TPs-bloque I

Transcription

TPs-bloque I

GUIAS DE TRABAJO PRÁCTICO
BLOQUE I
Cronograma de Biología del Desarrollo 2015
TEORICAS
TRABAJOS PRACTICOS
Lunes 14-16 h Aula 72
20/4 Introducción al desarrollo
animal ES
27/4 Inducción y morfogénesis
ES
4/5 Desarrollo en Drosophila
ES
11/5 Desarrollo en peces, aves
y mamíferos ES
Miércoles 10-12 Aula 36
22/4 Equivalencia genómica ES
18/5 Desarrollo postembrionario ES
20/5 Regeneración ES
25 FERIADO
27/5 Ciclo de vida en plantas
EZ
3/6 Desarrollo Floral II EZ
1/6 Desarrollo Floral I EZ
2/49 Fertilización y desarrollo
temprano ES
6/5 Desarrollo en anfibios e
invertebrados ES
13/5 Organogénesis ES
Lunes
11/5 Desarrollo embrionario y
post-embrionario en
crustáceos RI
18/5 Interacción de gametas y
fertilización in vitro de ovocitos
de ratón GR-JB-AC
1/6 Desarrollo embrionario de
anfibios y mamíferos JB-GR-AC
8/6 Desarrollo embrionario tardío
y anexos extraembrionarios en
mamíferos y aves JB-GR-AC
8/6 Meristemas Vegetativo,
de Inflorescencia y Floral EZ
10/6 Embriogénesis EZ
15/6 Órganos laterales EZ
17/6 Diferenciación frutos EZ
15/6 Especificación de gametas y
fertilización en plantas GP
22/6 Fotomorfogénesis EZ
24/6 Epigenética. EZ
29/6 Evolución y desarrollo. I AV
6/7 Relaciones entre ambiente y
1/7 Evolución y desarrollo. II AV
8/7 Relaciones entre ambiente y
22/6 Regulación hormonal y
desarrollo embrionario en plantas
GP
29/6 Seminarios plantas GP y EZ
6/7 Seminario de Modularidad AV
desarrollo. I DA
13/7 Seminario de Imprinting y
comportamiento II AV
desarrollo. II DA
15/7 PARCIAL II
13/7 Seminario de Imprinting y
comportamiento I
AV
Docentes
ES: Eduardo Spivak
EZ: Eduardo Zabaleta
DA: Daniel Antenucci
AV: Aldo Vassallo
Primer parcial: 29 o 30 de mayo, a convenir
AC: Andreina Cesari
GR: Glenda Rios
JB: Jorgelina Buschiazzo
LB: Laura Biondi
RI: Romina Ituarte
GP: Gabriela Pagnussat
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Trabajos Prácticos Biología del Desarrollo UNMdP
│ 2015
DESARROLLO EMBRIONARIO Y POST-EMBRIONARIO EN CRUSTÁCEOS
ACUÁTICOS
Responsable: Romina Ituarte
Resumen
En esta práctica se estudiarán los procesos del desarrollo embrionario y postembrionario en referencia a dos órdenes de crustáceos, Decapoda (cangrejos y
camarones) y Anostraca. En cuanto al desarrollo embrionario temprano se hará
hincapié en los estadios de blástula, gástrula y diferenciación. En cuanto al desarrollo
post-embrionario se discutirá el concepto de desarrollo anamórfico y variaciones en el
patrón de desarrollo. Además, se observarán fotografías de órganos y tejidos
relacionados con la ontogenia de la osmorregulación en un camarón de agua dulce.
Palabras claves
Embriones. Segmentación. Blástula. Gástrula. Larvas. Osmorregulación
Objetivos
Reconocer las etapas del desarrollo embrionario inicial basándose en observaciones
morfológicas e introducir la idea de estadios discretos a partir de un desarrollo continuo
(“estadificación”).
Reconocer distintos tipos larvales, introducir el concepto de desarrollo anamórfico y de
egg-nauplius (Artemia spp, como ejemplo)
Familiarizarse con el material biológico y obtener cierta destreza en el manejo del
mismo.
Material biológico
Embriones fijados en distintos estadios del desarrollo (Palaemonetes argentinus,
Palaemon macrodactylus y Neohelice granulata)
Larvas fijadas (desarrollo larval completo) Cyrtograpsus angulatus
macrodactylus
y Palaemon
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Material vivo (sujeto a la disponibilidad del mismo)
Cultivos de Artemia spp in vivo
Material didáctico
Fotografías de embriones en distintos estadios del desarrollo.
Fotografías de cortes histológicos y de inmunofluorescencia de órganos en desarrollo
involucrados en la osmo-iono-regulación.
