar – Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, USA
ADAM13 controls PCNS function during cranial neural crest cell migration
O24 Laura Ann Lowery – Biology, Boston College, Chestnut Hill, USA
Regulation of microtubule plus-end dynamics by TACC3 during axon guidance
S26 Amy Sater - Department of Biology and Biochemistry, University of Houston, Houston Texas, USA
Dual roles for miR-199 in early eye development
13.30 - 14.30
14.30 - 16.30
16.30 - 17.00
17.00 - 19.30
Session 10
Session Chair: Thomas Hollemann
17.00 - 17.25
S27 Andre Brandli - Walter-Brendel-Center of Experimental Medicine, Ludwig-Maximilians-University Munich, Planegg-Martinsried, Germany
Rare disease modeling in Xenopus: the example of alkaptonuria
17.25 - 17.40
O25 Marcela Torrejon – Department of Biochemistry and Molecular Biology, University of Concepcion, Concepcion, Chile
Ric-8A/Gα13 signaling pathway is required to proper cranial neural crest migration in Xenopus
17.40 - 18.05
S28 Dan Buchholz - University of Cincinnati, Cincinnati
The role of thyroid hormone receptor alpha during larval growth and intestinal stem cell development
18.05 - 18.20
O26 Aldo Ciau-Uitz - MRC Molecular Haematology Unit, University of Oxford, Oxford, UK
A common transcriptional network confers stemness on nascent stem cells
18.20 - 18.35
O27 Peter Walentek – Molecular and Cell Biology, University of California Berkeley, Berkeley, USA
Ciliary transcription factors and miRNAs precisely regulate Cp110 levels at basal bodies required for ciliogenesis
18.35 - 19.00
S29 Sally Moody - George Washington University, Washington DC
Novel Six1 Co-factors are Candidates for Branchiootorenal Syndrome
20.30
Gala Dinner and Prize Giving
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Thursday 1 September, 2016
08.00 - 11.00
PI Meeting
Session Chair: Sally Moody
08.00 - 08.30
Resource Centers (Matt Guille, Marko Horb and Atsushi Suzuki)
08.30 - 08.45
Xenbase (Aaron Zorn)
08.45 - 09.00
Training Courses Summary (Marko Horb)
09.00 - 09.30
Regent Pipelines - Genomics (TBA)
09.30 - 10.00
Regent Pipelines - Proteomics (Todd Stukenberg)
10.00 - 11.00
International Xenopus Board and New Business (Sally Moody/Amy Sater)
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SPONSORS AND EXHIBITORS
The Organisers would like to thank the following organisations for their generous
support of the 16th International Xenopus Conference.
Aquaneering Inc.
Aqua Schwarz GmbH
Cytoskeleton
Elsevier
Gene Tools
Intavis Bioanalytical Instruments AG
Karger
Nasco
Pentair Aquatic Eco-Systems Inc.
Society for Development Biology
The Company of Biologists
The Journal of Genetics and Development
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SPONSORS
The Organisers would like to thank the following sponsors for their support.
GOLD SPONSOR:
Aqua Schwarz GmbH
SILVER SPONSOR:
Elsevier
SPONSORS:
Cytoskeleton
Nasco
Gene Tools
The Journal of Genetics
and Development
13
The Organisers would like to give special thanks to the below sponsors for
their continued support.
The Company of Biologists
The Society for Developmental Biology
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EXHIBITOR INFORMATION
Aquaneering Inc.
7960 Stromesa Court
San Diego
California
92126
Contact Name: Caitlin Coronel
Email: [email protected]
Telephone: 858 578 2028
Website: http://aquaneering.com/
Aquaneering is an internationally recognized leader in the manufacture of aquatic
housing used in medical research for zebrafish, Xenopus, and other species.
Aquaneering offers unmatched knowledge of highly advanced filtration technologies
pioneered within the aquaculture industry and provide no-maintenance filters with
undetectable levels of ammonia and nitrites. Aquaneering is the manufacturer of the
largest zebrafish systems in the world.
Aqua Schwarz GmbH
Maschmühlenweg 40/42
Göttingen
Germany
37081
Contact Name: Dunja Schwarz
Telephone: +49 551 3850780
Website: www.aquaschwarz.com
With the experience of 45 years AQUA SCHWARZ GmbH develops and manufactures
aquatic research systems which are individual, customer-specific and adjusted to onsite local conditions and circumstances (fresh & salt water, brackish water...). We
deliver and install aquatic systems with our own in-house technicians and mechanics –
worldwide. Our huge range of accessories, consumables and spare parts (food, water
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supplies, filter material, care products, spare parts, further equipment…) completes our
offering.
Elsevier
Radarweg 29
1043NX
Amsterdam
The Netherlands
Contact Name: Valentina Sasselli
Telephone: +31204853504
Website: http://www.journals.elsevier.com/developmental-biology/
Developmental Biology, the official journal of the Society for Developmental Biology,
publishes original research on mechanisms of development, differentiation and growth
in animals and plants at the molecular, cellular, genetic and evolutionary levels. DB
offers its authors a broad scope of article types, transparent review process handled by
editors who are leading scientists in the field, no limits and charges for pages and color
figures and immediate open access. For more information:
http://www.journals.elsevier.com/developmental-biology/ and www.elsevier.com
Intavis Bioanalytical Instruments AG
Widdersdorferstr. 248-252
Köln
Germany
50933
Contact Name: Andreas Bubis
Telephone: 0049221502946811
Website: www.intavis.com
Next-generation instruments for
immunohistochemistry on Xenopus
the
automation
of
in-situ
detection
and
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Pentair Aquatic Eco-Systems, Inc.
2395 Apopka Blvd.
Apopka FL
United States
32703
Contact Name: Mitch Manning
Telephone: +1 407 886 3939
Website: www.pentairaes.com
Pentair Aquatic Eco-Systems, Inc. is the largest source of aquatic products and systems
worldwide. PAES offers solutions and expertise to improve your aquatic research
project. As pioneers in the industry, we provide the best possible solutions for facilities,
from universities to laboratories and everything in between.
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OVERVIEW OF SHORT TALK AND SPEAKER ABSTRACTS
Sunday 28 August, 2016
Session 1
Main Meeting Room 18.00 - 20.00
S01 18.10 - 18.35
Frog genomes
R M Harland, D S Rokhsar and the Xenopus Genome Consortium
S02 18.35 - 19.00
The Xenopus laevis genome project reveals the features of subgenomes in the
allotetraploid
M Taira and the International Consortium for the Xenopus laevis genome project
PL01 19.00 - 19.50
Maternal Messages to Live By: past, present, future
M L King
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Session 2
S03 08.30 - 08.55
Z Shi, F Wang, H Zhao, Y Chen
O01 08.55 - 09.10
Poster Number: P112
Whole-embryo single-cell analysis of early Xenopus Tropicalis development
J A Briggs, L Peshkin, A M Klein, M W Kirschner
S04 09.10 - 09.35
Reconstitution of centromeric DNA replication reveals suppression of the ATR
dependent checkpoint and formation of DNA loops
V Costanzo
O02 09.35 - 09.50
Poster Number: P109
A S Tseng, C X Kha, Z Li, J Lauper
O03 09.50 - 10.05
Poster Number: P002
R E Stephenson, T Higashi, B Coy, T Arnold, A L Miller
S05 10.05 - 10.30
AmphiBase: Comprehensive Genomic Resource of Amphibians And Its Application to
Characterize ‘unnamed’ Xenopus Genes
T Kwon
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Session 3
S06 11.00 - 11.25
Discovery Mass Spectrometry Finds Metabolic
Blastomeres in the Early Xenopus laevis Embryo
P Nemes, R M Onjiko, E P Portero, S A Moody
Activity
Differences
between
O04 11.25 - 11.40
Poster Number: P083
Isolation of novel cell lines from standard laboratory strains of Xenopus laevis and
Xenopus tropicalis embryos
G J Gorbsky, W Ratzan, M E Horb
S07 11.40 - 12.05
The evolution of advertsiement calls in Xenopus; genomic, developmental and
functional insights
D B Kelley, B J Evans, E C Leininger, C L Barkan, E Zornik
O05 12.05 - 12.20
Poster Number: P010
M V Danilchik, C Wulff
S08 12.20 - 12.45
L Marshall, C Vivien, F Girardot, L Péricard, N Chai, P Scerbo, K Palmier, B A
Demeneix, L Coen
O06 12.45 - 13.00
Poster Number: P091
Development of a fast and efficient genetic platform in Xenopus tropicalis for modeling
human congenital limb malformations
H Thi Tran, L Vlaeminck, T Van Nieuwenhuysen, T Naert, R Noelanders, K Vleminckx
S09 13.00 - 13.25
Budgett’s frog (Lepidobatrachus) embryos reveal mechanisms of left-right asymmetric
organ morphogenesis and new left-right asymmetry genes
N M Nascone-Yoder
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Session 4
S10 17.00 - 17.25
J B Wallingford, J Sedzinski
O07 17.25 - 17.40
Poster Number: P052
12-time point proteomics of Xenopus laevis allows for broad understanding of proteomic
expression file emerging from a mature oocyte to late neurala stage embryo quantifying
more than 6,100 protein profiles
E H Peuchen, L Sun, O F Cox, P Huber, N J Dovichi
O08 17.40 - 17.55
Poster Number: P005
Identification of methylated deoxyadenosines in vertebrates reveals diversity in DNA
modifications
M J Koziol, C R Bradshaw, G E Allen, A S H Costa, C Frezza, J B Gurdon
O09 17.55 - 18.10
Poster Number: P107
The heterochronic gene Lin28 regulates metamorphosis by inhibiting the thyroid
hormone axis
F Faunes, D Guzmán, R Muñoz, J Larraín
PL02 18.20 - 19.00
Measurement of force field during the collective cell migration of Xenopus embryonic
cells
T Negishi, A Miyagi, H Ninomiya, N Ueno
PL03 19.00 - 19.50
Morphogenic Machines of Early Embryogenesis: How embryos put their players on the
field
R Keller, C Chang, K Pfister, D Shook, S Skoglund, J Wen, R Winklbauer
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Session 5
S11 08.30 - 08.55
FGF mediated MAPK and PI3K/Akt Signaling make distinct contributions to the control
of pluripotency and the establishment of the Neural Crest state
C LaBonne
O10 08.55 - 09.10
Poster Number: P055
M Tisler, T Thumberger, I Schneider, A Schweickert, M Blum
S12 09.10 - 09.35
P Tandon, F L Conlon
O11 09.35 - 09.50
Poster Number: P111
H Davaapil, P B Gates, M H Yun
O12 09.50 - 10.05
Poster Number: P058
Identification of new regulators of embryonic patterning and morphogenesis in Xenopus
gastrulae by RNA sequencing
I Popov, T Kwon, D K Crossman, M R Crowley, J B Wallingford, C Chang
S13 10.05 - 10.30
J Chang, H Arbach, M Singh, P Greenside, A Kundaje, J Baker, A Wills
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Session 6
S14 11.00 - 11.25
S Zitouni, M Francia, M Lince-Faria, F Leal, S Gouveia, C Nabais, T Lorca, E Karsenti,
S Kandel-Lewis, M Bettencourt-Dias, A Holland, T Moyer, M Ohta, D Kitagawa
O13 11.25 - 11.40
Poster Number: P037
S Xue, C T Gordon, A Javed, G Yigit, K Chen, A Hillmer, M Blewitt, B Wollnik, J Amiel,
B Reversade
S15 11.40 - 12.05
M C Good
O14 12.05 - 12.20
Poster Number: P024
N Suzuki, T Kumada, H Ogino, H Ochi
S16 12.20 - 12.45
M Kaminski, J Tosic, H Engel, J Klockenbusch, C Kresbach, O Kretz, T Huber, G Walz,
S Arnold, S S Lienkamp
O15 12.45 - 13.00
Poster Number: P012
H Danno, Y Sasagawa, I Nikaido
S17 13.00 - 13.25
Y E Gutkovich, H Polevoy, A Michaelov, D Frank
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Session 7
S18 17.00 - 17.25
D Houston
O16 17.25 - 17.40
Poster Number: P054
P Mancini, J Vardanyan, A M Zorn
S19 17.40 - 18.05
A Nestor-Bergmann, G Stooke-Vaughan, G Goddard, O Jensen, S Woolner
O17 18.05 - 18.20
Poster Number: P050
The newly identified ephrinB2 binding partner, TBC1d24, plays a role in neural crest cell
migration
J Yoon, Y S Hwang, M S Lee, J Sun, K Soria, L Knapik, I Daar
S20 18.20 - 18.45
A role for reactive oxygen species during appendage regeneration and early embryonic
development
N Love, S Ishibashi, Y Han, J Iglesias, Y Y Chen, C Thomson, E Amaya
PL04 19.00 - 19.50
The mechanism of bipolar spindle assembly: Using the Xenopus egg extract system to
unravel the chromosome RanGTP dependent microtubule assembly pathway and its
integration with the centrosomal pathway in animal cells
I Vernos
24
Session 8
S21 08.30 - 08.55
The Ska complex is recruited to kinetochores by Ndc80 proteins clustered along
microtubule protofilaments
T Stukenberg
O18 08.55 - 09.10
Poster Number: P046
Distribution and signaling of Wnt and BMP ligands are modulated by secreted
antagonists and heparosan/heparan sulfate proteoglycans in Xenopus embryos
T Yamamoto, Y Mii, S Takada, M Taira
S22 09.10 - 09.35
A new view of cell state transitions and their timing, based on the dynamics of gene
expression
N Papalopulu
O19 09.35 - 09.50
Poster Number: P097
Xenopus epidermal mucin forms a barrier to infection and is important for
osmoregulation
E Dubaissi, K Rousseau, R K Grencis, I S Roberts, D J Thornton
O20 09.50 - 10.05
Poster Number: P116
A M Butler, L Wang, D A Owens, K M Newman, M L King
S23 10.05 - 10.30
A Gambus, S Priego Moreno
25
Session 9
S24 11.00 - 11.25
M E Pownall, S W Fellgett, R J Maguire
O21 11.25 - 11.40
Poster Number: P072
A Novel GTPase System Regulates β-Catenin Nuclear Transport in Development and
Disease
J N Griffin, A R Duncan, F del Viso, A Robson, S Kulkarni, K J Liu, M K Khokha
O22 11.40 - 11.55
Poster Number: P106
Answer, the gene lost during evolution in higher vertebrates regulates regeneration and
early forebrain development in Xenopus laevis
D D Korotkova, A S Ivanova, V A Lubetsky, A V Seliverstov, M B Tereshina, A M
Nesterenko, A G Zaraisky
S25 11.55 - 12.20
Tissue- and stage-specific Wnt target gene expression is controlled subsequent to betacatenin recruitment to cis-regulatory modules
Y Nakamura, E de Pavia Alves, G J C Veenstra, S Hoppler
O23 12.20 - 12.35
Poster Number: P065
V Khedgikar, K Mathavan, H Cousin, D Alfandari
O24 12.35 - 12.50
Poster Number: P040
B Erdogan, G Cammarata, A Francl, L A Lowery
S26 12.50 - 13.15
R A Ritter, B Breszinska, L Kelly, H M El-Hodiri, A K Sater
26
Session 10
S27 17.00 - 17.25
A W Brandli
O25 17.25 - 17.40
Poster Number: P017
Ric-8A/Gα13 signaling pathway is required to proper cranial neural crest migration in
Xenopus
G Toro-Tapia, A Beyer, S Villaseca, J I Leal, R Mayor, M Torrejon
S28 17.40 - 18.05
The role of thyroid hormone receptor alpha during larval growth and intestinal stem cell
development
J Choi, D R Buchholz
O26 18.05 - 18.20
Poster Number: P031
A Ciau-uitz, A Kirmizitas, R Stephenson, R Patient
O27 18.20 - 18.35
Poster Number: P063
Ciliary transcription factors and miRNAs precisely regulate Cp110 levels at basal bodies
required for ciliogenesis
P Walentek, I Quigley, D I Sun, U K Sajjan, C Kintner, R M Harland
S29 18.35 - 19.00
S A Moody, K M Neilson, D Alfandari
27
OVERVIEW OF ABSTRACTS SELECTED FOR POSTERS
Poster Number: P1
POSTER WITHDRAWN
Poster Number: P2
Poster Number: P3
Mechanisms of Cell Division and Size Scaling in Pipid Frogs
K Miller, R Heald
Poster Number: P4
Anillin regulates epithelial tension at tissue and cellular scales
T R Arnold, K Dinshaw, T Higashi, R E Stephenson, A L Miller
Poster Number: P5
modifications
M J Koziol, C R Bradshaw, G E Allen, A S H Costa, C Frezza, J B Gurdon
Poster Number: P6
Characterization of oxidized Cytosine derivatives in Xenopus development
V Hatch, D Han, M Musheev, C Niehrs
Poster Number: P7
Neil2 stimulates Tdg-mediated BER in active DNA demethylation in Xenopus
embryogenesis
D Han, M Musheev, C Niehrs
Poster Number: P8
The regulatory role of Transmembrane Protein Tyrosine Phosphatase in Wnt signaling
L Chang, M Kim, A Glinka, C Niehrs
Poster Number: P9
The role of adsorption on the extracellular matrix in the diffusion of morphogens within
the intercellular space
A G Zaraisky, A M Nesterenko, A V Bayramov, F M Eroshkin, N Y U Martynova, G V
Ermakova, D D Korotkova
Poster Number: P10
M V Danilchik, C Wulff
28
Poster Number: P11
Identifying Phenotypic Convergence Among Autism-Associated Genes in X. tropicalis
H R Willsey, A J Willsey, R M Harland
Poster Number: P12
Poster Number: P13
Tissue- and stage-specific Wnt target gene expression is controlled subsequent to βcatenin recruitment to cis-regulatory modules
Y Nakamura, E de Paiva Alves, G J Veenstra, S Hoppler
Poster Number: P14
Maternally expressed transcription factor Foxh1 bookmarks the genome before the
onset of zygotic gene activation and regulates endoderm programming
R Le-Charney, J S Cho, M Fish, E Forouzmand, J Cheung, X Xie, I L Blitz, K W Y Cho
Poster Number: P15
Human Serotonin type 3 receptor (HT3): Evidences for interactions with canonical Wnt
signaling
A Schweickert, M Maerker, S Schmitteckert, S Bogusch, B Niesler
Poster Number: P16
MicroRNAs in Neural Crest development
N Ward, G N Wheeler
Poster Number: P17
Xenopus
G Toro-Tapia, A Beyer, S Villaseca, J I Leal, R Mayor, M Torrejon
Poster Number: P18
Uch37 mediates DNA binding of Tcf1 through its deubiquitinating activity during
Xenopus gastrulation
W Han, B R Keum, J K Han
Poster Number: P19
Elucidating the Role of Epigenetic Regulation in Neural Crest Cells Formation
M Marin-Barba, G N Wheeler
Poster Number: P20
Nodal is a conserved neural inducer in chordates
L Kodjabachian, G Luxardi, P Scerbo, M Cibois, Y Le Petillon, H Escriva, S Bertrand
29
Poster Number: P21
Gremlin and Bone Morphogenetic Protein signalling in Xenopus dorsoventral patterning
J Pegge, A J Tatsinkam, M Dhomeja, C C Rider, E Bell
Poster Number: P22
XDSCR6 (Xenopus Homologue of Human Down Syndrome Critical Region protein 6)
and its partner XEZH2 regulate XSTAT3 activity during embryonic axis formation of
Xenopus laevis
M Loreti, D L Shi, C Carron
Poster Number: P23
Coordinated transcriptional regulation of myogenesis
C McQueen, R J White, M E Pownall
Poster Number: P24
N Suzuki, T Kumada, H Ogino, H Ochi
Poster Number: P25
Leapfrogging: Gene knockout phenotypes in the F1 generation
I L Blitz, M B Fish, K W Y Cho
Poster Number: P26
Dissecting the pre-placodal transcriptome to reveal direct targets of Six1 and Eya1
affecting neurogenesis in cranial placodes
N Riddiford, G Schlosser
Poster Number: P27
Transcriptomic Insights into Genetic Diversity of Protein-Coding Genes in X. laevis
V Savova, E Pearl, M Horb, A Nag, A Gimelbrant, L Peshkin
Poster Number: P28
Structural and expression analyses of the Xenopus laevis Hox clusters
M Kondo, T Yamamoto, S Takahashi, M Taira
Poster Number: P29
Using ATAC-seq to detect novel gene regulatory elements in development and its
application for studying lens formation in Xenopus tropicalis
S Manohar, T Nakayama, A R Bright, M Fisher, G J C Veenstra, R M Grainger
Poster Number: P30
Identification and functional analysis of human RALDH2 genetic variants with reduced
activity using Xenopus embryos
Y Shabtai, S Liu, G G Hicks, A Fainsod
30
Poster Number: P31
Poster Number: P32
Identifying Convergent Transcriptional Signatures Following Loss of Autism-Associated
Genes
A J Willsey, H R Willsey, R M Harland
Poster Number: P33
Durian, a brain-specific secreted peptide impacting behavior
G H Goh, P M Wong, M Garcia-Miralles, M Pouladi, L Ho, B Reversade
Poster Number: P34
Ouro proteins are not essential to tail regression during Xenopus metamorphosis
Y Yaoita, Y Nakai, J Robert, K Nakajima
Poster Number: P35
Molecular and Cellular Mechanisms of Spinal cord Regeneration
J Larrain, G Edwards, D Lee-Liu, E Méndez, R Muñoz, J Peñailillo, V Tapia, E De
Domenico, M Gilchrist, L L Sun, A Cebrian-Silla
Poster Number: P36
Suppression of vascular network formation by chronic hypoxia and prolyl-hydroxylase 2
(phd2) deficiency during Xenopus development
S Metikala, H Neuhaus, T Hollemann
Poster Number: P37
S Xue, C T Gordon, A Javed, G Yigit, K Chen, A Hillmer, M Blewitt, B Wollnik, J Amiel,
B Reversade
Poster Number: P38
Analysis of the in vivo role of late endolysosomal transport function for early
development in Xenopus
J Kreis, P Vick
Poster Number: P39
The unique and collective functions of Transforming Acidic Coiled Coil (TACC) family
members in regulating microtubule plus-end dynamics in vivo
E R Rutherford, L Carandang, P Ebbert, M Evans, C Lucaj, L A Lowery
Poster Number: P40
31
Poster Number: P41
Characterisation of the ADAMTS family in Xenopus
I Desanlis, G N Wheeler, D R Edwards
Poster Number: P42
Downregulation of the cytoskeletal protein Zyxin functioning in cells of the Xenopus
laevis midneurula axial tissues leads to activation of stem cells markers
N Y Martynova, F M Eroshkin, E E Orlov, A G Zaraisky, E B Prokhortchouk
Poster Number: P43
March2 E3 ubiquitin ligase antagonizes canonical Wnt signaling for Xenopus head
formation
H Lee, B R Keum, S M Cheong, J K Han
Poster Number: P44
Emergence of primitive myeloid cells at the mesonephric rudiment in early Xenopus
tadpole
Y Imai, K Ishida, M Nemoto, K Nakata, T Kato, M Maeno
Poster Number: P45
A role of JunB proto-oncogene in tailbud induction and tail regeneration during early
Xenopus embryogenesis
H Yoshida, M Okada, K Takebayashi-Suzuki, N Ueno, A Suzuki
Poster Number: P46
T Yamamoto, Y Mii, S Takada, M Taira
Poster Number: P47
Permanent Blastula-type embryos in Xenopus destined for cell death are rescued by
signals from vegetal cells
M Sakai, H Nodono
Poster Number: P48
A retinoic acid - hedgehog cascade coordinates mesoderm inducing signals and
endoderm competence during lung specification
S A Rankin, L Han, K W McCracken, A P Kenny, C T Anglin, E A Grigg, C W Crawford,
J M Wells, J M Shannon, A M Zorn
Poster Number: P49
Germ line-specific activation of Xenopus tropicalis histone B4 through the proximal
promoter sequence
M Nakamigawa, T Kondo, M Maeno
32
Poster Number: P50
migration
Poster Number: P51
Xenopus CIC: A putative downstream regulator of FGF dependent transcription
M King, P Genever, H V Isaacs
Poster Number: P52
Poster Number: P53
Transcriptional factor Ets1 in heart development and disease
S Nie, L Lin, P Grossfeld
Poster Number: P54
Poster Number: P55
Poster Number: P56
Functional analysis of a novel placode gene Fam46a identified by new placode
induction system under the control of BMP signaling
T Watanabe, Y Ito, Y Onuma, T Michiue
Poster Number: P57
Slco1a2, an ion transporter, is a novel heterotaxy candidate gene that regulates leftright patterning and heart development through a potential role in midline signaling
D Bhattacharya, M K Khokha
Poster Number: P58
I Popov, T Kwon, D K Crossman, M R Crowley, J B Wallingford, C Chang
33
Poster Number: P59
Role of heterotrimeric G-protein Gα13 and Leukemia-associated Rho guaninenucleotine exchange factor (LARG) in RhoA activation and radial intercalation
movements in Xenopus laevis epiboly
D O Kiryukhin, N N Luchinskaya, L A Shustikova, Y Y Kopantseva, M V Zinovyeva, A
V Belyavsky
Poster Number: P60
The difference of shape and tension between neural and epidermal ectodermal cells in
Xenopus
S Yamashita, N Ishinabe, T Ide, T Michiue
Poster Number: P61
The T-box gene Brachyury and its regulative functions during left-right axis development
in Xenopus laevis
S Kurz, P Andre, A Schweickert, M Blum
Poster Number: P62
POSTER WITHDRAWN
Poster Number: P63
P Walentek, I Quigley, D I Sun, U K Sajjan, C Kintner, R M Harland
Poster Number: P64
The optimisation of sperm cryopreservation in Xenopus and analysis of the damage that
it causes
S Morrow, E Pearl, A Noble, A Lerebours, C Sharpe, M Horb, M Guille
Poster Number: P65
Poster Number: P66
Novel genes in congenital heart disease (CHD): using Xenopus to understand heart
development
E K Mis, M K Khokha
Poster Number: P67
Aquaporin3b acts in noncanonical Wnt signaling and convergent extension during
K See, C S Merzdorf, J Forecki
34
Poster Number: P68
Identifying Mink1, a novel CHD target gene, role in development of left-right patterning
and heart formation
V D Colleluori, M K Khokha
Poster Number: P69
Role of Pou3f transcription factors during kidney development
C Cosse-Etchepare, I Gervi, I Buisson, J F Riou, M Umbhauer, R Le Bouffant
Poster Number: P70
Unraveling the role of potassium in early embryo morphogenesis and left-right
patterning
E Sempou, M K Khokha
Poster Number: P71
Determining the Role of Cadherin-11 Cleavage in Cranial Neural Crest Migration
K Mathavan, G Abbruzzese, D Alfandari
Poster Number: P72
Disease
J N Griffin, A R Duncan, F del Viso, A Robson, S Kulkarni, K J Liu, M K Khokha
Poster Number: P73
The chromatin modifier, WDR5, is a multifunctional protein that has dual roles in
ciliogenesis and left-right patterning
S S Kulkarni, J Griffin, K Liem, M Khokha
Poster Number: P74
The role of dashsous2 in congenital heart disease
E D Deniz, A R Robson, N A Al-Mahmoud, M B Brueckner, M K Khokha
Poster Number: P75
Musculocontractural-Ehlers-Danlos-syndrome: Dermatan sulfate
Xenopus neural crest cells to migrate and adhere to fibronectin
N Gouignard, M Maccarana, I Strate, A Malmström, E M Pera
is
required
for
Poster Number: P76
Motile Cilia: Characterization of Novel foxj1 Target Genes in Xenopus and Mouse
T Ott, L Alten, A Beckers, C Adis, A Gossler, M Blum
Poster Number: P77
LRPPRC, a novel role in L/R patterning of the Xenopus embryo
A E MacColl Garfinkel, M Khokha
35
Poster Number: P78
Technical innovations for InDrops single-cell transcriptomics of Xenopus cells
Poster Number: P79
Primitive Myeloid Cells from the Blood Island are Necessary and Sufficient Inducers of
Foregut Progenitors and BMP signaling in Xenopus laevis and Musculus musculus
Z N Agricola, A K Jagpal, S A Rankin, S W Cha, A M Zorn, A P Kenny
Poster Number: P80
Novel secreted protein AFRO regulates anterior formation in amphibian embryos
Y Sato, H Kuroda
Poster Number: P81
Loss-of-function analysis of a large family of nonclassical MHC genes in Xenopus laevis
by the CRISPR/Cas9 system
M Banach, E S Edholm, J Robert
Poster Number: P82
Modelling human diseases in Xenopus; Celf3 protein complex enhances translation
rather than repressing
S Metikala, L Horb, W Thomas, B Suh, N Shaidani, C Collins, M Horb
Poster Number: P83
G J Gorbsky, W Ratzan, M E Horb
Poster Number: P84
Insights into conservation of early developmental program from sequence and
transcriptional comparison between Xenopus and Sturgeon embryogenesis throughout
blastula, gastrula and neurula stages
L Peshkin, A Zaraisky, F Eroshkin, M Kirschner
Poster Number: P85
Development of novel Xenopus-specific single-chain antibodies
M Z Piccinni, M J Guille, V J Allan, C R Sharpe, A Noble
Poster Number: P86
Xenopus laevis developmental proteomics: an upgraded resource
L Peshkin, M Wuehr, J Briggs, M Kirschner
Poster Number: P87
RNA whole-mount In situ Hybridisation Proximity Ligation Assay (rISH-PLA), an assay
for detecting RNA-protein complexes in intact cells
I M Roussis, M J Guille, F A Myers, G P Scarlett
36
Poster Number: P88
Absolute Protein and Phospho-Site Stoichiometry Dynamics during Vertebrate
Fertilization
M Presler, M Wühr, A M Klein, E Van Itallie, J Ingraham, R C Kunz, M Coughlin, L
Peskin, T J Mitchison, S P Gygi, M W Kirschner
Poster Number: P89
RNA species whose transcription is totally silent in pre-MBT stage is not mRNA but
rRNA and possible involvement of weak bases in the transcriptional silence of rRNA
genes during the pre-MBT stage in Xenopus embryogenesis
K Shiokawa, Y Misumi, K Tashiro
Poster Number: P90
Inflammation-mediated blood brain barrier disruption and macrophage infiltration
contribute to disseminate Frog Virus 3 into the brain of X. laevis tadpoles
J Robert, J Wang, F De Jesús Andino
Poster Number: P91
H Thi Tran, L Vlaeminck, T Van Nieuwenhuysen, T Naert, R Noelanders, K Vleminckx
Poster Number: P92
Mitochondrial transport protein Rhot1 is involved in the aggregation of germinal granule
components during primordial germ cell formation in Xenopus
H Tada, Y Taira, K Morichika, T Kinoshita
Poster Number: P93
National Xenopus Resource – serving the Xenopus research community
M Wlizla, R Falco, S Mcnamara, M E Horb
Poster Number: P94
Improving Animal Husbandry Conditions and Care in a Xenopus Laboratory to Facilitate
Frog Development in an Abbreviated Timeframe
S McNamara, M Wlizla, M Horb
Poster Number: P95
Modeling molecular subgroups of medulloblastoma, in Xenopus tropicalis by
CRISPR/Cas9
D Dimitrakopoulou, R Noelanders, T V Nieuwenhuysen, T Naert, K Vleminckx
Poster Number: P96
Xenopus embryos as a model for alcohol-induced developmental growth restriction
N Shukrun, Y Shabtai, A Fainsod
37
Poster Number: P97
osmoregulation
Poster Number: P98
Xenbase: the Xenopus bioinformatics database supports your research
C James-Zorn, V G Ponferrada, M E Fisher, K A Burns, K Karimi, V Lothay, J D
Fortriede, E Segerdell, P Vize, A M Zorn
Poster Number: P99
POSTER WITHDRAWN
Poster Number: P100
SLiM acquisition and alternative splicing mediate the diversity of NCoR-family
corepressors
C R Sharpe, T Peterkin, S Short, R Patient, M Guille
Poster Number: P101
RAF1 loss-of-function mutation causes acro-cardio-facial syndrome in humans by
blocking FGF signaling
N Escande-Beillard, S Wong, A Loh, H Kayserili, B Reversade
Poster Number: P102
POSTER WITHDRAWN
Poster Number: P103
Analysis of Novel Candidates for Short Rib Thoracic Dysplasia (SRTD) Associated
Genes
M Getwan, S Lienkamp
Poster Number: P104
Thyroid hormone transporters in Xenopus and their susceptibility to xenobiotics
B Mughal, M Leemans, L Marshall, S Le Mevel, T Visser, B Demeneix, J B Fini
Poster Number: P105
POSTER WITHDRAWN
Poster Number: P106
D D Korotkova, A S Ivanova, V A Lubetsky, A V Seliverstov, M B Tereshina, A M
Nesterenko, A G Zaraisky
38
Poster Number: P107
hormone axis
F Faunes, D Guzman, R Muñoz, J Larraín
Poster Number: P108
Characterizing the role of foxm1 during tail regeneration in Xenopus tropicalis
D Pelzer, K Dorey
Poster Number: P109
Poster Number: P110
Investigating how thyroid hormone impedes cardiac regeneration with CRISPR/Cas9
L Marshall, C Vivien, N Chai, B Mughal, F Girardot, L Péricard, P Scerbo, K Palmier, S
le Mevel, J B Fini, J P Concordet, B Demeneix, L Coen
Poster Number: P111
Poster Number: P112
Poster Number: P113
FGF-mediated activation of MAPK and PI3K/Akt controls the lineage restriction of
pluripotent blastula stem cells
L Geary, C LaBonne
Poster Number: P114
Exploiting CRISPR/Cas9-mediated Xenopus tropicalis cancer models for identification
of novel drug targets and pre-clinical therapeutic compound validation
T Naert, T Van Nieuwenhuysen, R Colpaert, R Noelanders, D Dimitrakopoulou, H T
Tran, D Creytens, A Boel, K Vleminckx
Poster Number: P115
Snail1 is essential for the maintenance of pluripotency in blastula animal pole cells
A N Rao, C LaBonne
Poster Number: P116
A M Butler, L Wang, D A Owens, K M Newman, M L King
39
Poster Number: P117
High Throughput Analysis Reveals Novel Maternal Germline RNAs Critical for PGC
Preservation and Proper Migration
D A Owens, A M Butler, K M Newman, T H Aguero, D Van Booven, M L King
Poster Number: P118
The Tumor-Suppressor BAP1 Promotes Expression of Differentiation Genes in
Ectodermal Derivatives
M L King, J Kuznetsov, T H Aguero, W Harbour
Poster Number: P119
A comparison of junction-centric versus exon-centric analysis for identification of Ptbp1dependent splicing events in Xenopus laevis
M Noiret, A Mereau, G Angrand, M Bervas, C Gautier-Courteille, V Legagneux, S
Deschamps, H Lerivray, J Viet, S Hardy, L Paillard, Y Audic
Poster Number: P120
The necessity of nitric oxide during the embryonic epidermis development
S Tomankova, P Abaffy, R Sindelka
Poster Number: P121
PAWS1/FAM83G is a positive regulator of the canonical WNT pathway
K S Dingwell, P Bozatzi, T Cummins, G P Sapkota, J C Smith
Poster Number: P122
States of the heart: insights from the cardiac transcriptome during post-embryonic
development, aging and regeneration
F Girardot, L Marshall, L Pericard, C Vivien, L Coen, B Demeneix
Poster Number: P123
Implications of Knockout versus Knockdown in Xenopus Embryos
T Spruce, R S Monteiro, J C Smith, G E Gentsch
Poster Number: P124
A new regulator of Groucho/TLE activity in fate determination of the Spemann organizer
B C Durand, N Rocques, E Sena
Poster Number: P125
Improving the usefulness of the European Xenopus Resource Centre (EXRC) to the
research community
A Noble, M Piccinni, A Jafkins, G Nicholson, L Nazlamova, V Allan, C Sharpe, M Guille
40
Poster Number: P126
National BioResource Project (NBRP) for Xenopus: recent developments at the Asian
hub for the international Xenopus research community
A Suzuki, K Kashiwagi, H Hanada, N Furuno, I Tazawa, A Kurabayashi, K Nakajima, K
Takebayashi-Suzuki, T Igawa, M Sumida, H Yoshida, S Murakami, K Oriha, T Mido, M
Masumoto, K Kawaguchi, A Miura, A Kashiwagi
41
SHORT TALK AND SPEAKER ABSTRACTS
Date: Sunday 28 August, 2016
Session: Session 1
Time: 18.00 - 20.00
Venue: Main Meeting Room
S01 18.10 - 18.35
Frog genomes
R M Harland, D S Rokhsar and the Xenopus Genome Consortium
Molecular and Cell Biology and Center for Integrative Genomics, University of California,
Berkeley, CA, USA
The progress in sequencing, assembly and annotation of frog genomes will be presented. A
high quality genome assembly of X tropicalis and X. laevis is complete, and other Xenopus
species are being added. In addition outgroups that have been solicited by the commnity are in
progress.
S02 18.35 - 19.00
The Xenopus laevis genome project reveals the features of subgenomes in the
allotetraploid
M Taira and the International Consortium for the Xenopus laevis genome project
Department of Biological Sciences, University of Tokyo, Tokyo, Japan
One main issue of the X. laevis genome project is to elucidate the structure of its allotetraploid
genome and to evaluate the evolutional consequence of the subgenomes derived from the
hybridization of the parental diploid species 17 million years ago. Based on whole genome
sequences, BAC-FISH, phylogenetic relationships with X. tropicalis, and karyotypic
observations (Long and Short homeologous chromosome sets), the X. laevis chromosomes
(XLA) are re-numbered as XLA1L, XLA1S, XLA2L, XLA2S, and so on. Using “fossil” DNA
transposons and unique repetitive sequences, we successfully identified the two subgenomes,
each of which turned out to reside on the L or S chromosome set. Therefore, homeologous
genes are now referred to as “gene.L” and “gene.S,” which were previously arbitrarily called "a"
and "b" genes. The identification of the subgenomes now allows us to investigate how
allotetraploidization differentially affected each subgenome in chromosome stability, gene
contents, subfunctionalization of genes, etc.
42
PL01 19.00 - 19.50
Maternal Messages to Live by: past, present, future
M L King
Plenary/Prospectives Talk
43
Date: Monday 29 August, 2016
Session: Session 2
Time: 08.30 - 10.30
S03 08.30 - 08.55
Z Shi1, F Wang1, H Zhao2, Y Chen1
1
Department of Biology, South University of Science and Technology of China, Shenzhen,
China; 2School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong,
China
Xenopus tropicalis is an emerging vertebrate genetic model. So far gene knock-in method has
not been reported in this species. Here, we report that heritable targeted integration can be
achieved in this diploid frog using a concurrent cleavage strategy mediated by the clustered
regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated 9
(CRISPR/Cas9) system. For the three distinct loci tested, all showed efficient targeted
integration that was verified by both germ line transmission and Southern blot analyses. By
designing the target sites in introns, we were able to get precise editing of the tyrosinase coding
sequence and GFP expression from endogenous n-tubulin promoter and enhancers. Last but
not least, we were unable to detect off-target effects with the T7EI assay. Precise editing of
protein coding sequences in X. tropicalis expands the utility of this diploid frog, such as for
establishing models to study human inherited diseases.
O1 08.55 - 09.10
Poster Number: P31
Department of Systems Biology, Harvard Medical School, Cambridge, USA
We have applied InDrops, a droplet-based single-cell transcriptomic technology developed in
our lab, to profile 30,000 single-cell transcriptomes sampled over a timecourse of early Xenopus
Tropicalis development. Our data span activation of the zygotic genome (NF stage 8) to the
formation of progenitors for most major organ systems (NF stage 22). Unsupervised analysis of
the resulting data reveals a comprehensive atlas of embryonic cell types. For every cell type we
measure its genome wide transcriptional profile, and thus discover gene expression modules
containing dozens of new marker genes that are specific to each cell state. We are developing
novel bioinformatics strategies to connect cell states over time into branching gene expression
topologies to study transcriptional principles of cell fate choices in the embryo. In a single
experiment our data reveal the gene expression dynamics associated with every cell fate choice
during early embryogenesis.
44
S04 09.10 - 09.35
Reconstitution of centromeric DNA replication reveals suppression of the ATR
dependent checkpoint and formation of DNA loops
V Costanzo
DNA Metabolism, IFOM, Milan, Italy
Over the past few years we have successfully used Xenopus laevis egg cell-free extract to
recapitulate the function of DNA repair and checkpoint proteins. However, it is still unclear how
these proteins work at specific chromosome loci, especially the ones containing DNA
sequences difficult to replicate. Centromeric DNA in many eukaryotic organisms contains large
regions of alpha satellite DNA. We reconstituted replication of centromeric DNA in Xenopus
laevis egg extract. The replicating centromeric chromatin proteome revealed the enrichment of
several DNA repair factors. However, activation of the ATR dependent checkpoint monitoring
replication origin firing was completely suppressed. Using electron microscopy based analysis
of replication intermediates we revealed that replicating centromeric DNA forms secondary
structures resembling DNA loops arranged in spring-like structures, which might play a
functional role in centromere organization. These findings have profound implications on our
understanding of repetitive DNA metabolism and centromere assembly under normal and
stressful conditions.
O2 09.35 - 09.50
Poster Number: P109
School of Life Sciences, University of Nevada Las Vegas, Las Vegas, USA
Vertebrate eye development is complex and requires early interactions between neuroectoderm
and epidermis. In Xenopus, individual eye tissues such as the retina, lens and cornea, can
undergo regeneration. However, partial removal of the specified eye field during neurulation or
the tadpole stage does not induce replacement. Here we describe a model for investigating eye
regrowth. We found that tailbud embryos can readily regrow eyes after removal of the specified
eye tissues. This is a rapid process as an eye of similar size to the control is seen by 4 days and
development is normal. The regrown eye contains a full complement of eye cell types, connects
to the brain and is functional. Eye regrowth also requires the same early mechanisms
(apoptosis and bioelectrical signaling) as appendage regeneration. Together, our findings
indicate that frog embryos can re-initiate development of the eye after tissue loss and that this
process requires regenerative mechanisms.
45
O3 09.50 - 10.05
Poster Number: P002
Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann
Arbor, MI, USA
The small GTPase RhoA is an important regulator of cell-cell junctions. We recently described
transient accumulations of active RhoA (“Rho flares”) at junctions in the epithelium of gastrulastage Xenopus laevis embryos. In order to investigate the cause and consequence of Rho
flares, we co-imaged active Rho with fluorescently-tagged junction proteins. Intriguingly, the
tight junction proteins ZO-1 and Occludin are locally decreased prior to the Rho flare and are
increased, or reinforced, following the flare. Additionally, Rho flares are accompanied by
deformation of the plasma membrane, and F-actin accumulates on either side of the membrane
deformation. Here, I will present data that tests the mechanism through which Rho flares
reinforce cell-cell junctions (via actin polymerization and/or junction contraction) and
investigates the signal that triggers the flare. Our data suggest that localized activation of Rho is
needed to repair and remodel junctions to maintain the barrier function and integrity of
developing epithelia.
S05 10.05 - 10.30
AmphiBase: Comprehensive Genomic Resource of Amphibians and Its Application to
Characterize ‘unnamed’ Xenopus Genes
T Kwon
Department of Biomedical Engineering, School of Life Sciences, Ulsan National Institute of
Science and Technology, Ulsan, Republic of Korea
Recently finished Xenopus laevis genome project reported 45,099 protein-coding genes, “the
largest gene set of any vertebrate so far sequenced”. However, based on sequence similarity,
more than ten thousand X. laevis genes, and similarly about 5,000 X. tropicalis genes, do not
have orthologous gene candidates in any other vertebrates. Many of them showed specific
expression patterns in various conditions, so it is speculated that they are functional. However,
because of similar annotation result, it is difficult to infer their roles just with the genomic
resources of two closed Xenopus species.
Here, I present AmphiBase, the collection of de novo assembled transcripts derived from RNAseq data of 12 amphibian species. Together with over 25 million de novo assembled Xenopus
transcripts and their expression patterns, I will present how we can predict their functions, and
distinguish them to mis-annotated genes or pseudogenes.
46
Session: Session 3
Time: 11.00 - 13.30
S06 11.00 - 11.25
Discovery Mass Spectrometry Finds Metabolic Activity Differences between Blastomeres
in the Early Xenopus laevis Embryo
P Nemes1, R M Onjiko1, E P Portero1, S A Moody2
1
Department of Chemistry, The George Washington University, Washington, DC, USA;
Department of Anatomy and Regenerative Biology, The George Washington University,
Washington, DC, USA
2
Discovery (untargeted) characterization of biomolecular networks provides a powerful
opportunity to help better understand normal embryonic development. How small molecules
(metabolites) are implicated in the establishment of tissue fates is less known because it has not
been technologically feasible to measure a broad spectrum of metabolites with single-cell
sensitivity. We herein describe a custom-built high-resolution mass spectrometry (HRMS)
instrument with sufficient sensitivity to enable discovery analysis of single blastomeres. We use
this platform to uncover previously unknown metabolic activity differences between blastomeres
that have different tissue fates in the cleavage stage Xenopus laevis embryo. Furthermore, in
combination with fluorescence cell tracking and microinjection, we discover metabolites that are
able to alter the developmental fate of blastomeres. These results suggest exciting new
functional roles for metabolites at an early stage of embryonic development, long before
transcription of the embryo’s own genome is known to begin.
O4 11.25 - 11.40
Poster Number: P083
G J Gorbsky1, W Ratzan2, M E Horb2
1
Cell Cycle and Cancer Biology Research Program, Oklahoma Medical Research Foundation,
Oklahoma City, USA; 2National Xenopus Resource and Eugene Bell Center for Regenerative
Biology, Marine Biological Laboroatory, Woods Hole, USA
We are developing novel cell lines from the standard Xenopus laboratory strains, X. laevis J
strain and X. tropicalis Nigerian strain, using digested embryos. Thus far, we have isolated and
cloned three new X. laevis and five new X. tropicalis cell lines. The lines have been passaged
multiple times and grow without limit. They can be frozen, and regenerate new cultures when
thawed. Most chromosome spreads prepared from all three X. laevis lines and four of the five X.
tropicalis lines exhibit normal ploidy. We anticipate that these lines will be amenable to modern
gene editing techniques allowing the generation of homozygous mutant cells, which can then be
cloned to produce isogenic populations. We expect that nuclei from these cells, transferred to
enucleated eggs, will generate F0 mutant embryos. This approach has the potential to radically
transform and simplify the production of mutant Xenopus embryos, tadpoles, and adults.
47
S07 11.40 - 12.05
The evolution of advertsiement calls in Xenopus; genomic, developmental and functional
insights
D B Kelley1, B J Evans2, E C Leininger3, C L Barkan1, E Zornik4
1
Biological Sciences, Columbia University, New York, USA; 2Biology, McMaster University,
Hamilton, Canada; 3Biology, St. Mary's College of MD, St. Mary's City, USA; 4Biology, Reed
College, Portland, USA
Twenty-nine Xenopus species have been described using molecular variation, morphology,
karyotypes, geographic ranges and vocalizations (e.g. Evans et al., 2015). While acoustic
features of the release call are shared (Tobias et al., 2014), characteristics of the male
advertisement call - sound pulse temporal patterns (call type) and component frequencies - are
unique species identifiers (Tobias et al., 2011). Using "fictive" calling evoked in the isolated
brain (Rhodes et al., 2007) and larynx (Kelley & Tobias, 1995), we determined that convergence
on a click-type call in two genetically distant species uses divergent neural and muscular
mechanisms (Leininger & Kelley, 2013). In one - X. borealis - a subset of sexually differentiated
laryngeal features was lost (Leininger et al., 2015). In two genetically similar species - X. laevis
and X. petersii - membrane properties contribute to trill-type call length. Crosses producing
fertile hybrids will test genome-wide associations between loci and call features.
O5 12.05 - 12.20
Poster Number: P010
M V Danilchik1, C Wulff1,2
1
Integrative Biosciences, Oregon Health and Science University, Portland OR, USA; 2Biology,
Mountain View High School, Vancouver WA, USA
Unexpectedly, the perivitelline (PV) fluid of the pregastrulation Xenopus embryo was found to
contain an abundance of microvesicles. Proteomic analysis of PV fluid via mass spectroscopy
indicates an enrichment of exosome-associated proteins, and electron microscopy confirms the
presence of large numbers of particles in a size range typical of exosomes (~50-150 nm).
Exosomes are known to carry on their surfaces a complement of proteins involved with cell-cell
recognition and signaling, and appear to be effective in transferring mRNAs and mRNAs
between cells. Thus, as demonstrated in various tissue-culture and pathogenic situations,
exosomes have the potential to alter gene expression patterns of cells receiving them. Whether
this exciting property is utilized during normal embryonic development is largely unexplored.
Here, we report that embryos deprived of PV-borne exosomes develop with a significant
reduction in hematopoiesis, suggesting that early tissue specification involves exosomemediated signaling across extracellular spaces in the embryo. Support: NSF IOS-1557527.
48
S08 12.20 - 12.45
L Marshall1, C Vivien1,2, F Girardot1, L Péricard1, N Chai1, P Scerbo1,3, K Palmier1,4, B A
Demeneix1, L Coen1
1
UMR7221, MNHN - CNRS, Paris, France; 2UQ Center of Cardiac and Vascular Biology,
University of Queensland, Brisbane, Australia; 3IBDM, Aix-Marseille Universite, Marseille,
France; 4Microbiologie, I2BC, Orsay, France
Overcoming the pathological consequences of ischemic heart disease requires animal models
for cardiac regenerative studies. Zebrafish are of interest as they maintain robust lifelong
cardiac regeneration. Mice provide a mammalian model with characteristic loss of regenerative
capacity at seven days post-natal. Cardiac regeneration in amphibians, including the anuran
amphibian Xenopus, is less studied, even though this model has been highly used to explore
regenerative processes in other tissues. To gain such knowledge, we took advantage of the THinduced metamorphic period in Xenopus laevis to study the putative link between the thyroid
hormone (TH) status and heart regenerative capacity. We show that metamorphosis or short
exogenous TH exposure hinders cardiac regeneration.
O6 12.45 - 13.00
Poster Number: P091
H Thi Tran1, L Vlaeminck1, T Van Nieuwenhuysen1, T Naert1, R Noelanders1, K Vleminckx1,2
1
Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium; 2Center for
Medical Genetics, Ghent University, Ghent, Belgium
Malformations of the upper or lower limbs are rather common, affecting about 1 in 500 live
births. Despite the current improvements in finding the genetic defects underlying the
malformations, many cases remain unresolved. Not surprisingly, most mutations could be found
in genes known to mediate initiation, outgrowth and patterning of the early limb bud, such as
FGF, Wnt, Shh. However, mutations or genome rearrangements may also affect regulatory
regions in the non-coding genome. Improved methods for identification of structural defects,
copy number detection and whole exome and whole genome sequencing produce massive
amounts of data leading to the identification of new candidate disease-linked genes. Now, there
is a growing need for functional assays to assess the causality, where Xenopus tropicalis, as a
diploid tetrapod, is ideally positioned. Via CRISPR/Cas9 mediated genome editing, we hope to
substantially facilitate the discovery of new genes that are linked to limb malformations in
humans.
49
S09 13.00 - 13.25
Budgett’s frog (Lepidobatrachus) embryos reveal mechanisms of left-right asymmetric
organ morphogenesis and new left-right asymmetry genes
N M Nascone-Yoder
Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State
University, Raleigh, USA
Anatomical left-right (LR) asymmetries are integral to physiological function. The early
embryonic LR asymmetry pathway is well established, but little is known regarding the
downstream genes or cellular events that orchestrate asymmetric organ morphogenesis. To
address this issue, we have pioneered the use of a new model, the Budgett’s frog
(Lepidobatrachus). The extra-large cells and embryos of this species allow visualization of the
cellular morphogenetic processes that differ between contralateral sides of asymmetric organs.
In addition, the large size facilitates fine-scale manual microdissection of left and right subregions of early organs for RNAseq and proteomic profiling, providing a unique platform to
identify molecules involved in lateralized morphogenesis. Using this system, we identified new
organ-specific left- and right-sided factors, including signaling molecules, transcription factors
and ECM components. Analyses of the expression and function of these new genes in
Lepidobatrachus and Xenopus is revealing discrete roles in LR asymmetric morphogenesis.
50
Session: Session 4
Time: 17.00 - 20.00
S10 17.00 - 17.25
J B Wallingford, J Sedzinski
Molecular Biosciences, University of Texas, Austin, USA
Epithelial sheets are crucial components of all metazoan animals. Epithelial homeostasis poses
unique challenges, as addition of new cells and loss of old cells must be achieved without
disrupting the fluid-tight barrier and apicobasal polarity of the epithelium. While several studies
have identified cell biological mechanisms underlying extrusion of cells from epithelia, far less is
known of the converse mechanism by which new cells are added. We have combined
molecular, pharmacological, and laser dissection experiments with time-lapse imaging and
theoretical modeling to characterize the forces driving emergence of an apical surface as single
nascent cells are added to a vertebrate epithelium in vivo.
O7 17.25 - 17.40
Poster Number: P052
Chemistry and Biochemistry, University of Notre Dame, Notre Dame, USA
Proteomics is a relatively new endeavor of Xenopus laevis research with the first of a series of
papers being published in 2014. This study examines the proteomic changes taking part in
development starting with the mature oocyte and looking through the neurala stage (stage 22)
by analyzing 12-distinct time points. For a majority of the proteins, initial data shows either high
expression of the protein before fertilization or post fertilization, but not consistently high or
consistently low expression for both a mature oocyte and a fertilized egg. In this study, 6,148
quantifiable proteins and 58,485 peptide sequences (21.5 % average sequence coverage or 9.5
peptides/ protein) were identified in biological and technical duplicate. This study provides a
solid baseline of proteins essential for fertilization and early development.
51
O8 17.40 - 17.55
Poster Number: P005
modifications
M J Koziol1,2, C R Bradshaw1, G E Allen1, A S H Costa3, C Frezza3, J B Gurdon1,2
1
Wellcome Trust Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge,
UK; 2Department of Zoology, University of Cambridge, Cambridge, UK; 3Hutchison/MRC
Research Centre, University of Cambridge, Cambridge, UK
Methylation of cytosine deoxynucleotides (dC5m) is a well-established epigenetic mark, but in
higher eukaryotes much less is known about modifications affecting other deoxynucleotides. We
report the detection of N-6-methyl-deoxyadenosine (dA6m) in vertebrate DNA, specifically in
Xenopus laevis, but also in other species including mouse and human. Our methylome analysis
reveals that dA6m is widely distributed across the eukaryotic genome, is present in different cell
types, but commonly depleted from gene exons. Thus, direct DNA modifications might be more
widespread than previously thought.
O9 17.55 - 18.10
Poster Number: P107
hormone axis
Center for Aging and Regeneration, Millennium Nucleus in Regenerative Biol, P. Universidad
Catolica de Chile, Santiago, Chile
The link between hormones and heterochronic genes in regulating developmental transitions
has only recently been explored. In Xenopus, the thyroid hormones (TH) are crucial for
metamorphosis. We studied the role of the heterochronic gene Lin28 during metamorphosis.
Lin28 levels decreased before the increase of the TH-target genes thr-b and klf9. To study the
role of Lin28 we generated transgenic animals that overexpress Lin28 under the control of a
heat-shock promoter. Overexpression of Lin28 from pre-metamorphosis significantly delayed
metamorphosis compared to controls. This delay was correlated with a lower endogenous
activation of thr-b and klf9 in Lin28 overexpressing animals. Exogenous administration of TH
rescued the delay induced by Lin28. Transcriptome and proteome analyses are currently been
carried out to determine cellular processes downstream Lin28.
These results indicate that Lin28 regulates metamorphosis by inhibiting TH signaling, linking the
heterochronic gene network with the hormonal axis in vertebrates.
FUNDINGS: FONDECYT 11130564 (FF), CARE-PFB12/2007 (JL), MILENIO RC120003 (JL)
52
PL02 18.20 - 19.00
Measurement of force field during the collective cell migration of Xenopus embryonic
cells
T Negishi, A Miyagi, H Ninomiya, N Ueno
Developmental Biology, National Institute for Basic Biology, Okazaki, Japan
During gastrulation of Xenopus, the leading edge mesoderm (LEM) cells crawl on the substrate
of blastocoel roof (BCR) exhibiting a coordinated cell movement toward the anterior. This
oriented LEM movement can be partially reproduced on a glass slide on which ECMs are
transferred from BCR. We have found that cells of LEM explants migrating show intriguing
movements within the explant vigorously changing their relative positions in the explant. We
hypothesized that in this particular tissue movement, the convective motion of cells
cooperatively generate a force that drives the oriented movement as a whole explant and
therefore attempted to measure force field of the migrating explant by traction force microscopy
(TFM). Our preliminary results suggest that force is not evenly produced within the explant and
that cells may share roles for the force generation depending on the position within the explant.
PL03 19.00 - 19.50
Morphogenic Machines of Early Embryogenesis: How embryos put their players on the
field
R Keller1,2, C Chang3, K Pfister2, D Shook1, S Skoglund1, J Wen4, R Winklbauer4
1
Department of Biology, University of Virginia, Charlottesville, Virginia, USA; 2Department of Cell
Biology, University of Virginia, Charlottesville, Virginia, USA; 3Department of Cell Biology,
University of Alabama, Birmingham, Alabama, USA; 4Department of Cell and Systems Biology,
University of Toronto, Toronto, Ontario, Canada
I will discuss new findings on three major morphogenic machines, Convergent Extension,
Tissue Subduction, and Convergent Thickening. All generate similar large, long-range tensile
forces that shape the body plan of amphibians, but each uses different cell behaviors.
Convergent Extension develops tensile force by oriented, actomyosin-driven cell motility and
cell intercalation. Tissue Subduction uses an unpolarized apical actomyosin contraction that
produces a polarized result due to its mechanical context. Convergent Thickening is driven by a
decrease in tissue affinity, resulting in surface tension-driven thickening, an isotropic process
that is again polarized by mechanical context. All are used in varying degrees, combinations,
and spatial configurations, with varying dynamics, and in different mechanical contexts in
different amphibians. A systems-level mechanobiology, the "mechanome" of embryonic
morphogenesis, is emerging from these and the inspiring studies of others. Support:NIH
R37HD025594 MERIT Award and S1ARRA(R.K.), NIH TG GM008136(K.P.), NIH
HD069297(C.C.), NIH GM099108(P. S.) and CIHR Grant MOP-53075(R.W.)
53
Date: Tuesday 30 August, 2016
Session: Session 5
Time: 08.30 - 10.30
S11 08.30 - 08.55
FGF mediated MAPK and PI3K/Akt Signaling make distinct contributions to the control of
pluripotency and the establishment of the Neural Crest state
C LaBonne
Molecular Biosciences, Northwestern University, Evanston, IL, USA
Blastula embryos possess cells that are pluripotent. This potential is progressively lost as
lineage restriction initiates, however, neural crest cells retain broad developmental potential.
Here we provide insights into the signals that are essential for the pluripotency of blastula cells
and subsequent retention of this potential in neural crest cells. We show that FGF signaling
controls the expression of genes expressed by pluripotent blastula cells, and subsequent
lineage restriction of these cells. Remarkably, the signaling cascades that mediate FGF
signaling change as cells lose pluripotency and commence lineage restriction. Pluripotent cells
display robust Map Kinase signaling, whereas PI3 Kinase-mediated AKT signals increase as
developmental potential is restricted. We provide evidence that Map Kinase is essential for
pluripotency, and that PI3 Kinase regulates transit to lineage restricted states while opposing
establishment of neural crest. These findings shed important new light on signal-mediated
control of pluripotency, and the molecular mechanisms governing the genesis of neural crest.
O10 08.55 - 09.10
Poster Number: P055
Zoology, University of Hohenheim, Stuttgart, Germany
Experimental analysis of laterality started in 1918, when Spemann and Falkenberg
demonstrated that organ situs in ligature-induced conjoined newt twins was normal in the left
and randomized in the right twin. We analyzed Wnt pathway-induced Siamese twins in
Xenopus, which displayed two adjacent gastrocoel roof plates with wildtype ciliation. Flow was
continuous from the right side of the right to the left side of the left twin. Motile cilia were
required for situs in twins, as Nodal cascade induction was abrogated in flow-impaired twins;
Nodal was rescued by parallel knockdown of Dand5 on the left side of the left twin, and the
cascade was inverted when ablation of flow was combined with Dand5-MOs on the right side of
the right twin. Together these experiments demonstrate that GRP cilia and flow determine organ
laterality in conjoined frog twins, which is likely the case in human siamese twins as well.
54
S12 09.10 - 09.35
P Tandon1,2,3, F Conlon1,2,3
1
Department of Biology, UNC at Chapel Hill, Chapel Hill, USA; 2Department of Genetics, UNC at
Chapel Hill, Chapel Hill, USA; 3McAllister Heart Institute, UNC at Chapel Hill, Chapel Hill, USA
The development of the vertebrate embryonic heart occurs by hyperplastic growth as well as the
incorporation of cells from tissues outside of the initial heart field. Amongst these tissues is the
epicardium, a cell structure that develops from the precursor proepicardial organ on the right
side of the septum transversum caudal to the developing heart. During embryogenesis, cells of
the proepicardial organ migrate, adhere and envelop the maturing heart, forming the
epicardium. As a resident source of pluripotent cells and mitogenic stimulation, the epicardial
layer is critical for the continued growth and function of the heart. Through a systems-based
approach we recently identified a new panel of epicardial genes whose expression is dependent
on the transcription factor Tcf21. We have used genome editing to generate Xenopus laevis that
are genetically null for a number of these genes including the kielin/chordin-like protein (kcp), a
novel modifier of the TGFβ signaling pathway. Our analysis shows that kcp function is essential
with null animals dying post-metamorphosis; a stage equivalent to mammalian adolescence. By
adapting ultrasound Doppler imaging to live Xenopus froglets we demonstrated that animals
lacking kcp have reduced blood flow and impaired cardiac function. Further analysis has
identified a requirement for kcp during atrioventricular valve formation. Collectively, these gnetic
studies establish a role for kcp in the epicardium and its derivatives and provides mechanistic
insights into the role of epicardial paracrine signaling during valve morphogenesis.
O11 09.35 - 09.50
Poster Number: P111
Structural and Molecular Biology, University College London, London, UK
Cellular senescence is an anti-tumourigenic mechanism which can lead to disruption of tissue
structure and function. Indeed, accumulation of senescent cells contributes to age-related
disorders in mammals. Nevertheless, senescent cells can also contribute to physiological
processes, as illustrated by the recent finding that cellular senescence plays functions during
mammalian development. The degree of conservation of developmental senescence through
phylogeny, and the extent of its functions during development, remains unknown.
Here, we show that cell senescence is an intrinsic part of the developmental programme in
amphibians. Programmed senescence occurs in specific structures at defined time-windows
during amphibian development. It contributes to the physiological degeneration of the
pronephros and to the development of the cement gland and neighbouring regions, such as the
oral cavity. Our findings uncover conserved and new roles of senescence in vertebrate
organogenesis and support the view that cellular senescence may have arisen in evolution as a
developmental mechanism.
55
O12 09.50 - 10.05
Poster Number: P058
I Popov1, T Kwon2, D K Crossman1, M R Crowley1, J B Wallingford3, C Chang1
1
Cell, Developmental and Integrative Biology, University Alabama at Birmingham, Birmingham,
USA; 2Biomedical Engineering, Ulsan National Institute of Science and Technology, Repulic of
Korea; 3Molecular Biosciences, University of Texas at Austin, Austin, USA
Cell fate specification is often coupled with specific cell behaviors during development. Though
many genes with embryonic inducing ability have been identified, factors that control cell
movements are less understood. To uncover new regulators of embryonic patterning and
motility, we performed RNA sequencing to investigate differentially expressed genes in early
organizer, the dorsal and the ventral marginal zone of Xenopus gastrulae. Upon confirmation of
a panel of differentially expressed genes by RT-PCR, we examined a selected subset for their
ability to block activin-induced animal cap elongation. Several genes interfered with cap
elongation without affecting mesodermal induction by activin, but an ECM protein, EFEMP2,
inhibited activin signaling. We further showed that a secreted protein kinase PKDCC1 regulated
gastrulation movements as well as anterior neural patterning. Overall, our studies identify many
differentially expressed signaling and cytoskeleton regulators in Xenopus gastrulae and imply
their functions in regulating cell fates and/or behaviors during gastrulation.
S13 10.05 - 10.30
J Chang1, H Arbach2, M Singh2, P Greenside1, A Kundaje1, J Baker1, A Wills2
1
Genetics, Stanford University, Stanford, CA, USA; 2Biochemistry, University of Washington,
Seattle, WA, USA
Regeneration represents a unique challenge with respect to gene regulation. The complex
inputs of stress responses, proliferation, apoptosis, differentiation and patterning cues are all
critical to successful regeneration. This dense gene regulatory environment is difficult to
understand through isolated studies of individual genes. To generate a coherent picture of gene
regulation during regeneration, we generated two time series datasets during tail regeneration in
Xenopus tropicalis: RNA-Seq transcriptional data and ATAC-Seq chromatin accessibility data.
By integrating these datasets we identify differentially expressed transcription factors that are
responsible for regulating a large proportion of changes in chromatin accessibility during
regeneration. Primary drivers of regeneration identified using this approach include bHLH, FoxO
and myeloid-specific transcription factors. Unexpectedly, we also find that regeneration is
characterized by rapid synchronous remodeling of the entire chromatin landscape, which is
evident at both the scale of small regulatory motifs and overall nuclear morphology.