Desarrollo del práctico

Identificar en el material vivo disponible y en las fotografías (Fig. 1) blástula
inicial, blastómeros, blástula tardía, gástrula, vitelo embrionario, ojos, latido cardíaco
(material vivo). Ejes antero-posterior, dorsal-ventral.
Figura 1. Embriones de Palaemonetes argentinus en distintos
estadios del desarrollo
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• Identificar la larva nauplius y distintos estadios del desarrollo en cultivos in vivo de
Artemia spp. Observar tipos de larvas de crustáceos (nauplius, zoea,
decapodito/megalopa). Discutir las variaciones en el patrón de desarrollo.
• Observación de fotografías de órganos/tejidos relacionados con la tolerancia a la
salinidad en un camarón dulceacuícola en las fases adulta, larval y embrionaria.
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GAMETOGÉNESIS Y FECUNDACIÓN EN VERTEBRADOS
Objetivo general
Identificar y comprender las distintas etapas del proceso de fecundación en mamíferos, desde la
gametogénesis a la formación del cigoto, poniendo particular énfasis en la interacción de gametas.
Objetivos particulares




Reconocer y comprender las distintas etapas de la espermatogénesis tomando como modelo el
mamífero.
Diferenciar y reconocer las distintas características de los ovarios y ovocitos de mamífero y anfibio.
Identificar y comprender cada una de las etapas de la ovogénesis tomando como modelo el
mamífero.
Comprender los principales eventos que ocurren en la interacción de gametas y la fecundación.
OBSERVACION DEL MATERIAL BIOLÓGICO Y ACTIVIDADES
I. ESPERMATOGÉNESIS
1) Observar al microscopio cortes histológicos de testículo de ratón. Identificar desde la periferia hacia el
lumen, células de Sertoli, espermatogonias, espermatocitos, espermátides y espermatozoides. Relacionar
los estadios con las etapas de la meiosis (Ver Anexo I).
2) En base a lo observado señalar en el esquema las distintas estructuras.
Figura 1. Esquema de una sección de epitelio seminífero que muestra la relación entre las células de Sertoli y células
espermáticas en desarrollo.
3) Obtener espermatozoides de epidídimo de ratón, capacitarlos y realizar el conteo de los mismos en
cámara cuenta célula (utilizar como guía el Anexo I).
4) Observar y esquematizar la morfología de espermatozoides de anfibio, bovino, y ratón (epididimarios),
señalando los principales componentes del mismo. En preparados teñidos con Coomasie blue, identificar
el acrosoma y evaluar su integridad (Anexo I).
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II. OVOGÉNESIS
1) Observar y esquematizar ovarios de anfibio y bovino. En el bovino, identificar los folículos, cuerpo lúteo, y
corpus albicans.
2) a) Observar al microscopio cortes histológicos de ovario de ratón e identificar:
a.
b.
c.
d.
estructura del ovario, estroma y corteza.
los distintos estadios de maduración de los folículos (¿dónde se ubican?).
estructura del folículo.
ovocitos.
(Esquematizar tratando de guardar las proporciones)
b) Relacionar cada estadio con la etapa de la meiosis en la que se encuentra el ovocito (utiliza como guía
los esquemas del Anexo II).
3) Obtener complejos cumulus-ovocito (COCs) bovinos por punción folicular de ovarios provenientes de
frigorífico. Esquematizar los ovocitos y definir en qué estadio se encuentran.
4) Observar y diferenciar al microscopio óptico COCs bovinos inmaduros (estadio de vesícula germinal) y
madurados in vitro (estadio de Metafase II).
5) Observar y comparar con los ovocitos de anfibio, bovino y ratón teniendo en cuanta: el tamaño de los
ovocitos, cubiertas y la cantidad de vitelo. Relacionarlo con la estrategia reproductiva de ambos grupos
(AnexoII)
III. INTERACCIÓN DE GAMETAS Y FECUNDACIÓN
1) Observar al microscopio de epifluorescencia ovocitos bovinos fecundados teñidos con Vectashield-DAPI.
2) La fecundación involucra la unión de las gametas para generar un nuevo organismo. Este es un proceso
complejo que se desarrolla en etapas:
a. Liberación y transporte de los gametos.
b. Reconocimiento y contacto entre el espermatozoide y el ovocito.
c. Regulación de la entrada del espermatozoide en el ovocito.
d. Activación del metabolismo del cigoto y comienzo del desarrollo.
e. Fusión del material genético.
Describe brevemente cada una de las etapas. En el siguiente esquema se observan algunas de ellas. Señalar y
describir qué ocurre en cada uno de los puntos esquematizados.
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ANEXO I
Figura 1.Resumen de los principales fenómenos durante la espermatogénesis humana
Concentración espermática
El conteo de espermatozoides puede realizarse en forma manual, mediante cámaras cuenta células. Existen
diferentes tipos de cámaras (Thoma, Neubauer, entre otras) las cuales se diferencian entre sí básicamente por
las características de dichas cuadrículas. A modo de ejemplo, describiremos la cámara de Neubauer (Fig. 2).
Esta cámara puede contener una o dos cuadrículas. Cada una de ellas está constituida por un cuadrado
primario dividido a su vez en 9 cuadrados secundarios (Fig. 2, 1). El cuadrado secundario central, que
utilizaremos para el conteo de espermatozoides, está dividido en 25 cuadrados medianos de 0,2 mm de lado
(Fig.2, 2), y cada uno de estos cuadros se subdivide a su vez en 16 cuadrados pequeños (Fig 2,3).
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Procedimiento: Para poder contabilizar los espermatozoides, los mismos deben estar estáticos, y en una
concentración tal que sea posible individualizarlos. Se recomienda utilizar como medio de dilución una
solución salina formolada. Para el conteo, en general se necesita realizar una dilución. Por ejemplo, si
trabajamos con una dilución de 1:400 (10 µL de semen en 3,99 mL de solución salina formolada).
Colocaremos, 10 µL de la muestra diluida por debajo del cubreobjetos colocado sobre la cámara. Luego se
procederá a contar 5 cuadros medianos (Fig. 2,4) de triple línea.
Figura 3. Espermatozoides ovinos teñidos con Comassie Blue. A: espermatozoides con acrosomas íntegros
(tinción azul uniforme superpuesto en toda la región acrosomal). B: espermatozoides con acrosomas no
íntegros, presentan un patrón de tinción muy claro o irregular por pérdida del acrosoma o proceso de
reacción acrosomal. Las imágenes se encuentran a 100X.
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ANEXO II
Figura 4. Clasificación de ovocitos en función de la cantidad y distribución de vitelo (deutoplasma),
A.
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B.
Figura 5 . A) Esquema de un corte histológico de ovario de anfibio. B) corte de ovario de rana, hematoxiliaeosina 4X. Los ovocitos inmaduros y maduros se sitúan en una bolsa epitelial (túnica) de aspecto irregular. Los
ovocitos inmaduros en la periferia y los maduros en el interior.
Figura 6. Esquema un corte de ovario de mamífero que muestra la secuencia de fenómenos de origen,
crecimiento y ruptura del folículo de Graaf así como la formación y retrogresión del cuerpo amarillo.
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Figura 7. Resumen de los principales fenómenos durante la ovogénesis humana.
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Figura 8. Etapas de desarrollo del ovocito humano y del folículo ovárico. A-D, folículos primarios, E-F, folículos
secundarios, con la formación del antro, G-H, folículos de Graaf.
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DESARROLLO EMBRIONARIO DE ANFIBIOS Y MAMÍFEROS
Objetivo general
Reconocer los tipos de segmentación, identificar los distintos estadios embrionarios y comprender los
mecanismos que operan en la gastrulación en ambas clases del reino animal.
Objetivos particulares
•
•
•
•
•
Reconocer características anatómicas y comportamentales propias de cada sexo en el anfibio (sapo).
Identificar las distintas modalidades de segmentación de acuerdo con la cantidad y distribución de
vitelo y relacionarla con el tipo de ovocito considerado.
Reconocer los tipos de blástulas formadas de acuerdo con el tipo de huevo y segmentación.
Diferenciar los estadios embrionarios en anfibios (sapo) y en mamíferos (bovino).
Interpretar los principales cambios que ocurren en la gastrulación, en particular, los movimientos
morfogenéticos que llevan a la formación de las capas germinales y cavidades.
OBSERVACIÓN DE MATERIAL BIOLÓGICO Y ACTIVIDADES
1) Observar ejemplares vivos de Rhinella arenarum (sapo común de jardín). Reconocer características
anatómicas y comportamentales propias de cada sexo.
2) Observar a la lupa binocular e identificar los distintos estadios embrionarios en anfibios (sapo).
Comparar el tamaño relativo del embrión y el aspecto de la ganga a medida que avanza el desarrollo.
Esquematizar los distintos estadios usando como guía el Anexo I. El material biológico está conservado
en formol tamponado al 10 %.
Ovocitos no fertilizados, identificar los polos animal y vegetativo.
Formación del surco I, observar su orientación respecto de los polos vegetativo y animal con la
formación de 2 células iguales.
Formación del surco II, observar su orientación respecto del I con la formación de 4 células
iguales.
Formación del surco III, observar su orientación respecto de los anteriores y la formación de 8
células de distinto tamaño, denominadas macrómeras y micrómeras.
Estadio de mórula, observar la masa de células y las diferencias entre los polos del embrión.
Estadio de blástula, se distinguen los límites celulares y en el interior se ha formado el blastocele.