56
Session: Session 6
Time: 11.00 - 13.30
S14 11.00 - 11.25
S Zitouni1, M Francia1, M Lince-Faria1, F Leal1, S Gouveia1, C Nabais1, T Lorca2, E Karsenti3, S
Kandel-Lewis3, M Bettencourt-Dias1, A Holland4, T Moyer4, M Ohta5, D Kitagawa5
1
CCR, Instituto Gulbenkian de Ciencia, Oeiras, Portugal; 2CNRS, CRBM, Montpellier, France;
Directors Research, EMBL, Heidelberg, Germany; 4John Hopkins University, Baltimore,
Maryland, USA; 5National Institute of Genetics, Japan
3
Centrioles are essential for the assembly of both centrosomes and cilia. Centriole biogenesis
occurs once and only once per cell cycle and is temporally coordinated with cell-cycle
progression, ensuring the formation of the right number of centrioles at the right time.The
PLK4/STIL complex triggers centriole biogenesis. Here we show that in mitosis, the mitotic
kinase CDK1-CyclinB binds STIL and prevents formation of the PLK4-STIL complex and STIL
phosphorylation by PLK4, thus inhibiting untimely onset of centriole biogenesis. After CDK1CyclinB inactivation upon mitotic exit, PLK4 can bind and phosphorylate STIL in G1, allowing
pro-centriole assembly in the subsequent S phase. Our work shows that complementary
mechanisms, such as mother-daughter centriole proximity and CDK1-CyclinB interaction with
centriolar components, ensure that centriole biogenesis occurs once and only once per cell
cycle, raising parallels to the cell-cycle regulation of DNA replication and centromere formation.
O13 11.25 - 11.40
Poster Number: P037
S Xue1, C T Gordon2, A Javed3, G Yigit4, K Chen5, A Hillmer3, M Blewitt5, B Wollnik4, J Amiel2, B
Reversade1
1
Laboratory of Human Genetics and Embryology, Institute of Medical Biology (A*STAR),
Singapore; 2Laboratory of Embryology and Genetics of Congenital Malformations, Institut
Imagine (INSERM), Paris, France; 3Cancer Therapeutics and Stratified Oncology, Genome
Institute of Singapore (A*STAR), Singapore; 4Institute of Human Genetics, University of
Cologne, Cologne, Germany; 5The Walter and Eliza Hall Institute of Medical Research,
University of Melbourne, Melbourne, Australia
Nose shape varies between species, the longest being that of the elephant. An extreme aplasia
of the nose is seen in congenital arhinia, a rare condition characterized by a complete absence
of the nose. With less than 50 cases reported in the literature, its genetic and molecular basis
remains unknown. Through trio whole-exome sequencing of 12 unrelated patients with varying
degrees of arhinia ranging from nose hypoplasia to Bosma syndrome (a severe disease
with ear, eye, palate abnormalities and absent nose), we identified 10 independent de
novo mutations in the same gene. Its product, an enzyme with DNA binding activity, is known to
be important for X-inactivation and genomic imprinting in mice. We hypothesize that by
modifying DNA structure and methylation, this epigenetic regulator serves as a master controller
57
of a gene network involved in nose development. Using patient cells and Xenopus embryos, we
propose that the uncovered heterozygous mutations behave as gain-of function alleles with
neomorphic activity.
S15 11.40 - 12.05
M C Good
Cell and Developmental Biology, University of Pennsylvania, Philadelphia, PA, USA
To grow and survive, cells must adapt to fluctuations. For example, cells sense and respond to
external chemical stimuli, such as growth factors, using receptors and intracellular signaling
pathways. However,little is known about how these same pathways adapt to fluctuations in
cellular dimensions. Cell size and shape vary broadly, from one-millimeter eggs to elongated
neurons with sub-micron extensions. In silico studies suggest that the rates of intracellular
signaling are sensitive to cell geometry but it has proven challenging to test these models in
vivo. Additionally, extreme cell sizes, such as those found in Xenopus blastomeres, present
separate, unique challenges to the internal reactions of a cell. My lab investigates the
specialized adaptations necessary to facilitate signaling in the giant, millimeter-size cells of
amphibian embryos. Using synthetic cells and Xenopus egg extracts we are exploring the
impacts of cell geometry and spatial patterning of signaling molecules on intracellular signal
transmission.
O14 12.05 - 12.20
Poster Number: P024
N Suzuki1, T Kumada1, H Ogino2, H Ochi1
1
Institute for Promotion of Medical Science Research, Faculty of Medicine, Yamagata
University, Yamagata, Japan; 2Department of Animal Bioscience, Nagahama Institute of BioScience and Technology, Nagahama, Japan
Amphibians and fish can regenerate many tissues and recover normal function after injury,
whereas mammals have a limited regenerative capacity. Currently, it is speculated that tissue
regeneration recapitulates developmental programs because the expression of developmental
genes is reactivated during regeneration in many cases. However, it is unclear whether the
unique cis-regulatory elements specific to tissue regeneration are activated during tissue
regeneration or whether mammals have hidden cis-regulatory elements that can be activated in
regenerating tissues and merely suppress their function. Here, we searched for regeneration
signal response enhancers in lhx1 locus, and found that the noncoding elements conserved
between only highly regenerative species do not show strong enhancer activities in the
regenerating amphibian pronephros. Instead, the noncoding elements conserved from fish to
human function as enhancers in the regenerating pronephros. This result suggests that
mammals whose regenerative abilities are limited still retain regeneration signal response
enhancers in their genome.
58
S16 12.20 - 12.45
M Kaminski1, J Tosic1,2, H Engel1, J Klockenbusch1, C Kresbach1, O Kretz1, T Huber1,3, G
Walz1,3, S Arnold1,3, S S Lienkamp1,3
1
Renal Division, University Hospital Freiburg, Freiburg, Germany; 2Spemann Graduate School
of Biology and Medicine (SGBM), Alber-Ludwigs-University Freiburg, Freiburg, Germany;
3
BIOSS Centre of Biological Signalling Studies, Albert-Ludwigs-University Freiburg, Freiburg,
Germany
Recently, enormous progress has been made to generate kidney tissue in vitro. Induced
pluripotent stem cells were differentiated into kidney tissue by replicating conditions of
embryonic renal development. This method, however, requires stem cells as source material.
An alternative technique is direct reprogramming, where expression of transcription factors
converts differentiated cell types without prior induction of pluripotency. Fibroblasts have been
converted to many cell types (cardiomyocytes, neurons, hepatocytes) but not yet to renal tissue.
We use both Xenopus embryos and mammalian cells to identify transcription factors that induce
a renal tubule cell specific identity. We determined the minimum set of factors with an
evolutionary conserved expression and role in renal development to induce renal cell types.
Partially overlapping sets of factors drive either embryonic tissue of Xenopus or mouse
fibroblasts towards a tubular cell fate. These results may also elucidate core transcriptional
programs active in kidney development and disease.
O15 12.45 - 13.00
Poster Number: P012
Advanced Center for Computing and Communication, RIKEN, Wako, Japan
In vertebrate embryos, neural induction starts during gastrulation. Simultaneously, induced
neuroectoderm is regionalized along its anterior-posterior, medial-lateral and apical-basal axes,
and starts differentiation into many cell types that have specific functions in future central
nervous system. However, the mechanism by which dozens of cell type are robustly generated
from almost-uniform cell population is not understood. In this study, we approached this
question with high-throughput single-cell RNA-seq. Hundreds of single cells from presumptive
ectoderm of Xenopus laevis early neurula were analyzed with Drop-seq. Quantification of
mRNA molecules in each cell led to identification of cell type clusters and rare populations.
Furthermore, combining this data with whole-mount in situ hybridization images of several
genes enabled us to infer spatial position of analyzed cells and spatial expression pattern of
hundreds of genes. Using these, we construct a model for regionalization of neuroectoderm.
59
S17 13.00 - 13.25
Y E Gutkovich, H Polevoy, A Michaelov, D Frank
Biochemistry, Technion - Israel Institute of Technology, Haifa, Israel
The paradigm of neural AP patterning suggests a gradient in which high caudalizing Wnt-activity
induces spinal cord, intermediate levels induce hindbrain, while lowest Wnt-levels enable
forebrain formation. We suggest reevaluating this paradigm.
1. Anteriorized embryos (inhibited Wnt-activity) lose the hindbrain, but have remarkably
normal spinal cord marker expression and morphology.
2. Knockdown of Wnt-downstream Zic proteins disrupts hindbrain fates, while expanding
forebrain and spinal cord marker expression domains.
3. In animal caps/ACs, spinal cord markers are not induced by BMP-antagonists and
neural caudalizers, while hindbrain markers are induced. BMP induces spinal cord
markers, while inhibiting hindbrain markers in embryos and ACs. BMP induction of spinal
cord markers is FGF-dependent.
Interactions between canonical Wnt-signaling and BMP-activity/antagonism in neural patterning
must be reassessed. A high-to-low gradient of Wnt-activity appears incompatible with spinal
cord and hindbrain specification. Low-moderate BMP-signaling (+FGF-activity) may induce
spinal cord. These findings challenge present paradigms of neural AP patterning.
60
Session: Session 7
Time: 17.00 - 20.00
S18 17.00 - 17.25
D W Houston, D Oh
Department of Biology, The University of Iowa, Iowa City, USA
In Xenopus and zebrafish, transport of dorsal determinants in the zygoteis thought to require
syntabulin (sybu), which encodes a kinesin linker protein localized to the oocyte germ plasm.
This germ plasm consists of mitochondria, germ line granules, and various RNAs, and is critical
for primordial germ cell (PGC) specification. Although sybu mRNA is lost from zygotic germ
plasm in fish, it persists in the germ plasm of early Xenopus embryos through the gastrula
stage, suggesting an additional role in PGC specification. Here we show that maternal
antisense inhibition of sybu results in embryos lacking PGCs by the tailbud stage. Analysis of
early embryos revealed altered germ plasm dynamics, resulting in mislocalized and reduced
aggregation of germ plasm along cleavage furrows of the four vegetal cells and failure of
perinuclear germ plasm accumulation. We further present experiments elucidating specific
kinesin-like protein interactions and mechanisms regulating Sybu function in the germline.
O16 17.25 - 17.40
Poster Number: P054
Division of Developmental Biology, Cincinnati Children's Hospital Research Foundation,
Cincinnati, OH, USA
Congenital birth defects of the trachea and esophagus are quite common (~1/3000) and often
life-threatening conditions caused by disrupted foregut tube morphogenesis. Despite this, the
cellular and molecular mechanisms underlying normal and defective tracheo-esophageal (TE)
morphogenesis are poorly understood. Using Xenopus as a complement to mouse genetic we
showed that TE morphogenesis occurs in five conserved steps: 1) D/V foregut patterning, 2)
medial constriction, 3) epithelial fusion and septation, 4) tube elongation and 5) recanalization.
Medial constriction is the result of localized mesenchymal proliferation. Epithelial fusion and
septation are regulated by dynamic cell-adhesion and ECM remodeling that drive an
“unzippering” process that is concurrent with a mesenchymal invasion. Finally we showed that
loss of Hedgehog/Gli signaling, which is mutated in some human TE birth defect patients,
disrupts medial constriction and epithelial septation, leading to tracheal atresia and tracheoesophageal fistula, modeling the human phenotypes.
61
S19 17.40 - 18.05
A Nestor-Bergmann1,2, G Stooke-Vaughan1, G Goddard1, O Jensen2, S Woolner1
1
Faculty of Life Sciences, University of Manchester, Manchester, UK; 2School of Mathematics,
University of Manchester, Manchester, UK
Cell division timing and orientation must be carefully regulated in order to shape tissues and
determine cell fate, preventing defective embryonic development and diseases such as cancer.
In single cells, mechanical cues (e.g. tensile forces) are known to influence cell division rate and
orientation, but we know very little about how this applies to cells in tissues. Using Xenopus
laevis as our experimental model, we aim to reveal how mechanical cues influence cell division
in a multi-layered, developing tissue. Subjecting animal cap tissue explants to reproducible
tensile (stretching) forces, we observe that division rate increases upon stretch and cells divide
oriented along the axis of stretch. We are now using this system to investigate potential
regulators of division response to tensile force, including non-muscle myosin-2, vinculin and
FAK. Furthermore, we are utilising this data to build mathematical models, exploring how cells
respond to mechanical forces transmitted across complex tissue environments.
O17 18.05 - 18.20
Poster Number: P050
migration
Laboratory of Cell and Developmental Signaling, National Cancer Institute, Frederick, MD, USA
Although Eph-ephrin signaling contributes to the migration of cranial neural crest (CNC) cells, it
is still unclear how ephrinB transduces signals affecting this event. Using ephrinB2
immunoprecipitation and mass spectrometric analysis, we identified an interaction between
ephrinB2 and TBC1d24 that is mediated by Dishevelled. Both ephrinB2 and TBC1d24 morphant
embryos display abnormal CNC cell migration, which is rescued by expressing their wild type
counterparts. However, a TBC1d24 mutant that cannot interact with ephrinB2 fails to rescue the
TBC1d24 morphant defect. TBC1d24 is known as a GAP for Rab35 that regulates cell-cell
adhesion and cell migration through regulating cadherin recycling. Both ephrinB2 and TBC1d24
morphants display increased E-cadherin levels that may disrupt normal CNC migration. In
addition, binding of the EphB4 receptor, decreases the interaction between ephrinB2 and
TBC1d24, and thus inhibits CNC cell migration. Our results indicate that TBC1d24 is a critical
player in ephrinB2 control of CNC cell migration.
62
S20 18.20 - 18.45
A role for reactive oxygen species during appendage regeneration and early embryonic
development
N Love, S Ishibashi, Y Han, J Iglesias, Y Y Chen, C Thomson, E Amaya
Healing Foundation Centre, University of Manchester, Manchester, UK
We are investigating the molecular and cellular mechanisms responsible for scarless wound
healing and tissue regeneration in frog embryos and tadpoles. We have shown that tadpole tail
amputation induces a sustained production of reactive oxygen species (ROS), which is
necessary for tail regeneration. More recently we have found remarkable parallels in the
induction, maintenance and role of ROS during tissue regeneration with those seen during
embryonic development. Intriguingly, both injury and fertilization seem to set in motion a similar
series of events, and as such, we have begun to think of fertilization as an injury, which induces
development, in much the same way that injury induces a regenerative response, at later
stages. Thus, we postulate that a sustained production of ROS following injury promotes a
return to an embryonic-like state of cellular oxidation, which facilitates growth factor signaling
and cellular metabolism, thereby promoting tissue specification, patterning and growth.
PL04 19.00 - 19.50
The mechanism of bipolar spindle assembly: Using the Xenopus egg extract system to
unravel the chromosome RanGTP dependent microtubule assembly pathway and its
integration with the centrosomal pathway in animal cells
I Vernos1,2,3
1
Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology,
Barcelona, Spain; 2Universitat Pompeu Fabra (UPF), Barcelona, Spain; 3Institucio Catalana de
Recerca i Estudis Avancats (ICREA), Barcelona, Spain
Cell division involves the assembly a microtubule-based apparatus, the bipolar spindle that
segregates the duplicated chromosomes to the two daughter cells. Spindle assembly relies on
acentrosomal microtubule nucleation around the chromosomes by a RanGTP regulated
complex that we recently identified. Another essential RanGTP-regulated complex interacts
specifically with the minus-ends of these microtubules and defines K-fiber MT minus-end
dynamics.
The chromosome-dependent microtubule assembly pathway is sufficient for bipolar spindle
assembly in cells lacking centrosomes like the vertebrate oocyte. However, the male gamete
provides a centrosome to the egg upon fertilization and therefore all subsequent cell divisions
occur in the presence of centrosomes that act as dominant microtubule organizing centers. We
have used Xenopus egg extracts to study the necessary adaptation required to integrate the
chromosome-dependent spindle assembly pathway that is dominant in the egg cytoplasm with
the activity of the duplicated centrosomes during the assembly of the first embryonic bipolar
spindle.
63
Date: Wednesday 31 August, 2016
Session: Session 8
Time: 08.30 - 10.30
S21 08.30 - 08.55
The Ska complex is recruited to kinetochores by Ndc80 proteins clustered along
microtubule protofilaments
P Janczyk1, K Skorupka1, J Tooley1, D Matson1, S Sivakuma2, G Alushin3, E Nogales3, G
Gorbsky2, O Pornillos1, T Stukenberg1
1
University of Virginia, School of Medicine, Virginia, USA; 2Oklahoma Medical Research
Foundation, Oklahoma City, USA; 3University of California, Berkeley, USA
Kinetochores bind microtubules of the spindle and couples this attachment to the silencing of
the spindle checkpoint (SAC) signal that blocks the metaphase to anaphase transition. This is in
part accomplished by having the same domain of the Ndc80 protein both directly bind
microtubules and recruit the MPS1 kinase that generates the signal. To silence the SAC the Ska
protein must also recruit the PP1 phosphatase to kinetochores. It is not understood how Ska is
recruited to kinetochores and how this is linked to microtubule attachments. We characterize a
novel mutant of the Ndc80 unstructured tail that allow proper kinetochore microtubule
attachments and the generation of full pulling forces, but is unable to exit mitosis. Ska is not
recruited to the kinteochore in cells expressing this mutant. The mutant can bind microtubules
with similar affinity as the wild type protein in vitro, but poorly clusters along protofilaments of
bound microtubules. In addition, it displays negative cooperativity in equilibrium binding assays
consistent with its inability to cluster on microtubules. Using EM tomography we have identified
a structure on microtubules that forms only when both the Ska and Ndc80 complexes are
present and we hypothesize that this structure is the core kinetochore-microtubule linkage of
metazoans. Insights gained from this structure integrate the structure, cell biology and
biochemistry of the Ska complex to explain how Ska works with multiple Ndc80s to build the
“foot” of the metazoan kinetochore. Our data identifies a new function of the Ndc80 tail to
regulate the recruitment of Ska protein to control the exit from mitosis. In addition, our in vivo
and in vitro data suggest that the clustering of Ndc80 calponin homology subunits along
protofilaments is required to recruit Ska. We suggest that these mechanisms act downstream of
MPS1 displacement to allow kinetochores to mature end-on attachments and couple the binding
of microtubules to spindle checkpoint silencing.
64
O18 8.55 - 9.10
Poster Number: P046
T Yamamoto1, Y Mii1,2, S Takada2, M Taira1
1
Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo,
Japan; 2Division of Molecular and Developmental Biology, National Institute for Basic Biology,
Okazaki, Japan
Morphogens including Wnt and BMP confer regional identities along the body-axes in a
concentration-dependent manner, and heparan sulfate (HS) is a key component for distribution
and reception of morphogens. However, the molecular basis understanding of graded
distribution of ligands remains to be clarified. Here we discovered two discrete punctate
clusters of HS and its nascent chain heparosan (Hn) on the cell surface. Notably, HS and Hn
clusters have distinct properties: (i) association preferences (HS for Wnt and Hn for Frzb and
Wnt/Frzb complexes), and (ii) internalization tendency (HS is higher than Hn), and thereby HS is
necessary for short-range distribution and signalling of Wnt, whereas Hn is necessary for longrange distribution for Wnt/Frzb complexes. Furthermore, we show that secreted BMP antagonist
Cerberus is colocalized with Hn, and expands BMP distribution and signalling. These results
suggest that HS/Hn clusters are a fundamental platform essential for morphogen distribution
and signalling.
S22 9.10 - 9.35
A new view of cell state transitions and their timing, based on the dynamics of gene
expression
N Papalopulu
Faculty of Life Sciences, University of Manchester, Manchester, UK
Our understanding of how cells make cell state transitions has been transformed by the
application of single cell molecular technologies. In neural progenitors, single cell imaging with
unstable reporters has revealed asynchronous oscillations in regulatory gene expression, which
is masked by static measurements of population averages. Using single cell quantitative
approaches, live imaging, multiple experimental model systems and mathematical modeling, I
will show how a relatively simple transcription factor/miR regulatory network is capable of
transitioning from pulsatile gene expression to a stable state, autonomously but also in a timecontrolled manner. Although autonomous, the timing can be “tuned” by external influences such
as a change in parameters or the initial conditions. Finally, I will present some unpublished data
on how intrinsic stochasticity, due to finite number of interacting molecules, may be beneficial
for spreading the timing of differentiation, conferring robustness to the progenitor state and
tolerance to cell division asymmetries.
65
O19 9.35 - 9.50
Poster Number: P097
osmoregulation
Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK
The native freshwater habitat of Xenopus tropicalis is microbe-rich, but before developing
adaptive immunity, tadpoles must employ robust innate defence mechanisms to protect
themselves against infection. The skin is the principal route of infection in young tadpoles and
so must be protected. One of the ways the tadpole does this is to generate a mucus barrier on
its skin surface. Through proteomic analysis, we have identified a major mucin glycoprotein.
This mucin is similar in sequence to human mucins, which form the structural basis of mucus
barriers. Through knockdown studies, we have shown that the epidermal mucin is critical for
protection against infection from a known opportunistic pathogen, Aeromonas hydrophila, and
have uncovered a potential role for the mucin in osmoregulation. We have also identified a
number of mucin-interacting molecules (e.g. FCGBP) conserved in human mucosa. We propose
the tadpole skin as a powerful model to study live mucus biology.
O20 9.50 - 10.05
Poster Number: P116
A M Butler1, L Wang2, D A Owens1, K M Newman1, M L King1
1
Department of Cell Biology, University of Miami Miller School of Medice, Miami, USA;
Department of Biology, University of Miami, Coral Gables, USA
2
Germ plasm contains the genetic information that protects Xenopus PGCs from somatic
differentiation and initiates a unique gene expression program that preserves totipotent
potential. Here, we utilized RNA-sequencing to comprehensively interrogate PGC and
neighboring endoderm cell RNAs after segregation from the endoderm lineage. Over 3,000
transcripts were upregulated in PGCs. The top 300 genes upregulated in PGCs were analyzed
and the following gene ontology processes were identified: cell cycle regulation, meiosis,
reproduction, progesterone signaling, motility, protein folding, and apoptosis. Network analysis
revealed 18 hubs linking 125/300 genes in a network including the transcription factors e2f1,
pou5f3.3 (oct60, the Oct3/4 homologue), and sox7. Interestingly, initial functional studies show
that PGC-directed oct60 inhibition increases the number of PGCs in the early embryo, whereas
over-expression or inhibition of sox7 reduces PGC number. These data suggest that sox7 and
oct60 likely mediate germline specification in the early embryo. NIH GM102397 and
HD072340 (MLK).
66
S23 10.05 - 10.30
A Gambus, S Priego Moreno
Institute for Cancer and Genomic Sciences, University of Birmingham, Birmingham, UK
To ensure duplication of the whole genome DNA replication initiates from thousands of origins.
The pre-loaded inactive helicase (Mcm2-7) becomes activated during replication initiation. The
progressing replication fork moves through the chromatin until it encounters fork from the
neighboring origin, which leads to replication forks termination.
Using the Xenopus laevis egg extract system, we have shown (Priego Moreno et al. Science
2014) that blocking polyubiquitylation results in the prolonged association of the active helicase
with chromatin. It was accompanied by a defect in replication forks termination. Mcm7 was the
only helicase subunit polyubiquitylated during S-phase and only when forks can terminate.
Finally, disassembly of the helicase was dependent on p97/VCP/Cdc48 segregase activity.
This process of replisome dissolution is conserved through evolution (Maric et al. Science
2014), however, the ubiquitin ligase driving it is not. I will present our recent findings on the
identity of this enzyme in higher eukaryotes.
67
Session: Session 9
Time: 11.00 - 13.30
S24 11.00 - 11.25
M E Pownall1, S W Fellgett2, R J Maguire2
1
Biology, University of York, York, UK; 2The Bateson Centre, University of Sheffield, Sheffield,
UK
An important regulator of Wnt signalling is the heparan sulfate specific 6-O-endosulfatase Sulf1.
Sulf1 acts extracellularly to modify the structure of heparan sulfate chains to affect the bioavailability of Wnt ligands. We have shown that Sulf1 influences the formation of Wnt signalling
complexes to modulate the activation of both canonical and non-canonical pathways by
employing well-established assays in Xenopus to investigate the ability of Sulf1 to modify
canonical and non-canonical Wnt signalling. In addition, we model the ability of Sulf1 to
influence morphogen gradients using fluorescently tagged Wnt ligands in ectodermal explants.
We show that Sulf1 overexpression has ligand-specific effects on Wnt signalling: it affects
membrane accumulation and extracellular levels of tagged Wnt8a and Wnt11b ligands
differently, and inhibits the activity of canonical Wnt8a but enhances the activity of noncanonical Wnt11b.
O21 11.25 - 11.40
Poster Number: P072
Disease
J N Griffin1,2, A R Duncan1, F del Viso1, A Robson1, S Kulkarni1, K J Liu2, M K Khokha1
1
Pediatrics, Yale University School of Medicine, New Haven, USA; 2Craniofacial Development
and Stem Cell Biology, King's College London, London, UK
Canonical Wnt signaling coordinates many critical aspects of embryonic development, while
dysregulated Wnt signaling contributes to numerous common diseases, including congenital
malformations and cancer. The nuclear localization of β-catenin is fundamental in pathway
activation. However, despite intensive investigation, the mechanisms regulating β-catenin
nuclear transport remain undefined. β-catenin nuclear transport is energy (GTP) dependent but
β-catenin lacks a classic “NLS” nuclear localization signal and does not require the
karyopherin/Ran GTPase transport system. Here, we describe a nuclear localized guanine
nucleotide exchange factor (GEF) that is an unexpected regulator of β-catenin nuclear transport.
Identified in a patient with congenital heart disease and heterotaxy, we show that this GEF
alters left-right patterning via Wnt signaling and the nuclear localization of β-catenin, rather than
β-catenin degradation. Together, our results define a novel GTPase based system that
facilitates nuclear transport of β-catenin, and suggest new targets for the modulation of Wnt
signaling in disease.
68
O22 11.40 - 11.55
Poster Number: P106
D D Korotkova1,2, A S Ivanova1, V A Lubetsky3, A V Seliverstov3, M B Tereshina1, A M
Nesterenko4, A G Zaraisky1
1
Laboratory of Molecular Bases of Embryogenesis, Shemyakin-Ovchinnikov Institute of
Bioorganic Chemistry, Russian Academy of, Moscow, Russia; 2Department of Embryology,
Faculty of Biology, Lomonosov Moscow State University, Moscow, Russia; 3The Institute for
Information Transmission Problems, Russian Academy of Sciences, Moscow, Russia;
4
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University,
Moscow, Russia
It has been shown recently that reduction of regenerative capacity in higher vertebrates could
be explained by extinction of some genes that control regeneration in lower vertebrates. We
have investigated now expression and physiological function of one of such genes, named
Answer (Anamniotic specific wound epithelium receptor). This gene has been identified
previously during bioinformatics screening of vertebrate genomes for genes lost in higher
vertebrates. Answer encodes transmembrane protein and probably operates as a receptor of
unknown ligand(s). We have shown that in Xenopus laevis embryo Answer is expressed in
dorsal ectoderm, including neural plate, and it is sharply activated during regeneration in the
wound epithelium. The inhibition of Answer function leads to increase of the forebrain and
decrease in regeneration. Our data indicate that loss of Answer in higher vertebrates could
result in the reduction of regenerative capacity in exchange for the progressive evolution of the
forebrain.
S25 11.55 - 12.20
Tissue- and stage-specific Wnt target gene expression is controlled subsequent to betacatenin recruitment to cis-regulatory modules
Y Nakamura1, E de Pavia Alves2, G J C Veenstra3, S Hoppler1
1
Institute of Medical Sciences, University of Aberdeen, Aberdeen, Scotland, UK; 2Centre for
Genome-Enabled Biology and Medicine, University of Aberdeen, Aberdeen, Scotland, UK;
3
Department of Molecular Developmental Biology, Radboud University, Nijmegen, The
Netherlands
Recruitment of nuclear beta-catenin to target genomic loci serves as the hallmark of target gene
regulation by canonical Wnt signalling. A direct comparison in Xenopus embryos of genomewide beta-catenin occupancy with a stage-matched wnt8a-regulated transcriptome reveals that
only a subset of beta-catenin-bound genomic loci is transcriptionally regulated by wnt8a. We
uncover that Wnt signalling regulates beta-catenin binding to Wnt targets not only when they are
transcriptionally regulated, but also in contexts in which their transcription remains unaffected.
The transcriptional response to wnt8a signalling in Xenopus depends on additional mechanisms
such as BMP or FGF signalling, which, however, do not influence beta-catenin recruitment. Our
findings suggest a new, more general paradigm for Wnt-regulated transcriptional mechanisms.
Chromatin association of beta-catenin, even to functional Wnt-response elements, can no
longer be considered a proxy for identifying transcriptionally Wnt-regulated genes. Context-
69
dependent mechanisms are crucial for transcriptional activation of Wnt target genes subsequent
to beta-catenin recruitment.
O23 12.20 - 12.35
Poster Number: P065
Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, USA
The cranial neural crest cells (CNC) are migratory pluripotent stem cell population that
contribute to craniofacial development. Our results show that ADAM13 regulates the
protocadherin PCNS, a protein essential for CNC migration, at both transcriptional and posttranscriptional levels. This regulation depends on both the proteolytic activity and the presence
of the ADAM cytoplasmic domain. Once translated, PCNS is cleaved by ADAM13 releasing an
extracellular fragment (EC1-4). Overexpression of PCNS inhibits CNC migration and is rescued
by overexpression of ADAM13. Conversely reduction of ADAM13 is significantly rescued by
PCNS re-expression and EC1-4. In contrast, a mutant form of PCNS with an extra EGF repeat
immediately before the transmembrane domain, designed to misaligned the ADAM13 cleavage
site, does not rescue CNC migration. Thus, ADAM13 regulate CNC migration by regulating
PCNS expression and cleaving its extracellular domain. Our goal is to understand how ADAM13
regulates gene expression.
O24 12.35 - 12.50
Poster Number: P040
Biology, Boston College, Chestnut Hill, USA
A fundamental question in neuronal development is how growth cone cytoskeletal dynamics are
coordinated to promote accurate axonal navigation. To address this question, we focus on
microtubule plus-end tracking proteins (+TIPs), which may play a key role in axon guidance. We
determined that TACC3 is a +TIP that promotes microtubule polymerization and axon
outgrowth. We have begun to test the hypothesis that TACC3 spatially restricts microtubule
polymerization in response to guidance signals in Xenopus laevis axons in vivo and ex vivo. We
demonstrate that TACC3 is required to promote axon outgrowth and prevent spontaneous
retractions. Additionally, we find that manipulation of TACC3 levels interferes with the growth
cone response to axon guidance cues. Finally, we observe that ablation of TACC3 causes
pathfinding defects in Xenopus laevis embryos. Together, our findings suggest that TACC3
functions as an axon guidance-regulating factor in embryonic neurons by spatially promoting
microtubule polymerization dynamics.
70
S26 12.50 - 13.15
R A Ritter1, B Breszinska2, L Kelly2, H M El-Hodiri2, A K Sater1
1
Deptartment of Biology and Biochemistry, University of Houston, Houston TX, USA; 2Center for
Molecular and Human Genetics, The Research Institute at Nationwide Children's Hospital,
Columbus OH, USA
To investigate roles of microRNAs (miRs) in eye field specification, we expressed a myc-tagged
Argonaute (Ago) under the control of the rax1 promoter and asked whether the Eye Field
Transcription Factors (EFTFs) were associated with Ago and thus presumably regulated
by miRs. Transcripts for all major EFTFs co-immunoprecipitated with Ago in late neurula
embryos. Predictions of miR binding sites within EFTF 3’UTRs suggested that miR-199a-3p
(“miR-199”) was a candidate regulator of eye development. Overexpression of miR-199 causes
a reduction in eye size and coloboma in over 75% of embryos. Excess miR-199 alters
partitioning between the eye field and forebrain, and reduces cell proliferation within the eye
field. Unexpectedly, targeted knockdown of miR-199 inhibits eye formation. While miR-199
knockdown leads to decreased EFTF expression, the eye field is unchanged in late neurulae.
Current studies pursue the identification of miR-199-targeted regulators of eye development,
based on transcriptome-wide identification of miR-199 predicted targets.
71
Session: Session 10
Time: 17.00 - 19.30
S27 17.00 - 17.25
A W Brandli
Walter-Brendel-Center of Experimental Medicine, Ludwig-Maximilians-University Munich,
Planegg-Martinsried, Germany
Alkaptonuria (AKU) is a rare metabolic disease caused by mutations in the homogentisate 1,2dioxigenase (HGD) gene, which encodes an enzyme required for the catabolism of tyrosine.
Patients carrying HGD mutations accumulate homogentisic acid (HGA) in the blood circulation
and excrete large amounts in the urine. Progressive accumulation of HGA polymers damages
cartilages, heart valves, and kidneys. Presently, no approved treatment modality is available for
AKU. Here, we report the development of a Xenopus model of AKU. As in mammals, Xenopus
embryos express HGD in the liver and pronephric kidneys. HGD-deficient embryos excrete HGA
indicating that the Xenopus AKU model accurately recapitulates the early pathophysiology of
AKU patients. Furthermore, HGA excretion in Xenopus can be efficiently suppressed by
treatment with nitisinone, a promising drug candidate for the treatment of AKU. In summary, the
Xenopus AKU model represents a novel tool for the screening, identification, and preclinical
testing of AKU drug candidates.