Estadio de gástrula, la primera evidencia externa es la aparición de un surco ligeramente curvo, el
blastoporo, que representa el sitio en el que las células comienzan a desplazarse hacia el interior
del embrión. El margen superior del surco recibe el nombre de labio dorsal del blastoporo. A
medida que ocurre la gastrulación, los labios blastoporales se extienden gradualmente hacia el
polo vegetativo, rodeando a las células vitelinas y formando el tapón vitelino. Identificar el tapón
vitelino y observar el avance en su reducción.
Estadio de néurula, observar la placa y surco neural así como el alargamiento del embrión.
Estadio de yema caudal, la parte posterior del embrión se alarga más rápidamente que el resto,
formándose el esbozo de la cola del futuro renacuajo o yema caudal.
Estadio de larva con yemas branquiales, formación del órgano adhesivo (protuberancia en la
zona medio ventral del cuerpo). En este estadio se produce la eclosión de sus envolturas.
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Estadio de larva con branquias externas, al final del período se puede observar el opérculo, la
regresión del +órgano adhesivo y la perforación de la boca, formada por un pico córneo.
3) Observar a la lupa binocular e identificar los distintos estadios embrionarios en mamíferos (bovino).
Esquematizar los distintos estadios usando como guía el Anexo II. El material biológico está fijado en
paraformaldehído al 4%.
Embriones de 2 a 8 células, relacionar el tamaño y cantidad de blastómeras.
Estadio de mórula.
Blastocisto y blastocisto protruido: identificar la masa celular interna, blastocele y trofoblasto.
4) a) Confeccionar un cuadro comparativo que contemple las distintas modalidades de segmentación de
acuerdo con la cantidad y distribución de vitelo dentro del citoplasma y por consiguiente con el tipo de
ovocito considerado.
b) Reconocer el tipo de blástula en los esquemas.
Tipo de
Ovocito
Tipo de
Segmentación
Tipo de
Blástula
Cavidad
blastocélica
Grupos
(ejemplos)
Oligolecítico
Heterolecítico
Telolecítico
Centrolecítico
---------------------
---------------------
---------------------
---------------------
---------------------
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ANEXO I
Desarrollo embrionario de anfibios
Figura 1. Esquema del huevo pigmentado de anfibio anuro en segmentación. a, nótese la acumulación de
pigmento en el polo animal; b, primer surco de segmentación; c, estadio de 4 blastómeras.
Figura 2. Formación del tercer surco en un anfibio anuro y estadios sucesivos. a, el tercer surco de
segmentación se formará paralelo al ecuador, orientado perpendicularmente al eje polar pero desplazado
hacia el casquete animal en función de la cantidad de vitelo que queda acumulado en la parte inferior del
huevo; b, se formarán cuatro voluminosas células vitelinas o macrómeras en el polo vegetativo, en tanto que
en el polo animal se encontrarán las células de menor volumen o micrómeras, a menudo más pigmentadas; c,
estadio de mórula.
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Figura 3. Blástula de anfibio anuro. a, vista in toto; b, corte sagital de la blástula (celoblástula desigual).
Figura 4. Estadio más adelantado de la blastulación en anfibio anuro.
b
Figura 5. Inicio de la gastrulación en anfibio anuro. a, comienzo de la epibolia; b, corte sagital: el blastocele
bastante amplio todavía se encuentra ubicado en el polo animal.
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Estadios del desarrollo del embrión bovino
bovino
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b
Figura 6. Formación del labio dorsal del blastoporo. a, vista in toto del comienzo de la embolia; b, corte
sagital.
b
Figura 7. Estadio de la gastrulación en anfibio. a, vista in toto; b, corte sagital. Nótese la reducción del
blastocele y el inicio del arquenterón.
tapón vitelino
b
Figura 8. Proceso gastrular más avanzado en anfibio. Nótese el arquenterón más amplio y el blastocele más
reducido.
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a
Figura 9. Ampliación del arquenterón y su formación definitiva.
Figura 10. Vista externa de la reducción del tapón vitelino.
Figura 11. Gástrula diseccionada. Nótese el piso del arquenterón y la correspondiente masa vitelina.
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Figura 12. Néurula de anfibio. a, placa neural; b, corte sagital; c, corte transversal.
Figura 13. Embrión de anuro en organogénesis temprana en corte sagital.
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ANEXO II
II
Desarrollo embrionario de mamíferos
Figura 1. Huevo de mamífero (oligolecítico) en el estadio de primer surco de segmentación (estadio de dos
células).
Figura 2. Segmentación irregular del huevo de mamífero.
Figura 3. Formación de la mórula en mamífero. En b, corte sagital.
Figura 4. Formación del blastocisto en mamíferos.