O25 17.25 - 17.40
Poster Number: P017
Xenopus
G Toro-Tapia1,2, A Beyer1, S Villaseca1, J I Leal1, R Mayor2, M Torrejon1
1
Department of Biochemistry and Molecular Biology, University of Concepcion, Concepcion,
Chile; 2Department of Cell and Developmental Biology, University College London, London, UK
Cell migration requires several signals to control cell movements, such as heterotrimeric G
protein pathway. Ric-8A, a GEF for Gα subunits plays an important role in neural crest (NC)
migration in Xenopus. However the Gα subunit activated by Ric-8A remains unknown. We
propose: Ric-8A, through Gα13 regulates NC migration. Loss and gain of function experiments
were performed. NC explants were analyzed in vivo by time-lapse microscopy and by
immunostaining to evaluate dispersion, protrusion stability and protein localization. Gα13 was
able to rescue the Ric-8A morphant phenotype and both interact and co-localize in protrusions
at the leading edge of NC cells. Ric-8A and Gα13 morphant cells display changes in the cortical
F-actin phenotype and protrusion stability, number and size of focal adhesions and subcellular
localization of polarity markers. These results suggest that Ric-8A regulates Gα13 signaling
pathway and together control cell polarity and focal adhesion properties required for proper cell
migration.
72
S28 17.40 - 18.05
The role of thyroid hormone receptor alpha during larval growth and intestinal stem cell
development
J Choi, D R Buchholz
Biological Sciences, University of Cincinnati, Cincinnati, USA
Thyroid hormone (TH) and TH receptor (TR) interaction is critical for normal development in all
vertebrates. Two genes encode TRs, TRα and TRβ, each with distinct expression patterns
during development. How these two genes work together or independently to orchestrate
developmental changes induced by TH throughout the body is not well understood. Here, we
use TRα knockout (TRαKO) animals to examine the impact of TRα on growth and development
during frog metamorphosis. Surprisingly, we found that, despite decreased responsivity to TH,
TRαKO tadpoles developed faster and achieved tail resorption earlier than wild-type tadpoles,
whereas larval growth was not affected. However, juvenile growth was reduced and was
associated with impaired intestinal remodeling during metamorphosis. Abnormal intestinal
morphology and expression profiles of TH-response genes in TRαKO animals suggest a
permanently reduced intestinal stem cell compartment. These data indicate a critical role for
TRα in tissue-specific, TH-dependent developmental regulation during in frog metamorphosis.
O26 18.05 - 18.20
Poster Number: P031
MRC Molecular Haematology Unit, University of Oxford, Oxford, UK
Stem cells are defined by their capacity to give rise to differentiated progeny while selfrenewing, properties which are referred to as stemness. What stemness is molecularly and how
it is established and maintained is not clear. Notch signalling is essential for the establishment
and maintenance of a number of tissue stem cells, including haematopoietic stem cells (HSCs),
but its molecular mechanism is not fully understood. Notch1 and Notch4 are expressed in the
dorsal aorta at the time HSCs are generated, their role was investigated and we defined the
Hes/Hey transcriptional effectors targeted by them to establish the gene regulatory network
(GRN) controlling the establishment of HSCs. Interestingly, Hes5, a gene essential for the
establishment and/or maintenance of other tissue stem cells, plays a central role. This suggests
that a common GRN might be shared amongst tissue stem cells and that Hes5 might be the key
molecule conferring stemness on them.
73
O27 18.20 - 18.35
Poster Number: P063
P Walentek1, I Quigley2, D I Sun1, U K Sajjan1, C Kintner2, R M Harland1
1
Molecular and Cell Biology, University of California Berkeley, Berkeley, USA;
Neurobiology Laboratory, Salk Institute for Biological Studies, La Jolla, USA
2
Molecular
Upon cell cycle exit, the mother centriole matures into a basal body to facilitate cilia formation.
The centriolar protein Cp110 is a key regulator of this process and inhibitor of cilia. Using
Xenopus we show that Cp110 also localizes to cilia forming basal bodies and is required in all
types of cilia. In multiciliated cells (MCCs), Cp110 promotes ciliary adhesion complex formation
and basal body-actin interactions. Two clusters of coiled-coil domains in Cp110 mediate
inhibition of ciliogenesis and other centriole-specific roles, suggesting that Cp110’s opposing
roles are generated through interactions with distinct protein complexes. Because of its dual
role, Cp110 levels must be precisely controlled. In MCCs, expression of cp110 is activated by
conserved ciliary transcription factors, which also activate miR-34/449s to repress cp110. Thus,
this transcriptional/posttranscriptional module generates optimal Cp110 levels for ciliogenesis.
Our data reveal novel possibilities through which Cp110 may contribute to development and
disease in vertebrates.
S29 18.35 - 19.00
S A Moody1, K M Neilson1, D Alfandari2
1
Anatomy and Regenerative Biology, George Washington University, Washington, DC, USA;
Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA, USA
2
Mutations in either SIX1 or one of its co-factors, EYA1, result in Branchiootorenal syndrome,
characterized hearing loss, craniofacial abnormalities and kidney defects. However, these
mutations only account for about half of the patients, indicating that there are additional
causative genes. In search for additional Six1-cofactors, we screened the Drosophila
interactome to identify Xenopus homologues to Sine oculis interacting proteins. We identified
many candidate cofactors that are expressed in the otocyst (precursor of the inner ear),
branchial arches (precursor of middle ear ossicles), and nephric mesoderm. Some of these
candidate co-factors directly bind to Six1 and thus may modify its transcriptional activity. When
the endogenous expression levels of some of these candidate co-factors are reduced, gene
expression in the branchial arches and otocyst are disrupted. These results indicate that
previously uncharacterized Six1-interacting partners have potential important roles in vertebrate
craniofacial development and congenital syndromes.
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POSTER ABSTRACTS
Poster Number: P1
POSTER WITHDRAWN
Poster Number: P2
Department of Molecular, Cellular, and Developmental Biology, University of Michigan,
Ann Arbor, MI, USA
The small GTPase RhoA is an important regulator of cell-cell junctions. We recently
described transient accumulations of active RhoA (“Rho flares”) at junctions in the
epithelium of gastrula-stage Xenopus laevis embryos. In order to investigate the cause
and consequence of Rho flares, we co-imaged active Rho with fluorescently-tagged
junction proteins. Intriguingly, the tight junction proteins ZO-1 and Occludin are locally
decreased prior to the Rho flare and are increased, or reinforced, following the flare.
Additionally, Rho flares are accompanied by deformation of the plasma membrane, and
F-actin accumulates on either side of the membrane deformation. Here, I will present
data that tests the mechanism through which Rho flares reinforce cell-cell junctions (via
actin polymerization and/or junction contraction) and investigates the signal that triggers
the flare. Our data suggest that localized activation of Rho is needed to repair and
remodel junctions to maintain the barrier function and integrity of developing epithelia.
Poster Number: P3
Mechanisms of Cell Division and Size Scaling in Pipid Frogs
K Miller, R Heald
UC Berkeley, Heald Lab, Berkeley, CA, USA
How the mitotic spindle scales to cell size across different cell types and organisms is
poorly understood. Recently, amphibians have emerged as a powerful system to study
size scaling relationships using two model frog species, the larger Xenopus laevis and
the smaller Xenopus tropicalis, which have correspondingly larger and smaller cells and
organelles, to identify spindle scaling factors. This project takes a comparative
evolutionary approach to investigate the conservation of spindle assembly and scaling
mechanisms across species by utilizing a tiny, distantly related member of the Pipid frog
family: Hymenochirus boettgeri. We have pioneered an H. boettgeri egg extract system
that recapitulates spindle assembly in vitro, similar to X. laevis and X. tropicalis egg
extracts, and are employing transcriptomic and proteomic approaches to identifying
75
candidate scaling factors in this frog. Our data suggests that H. boettgeri has evolved
mechanisms of spindle assembly and scaling which are distinct from those in Xenopus.
Poster Number: P4
Anillin regulates epithelial tension at tissue and cellular scales
T R Arnold, K Dinshaw, T Higashi, R E Stephenson, A L Miller
Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, USA
Dynamic changes in cell-cell junction tension, which are driven by actomyosin
contractility, are essential for establishing, maintaining, and rearranging cell-cell
contacts, while failures in these processes underlie birth defects and cancer cell
metastasis. Here, we investigated how the scaffolding protein Anillin contributes to
junction tension. At the tissue scale, Anillin expression level correlates with increased
junctional tension measured with a tension-sensing probe and Anillin knockdown
abolishes whole-embryo contraction following exogenous ATP application. At the
cellular scale, Anillin accumulates at sites of local transient RhoA activation at junctions
(“Rho flares”). Work in progress will test which of Anillin’s domains are required for its
recruitment to Rho flares and the mechanism by which Anillin regulates contractility at
Rho flares. Together, our data demonstrates that Anillin functions to regulate junctional
tension and contractility at both tissue and cellular scales.
Poster Number: P5
Identification of methylated deoxyadenosines in vertebrates reveals diversity in
DNA modifications
M J Koziol1,2, C R Bradshaw1, G E Allen1, A S H Costa3, C Frezza3, J B Gurdon1,2
1
Wellcome Trust Cancer Research UK Gurdon Institute, University of Cambridge,
Cambridge, UK; 2Department of Zoology, University of Cambridge, Cambridge, UK;
3
Hutchison/MRC Research Centre, University of Cambridge, Cambridge, UK
Methylation of cytosine deoxynucleotides (dC5m) is a well-established epigenetic mark,
but in higher eukaryotes much less is known about modifications affecting other
deoxynucleotides. We report the detection of N-6-methyl-deoxyadenosine (dA6m) in
vertebrate DNA, specifically in Xenopus laevis, but also in other species including
mouse and human. Our methylome analysis reveals that dA6m is widely distributed
across the eukaryotic genome, is present in different cell types, but commonly depleted
from gene exons. Thus, direct DNA modifications might be more widespread than
previously thought.
76
Poster Number: P6
Characterization of oxidized Cytosine derivatives in Xenopus development
V Hatch1, D Han1, M Musheev1, C Niehrs1,2
1
DNA Demethylation and Reprogramming, Institute of Molecular Biology gGmbH,
Mainz, Germany; 2Division of Molecular Embryology, DKFZ-ZMBH Alliance, Heidelberg,
Germany
DNA methylation is important in genome stability and the regulation of gene expression
during both development and disease. The presence of methylated cytosine (5mC)
within DNA was the first epigenetic mark to be identified, however a recent discovery
has uncovered three modified versions of 5mC; 5-hydroxymethlycytosine (5hmC), 5formylcytosine (5fC) and 5-carboxylcytosine (5caC). It is thought that these subsequent
oxidised methylation marks are important intermediate steps for DNA demethylation. It
is also possible that these newly discovered marks could be considered epigenetic
marks in their own right. This raises the question, what is their role in early
embryogenesis? To address this question Xenopus embryos were used as a model
system to investigate the spatio-temporal occurrence of 5mC, 5hmC, 5fC and 5caC
across different developmental stages. We will present data showing differential
occurrence of these genomic marks during Xenopus development.
Poster Number: P7
Neil2 stimulates Tdg-mediated BER in active DNA demethylation in Xenopus
embryogenesis
D Han1, M Musheev1, C Niehrs1,2
1
DNA Demethylation and Reprogramming, Institute of Molecular Biology gGmbH,
Mainz, Germany; 2Division of Molecular Embryology, German Cancer Research Center
(DKFZ), Heidelberg, Germany
DNA demethylation plays an important role in development and animal physiology. In
active DNA demethylation, 5-methylcytosine (5mC) is iteratively oxidized into 5hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-caboxylcytosine (5caC)
by Ten-eleven-translocation (TET) enzymes. For demethylation, 5fC and 5caC are
removed by Thymine DNA glycosylase and base excision repair (BER). Our recent in
vitro data indicate that Nei-like (NEIL) glycosylases play a crucial role in this context by
increasing the enzymatic turnover of TDG in BER. Here we analyzed the role of TET,
TDG, and especially NEILs in active DNA demethylation in Xenopus laevis
embryos. Expression analysis shows that tet3, tdg, and neils are expressed in the
embryonic central neural system. Antisense Morpholino knock down of tet3, tdg, neil2,
and neil3 induces microcephaly and neural crest defects. Analysis of 5mC and its
oxidative derivatives by mass spectrometry supports the cooperation of tet3, tdg, and
neil2 in active DNA demethylation in vivo.
77
Poster Number: P8
The regulatory role of Transmembrane Protein Tyrosine Phosphatase in Wnt
signaling
L Chang1, M Kim1, A Glinka1, C Niehrs1,2
1
Division
of
Molecular
Embryology,
DKFZ-ZMBH
Alliance,
Deutsches
Krebsforschungszentrum, Heidelberg, Germany; 2Institute of Molecular Biology, Mainz,
Germany
Wnt/ß-catenin signaling plays a crucial role in cellular events, such as cell proliferation,
and embryonic development, including Xenopus. To identify novel factors involved in
Wnt signaling, a genome-wide siRNA screen was performed and Transmembrane
Tyrosine Phosphatase (TMTP) was found as a putative regulator of Wnt signaling. To
clarify how TMTP regulates Wnt signaling in vivo, loss-of-function experiments were
performed using an antisense Morpholino oligonucleotide and CRISPR/Cas9 targeting
TMTP in Xenopus tropicalis. Loss of TMTP leads to a small-headed phenotype which
may result from increased Wnt activity. Moreover, depletion of TMTP upregulates
TopFlash reporter activity, which can be restored by overexpressing wild-type TMTP but
not enzymatic-dead mutants, indicating that the phosphatase activity is critical for Wnt
regulation. In addition, knockdown of TMTP reduces the expression of forebrain
markers, similar to Wnt gain-of-function. These results show that TMTP acts as a
negative Wnt regulator in Xenopus.
Poster Number: P9
The role of adsorption on the extracellular matrix in the diffusion of morphogens
within the intercellular space
A G Zaraisky1, A M Nesterenko1,2, A V Bayramov1, F M Eroshkin1, N Y U Martynova1,
G V Ermakova1, D D Korotkova1,3
1
Bioorganic Chemistry, Russian Academy of, Moscow, Russia; 2Belozersky Institute of
Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia;
3
Department of Embryology, Faculty of Biology, Lomonosov Moscow State University,
Moscow, Russia
On example of Noggin proteins we investigated the role of adsorption on the
extracellular matrix (ECM) in the diffusion of morphogens within the intercellular spaces
(IS) of the Xenopus laevis embryonic ectoderm. We have demonstrated that Noggin1
and Noggin2 are strongly adsorbed on ECM, which significantly retards their diffusion.
By contrast, Noggin4 is not adsorbed on ECM and its diffusivity exceeds that of two
other Noggins. Using Fluorescence Recovery After Photobleaching (FRAP) assay and
mathematical modelling, we developed an approach allowing to measure diffusivity of
the morphogens adsorbed on ECM directly within IS of living embryo. We also
demonstrated that accounting for the adsorption on ECM significantly increases specter
of selforganizing structures which can be generated by the reaction-diffusion systems in
78
embryonic tissues. In particular, we showed that gradual increase or decrease of
ECM concentration along some axis can lead to substantial changes in spatial
patterning along this axis.
Poster Number: P10
M V Danilchik1, C Wulff1,2
1
2
Integrative Biosciences, Oregon Health and Science University, Portland OR, USA;
Biology, Mountain View High School, Vancouver WA, USA
Unexpectedly, the perivitelline (PV) fluid of the pregastrulation Xenopus embryo was
found to contain an abundance of microvesicles. Proteomic analysis of PV fluid via
mass spectroscopy indicates an enrichment of exosome-associated proteins, and
electron microscopy confirms the presence of large numbers of particles in a size range
typical of exosomes (~50-150 nm). Exosomes are known to carry on their surfaces a
complement of proteins involved with cell-cell recognition and signaling, and appear to
be effective in transferring mRNAs and mRNAs between cells. Thus, as demonstrated
in various tissue-culture and pathogenic situations, exosomes have the potential to alter
gene expression patterns of cells receiving them. Whether this exciting property is
utilized during normal embryonic development is largely unexplored. Here, we report
that embryos deprived of PV-borne exosomes develop with a significant reduction in
hematopoiesis, suggesting that early tissue specification involves exosome-mediated
signaling across extracellular spaces in the embryo. Support: NSF IOS-1557527.
Poster Number: P11
Identifying Phenotypic Convergence Among Autism-Associated Genes in X.
tropicalis
H R Willsey1, A J Willsey2, R M Harland1
1
Molecular and Cell Biology, UC Berkeley, Berkeley, CA;
Francisco, San Francisco, CA
2
Psychiatry, UC San
Autism Spectrum Disorder (ASD) is a devastating genetic neurodevelopmental disorder
of undetermined etiology. Advances in genome technology have led to the identification
of 65 high-confidence ASD risk genes. These genes are co-expressed during human
fetal cortex development, and have been suggested to play critical roles in
glutamatergic neuron development. Here we present results from a F0 CRISPR/Cas9
screen of these ASD-associated genes in X. tropicalis. The effects of ASD gene loss
were assayed by imaging neurons throughout embryogenesis using fluorescent
reporters and by in situ RNA hybridization for neuronal cell fate specification markers.
Specific attention will be paid to the role of ASD genes in glutamatergic neuron
specification and proliferation. By combining the high-throughput capability of the
CRISPR/Cas9 system, a tractable vertebrate model organism, and a reliably-associated
79
set of ASD genes, this study aims to understand the neuropathology of ASD by focusing
on convergent phenotypes shared among genes.
Poster Number: P12
Disassembling regionalization of neuroectoderm by single-cell transcriptome
analysis
Advanced Center for Computing and Communication, RIKEN, Wako, Japan
In vertebrate embryos, neural induction starts during gastrulation. Simultaneously,
induced neuroectoderm is regionalized along its anterior-posterior, medial-lateral and
apical-basal axes, and starts differentiation into many cell types that have specific
functions in future central nervous system. However, the mechanism by which dozens
of cell type are robustly generated from almost-uniform cell population is not
understood. In this study, we approached this question with high-throughput single-cell
RNA-seq. Hundreds of single cells from presumptive ectoderm of Xenopus laevis early
neurula were analyzed with Drop-seq. Quantification of mRNA molecules in each cell
led to identification of cell type clusters and rare populations. Furthermore, combining
this data with whole-mount in situ hybridization images of several genes enabled us to
infer spatial position of analyzed cells and spatial expression pattern of hundreds of
genes. Using these, we construct a model for regionalization of neuroectoderm.
Poster Number: P13
Tissue- and stage-specific Wnt target gene expression is controlled subsequent
to β-catenin recruitment to cis-regulatory modules
Y Nakamura1, E de Paiva Alves2, G J Veenstra3, S Hoppler1
1
Institute of Medical Sciences, University of Aberdeen, Aberdeen, UK; 2Centre for
Genome-Enabled Biology and Medicine, University of Aberdeen, Aberdeen, UK;
3
Department of Molecular Developmental Biology, Radboud Institute for Molecular Life
Sciences, Nijmegen, The Netherlands
Canonical Wnt/β-catenin signalling operates repeatedly to regulate tissue- and stagespecific transcriptional responses during development. Although recruitment of nuclear
β-catenin to target genomic loci serves as the hallmark of canonical Wnt signalling,
mechanisms controlling stage- or tissue-specific transcriptional responses remain
elusive. Here, a direct comparison of genome-wide occupancy of β-catenin with a
stage-matched Wnt-regulated transcriptome reveals that only a subset of β-cateninbound genomic loci are transcriptionally regulated by Wnt signalling. We demonstrate
that Wnt signalling regulates β-catenin binding to Wnt target genes not only in the
developmental context in which they are transcriptionally regulated, but also in contexts
in which their transcription remains unaffected. The transcriptional response to Wnt
signalling depends on additional mechanisms, such as BMP or FGF signalling for the
80
particular genes we investigated, which do not influence β-catenin recruitment. Our
findings suggest that context-dependent mechanisms are crucial for transcriptional
activation of Wnt/β-catenin target genes subsequent to β-catenin recruitment.
Poster Number: P14
Maternally expressed transcription factor Foxh1 bookmarks the genome before
the onset of zygotic gene activation and regulates endoderm programming
R Le-Charney1, J S Cho1, M Fish1, E Forouzmand2, J Cheung1, X Xie2, I L Blitz1, K W Y
Cho1
1
Developmental and Cell Biology, University of California, Irvine, USA;
Science, University of California, Irvine, USA
2
Computer
The essential control feature in the process of development is gene regulation in time
and space and the regulatory logic controlling developmental processes is hardwired in
the genome. We would like to understand how the naive genome in the early embryo
progressively undergoes a series of modifications to control gene expression such that
proper cellular differentiation programs are correctly implemented. There is an intimate
connection between transcription factor interactions with the genome and epigenetic
states in gene regulation. We have examined the temporal order of events regulating
endoderm gene expression and found that Foxh1 transcription factor binds to the
regulatory regions of target endodermal genes long before the onset of target gene
transcription, prior to RNA polII and P300 binding. We have recently generated Foxh1
mutant Xenopus tropicalis using CRISPR/Cas9 approach and report on the effects of
Foxh1 on chromatin states, the onset of zygotic transcription, and germ layer
specification.
Poster Number: P15
Human Serotonin type 3 receptor (HT3): Evidences for interactions with canonical
Wnt signaling
A Schweickert1, M Maerker1, S Schmitteckert2, S Bogusch1, B Niesler2
1
Institute for Zoology, University of Hohenheim, Stuttgart, Germany; 2Department of
Human Molecular Genetics, University Hospital Heidelberg, Heidelberg, Germany
The serotonin type 3 receptor (5-HT3R) belongs to the class of ligand-gated ionchannels, consisting of five subunits. The human genome harbors five genes (HTR3AE) encoding five 5-HT3 subunits (5-HT3A-E). Subunits are characterized by a ligandbinding-domain (LBD) followed by four transmembrane regions. 5-HT3D differs and
lacks a signal-peptide and LBD. Human 5-HT3 subunits are broadly expressed,
particularly in the gastrointestinal tract. In addition, 5-HT3 receptors have been
implicated in various human diseases including anxiety, depression and irritable-bowelsyndrome. Recently, we have unraveled a role of 5-HT3 in canonical Wnt-signaling
during left-right development of Xenopus. Here we show evidences for a 5-HT3-Wnt
81
interface in humans. In Xenopus, we found that heterologous misexpression of human
HTR3D blocked Wnt3a induced double-axis, in contrast to HTR3A/B. We further show
that 5-HT3D interference on Wnt-signaling is located upstream of disheveled. Finally,
we demonstrate the presence of 5-HT3D in Wnt3a induced early endosomes,
suggesting a function in the Wnt-signalosome.
Poster Number: P16
MicroRNAs in Neural Crest development
N Ward, G N Wheeler
School of Biological Sciences, University of East Anglia, Norwich, UK
MicroRNAs (miRNAs) control various mechanisms during development by regulating
gene expression at the post-transcriptional level. The neural crest (NC) is a transient,
multipotent stem cell–like population of highly migratory embryonic cells found in
vertebrates. The induction and specification of NC is controlled by a complex gene
regulatory network. The role of miRNAs within this network is not known.
Using RNA-seq on animal caps induced to make NC as well as in situs on whole
embryos we have shown a number of known and novel miRs to be expressed in the
NC. We have confirmed expression of some of these miRNAs in NC using double in situ
hybridisations and Q-PCR. In addition, knocking down one of these microRNAs using a
morpholino shows a loss of neural crest structures and a decrease in the expression of
Sox10. We are further characterising the expression and function of these miRs in NC
development.
Poster Number: P17
Ric-8A/Gα13 signaling pathway is required to proper cranial neural crest
migration in Xenopus
G Toro-Tapia1,2, A Beyer1, S Villaseca1, J I Leal1, R Mayor2, M Torrejon1
1
Department of Biochemistry and Molecular Biology, University of Concepcion,
Concepcion, Chile; 2Department of Cell and Developmental Biology, University College
London, London, UK
Cell migration requires several signals to control cell movements, such as heterotrimeric
G protein pathway. Ric-8A, a GEF for Gα subunits plays an important role in neural
crest (NC) migration in Xenopus. However the Gα subunit activated by Ric-8A remains
unknown. We propose: Ric-8A, through Gα13 regulates NC migration. Loss and gain of
function experiments were performed. NC explants were analyzed in vivo by time-lapse
microscopy and by immunostaining to evaluate dispersion, protrusion stability and
protein localization. Gα13 was able to rescue the Ric-8A morphant phenotype and both
interact and co-localize in protrusions at the leading edge of NC cells. Ric-8A and Gα13
morphant cells display changes in the cortical F-actin phenotype and protrusion stability,
number and size of focal adhesions and subcellular localization of polarity markers.
82
These results suggest that Ric-8A regulates Gα13 signaling pathway and together
control cell polarity and focal adhesion properties required for proper cell migration.
Poster Number: P18
Uch37 mediates DNA binding of Tcf1 through its deubiquitinating activity during
W Han, B R Keum, J K Han
Life Sciences, Postech, Pohang, Republic of Korea
Tcf/Lef family mediates Wnt/β-catenin pathway, which plays diverse roles in biological
processes including embryogenesis and postnatal homeostasis of organism.
Posttranslational modifications including phosphorylation, sumoylation, and acetylation
have been suggested as crucial regulatory mechanisms for Tcf/Lef family to regulate
transcriptional activity or interaction with co-activators. However, ubiquitin-mediated
regulatory mechanism remains unknown. Here, we demonstrate that ubiquitin Cterminal hydrolase37 (Uch37) has a deubiquitinating activity for Tcf1. Uch37 directly
binds and deubiquitinates Tcf1. Furthermore, biochemical assays reveal that Uch37
regulates transcriptional activity of Tcf1 in an enzyme activity-dependent manner.
However, Uch37 does not affect steady-state level of Tcf1 protein. Chromatin
immunoprecipitation demonstrate that Uch37 is essential for binding of Tcf1 to target
promoter. Moreover, loss of function studies using Xenopus Uch37 MO revealed that
Xenopus Uch37 is required for ventrolateral mesoderm development by regulating
transcriptional activity of Xenopus Tcf1. Our study uncovers a novel mechanism for Tcf1
transcriptional activation through its deubiquitinating activity.
Poster Number: P19
Elucidating the Role of Epigenetic Regulation in Neural Crest Cells Formation
M Marin-Barba, G N Wheeler
School of Biological Sciences, University East Anglia, Norwich, UK
Neural Crest Cells (NCC) are a transient multipotent and migratory cell population
unique to vertebrates that give rise to several derivatives such as melanocytes, neurons
and craniofacial structures.. Although Neural Crest Cells are being widely studied in the
Xenopus animal model, their epigenetic regulation has not been deeply analysed.
In order to elucidate the importance of epigenetic regulation for Neural Crest formation,
we are taking advantage of the ATAC sequencing technology. ATAC sequence (Assay
for Transposase-Accessible Chromatin) is a high throughput technique that thanks to a
transposase enzyme reveals the open chromatin regions on the genome, known to be
enhancers and promoters. We are using ATAC sequencing in neural crest induced
animal caps to identify enhancers, and transcription factors bound to them, during the
process of the NCC formation.
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Poster Number: P20
Nodal is a conserved neural inducer in chordates
L Kodjabachian1, G Luxardi1, P Scerbo1, M Cibois1, Y Le Petillon2, H Escriva2, S
Bertrand2
1
IBDM, CNRS/Aix-Marseille Universite, Marseille, France;
Banyuls/mer, France
2
BIOM, CNRS/UPMC,
The leading model for neural induction states that BMP signal inhibition is necessary
and sufficient to confer neural identity to naive ectoderm cells. However, this model has
been challenged in Xenopus and other chordate model organisms. Thus, we reasoned
that additional signals produced by the Spemann organizer could contribute to neural
induction. In all vertebrates, Nodal is known for its key role in mesendoderm induction
and gastrulation. Strikingly, we found that when Nodal signals were delivered at the
mid-gastrula stage, ectoderm cells did not adopt a mesendodermal fate but instead
stably became neural. Loss-of-function assays confirmed the possibility that Nodal may
directly participate to neural induction. Collaborative work indicated that in the
amphioxus embryo, Nodal likely provides the main contribution to neural induction,
independently of BMP signalling. This study further reveals the diversity of neural
inducers selected during chordate evolution and advocates against a universal
molecular explanation for neural induction.
Poster Number: P21
Gremlin and Bone Morphogenetic Protein signalling in Xenopus dorsoventral
patterning
J Pegge1, A J Tatsinkam2, M Dhomeja1, C C Rider2, E Bell1
1
Centre for Developmental Neurobiology, King's College London, London, UK; 2Centre
for Biomedical Sciences, Royal Holloway University of London, Surrey, UK
The dorsoventral axis is patterned by a Bone Morphogenetic Protein (BMP) signalling
gradient – high ventrally to low dorsally. This is established by the diffusion of
antagonists from a dorsal signalling centre known as the organiser. Diffusion must be
tightly regulated in order for a sufficient gradient to form. We are investigating the role of
heparan sulfate proteoglycans (HSPGs) in this process. These cell surface
macromolecules have been shown to bind BMPs and BMP antagonists and may
regulate their diffusion. Mutations were introduced into the HSPG binding site of the
BMP antagonist Gremlin, reducing its affinity. Its function was then compared to the wild
type protein. Preliminary data suggest HSPG binding is required for its activity. The
BMP signalling gradient divides the ectoderm into epidermal, neural border and neural
plate regions. We are also investigating the role of Gremlin in this process.
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Poster Number: P22
XDSCR6 (Xenopus Homologue of Human Down Syndrome Critical Region protein
6) and its partner XEZH2 regulate XSTAT3 activity during embryonic axis
formation of Xenopus laevis
M Loreti, D L Shi, C Carron
Laboratory of Developmental Biology, UMR 7622, Sorbonne Universites, UPMC Paris
06, Institut de Biologie Paris-Seine, CNRS, Paris, France
The formation of the three germ layers and the establishment of embryonic axis are
fundamental events during early development. In Xenopus laevis, Xdscr6 triggers
mesoderm and embryonic axis formation through derepression of polycomb group
(PcG) proteins. Here we demonstrate that XSTAT3, a bona fide transcriptional
regulator, is a new partner of XDSCR6 and XEZH2 in xenopus embryos. We show that
these proteins are present in the three germ layers during gastrulation and form a
complex. Endogenous XSTAT3 is transcriptionally active during gastrulation and this
activity is drastically decreased when XDSCR6 is overexpressed or when XEZH2
function is lost. Moreover, loss of XEZH2 function in ventral region of the embryo also
induced axis duplication and ectopic expression of dorsal mesoderm genes, as
observed when XDSCR6 is overexpressed. Our results suggest that XDSCR6 could act
as an inhibitor of XSTAT3 activity in a XEZH2 dependent manner leading to tissue
dorsalisation.
Poster Number: P23
Coordinated transcriptional regulation of myogenesis
C McQueen, R J White, M E Pownall
Biology, University of York, York, UK
Recent advances in analysis of small RNAs and Protein-DNA interactions has enabled
the exciting opportunity to study RNA Polymerase III regulation at genome-wide scale.
This work has revealed a tightly regulated tRNA/mRNA interface in which RNA
Polymerase III transcription matches the demand set by mRNA codon usage during
development. This coordinate regulation indicates that transcriptional networks
important for development are likely to be more complex than previously thought. The
RNA Polymerase III specific subunit Polr3G exists in 2 distinct isoforms in mammals
and Xenopus. In mammals Polr3G is associated with proliferation and pluripotency, and
Polr3gL with differentiation. Data will be presented demonstrating the complementary
expression profiles of Polr3G and Polr3gL during Xenopus tropicalis development, and
the specific expression of Polr3G in skeletal muscle. The work presented will include
transcriptomic analyses of Polr3G CRISPR/Cas9 targeting and overexpression studies,
and data from a novel microarray designed to investigate specific tRNA transcription.
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Poster Number: P24
Evolutionary conserved regeneration signal response enhancers for renal
regeneration
N Suzuki1, T Kumada1, H Ogino2, H Ochi1
1
Institute for Promotion of Medical Science Research, Faculty of Medicine, Yamagata
University, Yamagata, Japan; 2Department of Animal Bioscience, Nagahama Institute of
Bio-Science and Technology, Nagahama, Japan
Amphibians and fish can regenerate many tissues and recover normal function after
injury, whereas mammals have a limited regenerative capacity. Currently, it is
speculated that tissue regeneration recapitulates developmental programs because the
expression of developmental genes is reactivated during regeneration in many cases.