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│ 2015
PARTE 1: DESARROLLO EMBIONARIO TARDÍO EN VERTEBRADOS
Objetivo general: Reconocer el desarrollo de los órganos en embriones en etapas
avanzadas del desarrollo embrionario-fetal en mamíferos.
Objetivos particulares:




Reconocer estructuras anatómicas en embriones de ratón de estadios
intermedios y hacia el final de la gestación (10-24 días).
Observar la disposición de fetos de ratón en los cuernos uterinos y las
estructuras extraembrionarias
Analizar la expresión de genes durante el desarrollo embrionario
Estudiar el desarrollo de algunos grupos de órganos reproductores durante el
desarrollo embrionario.
OBSERVACION DE MATERIAL BIOLOGICO Y ACTIVIDADES
1) Observar utilizando lupa estereoscópica embriones de ratón de estadios
intermedios y hacia el final de la gestación (10-24 días). Reconocer
estructuras, realizar mediciones e intentar determinar la edad fetal. Puede
obtener ayuda en http://sectional-anatomy.org/mouse-embryo/#
Estadios del desarrollo intrauterino completo en ratón comparado:
Especie Estadio 1
2
3
4
5
6
7
8
9
10
11
12
13
Humano Dia
1
2-3
4-5 5-6
7-12 13-15 15-17 17-19 20
22 24
28
30
Raton
Dia
1
2
3
4
5
6
7.0
8.0
9.0
9.5 10
10.5 11
Rata
Dia
1
3.5
4-5 5
6
7.5
8.5
9
10.5 11 11.5 12
14
33
15
36
11.5 12
12.5 13
16
40
17
42
12.5 13
13.5 14
18
44
19
48
13.5 14
14.5 15
20
52
21
54
14.5 15
15.5 16
22
55
58
15.5 16
16.5 17
1
23
17.5
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│ 2015
Figura 1: Comparación morfológica entre el desarrollo embrionario de ratones y humanos. Los estadios embrionarios
en el ratón están basados en el número de somitos y sus características. En total son 28 los estadios descriptos, los
cuales en el ratón abarcas hasta el día 20 post concepción. Las líneas punteadas señalan momentos homólogos de
organogénesis. (De: Xue et al. BMC Genomics 2013 14:568 doi:10.1186/1471-2164-14-568)
Vesícula ocular
E9.0: Día 9 post coito (evidenciado por el por la aparición del tapón
mucoso en la vagina de la hembra. Eventos mas importantes: formacion
del neuroporo posterior y brote de extremidades anteriores. Aparece el
brote de la cola y los 3ros y 4tos arcos braquilales. Comienza la
migración de las células primordiales germinales.
pairs.thttps://embryology.med.unsw.edu.au/embryology/index.php/Primor
dial_Germ_Cell_Migration_Movie
Fosa
auditiva
1er arco
branquial
Corazón
Brote de la extremidad anterior
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E10.5: Hinchamiento de los globos oculares. El 1er arco
branquial se divide en componentes maxilar y mandibular.
Desarrollo avanzado del tubo cerebral y elongación de la cola.
E11.5: Vesiculas oculars separadas de la superficie definiendo
la periferia del ojo. Comienzan a dividirse en dos regiones los
miembros anteriores. Los monticulos auditivos se hacen
visibles. Las celulas epiteliales coleomicas en ambos sexos
migran hacia las gonadas. Septado del corazón.
E17.5: Se engrosa la piel. Los dedos se hacen de miembros
inferiores y superiores se hacen paralelos. Los párpados se
fusionan. Bigotes visibles.
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│ 2015
2) Leer el trabajo “A High-Resolution Anatomical Atlas of the Transcriptome in
the Mouse Embryo” de Diez-Roux y col. PLOS Biology -January 18,
2011DOI: 10.1371/journal.pbio.1000582. El trabajo se discutira en clase.
3) Complemente la información obtenida en clase con bibliografía y complete el
siguiente cuadro referido al desarrollo del embrión de los estadios E7 y
E11.5. Establezca similitudes y diferencias.
Aspecto externo (relieves
que pueden observarse)
E7
E 11.5
Cara
Vesiculas que forman el
tubo neural
Corazon
Tubo digestivo
Glandulas anexas del
sistema digestivo (hígado y
páncreas)
Esbozos de los órganos de
los sentidos (vista, olfato y
audición).
4) Analice los siguientes experimentos:
Estudiando la ontogenia del desarrollo del embrión de ratón se observó que las
células de las crestas neurales tienen un patrón de migración muy específico a
cada uno de los arcos branquiales (AB). Asimismo se observó que los genes de
la familia HoxC se expresan en la región de las crestas 7 y 8 (R7 y R8),
inducidos por el ácido retinoico.