However, it is unclear whether the unique cis-regulatory elements specific to tissue
regeneration are activated during tissue regeneration or whether mammals have hidden
cis-regulatory elements that can be activated in regenerating tissues and merely
suppress their function. Here, we searched for regeneration signal response enhancers
in lhx1 locus, and found that the noncoding elements conserved between only highly
regenerative species do not show strong enhancer activities in the regenerating
amphibian pronephros. Instead, the noncoding elements conserved from fish to human
function as enhancers in the regenerating pronephros. This result suggests that
mammals whose regenerative abilities are limited still retain regeneration signal
response enhancers in their genome.
Poster Number: P25
Leapfrogging: Gene knockout phenotypes in the F1 generation
I L Blitz, M B Fish, K W Y Cho
Developmental and Cell Biology, University of California, Irvine, USA
CRISPR/Cas9 is revolutionizing genetic loss-of-function analysis but technical
limitations slow progress when creating mutant lines. In conventional genetic breeding
schemes, mosaic founder animals carrying mutant alleles are outcrossed to produce F1
heterozygotes. Intercrosses between heterozygotes allows for phenotypic analysis in
F2s. However, when targeting essential genes, efficient mutagenesis of founders is
often lethal, preventing the acquisition of mature F0 animals. To overcome this problem
we introduce “leapfrogging,” a method combining efficient CRISPR/Cas9 mutagenesis
with transplantation of mutated primordial germ cells into a wild-type host. Tested using
Xenopus tropicalis, we show that founders containing transplants transmit mutant
alleles with high efficiency. F1 offspring from intercrosses between F0s that carry
embryonic lethal alleles recapitulate loss-of-function phenotypes, circumventing an
entire generation of breeding. Leapfrogging will be especially useful for studying the
functions of maternal gene products, which are inaccessible by F0 analyses.
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Poster Number: P26
Dissecting the pre-placodal transcriptome to reveal direct targets of Six1 and
Eya1 affecting neurogenesis in cranial placodes
N Riddiford, G Schlosser
School of Natural Sciences and Regenerative Medicine Institute (REMEDI), National
University of Ireland Galway, Galway, Ireland
The pre-placodal ectoderm, marked by the expression of transcription factor Six1 and
its co-activator Eya1, gives rise to placodes and ultimately to many cranial sensory
organs and ganglia. Using RNA-Seq in Xenopus laevis we screened for direct placodal
target genes of Six1 and Eya1 by overexpressing hormone-inducible constructs of Six1
and Eya1 in pre-placodal explants, and blocking protein synthesis before hormoneinducing nuclear translocation of Six1 or Eya1. Comparing the transcriptome of explants
with non-induced controls, we identified hundreds of novel Eya1/Six1 target genes
including transcriptional regulators of progenitor fates (e.g. Sox2, Hes5/8) and
neuronal/sensory differentiation (e.g. Ngn1, Atoh1, Pou4f1, Gfi1). Gain and loss of
function studies showed that Eya1/Six1 (1) are required for placodal expression of these
genes and (2) repress/delay neuronal differentiation by directly activating Hes5/8
independent of Notch signaling. Our findings provide pioneering insights into the gene
regulatory network regulating placodal neurogenesis downstream of Six1 and Eya1.
Poster Number: P27
Transcriptomic Insights into Genetic Diversity of Protein-Coding Genes in X.
laevis
V Savova1, E Pearl2, M Horb2, A Nag3, A Gimelbrant3, L Peshkin1
1
Systems Biology, Harvard Medical School, Boston, USA; 2National Xenopus Resource,
Marine Biological Laboratory, Woods Hole, USA; 3Department of Genetics, Harvard
Medical School, Boston, USA
We characterize the genetic diversity of X.laevis using allele-specific RNA-seq data and
provide a catalogue of coding variation, which can be used for improving the genomic
sequence, probe design, and proteomics. We examine the genetic landscape of the
species by functionally annotating different classes of mutations as deleterious or
neutral. Further, we specifically compare the variation in crosses from the inbred
genomic J-strain, the popular occasionally outbred albino B-strain, and two hybrid
crosses. We identify a subset of mutations specific to the B-strain, which allows us to
investigate the selection pressures affecting duplicated genes in this pseudo-tetraploid.
We find the ratio of non-synonymous to synonymous mutations is lower in duplicated
genes, which therefore appear to be under greater purifying selection. Surprisingly,
function-altering ("damaging") mutations constitute a greater fraction of the nonsynonymous variants in this group, which may suggest a role for subfunctionalization in
coding variation affecting duplicated genes.
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Poster Number: P28
Structural and expression analyses of the Xenopus laevis Hox clusters
M Kondo1, T Yamamoto2, S Takahashi3, M Taira2
1
Misaki Marine Biological Station, The University of Tokyo, Kanagawa, Japan;
Department of Biological Sciences, The University of Tokyo, Tokyo, Japan; 3Institute
for Amphibian Biology, Hiroshima University, Hiroshima, Japan
2
From whole genome sequencing, two homeologous sets (L and S) of four Hox clusters
A through D (HoxA.L/S, HoxB.L/S, HoxC.L/S, and HoxD.L/S) and 13 paralogous groups
(PGs) were identified. There were 76 genes in total, including one pseudogene, which is
hoxb2p.L. Expression during development and in adult tissues was analyzed by RNAsequencing. The expression levels of most hox genes were similar between homeologs,
but in some pairs, large differences were observed and several of these were confirmed
by RT-PCR and whole mount in situ hybridization experiments. These results indicate
that subfunctionalization of hox genes may have occurred since allotetraploidization.
Furthermore, comprehensive analysis of hox gene expression during early development
led to an unexpected result that temporal collinearity is not apparent in genes belonging
to PG2 to PG10.
Poster Number: P29
Using ATAC-seq to detect novel gene regulatory elements in development and its
application for studying lens formation in Xenopus tropicalis
S Manohar1, T Nakayama1, A R Bright2, M Fisher1, G J C Veenstra2, R M Grainger1
1
Department of Biology, University of Virginia, Charlottesville, USA; 2Department of
Molecular Developmental Biology, Radboud University, Nijmegen, Netherlands
Using the recently developed method of ATAC-seq for in vivo genome analyses, we are
tracking the epigenetic landscape identifying stable or dynamic regions of accessible
chromatin during lens commitment in X. tropicalis. As expected, an ATAC-peak is
localized on a conserved enhancer (CE1) previously identified for the lens specification
gene foxe3 (Ogino et al., '08), indicating a link between ATAC-peak and enhancer
activity. We noticed many ATAC-peaks on regions not broadly conserved among
different organisms but actually conserved with X. laevis and theorized that such peaks
may correspond to unidentified enhancers. Among those, OCE1 (Open Chromatin
Element1) is located between CE1 and the foxe3 promoter and drives expression in the
lens when fused to a heterologous promoter-GFP reporter gene. Currently we are
identifying other enhancers based on the above-mentioned criteria, performing
functional studies. This strategy should be a generally powerful tool to find potential
enhancers to understand GRNs in development.
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Poster Number: P30
Identification and functional analysis of human RALDH2 genetic variants with
reduced activity using Xenopus embryos
Y Shabtai1, S Liu2, G G Hicks2, A Fainsod1
1
Developmental Biology and Cancer Research, Institute for Medical Research IsraelCanada, The Hebrew University, Jerusalem, Israel; 2Regenerative Medicine Program,
University of Manitoba, Winnipeg, Canada
Exposure of human embryos to ethanol induces a neurodevelopmental syndrome
known as Fetal Alcohol Spectrum Disorder (FASD). We demonstrated that ethanol
detoxification competes for enzymes dedicated to biosynthesis of retinoic acid (RA). We
identified RALDH2 as one of the earliest targets of alcohol in the early embryo. We
demonstrate that acetaldehyde, the oxidation product of ethanol, is a preferred
substrate of RALDH2 and competes with retinaldehyde. Human RALDH2 is fully
functional in Xenopus embryos exhibiting ethanol and acetaldehyde sensitivity.
FASD induction also has a genetic component. We analyzed naturally occurring human
Raldh2 SNPs generating single amino acid changes. Overexpression in embryos and
kinetic analysis identified alleles with reduced enzymatic activity. Exome sequencing of
samples from FASD children uncovered enrichment for one partially functional allele.
We conclude that this RALDH2 variant genetically predisposes the individual to FASD
following alcohol exposure and supports the competition between ethanol and retinoic
acid signaling.
Poster Number: P31
We have applied InDrops, a droplet-based single-cell transcriptomic technology
developed in our lab, to profile 30,000 single-cell transcriptomes sampled over a
timecourse of early Xenopus Tropicalis development. Our data span activation of the
zygotic genome (NF stage 8) to the formation of progenitors for most major organ
systems (NF stage 22). Unsupervised analysis of the resulting data reveals a
comprehensive atlas of embryonic cell types. For every cell type we measure its
genome wide transcriptional profile, and thus discover gene expression modules
containing dozens of new marker genes that are specific to each cell state. We are
developing novel bioinformatics strategies to connect cell states over time into
branching gene expression topologies to study transcriptional principles of cell fate
choices in the embryo. In a single experiment our data reveal the gene expression
dynamics associated with every cell fate choice during early embryogenesis.
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Poster Number: P32
Identifying Convergent Transcriptional Signatures Following Loss of AutismAssociated Genes
A J Willsey1, H R Willsey2, R M Harland2
1
Psychiatry, University of California, San Francisco, USA; 2Molecular and Cell Biology,
University of California, Berkeley, USA
Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder with complex
genetic underpinnings. Over 65 genes that confer ASD risk have been identified. Our
work applying systems biological approaches to transcriptional data collected over the
span of human brain development has identified convergence of ASD risk genes during
the development of midfetal deep layer cortical projection neurons. Excitingly, these
results generate hypotheses about ASD gene function in neurodevelopment that can be
tested in the Xenopus tropicalis model system. Here we present results from
transcriptional profiling of F0 CRISPR/Cas9 mutant brains of ASD-associated genes in
X. tropicalis. Co-expression network analysis characterized convergent transcriptional
signatures of ASD gene loss, and targeted mutagenesis validated the biological
pathways indicated by these convergent signatures. By integrating gene discovery and
systems biology along with a tractable model organism, this study provides insight into
the neurodevelopmental biology of ASD genes.
Poster Number: P33
Durian, a brain-specific secreted peptide impacting behavior
G H Goh, P M Wong, M Garcia-Miralles, M Pouladi, L Ho, B Reversade
Institute of Medical Biology, Agency of Science, Technology and Research, Singapore
We, and others, have shown that uncharacterized peptide hormones are still hiding in
plain sight within the human genome. To gain insight on the physiological function of
one such novel peptide, Durian, we developed gain-of-function assays in Xenopus
embryos and deleted it in mice.
Durian is conserved in all vertebrate species, encodes a brain-specific protein of less
than 80 amino-acids, and is readily secreted by cultured cells. Over-expression of
Durian in Xenopus embryos leads to a dose-dependent hyperpigmentation phenotype
that is indicative of increased serotonergic signalling to the pituitary gland.
Compared to wildtype littermates, behavioral studies performed on homozygous
knockout mice suggest that Durian may function as a potent neurotransmitter that
reduces anxiety-like phenotypes.
Along with several other uncharacterized hormones we have uncovered, our current
efforts seek to identify Durian's cognate cell surface receptor and establish possible
clinical indications for therapeutic intervention.
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Poster Number: P34
Ouro proteins are not essential to tail regression during Xenopus metamorphosis
Y Yaoita1, Y Nakai1, J Robert2, K Nakajima1
1
Institute for Amphibian Biology, Hiroshima University, Higashihiroshima, Japan;
Department of Microbiology and Immunology, University of Rochester Medical Center,
Rochester, USA
2
A tadpole tail that is twice as long as the tadpole trunk nearly disappears within three
days in Xenopus tropicalis. It is generally accepted that this phenomenon is driven by
immunological rejection of larval-skin-specific antigens, Ouro proteins. We generated
ouro-knockout tadpoles using TALEN method. Both ouro1- and ouro2-knockout
tadpoles expressed an undetectable level of Ouro protein encoded by a target gene and
a scarcely detectable level of the other Ouro protein from the untargeted ouro gene in
tail skin. Congenital athymic frogs were produced by Foxn1 gene modification. Flow
cytometry analysis revealed that mutant frogs lacked splenic CD8+ T cells. Skin allograft
rejection was dramatically impaired in mutant frogs. None of the knockout tadpoles
showed any significant delay in process of tail shortening during the climax of
metamorphosis, which demonstrates that Ouro proteins are not essential to tail
regression and suggests that regression is not executed by the immunological rejection.
Poster Number: P35
Molecular and Cellular Mechanisms of Spinal cord Regeneration
J Larrain1, G Edwards1, D Lee-Liu1, E Méndez1, R Muñoz1, J Peñailillo1, V Tapia1, E De
Domenico2, M Gilchrist2, L L Sun3, A Cebrian-Silla4
1
Cell and Molecular Biology, P Universidad Catolica, Santiago, Chile; 2Francis Crick
Institute, London, UK; 3Norman Dovichi Department of Chemistry and Biochemistry,
University of Notre Dame; 4José Manuel García-Verdugo, Laboratory of Comparative
Neurobiology, University of Valencia
Xenopus laevis at tadpole stages (stage 50-54, R-stages) regenerate in response to
spinal cord injury (SCI) a capability that is lost at the metamorphic climax (stage 56-66,
NR-stages). Here we will discuss three experimental approaches. i) Studies on the
function of neural progenitor cells in spinal cord regeneration. We have found that in Rstages Sox2+ cells have a rapid and transient activation in response to injury and almost
no activation of Sox2+ cells occurs in NR-stages. We have also found that Sox2+ cells
are necessary for proper regeneration. ii) Cell transplantation using cells isolated from
spinal cord at stage 50 and transplanted into non-regenerative animals. iii) Global
analysis of the transcriptome and the proteome deployed in response to SCI in R- and
NR-stages demonstrated extensive differences between those stages. Currently we are
testing the role in spinal cord regeneration of a subset of genes identified by these
global analyses.
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Poster Number: P36
Suppression of vascular network formation by chronic hypoxia and prolylhydroxylase 2 (phd2) deficiency during Xenopus development
S Metikala1,2, H Neuhaus1, T Hollemann1
1
Medical Molecular Biology, University Halle-Wittenberg, Halle, Germany; 2Eugene Bell
Center for Regenerative Biology and Tissue Engineering, Marine Biological Laboratory,
Woods Hole, USA
In the adult, new vessels form in response to hypoxia. Here, the oxygen-sensing system
(PHD–HIF) has been put into focus and is considered as therapeutic target. While the
oxygen-sensing system (PHD–HIF) has been studied intensively, only little is known
from developing vertebrate embryos since mutations within this pathway led to an early
decease of embryos. Xenopus provides an ideal experimental system to address these
processes in vivo. To this end, we adopted a computer-controlled four-channel system,
which allowed us to culture Xenopus embryos under defined oxygen concentrations.
Our data show that the development of vascular structures and blood cells is impaired
under hypoxia, while general development is less compromised. Interestingly,
suppression of Phd2 using specific antisense morpholinos or a chemical inhibitor
resulted in mostly overlapping vascular defects. Our results provide the first evidence
that oxygen via Phd2 has a decisive influence on the formation of the vascular network
during vertebrate embryogenesis.
Poster Number: P37
S Xue1, C T Gordon2, A Javed3, G Yigit4, K Chen5, A Hillmer3, M Blewitt5, B Wollnik4, J
Amiel2, B Reversade1
1
Laboratory of Human Genetics and Embryology, Institute of Medical Biology (A*STAR),
Singapore; 2Laboratory of Embryology and Genetics of Congenital Malformations,
Institut Imagine (INSERM), Paris, France; 3Cancer Therapeutics and Stratified
Oncology, Genome Institute of Singapore (A*STAR), Singapore; 4Institute of Human
Genetics, University of Cologne, Cologne, Germany; 5The Walter and Eliza Hall Institute
of Medical Research, University of Melbourne, Melbourne, Australia
Nose shape varies between species, the longest being that of the elephant. An extreme
aplasia of the nose is seen in congenital arhinia, a rare condition characterized by a
complete absence of the nose. With less than 50 cases reported in the literature, its
genetic and molecular basis remains unknown. Through trio whole-exome sequencing
of 12 unrelated patients with varying degrees of arhinia ranging from nose hypoplasia to
Bosma syndrome (a severe disease with ear, eye, palate abnormalities and absent
nose), we identified 10 independent de novo mutations in the same gene. Its product,
an enzyme with DNA binding activity, is known to be important for X-inactivation and
genomic imprinting in mice. We hypothesize that by modifying DNA structure and
methylation, this epigenetic regulator serves as a master controller of a gene network
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involved in nose development. Using patient cells and Xenopus embryos, we propose
that the uncovered heterozygous mutations behave as gain-of function alleles with
neomorphic activity.
Poster Number: P38
Analysis of the in vivo role of late endolysosomal transport function for early
development in Xenopus
J Kreis, P Vick
Institute of Zoology, University of Hohenheim, Stuttgart, Germany
Canonical Wnt is a major signaling pathway during development and has been
investigated extensively in Xenopus. In cell culture, it has been shown that Wnt
signaling and endolysosomal trafficking are tightly connected. Inhibiting endocytosis or
function of late multivesicular endosomes prevents receptor complex internalization or
signal propagation, and both inhibits Wnt target gene activation. Furthermore, interfering
with lysosome function results in enlarged late endosomes, coinciding with a multi-fold
amplification of Wnt/beta-catenin target gene activation. Classical Xenopus assays
indicated a potential in vivo requirement of late endosomes for Wnt signaling as well. To
test for in vivo functions of endolysosomal transport for Xenopus development, we
analyzed the activity of membrane trafficking regulators and performed knockdown
experiments. Interestingly, morphant phenotypes suggested an involvement of the
endolysosomal machinery during Xenopus gastrulation. Surprisingly, the obtained
phenotypes imply a potential role of late endolysosomal trafficking for early embryonic
morphogenesis related to non-canonical Wnt signaling.
Poster Number: P39
The unique and collective functions of Transforming Acidic Coiled Coil (TACC)
family members in regulating microtubule plus-end dynamics in vivo
E R Rutherford, L Carandang, P Ebbert, M Evans, C Lucaj, L A Lowery
Microtubule plus-end tracking proteins (+TIPs) play key roles in the regulation of
microtubule dynamics. Despite their importance, it is still unclear how various +TIPs
interact with each other and with plus-ends to control microtubule behaviors, particularly
during embryonic development. Our lab studies +TIP function by quantitative analysis of
microtubule behaviors using high-resolution live-maging data of cultured embryonic
Xenopus laevis cells and embryos. Previously, we demonstrated that the transforming
acidic coiled-coil (TACC) domain family member, TACC3, can function as a +TIP to
promote microtubule polymerization. More recently, we determined that TACC1 and
TACC2 can also act as +TIPs. However, each +TIP displays distinct localization profiles
on the microtubule plus-end, differential cell-type-specific effects on microtubule
dynamics, and embryonic cell type-specific expression patterns. In sum, our work
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highlights the unique and collective functions of TACC family members and how they
interact to regulate microtubule plus-end behaviors in different embryonic cell types
during development.
Poster Number: P40
A fundamental question in neuronal development is how growth cone cytoskeletal
dynamics are coordinated to promote accurate axonal navigation. To address this
question, we focus on microtubule plus-end tracking proteins (+TIPs), which may play a
key role in axon guidance. We determined that TACC3 is a +TIP that promotes
microtubule polymerization and axon outgrowth. We have begun to test the hypothesis
that TACC3 spatially restricts microtubule polymerization in response to guidance
signals in Xenopus laevis axons in vivo and ex vivo. We demonstrate that TACC3 is
required to promote axon outgrowth and prevent spontaneous retractions. Additionally,
we find that manipulation of TACC3 levels interferes with the growth cone response to
axon guidance cues. Finally, we observe that ablation of TACC3 causes pathfinding
defects in Xenopus laevis embryos. Together, our findings suggest that TACC3
functions as an axon guidance-regulating factor in embryonic neurons by spatially
promoting microtubule polymerization dynamics.
Poster Number: P41
Characterisation of the ADAMTS family in Xenopus
I Desanlis1, G N Wheeler1, D R Edwards2
1
BIO, UEA, Norwich, UK; 2MED, UEA, Norwich, UK
The ADAMTS (A Disintegrin and Metalloproteinase with Thrombospondin motifs)
enzymes are secreted, multi-domain matrix-associated zinc metalloendopeptidases that
have diverse roles in tissue morphogenesis and patho-physiological remodeling. The
human family includes 19 members. The role of the ADAMTS family in neural crest
development is not well known.
In this study Xenopus embryos are used as an in vivo model for studying the function of
ADAMTSs during development. We have identified the 19 members of the ADAMTS
family. A phylogenetic study with the genomes of vertebrates (Homo, Mus and
Xenopus), the chordate Ciona and protostome invertebrates (Drosophila and
Caenorhabditis) has shown strong conservation of the ADAMTS family and provided a
view of the evolutionary history.
We are focused on the aggrecanases/versicanases sub-family as versican has been
shown to be expressed in migrating neural crest cells during early Xenopus
development.
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We are now characterising the expression profile and the function of ADAMTSs in
Xenopus.
Poster Number: P42
Downregulation of the cytoskeletal protein Zyxin functioning in cells of the
Xenopus laevis midneurula axial tissues leads to activation of stem cells markers
N Y Martynova1, F M Eroshkin1, E E Orlov1, A G Zaraisky1, E B Prokhortchouk2
1
Bioorganic Chemistry of the Russian Academy, Moscow, Russia; 2Laboratory of
Genetics and Epigenetics of Vertebrates, Center of Bioengineering, Russian Academy
of Sciences, Moscow, Russia
Zyxin is a low abundant LIM-domain protein that binds to alpha-actinin and initiates
nucleation and assembly of actin filaments but is able to enter cell nuclei and interact
with proteins involved in the transcription machinery. Due to such ambivalence, Zyxin is
a good candidate for a mediator that couples, during the development, cell
morphogenetic movements with gene expression. We compared by using highthroughput sequencing transcriptomes of the axial tissues of the Xenopus laevis
midneurula, i.e. the neural plate with the underlying mesoderm, from the wild-type
embryos and embryos, in which translation of Zyxin mRNA was inhibited by
microinjection of anti-sense morpholino oligonucleotides. As a result of subsequent
bioinformatic analysis of the differentially expressed genes, confirmed by RT-PCR
analysis, we have established that downregulation of Zyxin functioning leads to
suppression of the pathways responsible for the neural and skeletal muscle
differentiation but enhances the expression of stem cells markers.
Poster Number: P43
March2 E3 ubiquitin ligase antagonizes canonical Wnt signaling for Xenopus
head formation
H Lee1,2, B R Keum1,2, S M Cheong1, J K Han1
1
Life Sciences, Pohang University of Science and Technology, Pohang, Republic of
Korea; 2BK21+ Program of Bio-Molecular Function, Pohang University of Science and
Technology, Pohang, Republic of Korea
Canonical Wnt signaling is crucial for vertebrate development including head formation.
As a key scaffolding protein, Dishevelled (Dvl) mediates activation of the Wnt signaling.
Recently, several Dvl binding partners have been suggested to regulate the stability of
Dvl. However, the detailed molecular mechanisms by which Dvl undergoes proteolysis
remain to be elucidated. Here we show that RING-type E3 ubiquitin ligase March2
antagonizes the Wnt signaling by regulating the turnover of Dvl protein via ubiquitinmediated lysosomal degradation. During Xenopus embryogenesis, March2 is
ubiquitously expressed and required for anterior head formation by maintaining low level
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of Wnt signaling activity in prospective head region. Our biochemical analysis and
phenotype analysis revealed that knockdown of March2 elevates the Wnt signaling and
induces head defects due to malformation of the head organizer. These results suggest
that March2 restricts cytosolic pools of Dvl protein and leads subsequent limitation of
Wnt signaling for vertebrate head development.
Poster Number: P44
Emergence of primitive myeloid cells at the mesonephric rudiment in early
Xenopus tadpole
Y Imai1, K Ishida1, M Nemoto2, K Nakata1, T Kato3, M Maeno1
1
Graduate School of Science and Technology, Niigata University, Niigata, Japan;
Department of Biology, Faculty of Science, Niigata University, Niigata, Japan;
3
Department of Biology, School of Education, Waseda University, Tokyo, Japan
2
Rabbit anti-serum against a myeloid cell-specific peroxidase (Mpo) in Xenopus laevis
has been generated to identify the myeloid cells in adult and larval animals. Double
staining of Mpo antibody with XL-2, mouse monoclonal antibody against a leukocyte
common antigen, enabled us to classify the leukocyte lineages (myeloid cells vs
lymphocytes). In immuno-histochemical analysis on the larval organs, we found a
cluster of XL-2+Mpo+ cells in the region of mesonephric rudiment. The frequency of XL2+Mpo+ cells out of XL-2+ cells in the mesonephric region was approximately 80%,
which was extremely higher than that found in other hematopoietic organs. We have
tried to identify the embryonic origin of the myeloid cells appeared in the mesonephric
region, and found that a tailbud region at the early neurula stage highly contributed to
the myeloid cluster at the mesonephric region. Therefore, there are at least four
independent origins of myeloid cell population in Xenopus embryo.
Poster Number: P45
A role of JunB proto-oncogene in tailbud induction and tail regeneration during
early Xenopus embryogenesis
H Yoshida1, M Okada1, K Takebayashi-Suzuki1, N Ueno2, A Suzuki1
1
Institute for Amphibian Biology, Graduate School of Science, Hiroshima University,
Higashi-Hiroshima, Japan; 2Division of Morphogenesis, National Institute for Basic
Biology, Okazaki, Japan
Integration of signaling pathways is important for the establishment of the body plan.
However, little is known about how the multiple signals interact to regulate
morphogenesis. Here, we show that junb is expressed in the posterior neural plate and
the caudal fin during Xenopus embryogenesis and that overexpression of junb induces
ectopic tail-like structures. A mutant form of JunB lacking GSK3 and MAPK
phosphorylation sites showed stronger effects than wild-type JunB. Moreover, mutant
JunB induced the expression of the tailbud and neural marker genes in ectopic tail-like
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structures. In ectodermal explants, overexpression of JunB increased the expression of
fgf3, wnt8 and xhox3. Embryos injected with JunB MO showed a reduction in tail-length.
These results suggest that JunB is likely to play important roles in tailbud formation by
integrating multiple morphogen signals. We are also currently studying a potential role
of JunB in tail regeneration.
Poster Number: P46
T Yamamoto1, Y Mii1,2, S Takada2, M Taira1
1
Department of Biological Sciences, Graduate School of Science, University of Tokyo,
Tokyo, Japan; 2Division of Molecular and Developmental Biology, National Institute for
Basic Biology, Okazaki, Japan
Morphogens including Wnt and BMP confer regional identities along the body-axes in a
concentration-dependent manner, and heparan sulfate (HS) is a key component for
distribution and reception of morphogens. However, the molecular basis understanding
of graded distribution of ligands remains to be clarified. Here we discovered two
discrete punctate clusters of HS and its nascent chain heparosan (Hn) on the cell
surface. Notably, HS and Hn clusters have distinct properties: (i) association
preferences (HS for Wnt and Hn for Frzb and Wnt/Frzb complexes), and (ii)
internalization tendency (HS is higher than Hn), and thereby HS is necessary for shortrange distribution and signalling of Wnt, whereas Hn is necessary for long-range
distribution for Wnt/Frzb complexes. Furthermore, we show that secreted BMP
antagonist Cerberus is colocalized with Hn, and expands BMP distribution and
signalling. These results suggest that HS/Hn clusters are a fundamental platform
essential for morphogen distribution and signalling.
Poster Number: P47
Permanent Blastula-type embryos in Xenopus destined for cell death are rescued
by signals from vegetal cells
M Sakai, H Nodono
Department of Chemistry and Bioscience, Kagoshoma University, Kagoshima, Japan
Xenopus permanent blastula-type embryos (PBEs) lacking vegetal part of the fertilized
egg and thus receiving neither mesodermalizing nor dorsalizing signals, form only
epidermal tissue. PBEs undergo entire cell degeneration due to apoptosis, as revealed
by caspase-3 assay, the DNA ladders and TUNEL assay. Here we developed a method
for combining the 8-cell stage PBE from the wild embryos and the four vegetal cells
from the albino embryos. The resulting developed to form normal-looking froglets,
showing the PBEs are rescued from cell death by the induction from the vegetal cells.
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Poster Number: P48
A retinoic acid - hedgehog cascade coordinates mesoderm inducing signals and
endoderm competence during lung specification
S A Rankin1, L Han1, K W McCracken1, A P Kenny3, C T Anglin1, E A Grigg1, C W
Crawford1, J M Wells1, J M Shannon2, A M Zorn1
1
Perinatal Institute, Division of Developmental Biology, Cincinnati Children’s Hospital
and the Department of Pediatrics, Cincinnati, OH, USA; 2Perinatal Institute, Division of
Pulmonary Biology, Cincinnati Children’s Hospital and the Department of Pediatrics,
Cincinnati, OH, USA; 3Perinatal Institute, Division of Neonatology, Cincinnati Children’s
Hospital and the Department of Pediatrics, Cincinnati, OH, USA
The respiratory system is induced in the embryonic foregut endoderm by paracrine
Wnt2/2b and Bmp from the surrounding mesoderm. However, the mechanisms that
regulate ligand expression and the competence of the endoderm to respond are
unknown. Using Xenopus embryology, mouse genetics, and human ES cells we
identified a conserved retinoic acid (RA) and hedgehog (Hh) signaling cascade that acts
upstream of Wnt2/2b initiating respiratory induction. Mesoderm-derived RA patterns the
lateral plate mesoderm into a foxf1+/hand1- domain overlying the presumptive lung
field. RA also promotes Hh-ligand expression in the foregut endoderm. Hh signals back
to the mesoderm via Gli2/3, which are required for Wnt2/2b and Bmp4 expression. In
addition, RA regulates the competence of the endoderm to activate the Nkx2-1+
respiratory program in response to Wnt and BMP. These data provide new insight into
early lung development and how mesenchymal signals are coordinated with epithelial
competence during organogenesis.
Poster Number: P49
Germ line-specific activation of Xenopus tropicalis histone B4 through the
proximal promoter sequence
M Nakamigawa, T Kondo, M Maeno
Graduate School of Science and Technology, Niigata University, Niigata, Japan
Histone B4 (HB4) is a linker histone specifically expressed in the Xenopus oocyte. To
date, the mechanism of tissue-specific expression of HB4 has not been elucidated. In
the present study, therefore, enhancer activity driven by HB4 flanking DNA was
examined. Among the genomic DNA of X. tropicalis from the 5’ flanking region to the
second intron of HB4 gene (HB4 -6273/+2915 bp), the promoter region (HB4 -3072/+29
bp) was able to drive the oocyte-specific transcriptional activity in the in vitro reporter
assay. In a transgenic frog line carring the gfp gene driven by HB4 -3072/+29 bp
sequence, expression of GFP was detected in both ovary and testis, but not in other
organs. Deletion mutants of the promoter DNA indicated that the proximal sequence
(HB4 -288/+29 bp) had a strong transcriptional activity, and we are currently examining
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a role of the oocyte-specific transcription factor, Nobox, in the proximal sequencedependent activation.
Poster Number: P50
The newly identified ephrinB2 binding partner, TBC1d24, plays a role in neural
crest cell migration
Laboratory of Cell and Developmental Signaling, National Cancer Institute, Frederick,
MD, USA
Although Eph-ephrin signaling contributes to the migration of cranial neural crest (CNC)
cells, it is still unclear how ephrinB transduces signals affecting this event. Using
ephrinB2 immunoprecipitation and mass spectrometric analysis, we identified an
interaction between ephrinB2 and TBC1d24 that is mediated by Dishevelled. Both
ephrinB2 and TBC1d24 morphant embryos display abnormal CNC cell migration, which
is rescued by expressing their wild type counterparts. However, a TBC1d24 mutant that
cannot interact with ephrinB2 fails to rescue the TBC1d24 morphant defect. TBC1d24 is
known as a GAP for Rab35 that regulates cell-cell adhesion and cell migration through
regulating cadherin recycling. Both ephrinB2 and TBC1d24 morphants display
increased E-cadherin levels that may disrupt normal CNC migration. In addition, binding
of the EphB4 receptor, decreases the interaction between ephrinB2 and TBC1d24, and
thus inhibits CNC cell migration. Our results indicate that TBC1d24 is a critical player in
ephrinB2 control of CNC cell migration.
Poster Number: P51
Xenopus CIC: A putative downstream regulator of FGF dependent transcription
M King, P Genever, H V Isaacs
Department of Biology, University of York, York, UK
FGF secreted signalling peptide play key roles in regulating germ layer specification in
Xenopus. We have considerable knowledge regarding the FGF dependent
transcriptome in Xenopus embryos, however, it is still unclear how signal transduction
downstream of FGF tyrosine kinase receptors leads to changes in target gene
transcription. The HMG-box transcription factor CIC, which was originally identified as
being post-translationally regulated by receptor tyrosine kinase signalling in Drosophila,
potentially provides such a link. Phosphorylation of CIC by MAPK inhibits its ability to
act as a transcriptional repressor. We hypothesis that transcription of a subset of FGF
target genes is dependent on MAPK mediated inhibition of CIC transcriptional
repression. We show that the two major isoforms of CIC are differentially expressed
during the maternal to early zygotic period of Xenopus development. We have also
begun to characterise the effects of CIC knockout and overexpression in early
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development.