Observe el esquema y responda:
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│ 2015
Figura 1: Distribución rostro-caudal de las crestas neurales (Rn) en el embrión de ratón y
patrones de migración de las células hacia los distintos arcos branquiales (ABn) durante el
desarrollo. Se indica las estructuras y funciones asociadas a cada arco branquial. AB1: Nervio
trigémino, mandíbula, músculos de la masticación, tensor del tímpano, tensor del velo del
paladar, martillo, yunque; AB2: Nervio facial, músculos de la expresión facial, estribo, musculo
estilohiodeo; AB3: faringe; AB4, 6: vago, elevador del paladar, constrictores de la faringe,
músculos del esófago
a) En un embrión de ratón knock-out (ausencia) del gen HoxC4, ¿qué
alteraciones en el desarrollo de los arcos branquiales esperaría encontrar?
b) En un embrión con un knock-out (ausencia) del elemento de respuesta al
ácido retinoico normalmente presente en la región promotora del gen HoxC4,
¿qué alteraciones en el desarrollo de los arcos branquiales esperaría usted
encontrar en este caso?
c) ¿Qué papel le atribuiría usted al ácido retinoico en el desarrollo craneofacial?
5) Ud. busca en la bibliografía y encuentra un trabajo donde los autores
demuestran que el esbozo hepático crece en respuesta al factor de crecimiento
hepático (HGF). Por lo tanto, decide realizar el siguiente experimento:
Cultiva esbozos hepáticos con mesodermo del septum transverso y agrega o no,
un anticuerpo (Ac) neutralizante del HGF. Los resultados obtenidos son:
Figura 1: Medida de la proliferación
del esbozo hepático (ramificaciones)
en función del tratamiento con células
del mesodermo del septum
transverso (ST), o con ST y
acticuerpo neutralizante de HGF (ac).
5
27
│ 2015
i) ¿Cuál fue la hipótesis al planear este experimento?¿Qué concluye?
ii) ¿Por qué cree que al agregar el anticuerpo el número de ramificaciones no es
igual al del control?
iii) ¿Qué resultados espera si repite el experimento a), pero utilizando esbozos
hepáticos provenientes de ratones con un knock-out del gen c-met (receptor del
HGF)? Justifique.
6) Ud. cuenta en su laboratorio con ratones normales y ratones portadores de
mutaciones de diferentes genes que afectan el desarrollo del páncreas. Los rótulos de
las jaulas se perdieron y es necesario volver a identificarlos. Para esto, toma como
parámetros del funcionamiento del páncreas exocrino la actividad de amilasa, y del
páncreas endocrino, los niveles de insulina y glucagon. Ud dispone de la siguiente
información de los ratones cuyos rótulos se perdieron: los ratones de la jaula A son los
ratones normales (sin mutaciones), los de la B tienen una mutación en el gen PDX (gen
de caja homeótica pancreática duodenal) que es un gen maestro para el desarrollo del
páncreas, los de la C tienen una mutación en el gen Pax4 y los de la D, una mutación
en el gen Pax6. Además, sabe que la expresión de los genes Pax es importante para la
diferenciación del páncreas endocrino: Pax6 para la diferenciación de las células α
(glucagon) y Pax4 + Pax6 para las células ß (insulina), γ (somatostatina) y δ
(polipéptido pancreático).
Figura 1: Cuantificación de actividades en
células pancreáticas de ratones control y
ratones obtenidos de las cajas I-IV
6
28
│ 2015
a) En base a los resultados, identifique los ratones A-D con los números I-IV.
b) ¿Qué niveles de somatostatina esperaría en cada grupo de ratones?
PARTE 2: ANEXOS EXTRAEMBRIONARIOS
Objetivo general: Reconocer la ubicación de los embriones dentro de las cavidades
uterinas en mamíferos y comprender el mecanismo de nutrición embrionaria en
mamíferos.
Objetivos particulares:



Diferenciar los tipos de nutrición del embrión a lo largo de su desarrollo.
Conocer la importancia de la placenta
Observar las envolturas embrionarias
OBSERVACION DE MATERIAL BIOLOGICO Y ACTIVIDADES
1)
Observar a la lupa estereoscópica úteros de hembras de ratón mantenidos en
10% formol. Analizar la disposición de los embriones en el cuerno uterino. Intentar
visualizar la placenta y las estructuras que rodean al embrión.
2)
Observar embriones conservados junto con la placenta en solución de formol al
10%. Analizar la estructura de las envolturas embrionarias y de la placenta.
3)
Observación de placenta fresca a termino de origen bovino. Describa sus
características macroscópicas.
Responder:
a- Cual es el significado del concepto “barrera placentaria” ?
b- Cuál es el origen de los vasos placentarios?
7
29
│ 2015
c- Describa brevemente las funciones placentarias.