Poster Number: P52
12-time point proteomics of Xenopus laevis allows for broad understanding of
proteomic expression file emerging from a mature oocyte to late neurala stage
embryo quantifying more than 6,100 protein profiles
Chemistry and Biochemistry, University of Notre Dame, Notre Dame, USA
Proteomics is a relatively new endeavor of Xenopus laevis research with the first of a
series of papers being published in 2014. This study examines the proteomic changes
taking part in development starting with the mature oocyte and looking through the
neurala stage (stage 22) by analyzing 12-distinct time points. For a majority of the
proteins, initial data shows either high expression of the protein before fertilization or
post fertilization, but not consistently high or consistently low expression for both a
mature oocyte and a fertilized egg. In this study, 6,148 quantifiable proteins and 58,485
peptide sequences (21.5 % average sequence coverage or 9.5 peptides/ protein) were
identified in biological and technical duplicate. This study provides a solid baseline of
proteins essential for fertilization and early development.
Poster Number: P53
Transcriptional factor Ets1 in heart development and disease
S Nie1, L Lin2, P Grossfeld2
1
Biology, Georgia Institute of Technology, Atlanta, USA; 2Cardiology, University of
California San Diego, San Diego, USA
Frog is placed at a unique position during heart evolution with a three-chambered heart
and spiral septum partially separating the outflow tract. While in amniotes, cardiac
neural crest is a major contributor to cardiac valves and septum, its derivatives in frog
are more restricted. To gain a better understanding of cardiac neural crest development
in frog, we looked at transcriptional factor Ets1, known to be important for neural crest
development. Despite Ets1’s role in neural crest specification and migration, we found
that Ets1 plays major roles in cardiac mesoderm versus cardiac neural crest during frog
heart development. Loss of Ets1 in the mesoderm significantly impairs heart formation,
leading to a thick-walled ventricle with no trabeculae formation. We are characterizing
the downstream signals of Ets1 and examining the interactions between myocardium
and endocardium, hoping to understand how Ets1 regulate the two cell populations in
heart development.
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Poster Number: P54
Division of Developmental Biology, Cincinnati Children's Hospital Research Foundation,
Cincinnati, OH, USA
Congenital birth defects of the trachea and esophagus are quite common (~1/3000) and
often life-threatening conditions caused by disrupted foregut tube morphogenesis.
Despite this, the cellular and molecular mechanisms underlying normal and defective
tracheo-esophageal (TE) morphogenesis are poorly understood. Using Xenopus as a
complement to mouse genetic we showed that TE morphogenesis occurs in five
conserved steps: 1) D/V foregut patterning, 2) medial constriction, 3) epithelial fusion
and septation, 4) tube elongation and 5) recanalization. Medial constriction is the result
of localized mesenchymal proliferation. Epithelial fusion and septation are regulated by
dynamic cell-adhesion and ECM remodeling that drive an “unzippering” process that is
concurrent with a mesenchymal invasion. Finally we showed that loss of Hedgehog/Gli
signaling, which is mutated in some human TE birth defect patients, disrupts medial
constriction and epithelial septation, leading to tracheal atresia and tracheo-esophageal
fistula, modeling the human phenotypes.
Poster Number: P55
Zoology, University of Hohenheim, Stuttgart, Germany
Experimental analysis of laterality started in 1918, when Spemann and Falkenberg
demonstrated that organ situs in ligature-induced conjoined newt twins was normal in
the left and randomized in the right twin. We analyzed Wnt pathway-induced Siamese
twins in Xenopus, which displayed two adjacent gastrocoel roof plates with wildtype
ciliation. Flow was continuous from the right side of the right to the left side of the left
twin. Motile cilia were required for situs in twins, as Nodal cascade induction was
abrogated in flow-impaired twins; Nodal was rescued by parallel knockdown of Dand5
on the left side of the left twin, and the cascade was inverted when ablation of flow was
combined with Dand5-MOs on the right side of the right twin. Together these
experiments demonstrate that GRP cilia and flow determine organ laterality in conjoined
frog twins, which is likely the case in human siamese twins as well.
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Poster Number: P56
Functional analysis of a novel placode gene Fam46a identified by new placode
induction system under the control of BMP signaling
T Watanabe1, Y Ito2, Y Onuma2, T Michiue1
1
Department of Life Science, The University of Tokyo, Tokyo, Japan; 2Organ
Development Research, Research Centre for Stem Cell Engineering, AIST, Tsukuba,
Japan
In the early development of Xenopus laevis, pre-placode region (PPR) is located in the
boundary between epidermis and neuroectoderm. PPR cells migrate and differentiate
into sensory nerves such as anterior pituitary and trigeminal nerve. In gastrula stage,
inhibition of BMP signaling is essential for neural differentiation. In this study, by
adjusting BMP signaling in animal cap explants, we have established a system to
induce placode-like cells.
Using microarray analysis, we identified a novel placode gene called Fam46a. Fam46a
is expressed in PPR at early neurula stage, in lateral line and epibranchial placodes at
tailbud stage. Experiments of placode and neural crest-like cells suggested that
Fam46a is required for upregulation of placode genes and downregulation of neural
crest genes.
It was reported that FAM46A is a cytosolic protein and possibly a SMAD signaling
related protein in human. Based on the available data, we are currently analyzing the
relevance of Fam46a to BMP signaling.
Poster Number: P57
Slco1a2, an ion transporter, is a novel heterotaxy candidate gene that regulates
left-right patterning and heart development through a potential role in midline
signaling
D Bhattacharya, M K Khokha
Pediatrics and Genetics, Yale School of Medicine, New Haven, USA
Congenital Heart Disease (CHD) is the most common birth defect affecting
approximately 1% of all live births in the US. Heterotaxy (Htx) is a severe form of CHD
that occurs when left-right patterning is not correctly established causing significant
morbidity and mortality. A recent genetic analysis of Htx patients identified numerous
candidate genes, including slco1a2. Slco1A2 is an ion transporter, previously implicated
in bile acid formation, but its role in left-right patterning and heart development is
completely novel. Knockdown of slco1a2 using a morpholino oligo or CRISPR/ Cas9
leads to abnormal heart looping, as well as defects in early left-right signaling cascade
(assayed by pitx2 and coco expression). Interestingly some embryos also have a
midline cyclopia phenotype strongly suggesting disruption of the midline signaling in
early development. Our aim is to investigate the the role of slco1a2 in left-right
patterning and heart development.
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Poster Number: P58
Identification of new regulators of embryonic patterning and morphogenesis in
Xenopus gastrulae by RNA sequencing
I Popov1, T Kwon2, D K Crossman1, M R Crowley1, J B Wallingford3, C Chang1
1
Cell, Developmental and Integrative Biology, University Alabama at Birmingham,
Birmingham, USA; 2Biomedical Engineering, Ulsan National Institute of Science and
Technology, Repulic of Korea; 3Molecular Biosciences, University of Texas at Austin,
Austin, USA
Cell fate specification is often coupled with specific cell behaviors during development.
Though many genes with embryonic inducing ability have been identified, factors that
control cell movements are less understood. To uncover new regulators of embryonic
patterning and motility, we performed RNA sequencing to investigate differentially
expressed genes in early organizer, the dorsal and the ventral marginal zone of
Xenopus gastrulae. Upon confirmation of a panel of differentially expressed genes by
RT-PCR, we examined a selected subset for their ability to block activin-induced animal
cap elongation. Several genes interfered with cap elongation without affecting
mesodermal induction by activin, but an ECM protein, EFEMP2, inhibited activin
signaling. We further showed that a secreted protein kinase PKDCC1 regulated
gastrulation movements as well as anterior neural patterning. Overall, our studies
identify many differentially expressed signaling and cytoskeleton regulators in Xenopus
gastrulae and imply their functions in regulating cell fates and/or behaviors during
gastrulation.
Poster Number: P59
Role of heterotrimeric G-protein Gα13 and Leukemia-associated Rho guaninenucleotine exchange factor (LARG) in RhoA activation and radial intercalation
movements in Xenopus laevis epiboly
D O Kiryukhin1, N N Luchinskaya2, L A Shustikova3, Y Y Kopantseva3, M V
Zinovyeva4, A V Belyavsky3
1
Berezov TT Department of Biochemistry, Medical Faculty, People`s Friendship
University of Russia, Moscow, Russia; 2Department of Emryology, Biological Faculty,
Moscow State University, Moscow, Russia; 3Laboratory of Stem and Progenitor Cell
Biology, Engelhardt Institute of Molecular Biology, Moscow, Russia; 4Laboratory of
Human Genes Structure and Functions, Shemyakin-Ovchinnikov Institute of Bioorganic
Chemistry, Moscow, Russia
Little is known about the regulation of the radial intercalation during the epiboly in X.
laevis. We examined the role of the signaling axis Gα13 – LARG – RhoA in this
process. Gα13 and LARG are expressed during the epiboly. Overexpression of the both
genes correlated with the activation of RhoA and the inhibition of the radial intercalation.
In Gα13-overexpressing embryos morpholinos against LARG blocked RhoA activation
while didn’t prevent inhibition of the radial intercalation. Thus, LARG is essential for
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RhoA activation through Gα13. In LARG-overexpressing embryos morpholinos against
Gα13 didn’t prevent RhoA activation, and the radial intercalation was inhibited.
Therefore the function of Gα13 in X. laevis epiboly is not solely dependent on RhoA
activation. Our results are the first evidence that Gα13 and LARG participate in
maintenance of RhoA activation state that is important for proper radial intercalation
during the epiboly of X. laevis.
Poster Number: P60
The difference of shape and tension between neural and epidermal ectodermal
cells in Xenopus
S Yamashita, N Ishinabe, T Ide, T Michiue
Graduate School of Arts and Sciences, The University of Tokyo, Tokyo 153-8902,
Japan
During gastrulation, ectodermal patterning is determined according to the signals from
mesendodermal cells. Though the shape of cells in presumptive neuroectoderm looks
similar to that in epidermal region at gastrula stage, these cells differently behave
according to their own properties. In this study, we aimed to distinguish these types of
cells by directly measuring the tension of these cells, as well as several features of cell
shape in Xenopus embryo. For measuring cell tension, we used FRET sensor probe
including Actinin and elastic linker. Our analysis made it possible to directly show the
level of tension in whole embryo without invasion and fixation. We further analyzed cell
shape (cell size, aspect ratio etc.) of mGFP-injected embryo at gastrula-neurula stage
by MATLAB software. Together with these results, we revealed that these types of cell
were obviously distinguishable.
Poster Number: P61
The T-box gene Brachyury and its regulative functions during left-right axis
development in Xenopus laevis
S Kurz1, P Andre2, A Schweickert1, M Blum1
1
Institute of Zoology, University of Hohenheim, Stuttgart, Germany;
Protection and System Technology, MAVIG GmbH, Munich, Germany
2
Radiological
The left-right (LR) axis is specified at neurula stages by a ciliated epithelium termed leftright organizer (LRO; GRP in frog, PNC/node in mouse). Motile cilia generate an
extracellular leftward fluid-flow, which finally results in asymmetric gene expression.
Brachyury (T) mutant embryos failed to develop a functional LRO and thereby exhibited
loss of asymmetry. In order to understand the underlying specification and
morphogenesis processes we analyzed Brachyury function in Xenopus laevis. Like in
the mouse Xbra was necessary for the development of frog LRO and hence for LR axis
formation. We were able to trace the origin of LRO malformation to early gastrula
stages, where the precursor tissue of the GRP, the superficial mesoderm (SM), was
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affected. Surprisingly, this Xbra activity was non cell-autonomous, suggesting a
complex gene network for SM specification. Using gain- and loss-of-function
experiments in an explant assay, we currently investigate Brachyury function in more
detail.
Poster Number: P62
POSTER WITHDRAWN
Poster Number: P63
Ciliary transcription factors and miRNAs precisely regulate Cp110 levels at basal
bodies required for ciliogenesis
P Walentek1, I Quigley2, D I Sun1, U K Sajjan1, C Kintner2, R M Harland1
1
2
Molecular and Cell Biology, University of California Berkeley, Berkeley, USA;
Molecular Neurobiology Laboratory, Salk Institute for Biological Studies, La Jolla, USA
Upon cell cycle exit, the mother centriole matures into a basal body to facilitate cilia
formation. The centriolar protein Cp110 is a key regulator of this process and inhibitor of
cilia. Using Xenopus we show that Cp110 also localizes to cilia forming basal bodies
and is required in all types of cilia. In multiciliated cells (MCCs), Cp110 promotes ciliary
adhesion complex formation and basal body-actin interactions. Two clusters of coiledcoil domains in Cp110 mediate inhibition of ciliogenesis and other centriole-specific
roles, suggesting that Cp110’s opposing roles are generated through interactions with
distinct protein complexes. Because of its dual role, Cp110 levels must be precisely
controlled. In MCCs, expression of cp110 is activated by conserved ciliary transcription
factors, which also activate miR-34/449s to repress cp110. Thus, this
transcriptional/posttranscriptional module generates optimal Cp110 levels for
ciliogenesis. Our data reveal novel possibilities through which Cp110 may contribute to
development and disease in vertebrates.
Poster Number: P64
The optimisation of sperm cryopreservation in Xenopus and analysis of the
damage that it causes
S Morrow1, E Pearl2, A Noble1, A Lerebours3, C Sharpe1, M Horb2, M Guille1
1
EXRC, University of Portsmouth, Portsmouth, UK; 2NXR, Marine Biological Laboratory,
Wood's Hole, USA; 3Biological Sciences, University of Portsmouth, Portsmouth, UK
Cryopreservation of Xenopus sperm is important for making the curation of genetically
altered lines cost efffective and to improve welfare of research animals. Whilst X.
tropicalis sperm cryopreservation is robust we have found that of X. laevis to be highly
variable due to high levels of plasma membrane damage in X. laevis sperm. We have
compared the published methods of cryopreservation on X. laevis and found that two
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are similarly effective. We have then tested the variables in the simpler method and
shown that alterations of these have little effect on the efficiency of fertilisation and
development rates with frozen-thawed sperm. We conclude the method of
cryopreservation is robust and the variability of X. laevis sperm cryopreservation is
animal dependent. Frozen-thawed X. tropicalis sperm has a population that can fertilise
but has DNA damage, we have analysed the effect of this on embryo development and
identified the DNA damage hotspots.
Poster Number: P65
Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst,
USA
The cranial neural crest cells (CNC) are migratory pluripotent stem cell population that
contribute to craniofacial development. Our results show that ADAM13 regulates the
protocadherin PCNS, a protein essential for CNC migration, at both transcriptional and
post-transcriptional levels. This regulation depends on both the proteolytic activity and
the presence of the ADAM cytoplasmic domain. Once translated, PCNS is cleaved by
ADAM13 releasing an extracellular fragment (EC1-4). Overexpression of PCNS inhibits
CNC migration and is rescued by overexpression of ADAM13. Conversely reduction of
ADAM13 is significantly rescued by PCNS re-expression and EC1-4. In contrast, a
mutant form of PCNS with an extra EGF repeat immediately before the transmembrane
domain, designed to misaligned the ADAM13 cleavage site, does not rescue CNC
migration. Thus, ADAM13 regulate CNC migration by regulating PCNS expression and
cleaving its extracellular domain. Our goal is to understand how ADAM13 regulates
gene expression.
Poster Number: P66
Novel genes in congenital heart disease (CHD): using Xenopus to understand
heart development
E K Mis, M K Khokha
Pediatrics, Yale University School of Medicine, New Haven, USA
Congenital heart disease (CHD) is the most common congenital malformation, affecting
approximately 1% of births per year in the US and Europe. Though common, the
genetic burden of CHD is still poorly described at the molecular level. Sequencing
analysis of patient-parent trios has identified over 300 genes that may be required for
heart development, the majority of which have not been described in development.
Utilizing a CRISPR/cas9 genome editing loss of function approach in Xenopus, we
screened 68 novel CHD genes for developmental defects. 62% of these novel genes
(42/68 genes) induced a developmental defect upon loss of function in the F0
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population, including cardiac looping defects, craniofacial defects, and preneurula
defects. Uncovering the developmental mechanism of these novel genes will inform our
understanding of congenital heart disease and development.
Poster Number: P67
Aquaporin3b acts in noncanonical Wnt signaling and convergent extension
during Xenopus gastrulation
K See, C S Merzdorf, J Forecki
Cell Biology and Neuroscience, Montana State University, Bozeman MT, USA
Aquaporins and aquaglyceroporins are membrane channel proteins that allow passive
movement of water and other small molecules like glycerol across plasma membranes.
Our results indicate that aquaporins play complex roles within tissues in addition to
allowing water movement. The aqp3b gene is expressed in the sensorial layer of the
Xenopus laevis animal cap and dorsal margin, which indicated a possible role in
gastrulation. Using aqp3b morpholinos, we identified a role for aqp3b in noncanonical
Wnt signaling. When aqp3b is inhibited in Keller explants, both convergence and
extension were impaired. Co-injected dvlDeltaDIX RNA rescued these CE defects,
demonstrating that Aqp3b acted through noncanonical Wnt signaling. Further rescue
experiments showed that Aqp3b exerts its influence specifically through the Wnt/Ca2+
pathway, rather than through Wnt/PCP or Wnt/Ror2 noncanonical Wnt signaling. We
are in the process of determining by which mechanism Aqp3b is able to interact
specifically with and/or regulate Wnt/Ca2+ signaling.
Poster Number: P68
Identifying Mink1, a novel CHD target gene, role in development of left-right
patterning and heart formation
V D Colleluori, M K Khokha
Pediatrics and Genetics, Yale School of Medicine, New Haven, USA
A recent exome sequencing project identified a number of candidate genes, such as
Mink1, for functional analysis regarding their involvement in the development of
heterotaxy. Heterotaxy is a severe form of congenital heart disease (CHD) caused by
abnormal establishment of left-right patterning. CHD is the most common birth defect
affecting 1% of all live births and is the leading cause of infant mortality in the US.
Previously, Mink1 has been found to phosphorylate Prickle1 and have implications in
planar cell polarity, but it’s a novel target for CHD research. I have shown that
knockdown by morpholino or CRISPR/Cas9 leads to significant heart looping
abnormalities resulting from defects in the signaling cascade known to establish leftright patterning. When stained for acetylated tubulin embryos also show a significant
loss of epidermal cilia. The aim of my research is to investigate the role of Mink1 in the
establishment of left-right patterning and heart development.
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Poster Number: P69
Role of Pou3f transcription factors during kidney development
C Cosse-Etchepare1,2, I Gervi1,2, I Buisson1,2, J F Riou1,2, M Umbhauer1,2, R Le
Bouffant1,2
1
Sorbonne Universites, UPMC University of Paris 06, IBPS, UMR 7622, Laboratoire de
Biologie du Developpement, Paris, France; 2CNRS, IBPS, UMR 7622, Laboratoire de
Biologie du Developpement, Paris, France
Although the high occurrence of kidney congenital diseases, the mechanisms involved
in kidney development remain largely unknown. Analyzing the expression profile of
Pou3f transcription factor family during embryonic development, we showed
that pou3f3 and pou3f4 are regionally and partially co-expressed in the developing
xenopus and mouse kidney. Taking advantage of the xenopus model and its simple and
conserved kidney organization, we studied the role of Pou3f3 and Pou3f4 during kidney
formation via gain and loss of function approaches. Our results highlight common
requirement of these two transcription factors for proper kidney formation. Pou3f3 or
Pou3f4 depletion prevents intermediate tubule formation. Pou3f4 overexpression leads
to an expansion of intermediate tubule marker expression. We further showed that
Pou3f3 and Pou3f4 are required for the expression of several terminal differentiation
markers of the tubule. Pou3f3 and Pou3f4 play crucial roles in tubule segmentation and
differentiation.
Poster Number: P70
Unraveling the role of potassium in early embryo morphogenesis and left-right
patterning
E Sempou, M K Khokha
Pediatrics, Yale University School of Medicine, New Haven, USA
The roles of potassium channels and potassium itself have been extensively studied in
excitable cells such as neurons and cardiomyocytes. However, little is known about how
potassium dynamics affect non-excitable cells during early stages of embryonic
development. Recently, KCNH6, a gene encoding an EAG related potassium channel,
was identified to carry a missense mutation in a congenital heart disease patient with
heterotaxy. In heterotaxy, defective embryonic left-right (LR) patterning results in
incorrect LR spatial distribution of visceral organs, including the heart. Editing of kcnh6
in Xenopus embryos using CRISPR/cas9 produced heterotaxy, recapitulating the
patient’s condition. Interestingly, the heterotaxy phenotype was linked to defective
morphogenesis in blastula/gastrula stages. Identical gastrulation and LR patterning
defects were observed in embryos treated with potassium channel blocker Barium and
non-physiological potassium levels in the embryonic medium. Altogether, our data
highlight novel and essential roles for kcnh6 and potassium in early vertebrate
development.
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Poster Number: P71
Determining the Role of Cadherin-11 Cleavage in Cranial Neural Crest Migration
K Mathavan, G Abbruzzese, D Alfandari
Molecular and Cellular Biology Program, Department of Veterinary and Animal
Sciences, University of Massachusetts, Amherst, USA
During development, cranial neural crest (CNC) cells emigrate from the neural plate
border to invade ventral tissues and form craniofacial structures. Migration of CNC cells
requires multiple ADAM metalloproteases. We have shown that knockdown of ADAM13
in Xenopus laevis impairs CNC migration. This phenotype can be rescued by
overexpression of EC1-3, a fragment of Cadherin-11 cleaved by ADAM13. Our current
work shows that overexpression of EC1-3 in CNC elicits phosphorylation of Akt, a
member of the PI3K pathway. We also show that EC1-3 interacts with multiple growth
factor receptors. However, Akt phosphorylation is only triggered by a small subset of
these receptors. Our goal is to determine the upstream and downstream effectors of
EC1-3/Akt that mediate CNC migration.
Poster Number: P72
A Novel GTPase System Regulates β-Catenin Nuclear Transport in Development
and Disease
J N Griffin1,2, A R Duncan1, F del Viso1, A Robson1, S Kulkarni1, K J Liu2, M K Khokha1
1
Pediatrics, Yale University School of Medicine, New Haven, USA;
Development and Stem Cell Biology, King's College London, London, UK
2
Craniofacial
Canonical Wnt signaling coordinates many critical aspects of embryonic development,
while dysregulated Wnt signaling contributes to numerous common diseases, including
congenital malformations and cancer. The nuclear localization of β-catenin is
fundamental in pathway activation. However, despite intensive investigation, the
mechanisms regulating β-catenin nuclear transport remain undefined. β-catenin nuclear
transport is energy (GTP) dependent but β-catenin lacks a classic “NLS” nuclear
localization signal and does not require the karyopherin/Ran GTPase transport system.
Here, we describe a nuclear localized guanine nucleotide exchange factor (GEF) that is
an unexpected regulator of β-catenin nuclear transport. Identified in a patient with
congenital heart disease and heterotaxy, we show that this GEF alters left-right
patterning via Wnt signaling and the nuclear localization of β-catenin, rather than βcatenin degradation. Together, our results define a novel GTPase based system that
facilitates nuclear transport of β-catenin, and suggest new targets for the modulation of
Wnt signaling in disease.
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Poster Number: P73
The chromatin modifier, WDR5, is a multifunctional protein that has dual roles in
ciliogenesis and left-right patterning
S S Kulkarni1,2, J Griffin1,2, K Liem1, M Khokha1,2
1
Pediatrics, Yale School of Medicine, New Haven, USA; 2Genetics, Yale School of
Medicine, New Haven, USA
Congenital heart disease (CHD) is a major cause of infant mortality and morbidity.
Histone modifiers including WDR5 are implicated in CHD, but the underlying
mechanisms remain unexplored. WDR5 is a crucial scaffolding subunit of H3K4methyltransferase (H3K4MT) complexes. Here, we demonstrate that Wdr5 depletion
leads to LR patterning and ciliogenesis defects in Xenopus, a phenotype seen in CHD.
We find that WDR5 is essential for LR patterning via its role in H3K4MT pathway,
specifically, via transcriptional regulation of FoxJ1. On the other hand, we find that Wdr5
is crucial for ciliogenesis in multiciliated cells (MCCs) but unexpectedly independently of
the H3K4MT pathway. Rather, Wdr5 is localized to the ciliary base and is critical for the
uniform distribution and polarization of basal bodies in MCCs. Further, Wdr5 is essential
for the apical organization of actin. Wdr5 interacts with actin and basal bodies, where it
plays a scaffolding role to pattern basal bodies across the apical cell surface.
Poster Number: P74
The role of dashsous2 in congenital heart disease
E D Deniz1, A R Robson1, N A Al-Mahmoud1, M B Brueckner1, M K Khokha1,2
1
Department of Pediatrics, Yale University, New Haven, USA; 2Department of Genetics,
Yale University, New Haven, USA
The genetics of congenital heart disease is an area of intense research. In Xenopus we
tested human CHD candidate genes for effects on cardiac development and
identified dachsous2 (DCHS2). When knockdown by morpholino oligonucleotides we
found that DCHS2 cause a loss of cardiac trabeculation and loss of epidermal cilia
resembling human phenotype closely. We found that DCHS2 protein localized to the
base of the cilium where it is essential for basal body patterning. Then we applied
microscale high speed imaging to morphant tadpole hearts and demonstrated loss of
cardiac trabeculation, a phenotype reminiscent of the cardiac non-compaction
phenotype seen in our DCHS2 patient. We successfully employed our “reverse
translation” approach, taking gene discovery from the bedside to the bench in order to
understand the underlying CHD disease process and showed that DCHS2 is a novel
gene within the planar cell polarity pathway related to cardiovascular defects in
humans.
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Poster Number: P75
Musculocontractural-Ehlers-Danlos-syndrome: Dermatan sulfate is required for
Xenopus neural crest cells to migrate and adhere to fibronectin
N Gouignard1, M Maccarana2, I Strate1, A Malmström2, E M Pera1
1
Lund Stem Cell Center, Lund University, Lund, Sweden; 2Department Experimental
Medicine Science, Lund University, Lund, Sweden
Musculocontractural-Ehlers-Danlos-syndrome (MCEDS) is a heritable disorder with
distinct craniofacial features and multiple congenital malformations that are caused by
dysfunction of dermatan sulfate (DS) biosynthetic enzymes, including DS epimerase-1
(DS-epi1). We show that DS-epi1 is important for generating isolated iduronic acid
residues in chondroitin sulfate (CS)-DS proteoglycans in early Xenopus embryos.
Knockdown of DS-epi1 impairs the correct activation of transcription factors involved in
epithelial-mesenchymal-transition (EMT) and causes a decrease in neural crest (NC)derived craniofacial skeleton, melanocytes, and dorsal fin structures. Transplantation
experiments demonstrate a tissue-autonomous role of DS-epi1 in cranial NC cell
migration in vivo. Cranial NC explant and single cell cultures show a requirement of DSepi1 in cell adhesion, spreading and extension of polarized cell processes on
fibronectin. Thus, our work reveals a functional link between DS and NC cell migration.
NC defects in EMT and cell migration might account for the craniofacial anomalies and
other congenital malformations in MCEDS.
Poster Number: P76
Motile Cilia: Characterization of Novel foxj1 Target Genes in Xenopus and Mouse
T Ott1, L Alten2, A Beckers2, C Adis2, A Gossler2, M Blum1
1
Institute of Zoology, University of Hohenheim, Stuttgart, Germany;
Molecular Biology, Hannover Medical School, Hannover, Germany
2
Institute for
Motile cilia are apically localized filamentous cell organelles projecting into the
extracellular space. Ciliary beating, in the plane or rotating, accelerate extracellular
fluids to regulate a variety of developmental processes and adult tissue homeostasis. In
humans loss of ciliary motility results in syndromes addressed as primary ciliary
dyskinesia, associated with male infertility, laterality defects, failure of mucociliary
clearance and hydrocephaly. The winged-helix transcription factor Foxj1 is a key
regulator of motile ciliogenesis. Its downstream program of motile ciliary biogenesis,
however, is largely unresolved. In a murine Foxj1 target screen we identified six so far
uncharacterized novel candidate genes, which were strictly associated with motile cilia
in mouse and frog. RNA and protein expression data will be presented as well as initial
functional characterizations by gene loss-of-function in both model systems. These
experiments should help to uncover the conserved role of foxj1 in motile ciliogenesis.
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Poster Number: P77
LRPPRC, a novel role in L/R patterning of the Xenopus embryo
A E MacColl Garfinkel, M Khokha
Genetics, Yale University School of Medicine, New Haven, USA
Congenital head disease is the leading cause of birth defects in the United States,
affecting 9 of every 1000 children born each year. One severe form of CHD is
heterotaxy, the rearagement of internal organs along the L/R axis. Although not all
patients born with CHD present with heterotaxy, the developmental mechanisms that
control L/R patterning are also required for proper heart morphology, and may be
implicated in the disrupted heart patterning in these patients. Through recent genetic
analysis of affected patients, novel candidate genes for CHD have been implicated,
however, thorough analysis of their underlying mechanisms is not complete. One of the
candidate genes is LRPPRC, previously implicated in French Canadian Leigh
Syndrome, however its role in development is not yet known. Through the use of
Xenopus as a powerful genetic model for human disease we have begun to elucidate its
role in embryological patterning.
Poster Number: P78
Technical innovations for InDrops single-cell transcriptomics of Xenopus cells
We describe a series of technical developments that enabled efficient and accurate
single-cell transcriptional profiling of Xenopus cells via InDrops, to minimize barrier to
entry for other interested Xenopus researchers. Major challenges were encountered
due to the unusually large size of Xenopus cells. New dissociation conditions were
developed that preserve viability in >95% of single cells for over two hours at room
temperature, while also allowing complete dissociation within 10-20 minutes, minimizing
technically induced transcriptional responses. New density-based washing strategies
were developed that eliminated pipetting of dissociated cells and thus do not cause cell
lysis. Neutral density buffers containing BSA were found to be a critical addition to all
microfluidic protocol steps. Together these innovations allow application of InDrops to
Xenopus embryos for barcoding of >10k single-cell transcriptomes per hour.
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Poster Number: P79
Primitive Myeloid Cells from the Blood Island are Necessary and Sufficient
Inducers of Foregut Progenitors and BMP signaling in Xenopus laevis and
Musculus musculus
Z N Agricola1,3, A K Jagpal1,3, S A Rankin2,3, S W Cha2,3, A M Zorn2,3, A P Kenny1,3
1
Neonatology, CCHMC, Cincinnati, USA; 2Developmental Biology, CCHMC, Cincinnati,
USA; 3Perinatal Institute, CCHMC, Cincinnati, USA
BMP2-requiring foregut endoderm and primitive myelocytes are directly neighboring
each other. Ablation of myelocytes cells with spib morpholino leads to normal cardiac
progenitors but abnormal cardiac morphogenesis, phenotypically reminiscent of hhexexpressing foregut endoderm progenitor ablation. We examined whether foregut
progenitor development was perturbed by loss of the primitive myeloid population.
spiba morpholino myeloid ablation resulted in loss of foregut progenitor cells and foregut
organ buds. Einsteck induction of foregut progenitors was achieved in host embryos
using myeloid-induced animal caps. BMP target gene sizzled expression in the
endoderm was reduced when primitive myeloid cells were ablated from the
aVBI. Csf1r-Cre driven diphtheria toxin ablation of primitive myeloid cells resulted in
significant
loss
of
hhex
expression
in
e9.5
mouse
foregut.
Primitive myeloid cells are essential for foregut endoderm progenitor in frog and mouse.
Thus coordinated, interdependent development occurs between primitive myeloid cells
in the vertebrate blood island and the foregut endoderm progenitor.
Poster Number: P80
Novel secreted protein AFRO regulates anterior formation in amphibian embryos
Y Sato1, H Kuroda2
1
Media and Governance, Keio University,
Information, Keio University, Kanagawa, Japan
Kanagawa,
Japan;
2
Environmental
Many proteins secreted from dorsal side of gastrula embryos in vertebrates have
important roles for the dorsoventral patterning. Here we describe a novel dorsal-specific
secreted protein, AFRO (amphibian-specific factor regulating optic formation), which
has 259 amino acids as secreted region in addition to 17 amino acids as signal peptide
motif.
Sequence similarity searching revealed that AFRO was only reported in Xenopus laevis
and tropicalis and consisted of no characterized domain inside. Using Xenopus laevis
embryos, we first checked its expression pattern by RT-PCR and in situ hybridization,
resulting that AFRO was localized at dorsal side of gastrula embryo and remarkably
detected at anterior neuroectoderm and midline in neurula stage. Functional analysis
revealed that AFRO could make severe effects on anterior neural and especially optic
formation.