4) Se hace un experimento en monos para estudiar el transporte de las siguientes
sustancias a través de la membrana placentaria. Para ello se inyectan a la mona
preñada vía i.v. 100 unidades marcadas (de forma de poder ser identificadas) de
c/u de las sustancias a estudiar. Luego de un tiempo, se hace el recuento
porcentual de las sustancias en sangre materna, sangre fetal y tejido placentario.
Los resultados fueron representados en los gráficos. Interprételos, justificando
su respuesta en función de sus conocimientos de fisiología placentaria.
8
30
│ 2015
ANEXOS
Figura 1. Esquema de placenta uterina. Se muestra la estructura del corion y su
relación con la pared uterina.
Figura 2. Aspecto general de los anexos embrionarios. A. estadío de línea primitiva en
corte sagital. B, desarrollo del alantoides que se une al corion, el cual a su vez se halla
en estrecho contacto con las criptas de la mucosa uterina.
31
│ 2015
TIPOS DE PLACENTA
-las placentas endocorial y hemocorial, deciduas
-las placentas epicorial y mesocorial, indeciduas
Figura 3 Esquemas de los distintos tipos de placenta. La disposición general del
conjunto de vellosidades se indica en la parte inferior derecha de cada esquema.
32
A High-Resolution Anatomical Atlas of the Transcriptome
in the Mouse Embryo
Graciana Diez-Roux1, Sandro Banfi1, Marc Sultan2, Lars Geffers3, Santosh Anand1, David Rozado2, Alon
Magen2, Elena Canidio4, Massimiliano Pagani4¤a, Ivana Peluso1, Nathalie Lin-Marq5, Muriel Koch6,
Marchesa Bilio1, Immacolata Cantiello1, Roberta Verde1, Cristian De Masi1, Salvatore A. Bianchi1, Juliette
Cicchini5, Elodie Perroud5, Shprese Mehmeti5, Emilie Dagand2, Sabine Schrinner2, Asja Nürnberger2,
Katja Schmidt2, Katja Metz2, Christina Zwingmann2, Norbert Brieske2, Cindy Springer2, Ana Martinez
Hernandez3, Sarah Herzog3, Frauke Grabbe3, Cornelia Sieverding3, Barbara Fischer3, Kathrin Schrader3,
Maren Brockmeyer3, Sarah Dettmer3, Christin Helbig3, Violaine Alunni6, Marie-Annick Battaini6, Carole
Mura6, Charlotte N. Henrichsen7, Raquel Garcia-Lopez8, Diego Echevarria8, Eduardo Puelles8, Elena
Garcia-Calero8, Stefan Kruse9, Markus Uhr3, Christine Kauck3, Guangjie Feng10, Nestor Milyaev10,
Chuang Kee Ong10, Lalit Kumar10, MeiSze Lam10, Colin A. Semple10, Attila Gyenesei10¤b, Stefan
Mundlos2, Uwe Radelof11¤c, Hans Lehrach2, Paolo Sarmientos4, Alexandre Reymond7, Duncan R.
Davidson10*, Pascal Dollé12*, Stylianos E. Antonarakis5,13*, Marie-Laure Yaspo2*, Salvador Martinez8*,
Richard A. Baldock10*, Gregor Eichele3*, Andrea Ballabio1,14,15,16*
1 Telethon Institute of Genetics and Medicine, Naples, Italy, 2 Max Planck Institute for Molecular Genetics, Berlin, Germany, 3 Genes and Behavior Department, Max Planck
Institute of Biophysical Chemistry, Goettingen, Germany, 4 Primm, Milan, Italy, 5 Department of Genetic Medicine and Development, University of Geneva Medical School,
Geneva, Switzerland, 6 Institut Clinique de la Souris, Illkirch, France, 7 Center for Integrative Genomics, University of Lausanne, Lausanne, Switzerland, 8 Experimental
Embryology Lab, Instituto de Neurociencias, Universidad Miguel Hernandez, San Juan de Alicante, Spain, 9 ORGARAT, Essen, Germany, 10 Medical Research Council
Human Genetics Unit, Western General Hospital, Edinburgh, United Kingdom, 11 RZPD—Deutsches Ressourcenzentrum für Genomforschung, Berlin, Germany, 12 Institut
de Génétique et de Biologie Moléculaire et Cellulaire, Inserm U 964, CNRS UMR 7104, Faculté de Médecine, Université de Strasbourg; Illkirch, France, 13 University
Hospitals of Geneva, Geneva, Switzerland, 14 Medical Genetics, Department of Pediatrics, Federico II University, Naples, Italy, 15 Department of Molecular and Human
Genetics, Baylor College of Medicine, Houston, Texas, United States of America, 16 Jan and Dan Duncan Neurological Research Institute, Texas Children’s Hospital,
Houston, Texas, United States of America
Abstract
Ascertaining when and where genes are expressed is of crucial importance to understanding or predicting the physiological
role of genes and proteins and how they interact to form the complex networks that underlie organ development and
function. It is, therefore, crucial to determine on a genome-wide level, the spatio-temporal gene expression profiles at
cellular resolution. This information is provided by colorimetric RNA in situ hybridization that can elucidate expression of
genes in their native context and does so at cellular resolution. We generated what is to our knowledge the first genomewide transcriptome atlas by RNA in situ hybridization of an entire mammalian organism, the developing mouse at
embryonic day 14.5. This digital transcriptome atlas, the Eurexpress atlas (http://www.eurexpress.org), consists of a
searchable database of annotated images that can be interactively viewed. We generated anatomy-based expression
profiles for over 18,000 coding genes and over 400 microRNAs. We identified 1,002 tissue-specific genes that are a source of
novel tissue-specific markers for 37 different anatomical structures. The quality and the resolution of the data revealed novel
molecular domains for several developing structures, such as the telencephalon, a novel organization for the hypothalamus,
and insight on the Wnt network involved in renal epithelial differentiation during kidney development. The digital
transcriptome atlas is a powerful resource to determine co-expression of genes, to identify cell populations and lineages,
and to identify functional associations between genes relevant to development and disease.