AFRO is an amphibian-specific but very strong regulator of optic formation, so it may
worth thinking evolutional meaning of it.
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Poster Number: P81
Loss-of-function analysis of a large family of nonclassical MHC genes in Xenopus
laevis by the CRISPR/Cas9 system
M Banach, E S Edholm, J Robert
Microbiology and Immunology, University of Rochester Medical Center, Rochester, USA
Xenopus nonclassical MHC class Ib (XNC) gene family determines Xenopus laevis
immunity and potentially its physiology. Using RNA interference, we previously
demonstrated that XNC10.1 gene is critical for the development and function of
specialized innate T (iT) cells. For more reliable and consistent functional studies of
XNC genes, we applied a CRISPR/Cas9-mediated gene disruption approach. We
specifically and efficiently generated single and double knockouts of different XNC
genes, including XNC10.1, XNC11, XNC1. The absence of transcripts for XNC10.1 and
Vα6-Jα1.43 invariant T cell receptor rearrangement in XNC10.1 knockouts X. laevis
tadpoles, indicated XNC10.1 loss-of-function and deficiency in innate Vα6 iT cells.
Surprisingly, XNC1 gene disruption induced mortality during developmental stage 47
suggesting some non-immune but essential function of this gene. These data
demonstrate that the CRISPR/Cas9 system can be successfully adapted for rapid
genetic analysis of XNC gene family.
Poster Number: P82
Modelling human diseases in Xenopus; Celf3 protein complex enhances
translation rather than repressing
S Metikala, L Horb, W Thomas, B Suh, N Shaidani, C Collins, M Horb
Bell Center for Regenerative Biology and Tissue Engineering, Marine Biological
Laboratory, Woods Hole, USA
Several Xenopus groups are reporting successful genome editing using CRISPR/Cas
system. Using this technique, we are working on generating 100-200 mutants for the
community in both laevis and tropicalis. In this poster, I present an update of the
progress on generating the mutants and discuss the challenges we face. I present some
of our most recent data regarding the various mutants we have growing here at the
NXR.
In the second part, I will present our work on Celf3, an RNA binding protein that we
have found is involved in translational stimulation in endoderm progenitor cells. We
used RIP-Chip to identify Celf3 targets and our efforts to understand the Celf3 protein
complex that enhances translation rather than represses translation, as is known with
most RNABP. Our preliminary data suggests that Celf3 bypasses the traditional eIF4
translational mechanism to enhance translation.
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Poster Number: P83
Isolation of novel cell lines from standard laboratory strains of Xenopus laevis
and Xenopus tropicalis embryos
G J Gorbsky1, W Ratzan2, M E Horb2
1
Cell Cycle and Cancer Biology Research Program, Oklahoma Medical Research
Foundation, Oklahoma City, USA; 2National Xenopus Resource and Eugene Bell
Center for Regenerative Biology, Marine Biological Laboroatory, Woods Hole, USA
We are developing novel cell lines from the standard Xenopus laboratory strains, X.
laevis J strain and X. tropicalis Nigerian strain, using digested embryos. Thus far, we
have isolated and cloned three new X. laevis and five new X. tropicalis cell lines. The
lines have been passaged multiple times and grow without limit. They can be frozen,
and regenerate new cultures when thawed. Most chromosome spreads prepared from
all three X. laevis lines and four of the five X. tropicalis lines exhibit normal ploidy. We
anticipate that these lines will be amenable to modern gene editing techniques allowing
the generation of homozygous mutant cells, which can then be cloned to produce
isogenic populations. We expect that nuclei from these cells, transferred to enucleated
eggs, will generate F0 mutant embryos. This approach has the potential to radically
transform and simplify the production of mutant Xenopus embryos, tadpoles, and adults.
Poster Number: P84
Insights into conservation of early developmental program from sequence and
transcriptional comparison between Xenopus and Sturgeon embryogenesis
throughout blastula, gastrula and neurula stages
L Peshkin1, A Zaraisky2, F Eroshkin2, M Kirschner1
1
Systems Biology, Harvard Medical School, Boston, USA; 2Laboratory for Molecular
Basis of Embryogenesis, Institute of Bioorganic Chemistry, Moscow, Russia
We present a three way comparison of the whole embryo transcriptional profile across
twenty early stages of embryogenesis -- from unfertilized egg to late neurula -- between
Xenopus laevis, Xenopus tropicalis and Sturgeon (Acipenser stellatus) separated from
amphibians by ~400 My. Time-aligned by developmental stage, the majority of
developmentally important genes exhibit striking conservation in the pattern of zygotic
expression. Surprisingly, we find many striking differences in the distribution of maternal
mRNA deposit, e.g. VegT is not at all found in Sturgeon egg. These differences are
undergoing “canalization” in the course of development.
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Poster Number: P85
Development of novel Xenopus-specific single-chain antibodies
M Z Piccinni1, M J Guille1, V J Allan2, C R Sharpe1, A Noble1
1
EXRC, University of Portsmouth, Portsmouth, UK;
Manchester, Manchester, UK
2
Life Sciences, University of
The lack of Xenopus antibodies is acknowledged as a limitation of the model by the
community. The development of single-chain antibodies promises not just the
antibodies themselves but a whole set of manipulated variants, for example inactivating
antibodies and fluorescently labelled antibodies that can be used in vivo. We have
worked with 2 libraries created against a set of Xenopus proteins, however these were
too complex to isolate useful antibody clones.
We are now expressing our own antigens, importantly in HEK cells to avoid non
mammalian protein contaminants, and plan to immunize with 1 or 2 immunogens per
llama. The llamas can be recycled after 6 months. Recently published studies have
shown this to be the most effective method.
We have a list of about 40 proteins of the community's interest but we are focusing on
only 4 for now. These can be tested for inactivation easily, and be used as proof of
principle.
Poster Number: P86
Xenopus laevis developmental proteomics: an upgraded resource
L Peshkin, M Wuehr, J Briggs, M Kirschner
Systems Biology, Harvard Medical School, Boston, USA
We substantially expanded our previously published survey of whole-embryo protein
expression in Xenopus laevis both in temporal coverage and depth of
measurement. Taken together with another emerging embryology resource – single cell
transcriptional profiling-- this data sheds new light onto morphogenesis and cell type
differentiation by allowing in-silico dissection and co-expression analysis. We present
initial analysis of the improved data and upgraded interface to our proteomics series
web server. Additionally, we refine our previous analysis of the protein homeostasis at
whole-genome scale through respective estimates of protein synthesis and degradation
rates.
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Poster Number: P87
RNA whole-mount In situ Hybridisation Proximity Ligation Assay (rISH-PLA), an
assay for detecting RNA-protein complexes in intact cells
I M Roussis1, M J Guille2, F A Myers1, G P Scarlett1
1
Biophysics Laboratories, University of Portsmouth, Portsmouth, UK; 2EXRC, University
of Portsmouth, Portsmouth, UK
Techniques for studying RNA-protein interactions lag behind those for DNA-protein
complexes because of the challenges of working with RNA. Here the method for In Situ
Hybridisation – Proximity Ligation Assay (ISH-PLA) protocol is adapted to the study of
RNA regulation (rISH-PLA). As proof of principle we used the interaction of Xenopus
laevis Staufen protein with Vg1 mRNA; these co-localise to the vegetal pole of oocytes.
The applicability of both the Stau1 antibody and the Locked Nucleic Acid probe (LNA)
recognising Vg1 mRNA were validated by whole-mount immunohistochemistry and
whole-mount in situ hybridisation assays respectively prior to combining them in the
rISH-PLA. The rISH-PLA allows identification of RNA-protein complexes at subcellular
and single cell levels, avoiding spatial resolution and sensitivity issues associated with
assaying heterogeneous cell populations. This technique will be useful for studying RNA
binding proteins (RBPs) in complex mixtures of cells, for example tissue sections or
whole embryos.
Poster Number: P88
Absolute Protein and Phospho-Site Stoichiometry Dynamics during Vertebrate
Fertilization
M Presler1, M Wühr1,2, A M Klein1, E Van Itallie1, J Ingraham1, R C Kunz2, M Coughlin1,
L Peskin1, T J Mitchison1, S P Gygi2, M W Kirschner1
1
Department of Systems Biology, Harvard Medical School, Boston, USA; 2Department
of Cell Biology, Harvard Medical School, Boston, USA
Fertilization of the vertebrate egg triggers a critical transition that initiates cell cycle
progression, the block to polyspermy, and embryogenesis. Rather than differential RNA
expression, fertilization is primarily governed by protein degradation and
phosphorylation. We present the first comprehensive resource of fertilization using
quantitative multiplexed proteomics in Xenopus laevis. We determine absolute protein
abundance and develop a new framework to calculate stoichiometry of multi-site
phosphorylation with confidence intervals. Surprisingly, the largest protein changes 20
minutes post-fertilization are from secretion of abundant proteins (3,600fmols).
Proteasome-mediated degradation is comparatively modest (~60fmols), limited to low
abundance proteins (<150nM). Contrastingly, the flux of phosphorylation is at least 10fold higher (720fmols) than degradation and regulates proteins present at 1000-fold
range of concentrations. Dephosphorylation (540fmol) associates with meiotic exit, cooccurring with phosphorylation (180fmols) associated with vesicle exocytosis. These
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broadly applicable analytical approaches lay the groundwork for systematic study of the
protein biochemistry of many embryonic processes.
Poster Number: P89
RNA species whose transcription is totally silent in pre-MBT stage is not mRNA
but rRNA and possible involvement of weak bases in the transcriptional silence
of rRNA genes during the pre-MBT stage in Xenopus embryogenesis
K Shiokawa1,2,3, Y Misumi1,4, K Tashiro1,2,5
1
Department of Biology, Faculty of Science, Kyushu University, Fukuoka, Japan;
Laboratory of Molecular Embryology, Zoological Institute, The University of Tokyo,
Tokyo, Japan; 3Department of Biosciences, School of Science and Engineering, Teikyo
University, Utsunomiya, Japan; 4Department of Cell Biology, Fukuoka University School
of Medicine, Fukuoka, Japan; 5Molecular Gene Technics, Genetic Resources
Technology, Kyushu University, Fukuoka, Japan
2
It has long been believed that zygotic nuclei are transcriptionally totally silent during preMBT stage in Xenopus embryogenesis. Recently, however, several papers reported
detection of mRNA transcription during the pre-MBT stage. We ourselves previously
(1987) detected incorporation of 3H-uridine into heterogeneous mRNA-like RNA in preMBT stage embryos. In our experiments to analyze 2'-O-methylation in high-molecularweight RNAs, we found extensive mRNA-cap methylation in pre-MBT stage
embryos. When we examined rRNA-specific 2'-O-methylation, however, totally no
activity was detected in embryos during cleavage and in the former half period of
MBT, although active methylation occurred in embryos in the latter half period
of MBT and in post-MBT stages. We searched substances which either stimulate or
inhibit rRNA synthesis. We unexpectedly found that weak bases such as amines and
ammonium salts quite specifically inhibit rRNA synthesis. Much higher levels of
ammonium ion, but not amines, were detected in pre-MBT embryos than in post-MBT
embryos. We, therefore, assume that weak bases, such as ammonium ion and possibly
amines, may be responsible for the transcriptional silence of rRNA genes during preMBT stage in Xenopus embryogenesis.
Poster Number: P90
Inflammation-mediated blood brain barrier disruption and macrophage infiltration
contribute to disseminate Frog Virus 3 into the brain of X. laevis tadpoles
J Robert, J Wang, F De Jesús Andino
Microbiology and Immunology, University of Rochester Medical Center, Rochester, USA
Monocytes/macrophages are central in amphibian host defenses, but are also important
in the dissemination and persistent infection caused by ranavirus emerging pathogens.
However, little is known about the fate of infected macrophages or if ranavirus exploits
immune privileged organs, such as the brain, to establish a reservoir. Xenopus laevis
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and Frog Virus 3 (FV3) were established as an experimental platform for investigating in
vivo whether ranavirus disseminate into the brain. We show that the FV3 infection alters
the BBB integrity, possibly mediated by an inflammatory response, which leads to viral
dissemination into the central nervous system in X. laevis tadpole but not adult. Using a
knock-in FV3 recombinant expressing GFP under an immediate-early viral promoter,
active infection into tadpole brain can be detected as early as 24 hr post-infection.
Notably, our findings indicate that the macrophages contribute to viral dissemination by
carrying the virus into the neural tissues.
Poster Number: P91
Development of a fast and efficient genetic platform in Xenopus tropicalis for
modeling human congenital limb malformations
H Thi Tran1, L Vlaeminck1, T Van Nieuwenhuysen1, T Naert1, R Noelanders1, K
Vleminckx1,2
1
2
Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium;
Center for Medical Genetics, Ghent University, Ghent, Belgium
Malformations of the upper or lower limbs are rather common, affecting about 1 in 500
live births. Despite the current improvements in finding the genetic defects underlying
the malformations, many cases remain unresolved. Not surprisingly, most mutations
could be found in genes known to mediate initiation, outgrowth and patterning of the
early limb bud, such as FGF, Wnt, Shh. However, mutations or genome rearrangements
may also affect regulatory regions in the non-coding genome. Improved methods for
identification of structural defects, copy number detection and whole exome and whole
genome sequencing produce massive amounts of data leading to the identification of
new candidate disease-linked genes. Now, there is a growing need for functional
assays to assess the causality, where Xenopus tropicalis, as a diploid tetrapod, is
ideally positioned. Via CRISPR/Cas9 mediated genome editing, we hope to
substantially facilitate the discovery of new genes that are linked to limb malformations
in humans.
Poster Number: P92
Mitochondrial transport protein Rhot1 is involved in the aggregation of germinal
granule components during primordial germ cell formation in Xenopus
H Tada1, Y Taira1, K Morichika2, T Kinoshita1
1
Department of Life Science, Rikkyo University, Tokyo, Japan; 2Computational Medicine
Center, Thomas Jefferson University, Pennsylvania, USA
In Xenopus, germ plasm contains germinal granules and abundant of mitochondria
(germline-Mt). It remains poorly understanding the role of germline-Mt in primordial
germ cell (PGC) formation. The germ plasm is distributed as many small islands at
vegetal pole and is gradually aggregated to form large mass in the most vegetal cells
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during early blastula. Polymerized microtubules and Kinesin protein are known to be
required for the aggregation of germ plasm. However, role of germline-Mt during the
germ plasm aggregation remains unknown. Here, we focused on mitochondrial specific
adaptor protein Rhot1. In the expression of Rhot1ΔC which lacks a C-terminal
mitochondrial transmembrane domain, the transport of germline-Mt was blocked. In the
Rhot1-inhibited embryos, germinal granule components did not aggregate during
cleavage stages, which caused a decrease in number of PGCs. These results suggest
that germline-Mt is involved in aggregation of germinal granule components, which are
essential for formation of PGCs.
Poster Number: P93
National Xenopus Resource – serving the Xenopus research community
M Wlizla, R Falco, S Mcnamara, M E Horb
National Xenopus Resource, Marine Biological Laboratory, Woods Hole, MA, USA
Since its establishment in 2010, the National Xenopus Resource (NXR) has focused on
development of three facets in particular: resources, services, and research, all of which
promote advancement of Xenopus research. Our resources include wild type and inbred
strains of both Xenopus laevis and Xenopus tropicalis as well as approximately 100
distinct mutant and transgenic lines. The NXR services include generation of custom
mutant and transgenic lines for Xenopus researchers who may not have the resources
or expertise to do so on their own, laboratory space available for visiting scientists, and
promoting dissemination of cutting edge techniques in Xenopus research through onsite held workshops. The research that the NXR does in-house focuses on developing
techniques that will be of considerable use to the Xenopus community. As we grow, we
will develop these facets further with the goal of serving the Xenopus community and
promoting Xenopus research.
Poster Number: P94
Improving Animal Husbandry Conditions and Care in a Xenopus Laboratory to
Facilitate Frog Development in an Abbreviated Timeframe
S McNamara, M Wlizla, M Horb
National Xenopus Resource (NXR), Marine Biological Laboratory, Woods Hole, USA
Recent development of highly efficient mutagenesis techniques, CRISPR/Cas and
TALEN, has allowed rapid generation of large number of F0 Xenopus mutant animals
for study of development and modeling of human disease, however the relatively long
time to reach sexual maturity has until now been an impediment to quick establishment
of mutant F1 lines. At the National Xenopus Resource (NXR), by altering standard
husbandry protocols and catering to the juvenile development we have decreased the
age of sexual maturity for both sexes in X. laevis and X. tropicalis. Water temperature,
frog density, and diet are the three factors that have the greatest impact on Xenopus
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growth and maturation. Under optimal husbandry conditions, 4 months old X. laevis
males are of sufficient age to produce healthy offspring via in vitro fertilization. This
decrease in generation time promotes the strength of Xenopus as a model system for
study of development and human disease.
Poster Number: P95
Modeling molecular subgroups of medulloblastoma, in Xenopus tropicalis by
CRISPR/Cas9
D Dimitrakopoulou, R Noelanders, T V Nieuwenhuysen, T Naert, K Vleminckx
Department of Biomedical Molecular Biology, Gent University, Gent, Belgium
Medulloblastoma (MB) is a pediatric malignancy originating in the cerebellum that
accounts for 20% of CNS tumors. MB is subdivided in Wnt-, Shh-, Group 3- and Group
4-, based on molecular signature. CRISPR/Cas9 is applied in our lab in Xenopus
tropicalis and demonstrated great efficiency in modeling human cancer, like Familial
Adenomatous Polyposis and retinoblastoma. Our direct aim is to establish models for all
MB subgroups. We found that co-injection of embryos with sgRNAs targeting apc and
tp53, induced tumors in the brain of developing tadpoles after 8 weeks. Our goal is to
optimize this Wnt tumor model and identify effector genes in MB formation. Furthermore
by targeting specific genes, we expect to obtain models for Shh-, Group 3- and Group
4- MB. These tumor models would expand the experimental portfolio for identification of
genes crucial in MB development and provide a platform for pre-clinical testing of
potential therapeutic compounds.
Poster Number: P96
Xenopus embryos as a model for alcohol-induced developmental growth
restriction
N Shukrun, Y Shabtai, A Fainsod
Department of Developmental Biology and Cancer Research, Institute for Medical
Research Israel-Canada, The Hebrew University, Jerusalem, Israel
In Intra Uterine Growth Restriction, fetuses are smaller than their developmental age.
IUGR is associated to stillbirth, cognitive defects and postnatal illnesses with a
prevalence of 8% of pregnancies. IUGR can be induced by alcohol exposure like in
FAS. We show that, ethanol and retinol (Vitamin A) compete for the retinoic acid (RA)
biosynthetic activity resulting in developmental defects growth restriction.
To study RA as a regulator of embryo size, we utilized RA biosynthesis inhibitors and
ethanol. Quantitating embryo size, we show that RA knock-down Xenopus embryos
show size decreases like in human IUGR. Size change correlates with treatment
strength and maternal genetic background modifies the alcohol sensitivity. RA knockdown affects anterior-posterior patterning, with only slight delay in developmental
progression and activates different growth factor pathways. We show that RA is a
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regulator of embryonic size and alcohol-treated Xenopus embryos are a reliably
experimental system for IUGR study.
Poster Number: P97
osmoregulation
Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK
The native freshwater habitat of Xenopus tropicalis is microbe-rich, but before
developing adaptive immunity, tadpoles must employ robust innate defence
mechanisms to protect themselves against infection. The skin is the principal route of
infection in young tadpoles and so must be protected. One of the ways the tadpole does
this is to generate a mucus barrier on its skin surface. Through proteomic analysis, we
have identified a major mucin glycoprotein. This mucin is similar in sequence to human
mucins, which form the structural basis of mucus barriers. Through knockdown studies,
we have shown that the epidermal mucin is critical for protection against infection from a
known opportunistic pathogen, Aeromonas hydrophila, and have uncovered a potential
role for the mucin in osmoregulation. We have also identified a number of mucininteracting molecules (e.g. FCGBP) conserved in human mucosa. We propose the
tadpole skin as a powerful model to study live mucus biology.
Poster Number: P98
Xenbase: the Xenopus bioinformatics database supports your research
C James-Zorn1, V G Ponferrada1, M E Fisher1, K A Burns1, K Karimi2, V Lothay2, J D
Fortriede1, E Segerdell1, P Vize2, A M Zorn1
1
Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center,
Research Foundation, Cincinnati, OH, USA; 2Department Zoology and Department of
Computer Science, University of Calgary, Calgary, AB, Canada
Xenbase (http://www.xenbase.org) is a web accessible, NIH funded, bioinformatics
database that integrates diverse genomic, expression and functional data for Xenopus,
an important tetrapod model in numerous, diverse fields of biomedical and basic
science research. The Xenbase website plays an indispensible role in making Xenopus
data accessible to the entire research community. Xenbase is a data portal for
researchers, accelerating scientific discovery by enabling novel connections between
Xenopus, humans and other model systems. Xenbase provides the latest genome
assemblies, functional data, expression profiles, relevant literature, specific
experimental reagents, and links to numerous resources including the Xenopus stock
centers. As theXenopus community hub, Xenbase provides a forum for up-to date
information on the community, events, funding, jobs, and the latest developments in
Xenopus research.
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Poster Number: P99
POSTER WITHDRAWN
Poster Number: P100
SLiM acquisition and alternative splicing mediate the diversity of NCoR-family
corepressors
C R Sharpe1, T Peterkin2, S Short3, R Patient2, M Guille1
1
EXRC, School of Biological Sciences, University of Portsmouth, Portsmouth, UK; 2The
Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK; 3School of
Biological Sciences, University of Cardiff, Cardiff, UK
Vertebrate NCoR-family co-repressors play central roles in the timing of embryo and
stem cell differentiation by repressing the activity of a range of transcription
factors. They interact with nuclear receptors using short linear motifs (SLiMs) termed
CoRNR boxes. The complete complement of CoRNR boxes arose in an ancestral
cephalochordate and was encoded in one large exon, which in the urochordates and
vertebrates is split between more than ten. In Xenopus, alternative splicing is prevalent
in NCoR2, but absent in NCoR1, however, for at least one NCoR1 exon, alternative
splicing can be recovered by a single point mutation. Patterns of NCoR2 alternative
splicing differ between Xenopus and zebrafish. Using splicing minigenes, we identify
that cellular context, rather than sequence, predominantly determines this
difference. The factors that have been identified to contribute to the diversity of NCoRfamily co-repressors across the deuterostomes can be used to quantify diversity in other
gene families.
Poster Number: P101
RAF1 loss-of-function mutation causes acro-cardio-facial syndrome in humans
by blocking FGF signaling
N Escande-Beillard1, S Wong1, A Loh1, H Kayserili2, B Reversade1
1
Institute of Medical Biology, A*STAR, Singapore;
Istanbul Medical Faculty, Istanbul, Turkey
2
Medical Genetics Department,
Acro-cardio-facial syndrome (ACFS) is a rare and severe genetic disorder characterized
by ectrodactyly, cardiac abnormalities, facial dysmorphisms and growth retardation.
Here we find that a neonatal lethal progeroid form of ACFS is caused by a germline
homozygous missense mutation in the serine-threonine kinase RAF1 (also known as
proto-ongene c-Raf).
In cultured cells, we found that this p.T543M mutant RAF1 behaves as a loss-offunction allele by reducing signaling via the MAPK/ERK pathway. Accordingly, upon
overexpression in Xenopus embryos and unlike the wildtype or gain-of-function p.S257L
variant, this p.T543M allele failed to recapitulate the effects of increased FGF signaling
such as ectopic mesoderm induction, neural differentiation or brain posterization.
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Our data provide the first genetic etiology for ACFS and highlight the power of the
Xenopus to test allele pathogenicity.
Poster Number: P102
POSTER WITHDRAWN
Poster Number: P103
Analysis of Novel Candidates for Short Rib Thoracic Dysplasia (SRTD)
Associated Genes
M Getwan, S Lienkamp
Nephrology, University Medical Center, Freiburg im Breisgau, Germany
Short Rib Thoracic Dysplasia (SRTD) is characterized by shortened bones, polydactyly
and often by a nephronophthesis-like phenotype (NPHP). Until now only few SRTD
genes have been identified, suggesting that additional disease genes still remained
unknown.
To shed light on the molecular pathogenesis of SRTD and its connection to NPHP, we
attempted to identify further SRTD candidate genes. We found 35 putative candidates
with an in silico analysis of published screens. Expression analysis demonstrated the
presence of a majority of these genes in ciliated tissues, especially the pronephros, like
it was the case for SRTD genes. Finally CRISPR/Cas LOF resulted in oedema, often
caused by a pronephros failure, and in cyclopia, the result of impaired Shh signaling.
To further describe the phenotypic consequences a comprehensive analysis of kidney
markers, Shh signaling and skeletal malformations will be presented to validate
Xenopus as a potent model organism to model human diseases.
Poster Number: P104
POSTER WITHDRAWN
Poster Number: P105
POSTER WITHDRAWN
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Poster Number: P106
Answer, the gene lost during evolution in higher vertebrates regulates
regeneration and early forebrain development in Xenopus laevis
D D Korotkova1,2, A S Ivanova1, V A Lubetsky3, A V Seliverstov3, M B Tereshina1, A M
Nesterenko4, A G Zaraisky1
1
Bioorganic Chemistry, Russian Academy of, Moscow, Russia; 2Department of
Embryology, Faculty of Biology, Lomonosov Moscow State University, Moscow, Russia;
3
The Institute for Information Transmission Problems, Russian Academy of Sciences,
Moscow, Russia; 4Belozersky Institute of Physico-Chemical Biology, Lomonosov
Moscow State University, Moscow, Russia
It has been shown recently that reduction of regenerative capacity in higher vertebrates
could be explained by extinction of some genes that control regeneration in lower
vertebrates. We have investigated now expression and physiological function of one of
such genes, named Answer (Anamniotic specific wound epithelium receptor). This gene
has been identified previously during bioinformatics screening of vertebrate genomes
for genes lost in higher vertebrates. Answer encodes transmembrane protein and
probably operates as a receptor of unknown ligand(s). We have shown that in Xenopus
laevis embryo Answer is expressed in dorsal ectoderm, including neural plate, and it is
sharply activated during regeneration in the wound epithelium. The inhibition of Answer
function leads to increase of the forebrain and decrease in regeneration. Our data
indicate that loss of Answer in higher vertebrates could result in the reduction of
regenerative capacity in exchange for the progressive evolution of the forebrain.
Poster Number: P107
hormone axis
Center for Aging and Regeneration, Millennium Nucleus in Regenerative Biol, P.
Universidad Catolica de Chile, Santiago, Chile
The link between hormones and heterochronic genes in regulating developmental
transitions has only recently been explored. In Xenopus, the thyroid hormones (TH) are
crucial for metamorphosis. We studied the role of the heterochronic gene Lin28 during
metamorphosis.
Lin28 levels decreased before the increase of the TH-target genes thr-b and klf9. To
study the role of Lin28 we generated transgenic animals that overexpress Lin28 under
the control of a heat-shock promoter. Overexpression of Lin28 from pre-metamorphosis
significantly delayed metamorphosis compared to controls. This delay was correlated
with a lower endogenous activation of thr-b and klf9 in Lin28 overexpressing animals.
Exogenous administration of TH rescued the delay induced by Lin28. Transcriptome
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and proteome analyses are currently been carried out to determine cellular processes
downstream Lin28.
These results indicate that Lin28 regulates metamorphosis by inhibiting TH signaling,
linking the heterochronic gene network with the hormonal axis in vertebrates.
FUNDINGS: FONDECYT 11130564 (FF), CARE-PFB12/2007 (JL), MILENIO
RC120003 (JL)
Poster Number: P108
Characterizing the role of foxm1 during tail regeneration in Xenopus tropicalis
D Pelzer, K Dorey
Faculty of Life Sciences, University of Manchester, Manchester, UK
In contrast to most vertebrates Xenopus tadpoles have the ability to regenerate their
central nervous system.
To investigate the molecular mechanism of regeneration, we used a bioinformatical
approach to uncover genes upregulated in the spinal cord during regeneration. This led
to the identification of foxm1,a transcription factor which is dynamically expressed
during regeneration. To investigate its role we are using knockdown and knockout
technologies. We have shown that Foxm1 promotes cyclinb3 expression leading to an
increased rate of proliferation specifically in the regenerating spinal cord. We are also
investigating the signals controlling the dynamic expression of foxm1 using chemical
inhibitors of signaling pathways acting early during regeneration, such as Sonic
Hedgehog (Shh) and Reactive Oxygen Species (ROS).
Combining these approaches will allow us to characterize a novel role for Foxm1 during
spinal cord regeneration, and to better understand the mechanisms controlling cell
proliferation upon amputation.
Poster Number: P109
School of Life Sciences, University of Nevada Las Vegas, Las Vegas, USA
Vertebrate eye development is complex and requires early interactions between
neuroectoderm and epidermis. In Xenopus, individual eye tissues such as the retina,
lens and cornea, can undergo regeneration. However, partial removal of the specified
eye field during neurulation or the tadpole stage does not induce replacement. Here we
describe a model for investigating eye regrowth. We found that tailbud embryos can
readily regrow eyes after removal of the specified eye tissues. This is a rapid process as
an eye of similar size to the control is seen by 4 days and development is normal. The
regrown eye contains a full complement of eye cell types, connects to the brain and is
functional. Eye regrowth also requires the same early mechanisms (apoptosis and
bioelectrical signaling) as appendage regeneration. Together, our findings indicate that
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frog embryos can re-initiate development of the eye after tissue loss and that this
process requires regenerative mechanisms.
Poster Number: P110
Investigating how thyroid
CRISPR/Cas9
hormone
impedes
cardiac
regeneration
with
L Marshall1, C Vivien2, N Chai1, B Mughal1, F Girardot1, L Péricard1, P Scerbo3, K
Palmier4, S le Mevel1, J B Fini1, J P Concordet5, B Demeneix1, L Coen1
1
Regulations, Developpement et Diversite Moleculaire, CNRS, MNHN, Sorbonne
Universities, Paris, France; 2School of Biomedical Sciences, The University of
Queensland, St Lucia, Queensland, Australia; 3IBDM, Aix-Marseille Universite,
Marseille, France; 4Departement de Microbiologi, Universite Paris-Sud, Orsay, France;
5
Inserm U1154, UMR 7196, MNHN, Sorbonne Universities, Paris, France
Cardiac insufficiency is caused by failure of the adult human heart to regenerate.
In mice, cardiac regenerative capacity is “lost” 7 days postnatally. Lost mammalian
heart regenerative capacity coincides with the post-natal peak of thyroid hormone (TH).
The neonatal period in mice is physiologically similar to TH-regulated
amphibian metamorphosis. Metamorphosis diminishes the capacity of Xenopus to
regenerate limbs, retina and nerve tissue, and may possibly affect heart regeneration.
We show that the tadpole heart completely regenerates, whereas post-metamorphosis,
the adult heart cannot. Further, exogenous TH impedes cardiac regeneration in the
regenerating
adult
zebrafish
and
Xenopus
tadpole.
To investigate TH availability in this process we applied CRISPR/Cas9, in Xenopus
laevis to disrupt the TH inactivating enzyme deiodinase 3 (dio3). Using multiple guideRNAs, we generated deletions in the dio3 locus. Further, using an existing Xenopus
transgenic reporter-line assessing TH availability, we obtained a hyperthyroid
phenotype in F0 tadpoles; providing a practical F0 mutant model. Forthcoming work will
explore the consequences of excessive TH signalling during cardiac regeneration.
Poster Number: P111
Structural and Molecular Biology, University College London, London, UK
Cellular senescence is an anti-tumourigenic mechanism which can lead to disruption of
tissue structure and function. Indeed, accumulation of senescent cells contributes to
age-related disorders in mammals. Nevertheless, senescent cells can also contribute to
physiological processes, as illustrated by the recent finding that cellular senescence
plays functions during mammalian development. The degree of conservation of
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developmental senescence through phylogeny, and the extent of its functions during
development, remains unknown.
Here, we show that cell senescence is an intrinsic part of the developmental programme
in amphibians. Programmed senescence occurs in specific structures at defined timewindows during amphibian development. It contributes to the physiological degeneration
of the pronephros and to the development of the cement gland and neighbouring
regions, such as the oral cavity. Our findings uncover conserved and new roles of
senescence in vertebrate organogenesis and support the view that cellular senescence
may have arisen in evolution as a developmental mechanism.
Poster Number: P112
MRC Molecular Haematology Unit, University of Oxford, Oxford, UK
Stem cells are defined by their capacity to give rise to differentiated progeny while selfrenewing, properties which are referred to as stemness. What stemness is molecularly
and how it is established and maintained is not clear. Notch signalling is essential for
the establishment and maintenance of a number of tissue stem cells, including
haematopoietic stem cells (HSCs), but its molecular mechanism is not fully understood.
Notch1 and Notch4 are expressed in the dorsal aorta at the time HSCs are generated,
their role was investigated and we defined the Hes/Hey transcriptional effectors targeted
by them to establish the gene regulatory network (GRN) controlling the establishment of
HSCs. Interestingly, Hes5, a gene essential for the establishment and/or maintenance
of other tissue stem cells, plays a central role. This suggests that a common GRN might
be shared amongst tissue stem cells and that Hes5 might be the key molecule
conferring stemness on them.
Poster Number: P113
FGF-mediated activation of MAPK and PI3K/Akt controls the lineage restriction of
pluripotent blastula stem cells
L Geary1, C LaBonne1,2
1
Department of Molecular Biosciences, Northwestern University, Evanston, USA;
Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Evanston,
USA
2
Central to vertebrate evolution, the neural crest is a stem cell population with multi-germ
layer potential, sharing remarkable molecular similarity to its ancestral cell population,
the pluripotent animal pole cells of the blastula. The extent to which there exists a
shared regulatory program controlling pluripotency and whether signaling inputs exhibit
conserved functions between these cell states remains unknown.
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FGF signaling is intricately involved in mediating embryonic stem cell pluripotency and
in the regulation of the neural crest state, yet how FGF signaling is used reiteratively
and elicits context-specific functions is unclear. Here, we show that FGF signaling is
necessary for proper lineage restriction, that differential activation of MAPK and
PI3K/Akt cascades orchestrates this process, and that this activation is modified in
neural crest cells maintaining a progenitor state. We present a novel mechanism in
which FGF signaling employs both MAPK and PI3K/Akt cascades to progressively
promote cellular restriction.
Poster Number: P114
Exploiting CRISPR/Cas9-mediated Xenopus tropicalis cancer models for
identification of novel drug targets and pre-clinical therapeutic compound
validation
T Naert1, T Van Nieuwenhuysen1, R Colpaert1, R Noelanders1, D Dimitrakopoulou1, H T
Tran1, D Creytens2, A Boel2, K Vleminckx1,2
1
Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium;
Center for Medical Genetics, Ghent University and Ghent University Hospital, Ghent,
Belgium; 3Pathology Department, Ghent University Hospital, Ghent, Belgium
2
We have recently shown that mosaic inactivation of the apc gene by TALENs or
CRISPR/Cas9 technology in Xenopus tropicalis leads to highly penetrant (>95%) and
fast (1-1.5 months) development of desmoid tumors [1] Next to this, we show that
mosaic inactivation of rb1 and rbl1 in Xenopus tropicalis leads to highly penetrant
(>70%) and fast (median 69 days) development of retinoblastoma (manuscript in
preparation). We aim to identify novel drug targets for both desmoid tumors and
retinoblastoma by multiplex CRISPR/Cas9 inactivation of the relevant tumor
suppressor(s) together with a modifier gene. We thus study the impact of inactivation of
the modifier gene on the relevant tumor phenotype. Potential novel drug targets will be
immediately validated within the relevant model by performing compound studies.
Within this scope, we have recently partnered up with industry to test a novel β-catenin
inhibitor within our desmoid tumor model.
Poster Number: P115
Snail1 is essential for the maintenance of pluripotency in blastula animal pole
cells
A N Rao1, C LaBonne1,2
1
Department of Molecular Biosciences, Northwestern University, Evanston, USA;
Robert H Lurie Comprehensive Cancer Center, Northwestern University, Evanston,
USA
2
The neural crest is a stem cell population that defines vertebrates. While they arise in
the ectoderm, these cells exhibit multi-germ layer developmental potential that has been
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linked to retention of the regulatory network that controls pluripotency in blastula animal
pole cells. Snail1, a key mediator of neural crest formation, is also expressed earlier in
pluripotent blastula cells. We hypothesize that Snail1 is integral to maintaining stem cell
attributes of a subset of blastula cells fated to become the neural crest. Here we show
that, Snail1 is essential for the pluripotency of blastula stem cells. Through loss of
function experiments, we identify Snail1 to be required for the expression of
pluripotency genes in blastula cells as well as the functional competency of these cells.
We describe a discrete interaction of Snail1 with the core pluripotency network, and
propose a novel mechanism for Snail1 dependent control of the stem cell state.
Poster Number: P116
A M Butler1, L Wang2, D A Owens1, K M Newman1, M L King1
1
2
Department of Cell Biology, University of Miami Miller School of Medice, Miami, USA;
Department of Biology, University of Miami, Coral Gables, USA
Germ plasm contains the genetic information that protects Xenopus PGCs from somatic
differentiation and initiates a unique gene expression program that preserves totipotent
potential. Here, we utilized RNA-sequencing to comprehensively interrogate PGC and
neighboring endoderm cell RNAs after segregation from the endoderm lineage. Over
3,000 transcripts were upregulated in PGCs. The top 300 genes upregulated in PGCs
were analyzed and the following gene ontology processes were identified: cell cycle
regulation, meiosis, reproduction, progesterone signaling, motility, protein folding, and
apoptosis. Network analysis revealed 18 hubs linking 125/300 genes in a network
including the transcription factors e2f1, pou5f3.3 (oct60, the Oct3/4 homologue), and
sox7. Interestingly, initial functional studies show that PGC-directed oct60 inhibition
increases the number of PGCs in the early embryo, whereas over-expression or
inhibition of sox7 reduces PGC number. These data suggest that sox7 and oct60 likely
mediate germline specification in the early embryo. NIH GM102397 and
HD072340 (MLK).
Poster Number: P117
High Throughput Analysis Reveals Novel Maternal Germline RNAs Critical for
PGC Preservation and Proper Migration
D A Owens1, A M Butler1, K M Newman1, T H Aguero1, D Van Booven2, M L King1
1
Department of Cell Biology, University of Miami Miller School of Medicine, Miami, USA;
Hussman Institute for Human Genomics, University of Miami Miller School of Medicine,
Miami, USA
2
During oogenesis hundreds of RNAs, including determinants of both somatic and
germline fates, are localized to either the animal or vegetal cortical region. Here, we
utilized RNA-sequencing to comprehensively interrogate cortically-localized RNAs in the
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Xenopus laevis oocyte. 411 RNAs were enriched at the vegetal pole and 27 at the
animal pole. Of these, 90/198 annotated transcripts were novel RNAs enriched at the
vegetal pole and 6/15 at the animal pole. Unlike mRNAs, microRNAs were not
asymmetrically distributed. WISH confirmed RNA localization. GeneGo analysis of
vegetally enriched transcripts identified protein-modifying enzymes, receptors, ligands,
RNA binding proteins and transcription factors with 5 defining hubs linking 47 genes in a
network. Initial functional studies show that sox7 plays an important role in primordial
germ cell (PGC) development and efnb1(ephrinB1) is required for proper PGC
migration. Additionally, we propose potential pathways operating at the vegetal pole for
future investigation. NIH GM102397 and HD072340 (MLK).
Poster Number: P118
The Tumor-Suppressor BAP1 Promotes Expression of Differentiation Genes in
Ectodermal Derivatives
M L King1, J Kuznetsov2, T H Aguero1, W Harbour2
1
Cell Biology, University of Miami Miller School of Medicine, Miami, Fl, USA; 2Bascom
Palmer Eye Institute, Sylvester Comprehensive Cancer Center and Interdisciplinary
Stem Cell Institute, University of Miami Miller School of Medicine, Miami, USA
Loss-of-function mutations in the BAP1 tumor suppressor gene are strongly associated
with human cancers, including melanoma and mesothelioma. In mice, Bap1 mutant
embryos die in utero preventing a molecular analysis of its role in development. BAP1 is
a mono deubiquitinase and well conserved between humans and Xenopus. A single
substitution in the deubiquitinase catalytic domain results in a Bap1 dominant negative
form functioning within the nucleus. Depletion of Bap1 protein leads to defects in
gastrulation and loss of differentiation of ectodermal derivatives. Differentiation markers
are down-regulated in the absent of Bap1 function, resembling key results found in
melanoma cell cultures. Alternations in gene expression were rescued by injection of
human or Xenopus Bap1 RNA. We present evidence that BAP1 functions at the
chromatin level interacting with the polycomb complex. We are developing a high
throughput screen to identify compounds able to revert the striking phenotypes of BAP1
mutants in Xenopus.
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Poster Number: P119
A comparison of junction-centric versus exon-centric analysis for identification of
Ptbp1-dependent splicing events in Xenopus laevis
M Noiret, A Mereau, G Angrand, M Bervas, C Gautier-Courteille, V Legagneux, S
Deschamps, H Lerivray, J Viet, S Hardy, L Paillard, Y Audic
Institute of Genetics and Development of Rennes, CNRS-Universite de Rennes,
Rennes, France
The regulation of alternative splicing is central for cell differentiation and development.
Repression of Ptbp1, a regulatory RNA-binding protein,leads to alteration of skin
development in Xenopus laevis. Assuming that developmental defects resulted from
alteration of splicing pattern we used RNAseq to identify differentially spliced RNA
following Ptbp1 depletion.
We systematically compared the classical exon-centric and junction-centric approaches
to detect differential splicing events using both our data and simulated human data
using different quality of annotations. We showed that the junction-centric approach
performs far better than the exon-centric approach in Xenopus laevis and was
drastically more robust to alteration of annotation than the exon centric approach in
human. We conclude that the junction-centric approach allows for a more complete and
informative description of splicing events, and propose that this finding might hold true
for other species with incomplete annotations.
Poster Number: P120
The necessity of nitric oxide during the embryonic epidermis development
S Tomankova1,2, P Abaffy1,2, R Sindelka1,2
1
Department of Gene Expression, Institute of Biotechnology - BIOCEV, Academy of
Science, Vestec, Czech Republic; 2Department of Genetics and Microbiology, Faculty of
Science, Charles University, Prague, Czech Republic
Nitric oxide (NO) is an important signaling molecule, which is involved in many
biological processes such as vasodilation and neurotransmission. Recently, we found
that NO is produced in the Xenopus embryonic epidermis. Xenopus epidermis is
composed of 4 cell types: goblet cells, multi-ciliated cells, small secretory cells and two
types of ionocytes and nicely reflects composition of specialized human epithelia. Our
results indicate that NO is produced only in ionocytes and multi-ciliated cells but NO
function during epidermis development remains unclear. We have studied the
molecular and cellular changes in the embryonic epidermis after NO inhibition. We
showed that NO inhibition causes defects especially in formation of multi-ciliated cell
development and NO functions through regulation of epidermal specific gene
expression. Altogether, our data indicates novel important role of NO during embryonic
development with potential impact in understanding of human mucociliary epithelia
development and its defects.
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Poster Number: P121
PAWS1/FAM83G is a positive regulator of the canonical WNT pathway
K S Dingwell1, P Bozatzi2, T Cummins2, G P Sapkota2, J C Smith1
1
Systems Biology, The Francis Crick Institute, Mill Hill Laboratory, London, UK; 2MRC
Protein Phosphorylation Unit, School of Life Sciences, University of Dundee, Dundee,
UK
PAWS1/FAM83G, a member of a family of proteins characterised by an N-terminal
DUF1669 domain, was originally identified as a modulator of SMAD4-independent BMP
signalling. However, RNA-seq experiments suggested that it modulated gene
expression independent of BMP signalling. Here we report that PAWS1 is a positive
regulator of the canonical Wnt pathway. PAWS1-injected embryos developed a
complete secondary axis and Xnr3 and Siamois was induced by PAWS1 in naïve
animal caps. Axis development in UV-treated embryos was also rescued by PAWS1.
Using a series of N- and C-terminal truncations, we mapped Wnt-activity to its Nterminal domain. GSK3ß, Axin, and C-cadherin inhibited PAWS1 activity, while in
contrast a dominant negative form of LRP6 was unable to block PAWS1 activity.
Together, these data support a role for PAWS1 in regulating the activity of the ß-catenin
destruction complex. We are currently using a variety of proteomic and biochemical
approaches to characterise PAWS1 effectors.
Poster Number: P122
States of the heart: insights from the cardiac transcriptome during postembryonic development, aging and regeneration
F Girardot1, L Marshall1, L Pericard1, C Vivien2, L Coen1, B Demeneix1
1
RDDM, MNHN, Paris, France; 2Centre for Cardiac Vascular Biology, University of
Queensland, Melbourne, Australia
Cardiac regenerative capacity is gradually lost during Xenopus post-embryonic
development. Late prometamorphic (NF55-57) tadpoles display a robust and efficient
response to ventricular resection, leading to scarless regeneration. Metamophic
tadpoles (NF61) as well as young juveniles (NF66+20days) display markedly slowed
regenerative response. In contrast, older juveniles (>6 months post metamorphosis) and
adults (>5 years old) do not show any obvious sign of ventricular regeneration.
To analyse the transcriptional state of ventricular tissue during metamorphosis and its
early responses to ventricular resection, we adapted the microfluidics-based highthroughput real-time-Quantitive-PCR approach to Xenopus. Multidimentional analysis of
the data confirms that Xenopus heart samples fall in 3 groups mirroring the functional
classes identified by the histochemical approach: high, low and no regenerative
capacity.
Further analysis allows the idenfication of genes that discriminate these functional
groups. The identification networks, potentially linked with modulation of cardiac
133
regenerative capacity is of obvious biomedical significance. Such likely candidates are
presented below.
Poster Number: P123
Implications of Knockout versus Knockdown in Xenopus Embryos
T Spruce, R S Monteiro, J C Smith, G E Gentsch
Developmental Biology Laboratory, The Francis Crick Institute, Mill Hill Laboratory,
London, UK
For almost two decades, antisense morpholino oligonucleotides (MO) have been an
indispensable tool for developmental biologists to block splicing or translation in an
effort to assess gene function in frog and fish embryos. More recently, genome editing
technologies such as the TALEN and type II CRISPR sytem made it possible to knock
out (KO) rather than only transiently knocking down (KD) genes. However, recent
studies in zebrafish claim low concordance between mutant and morphant phenotypes,
either because mutants are not null or the MO generates off target effects. Here we
report on the implications of knocking out versus knocking down Brachyury in X.
tropicalis. While both KO and KD affect most strongly the same core gene regulatory
network causing identical phenotypes, a small, but consistent group of genes shows
mis-regulation only in morphants, which may generate subtle off-target abnormalities in
development.
Poster Number: P124
A new regulator of Groucho/TLE activity in fate determination of the Spemann
organizer
B C Durand, N Rocques, E Sena
Signalisation Normale et Pathologique, Institut Curie - Université Paris Sud - U1021UMR3347, ORSAY 91405, France
Wnt/β-catenin induces formation of the Spemann Organizer (SO) but little is known on
what limits the size of the SO. The evolutionary conserved transcription factor Barhl2
disrupts the gradient of Shh and anti-BMP factors in the neural plate, promotes
apoptosis and limits Wnt activity. We investigated the role of Barhl2 and of the
apoptotic cascade on SO development. We demonstrated that the anti-apoptotic protein
Mcl1 promotes SO cells survival during gastrulation and that Barhl2 loss-of-function
(LOF) compensate for Mcl1 LOF. Barhl2 does not act on mcl-1, or of any other protein
of the apoptotic cascade, transcription but modulates Mcl1 stability. We demonstrated
that Mcl1 acts during gastrulation whereas Barhl2 acts at blastula stage and limits fate
determination of SO cells. Using Barhl2 mutants we showed that Barhl2 recruits the Wnt
pathway cofactor Groucho/TLE and consequently inhibits SO genes expression. Our
work reveals a new regulator of Groucho/TLE activity.
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Poster Number: P125
Improving the usefulness of the European Xenopus Resource Centre (EXRC) to
the research community
A Noble, M Piccinni, A Jafkins, G Nicholson, L Nazlamova, V Allan, C Sharpe, M Guille
EXRC, Institute of Biomedical and Biomolecular Research, School of Biological
Sciences, University of Portsmouth, Portsmouth, UK
The EXRC makes, collects, curates, holds and distributes animal and molecular
resources for researchers using the Xenopus model. Now in its tenth year, the EXRC
collaborates with the other Xenopus resources in an effort to provide the community
with all of the support possible at cost. Our current focus is: 1) on building a “clean”
Xenopus laevis colony using the protocols developed by the Grainger lab and adopted
at NXR; 2) on testing how nanobodies might be exploited to understand protein function
during Xenopus development; 3) on using sperm freezing to reduce animal numbers
used and costs for users and 4) on using CRIPSR/Cas to insert fluorescent protein
genes into endogenous loci to allow labelling of cells that have previously been
extremely difficult to target.
Thank you to all of those who contribute resources to the centres around the world –
please, if you have frog lines or key molecular resources that would be of use to the
community contact one of the centres to deposit them. Having strong resource centres
helps everyone’s research and strengthens our model at a time of funding pressure.
Poster Number: P126
National BioResource Project (NBRP) for Xenopus: recent developments at the
Asian hub for the international Xenopus research community
A Suzuki, K Kashiwagi, H Hanada, N Furuno, I Tazawa, A Kurabayashi, K Nakajima, K
Takebayashi-Suzuki, T Igawa, M Sumida, H Yoshida, S Murakami, K Oriha, T Mido, M
Masumoto, K Kawaguchi, A Miura, A Kashiwagi
Institute for Amphibian Biology, Graduate School of Science, Hiroshima University,
Kagamiyama 1-3-1, Higashihiroshima, Japan
The establishment of a Xenopus resource core facility in the Asia-Pacific region is
essential to promote Xenopus research around the globe and to make easily available
important genetic and live frog resources to the international Xenopus research
community. The National BioResource Project (NBRP) aimed at not only facilitating the
use of Xenopus tropicalis for a diverse array of genetic and physiological studies, but
also providing research and educational training opportunities for young investigators.
NBRP is also working together with EXRC, NXR, Xenbase and the international
Xenopus laevis genome project consortium to develop the system to share reagents
and information essential for basic research, such as the distribution of Xenopus laevis
BAC clones worldwide. We have recently completed the analysis of the genetic
135
backgrounds of Xenopus tropicalis strains commonly used in the US, Europe and Japan
through a collaborative efforts between three resource facilities, NBRP, EXRC and
NXR.
136
AUTHOR INDEX
A
Abaffy, P - P120
Abbruzzese, G - P71
Adis, C - P76
Agricola, Z N - P79
Aguero, T H - P117 & P118
Allan, V J - P85 & P125
Allen, G E - O08 & P05
Al-Mahmoud, N A - P74
Alten, L - P76
Amiel, J - O13 & P37
Andre, P - P61
Anglin, C T - P48
Angrand, G - P119
Arnold, T - O03 & P02
Arnold, T R - P04*
Audic, Y - P119*
B
Banach, M - P81*
Bayramov, A V - P09
Beckers, A - P76
Bell, E - P21
Belyavsky, A V - P59
Bertrand, S - P20
Bervas, M - P119
Beyer, A - O25 & P17
Bhattacharya, D - P57*
Blewitt, M - O13 & P37
Blitz, I L - P14 & P25*
Blum, M - O10*, P55*, P61 & P76
Boel, A - P114
Bogusch, S - P15
Bozatzi, P - P121
Bradshaw, C R - O08 & P05
Briggs, J - P86
Briggs, J A - O01*, P31* & P78*
Bright, A R - P29
Brueckner, M B - P74
Buisson, I - P69
Bullejos, M - P102*
Burns, K A - P98
Butler, A M - O20*, P116* & P117*
C
Cammarata, G - O24 & P40
Carandang, L - P39
Carron, C - P22
Cebrian-Silla, A - O09 & P107
Cha, S W - P79
Chai, N - P110
Chang, C - O12*, PL03 & P58*
Chang, L - P08*
Chen, K - O13 & P37
Cheong, S M - P43
Cheung, J - P14
Cho, J S - P14
Cho, K W Y - P14* & P25
Ciau-uitz, A - O26* & P112*
Cibois, M - P20
Coen, L - P110 & P122
Colleluori, V D - P68*
Collins , C - P82
Colpaert, R - P114
Concordet, J P - P110
Cosse-Etchepare, C - P69*
Costa, A S H - O08 & P05
Coughlin, M - P88
Cousin, H - O23 & P65
Cox, O F - O07 & P52
Coy, B - O03 & P02
Crawford, C W - P48
Creytens, D - P114
Crossman, D K - O12 & P58
Crowley, M R - O12 & P58
Cummins, T - P121
D
Daar, I - O17* & P50*
Danilchik, M V - O05* & P10*
Danno, H - O15* & P12*
Davaapil, H - O11 & P111
De Domenico, E - O09 & P107
137
De Jesús Andino, F - P90
de Paiva Alves, E - P13
del Viso, F - O21 & P72
Demeneix, B - O28, P104, P110 & P122
Deniz, E D - P74*
Desanlis, I - P41*
Deschamps, S - P119
Dhomeja, M - P21
Dimitrakopoulou, D - P95* & P114
Dingwell, K S - P121*
Dinshaw, K - P04
Dorey, K - P108
Dovichi, N J - O07 & P52
Dubaissi, E - O19* & P97*
Duncan, A R - O21 & P72
Durand, B C - P124*
E
Ebbert, P - P39
Edholm, E S - P81
Edwards, D R - P41
Edwards, G - O09 & P107
Erdogan, B - O24 & P40
Ermakova, G V - P09
Eroshkin, F - P84
Eroshkin, F M - P09 & P42
Escande-Beillard, N - P101*
Escriva, H - P20
Evans, M - P39
Furuno, N - P126
G
Garcia-Miralles, M - P33
Gates, P B - O11 & P111
Gautier-Courteille, C - P119
Geary, L - P113*
Genever, P - P51
Gentsch, G E - P123
Gervi, I - P69
Getwan, M - P103*
Gilchrist, M - O09 & P107
Gimelbrant, A - P27
Girardot, F - P110* & P122*
Glinka, A - P08
Goh, G H - P33*
Gorbsky, G J - O04* & P83*
Gordon, C T - O13 & P37
Gossler, A - P76
Gouignard, N - P75
Grainger, R M - P29
Grencis, R K - O19 & P97
Griffin, J - P73
Griffin, J N - O21* & P72*
Grigg, E A - P48
Grossfeld, P - P53
Guille, M - P64*, P100 & P125
Guille, M J - P85 & P87*
Gurdon, J B - O08 & P05
Guzman, D - P35
Gygi, S P - P88
F
Fainsod, A - P30* & P96
Falco, R - P93
Faunes, F - P35, O09* & P107*
Fini, J B - O28, P104 & P110
Fish, M - P14
Fish, M B - P25
Fisher, M - P29
Fisher, M E - P98
Forecki, J - P67
Forouzmand, E - P14
Fortriede, J D - P98
Francl, A - O24 & P40
Frezza, C - O08 & P05
H
Han, D - P06 & P07*
Han, J K - P18 & P43
Han, L - P48
Han, W - P18
Hanada, H - P126
Harbour, W - P118
Hardy, S - P119
Harland, R M - O27, P11, P32 & P63
Hatch, V - P06*
Hayes, M H - P99
Heald, R - P03
138
Hicks, G G - P30
Higashi, T - O03, P02 & P04
Hillmer, A - O13 & P37
Ho, L - P33
Hollemann, T - P36*
Hoppler, S - P13
Horb, L - P82
Horb, M - P27, P64, P82 & P94
Horb, M E - O04, P83 & P93
Houston, D W - P105*
Huber, P - O07 & P52
Hwang, Y S - O17 & P50
I
Ide, T - P60
Igawa, T - P126
Imai, Y - P44
Ingraham, J - P88
Isaacs, H V - P51
Ishida, K - P44
Ishinabe, N - P60
Ito, Y - P56
Ivanova, A S - O22 & P106
J
Jafkins, A - P125
Jagpal, A K - P79
James-Zorn, C - P98*
Javed, A - O13 & P37
K
Karimi, K - P98
Kashiwagi, A - P126
Kashiwagi, K - P126
Kato, T - P44
Kawaguchi, K - P126
Kayserili, H - P101
Kenny, A P - P48 & P79*
Keum, B R - P18* & P43*
Kha, C X - O02 & P109
Khedgikar, V - O23* & P65*
Khokha, M - P73 & P77
Khokha, M K - O21, P57, P66, P68,
P70, P72 & P74
Kim, M - P08
King, M - P51*
King, M L - O20, P116, P117 & P118*
Kinoshita, T - P92
Kintner, C - O27 & P63
Kirmizitas, A - O01 & P112
Kirschner , M - P84 & P86
Kirschner, M W - O26, P31, P78 & P88
Kiryukhin, D O - P59*
Klein, A M - O26, P31, P78 & P88
Knapik, L - O17 & P50
Kodjabachian, L - P20*
Kondo, M - P28*
Kondo, T - P49
Kopantseva, Y Y - P59
Korotkova, D D - O22*, P09 & P106*
Koziol, M J - O08* & P05*
Kreis, J - P38
Kulkarni, S - O21 & P72
Kulkarni, S S - P73*
Kumada, T - O14 & P24
Kunz, R C - P88
Kurabayashi, A - P126
Kuroda, H - P80
Kurz, S - P61*
Kuznetsov, J - P118
Kwon, T - O12 & P58
L
LaBonne, C - P113 & P115
Larraín, J - P35*, O09 & P107
Lauper, J - O02 & P109
Le Bouffant, R - P69
le Mevel, S - O28, P104 & P110
Le Petillon, Y - P20
Leal, J I - O25 & P17
Le-Charney, R - P14
Lee, H - P43
Lee, M S - O17 & P50
Lee-Liu, D - O09 & P107
Leemans, M - O28 & P104
Legagneux, V - P119
Lerebours, A - P64
139
Lerivray, H - P119
Li, Z - O02 & P109
Liem, K - P73
Lienkamp, S - P103
Lin, L - P53
Liu, K J - O21 & P72
Liu, S - P30
Loh, A - P101
Loreti, M - P22*
Lothay, V - P98
Lowery, L A - O24*, P39* & P40*
Lubetsky, V A - O22 & P106
Lucaj, C - P39
Luchinskaya, N N - P59
Luxardi, G - P20
M
Maccarana, M - P75
MacColl Garfinkel, A E - P77*
Maeno, M - P44* & P49
Maerker, M - P15
Malmström, A - P75
Mancini, P - O16* & P54*
Manohar, S - P29
Marin-Barba, M - P19*
Marshall, L - O28, P104, P110 & P122
Martinez-Cañas, J C - P102
Martynova, N Y - P42*
Martynova, N Y U - P09
Masumoto, M - P126
Mathavan, K - O23, P65 & P71*
Mayor, R - O25 & P17
McCracken, K W - P48
Mcnamara, S - P93 & P94*
McQueen, C - P23*
Méndez, E - O09 & P107
Mereau, A - P119
Merzdorf, C S - P67*
Metikala, S - P36 & P82*
Michiue, T - P56 & P60*
Mido, T - P126
Mii, Y - O18 & P46
Miller, A L - O03, P02 & P04
Miller, K - P03*
Mis, E K - P66*
Misumi, Y - P89
Mitchison, T J - P88
Miura, A - P126
Monteiro, R S - P123*
Morichika, K - P92
Morrow, S - P64
Muñoz, R - O09, P35 & P107
Mughal, B - O28*, P104* & P110
Murakami, S - P126
Musheev, M - P06 & P07
Myers, F A - P87
N
Naert, T - O06, P91, P95 & P114*
Nag, A - P27
Nakai, Y - P34
Nakajima, K - P34 & P126
Nakamigawa, M - P49*
Nakamura, Y - P13*
Nakata, K - P44
Nakayama, T - P29*
Nazlamova, L - P125
Nemoto, M - P44
Nesterenko, A M - O22, P09 & P106
Neuhaus, H - P36
Newman, K M - O20, P116 & P117
Nicholson, G - P125
Nie, S - P53*
Niehrs, C - P06, P07 & P08
Niesler, B - P15
Nieuwenhuysen, T V - P95
Nikaido, I - O15 & P12
Noble, A - P64, P85 & P125*
Nodono, H - P47
Noelanders, R - O06, P91, P95 & P114
Noiret, M - P119
O
Ochi, H - O14* & P24*
Ogino, H - O14 & P24
Oh, D - P105
Okada, M - P45
Onuma, Y - P56
140
Oriha, K - P126
Orlov, E E - P42
Ott, T - P76*
Owens, D A - O20, P116 & P117
Robson, A R - P74
Roco, A S - P102
Rocques, N - P124
Rousseau, K - O19 & P97
Roussis, I M - P87
Rutherford, E R - P39
P
Paillard, L - P119
Palmier, K - P110
Patient, R - O01, P100 & P112
Peñailillo, J - O09 & P107
Pearl, E - P27 & P64
Pegge, J - P21*
Pelzer, D - P108*
Pera, E M - P75*
Péricard, L - P110 & P122
Peshkin, L - O26, P27*, P31, P78, P84*
& P86*
Peskin, L - P88
Peterkin, T - P100
Peuchen, E H - O07* & P52*
Piccinni, M Z - P85* & P126
Ponferrada, V G - P98
Popov, I - O12 & P58
Pouladi, M - P33
Pownall, M E - P23
Presler, M - P88*
Prokhortchouk, E B - P42
Q
Quigley, I - O27 & P63
R
Rankin, S A - P48* & P79
Rao, A N - P115*
Ratzan, W - O04 & P83
Reversade, B - O13*, P33, P37* & P101
Riddiford, N - P26
Rider, C C - P21
Riou, J F - P69
Robert, J - P34, P81 & P90*
Roberts, I S - O19 & P97
Robson, A - O21 & P72
S
Sajjan, U K - O27 & P63
Sakai, M - P47*
Sapkota, G P - P121
Sasagawa, Y - O15 & P12
Sato, Y - P80*
Savova, V - P27
Scarlett, G P - P87
Scerbo, P - P20 & P110
Schlosser, G - P26*
Schmitteckert, S - P15
Schneider, I - O10 & P55
Schweickert, A - O10, P15*, P55 & P61
See, K - P67
Segerdell, E - P98
Seliverstov, A V - O22 & P106
Sempou, E - P70*
Sena, E - P124
Shabtai, Y - P30 & P96
Shaidani, N - P82
Shannon, J M - P48
Sharpe, C - P64 & P125
Sharpe, C R - P85 & P100*
Shi, D L - P22
Shiokawa, K - P89*
Short, S - P100
Shukrun, N - P96*
Shustikova, L A - P59
Sindelka, R - P120
Smith, J C - P121 & P123
Soria, K - O17 & P50
Spruce, T - P123
Stephenson, R - O01 & P112
Stephenson, R E - O03*, P02* & P04
Strate, I - P75
Suh, B - P82
Sumida, M - P126
Sun, D I - O27 & P63
141
Sun, J - O17 & P50
Sun, L - O07 & P52
Sun, L L - O09 & P107
Suzuki, A - P45 & P126*
Suzuki, N - O14 & P24
Visser, T - O28 & P104
Vivien, C - S08, P110 & P122
Vize, P - P98
Vlaeminck, L - O06 & P91
Vleminckx, K - O06*, P91*, P95 & P114
T
W
Tada, H - P92*
Taira, M - O18, P28 & P46
Taira, Y - P92
Takada, S - O18 & P46
Takahashi, S - P28
Takebayashi-Suzuki, K - P45 & P126
Tapia, V - O09 & P107
Tashiro, K - P89
Tatsinkam, A J - P21
Tazawa, I - P126
Tereshina, M B - O22 & P106
Thi Tran, H - O06 & P91
Thomas, W - P82
Thornton, D J - O19 & P97
Thumberger, T - O10 & P55
Tisler, M - O10 & P55
Tomankova, S - P120*
Toro-Tapia, G - O25 & P17
Torrejon, M - O25* & P17*
Tran, H T - P114
Tseng, A S - O02* & P109*
Wühr, M - P88
Walentek, P - O27* & P63*
Wallingford, J B - O12 & P58
Wang, J - P90
Wang, L - O20 & P116
Ward, N - P16
Watanabe, T - P56*
Weeks, D L - P99*
Wells, J M - P48
Wheeler, G N - P16*, P19 & P41
White, R J - P23
Willsey, A J - P11 & P32*
Willsey, H R - P11* & P32
Wlizla, M - P93* & P94
Wollnik, B - O13 & P37
Wong, P M - P33
Wong, S - P101
Wuehr, M - P86
Wulff, C - O05 & P10
X
U
Xie, X - P14
Xue, S - O13 & P37
Ueno, N - P45
Umbhauer, M - P69
Y
V
Van Booven, D - P117
Van Itallie, E - P88
Van Nieuwenhuysen, T - O06, P91 &
P114
Vardanyan, J - O16 & P54
Veenstra, G J - P13
Vick, P - P38*
Viet, J - P119
Villaseca, S - O25 & P17
Yamamoto, T - O18*, P28 & P46*
Yamashita, S - P60
Yaoita, Y - P34*
Yigit, G - O13 & P37
Yoon, J - O17 & P50
Yoshida, H - P45* & P126
Yun, M H - O11* & P111*
142
Z
Zaraisky, A - P84
Zaraisky, A G - O22, P09*, P42 & P106
Zinovyeva, M V - P59
Zorn, A M - O16, P48, P54, P79 & P98
143

16 International Xenopus Conference

Transcription

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