Citation: Diez-Roux G, Banfi S, Sultan M, Geffers L, Anand S, et al. (2011) A High-Resolution Anatomical Atlas of the Transcriptome in the Mouse Embryo. PLoS
Biol 9(1): e1000582. doi:10.1371/journal.pbio.1000582
Academic Editor: Gregory S. Barsh, Stanford University, United States of America
Received August 4, 2010; Accepted December 6, 2010; Published January 18, 2011
Copyright: ß 2010 Diez-Roux et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the EC VI Framework Programme contract number LSHG-CT-2004-512003. The authors also acknowledge the support of:
the Italian Telethon Foundation (AB, SB, and GD-R); the Swiss National Science Foundation (AR and SEA); the Max Planck Society (GE, M-LY, HL); MRC (RB, DD);
Association pour la Recherche sur le Cancer (PD); and Ingenio 2010 MEC-CONSOLIDER CSD2007-00023, DIGESIC-MEC BFU2008-00588, CIBERSAM/ISCIII (SM). The
funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Abbreviations: ABA, Allen Brain Atlas; AH, anterior hypothalamic; CNS, central nervous system; E[number], embryonic day [number]; EMAGE, Edinburgh Mouse
Atlas of Gene Expression; EMAP, Edinburgh Mouse Atlas Project; FIATAS, Fast Image Annotation Software; GO, Gene Ontology; HSC, hematopoietic stem cell; ISH,
in situ hybridization; MGI, Mouse Genome Informatics; TM, tuberomammillar
PLoS Biology | www.plosbiology.org
1
January 2011 | Volume 9 | Issue 1 | e1000582
High-Resolution Transcriptome33Atlas
* E-mail: [email protected] (DRD); [email protected] (PD); [email protected] (SEA); [email protected] (M-LY); [email protected] (SM);
[email protected] (RAB); [email protected] (GE); [email protected] (AB)
¤a Current address: Istituto Nazionale di Genetica Molecolare, Milan, Italy
¤b Current address: Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku, Finland
¤c Current address: Scienion, Berlin, Germany
organ architecture and function, and, in addition, stem cell
division and cell differentiation are still ongoing. Each gene was
analyzed on a set of 24 sagittal sections, which all together provide
a complete representation of all embryonic tissues [5]. We set up
semi-automated pipelines to design one appropriate probe per
gene (Figure S1), with the aim of capturing most of the isoforms
generated by alternative splicing. We also included a set of locked
nucleic acid (LNA) probes covering the mature sequences of 444
murine microRNAs in the analysis.
After ISH and automated microscopy image acquisition [16],
expression patterns were manually annotated by expert anatomists
using a revised version of the Edinburgh Mouse Atlas Project
(EMAP) anatomy ontology, which includes 1,420 anatomical terms.
The EMAP mouse anatomy ontology (http://www.emouseatlas.
org/Databases/Anatomy/new/theiler23.shtml) is widely accepted
and is used as the basis for annotating expression patterns in other
large-scale expression resources such as EMAGE and MGI. This
ontology supports annotation at different levels of resolution
through automatic inheritance of properties between levels. In
addition to identifying expression sites, our curated annotation
provided information on the expression pattern (homogeneous,
regional, or single cell) and on its strength (strong, moderate, or
weak), revealing detailed patterns even for genes expressed at low
levels. Compiling all ,15,500 annotated patterns allowed classifying them into three broad categories: 39% were ‘‘regional’’ (signal
detected in a limited number of discrete locations), 43% showed a
nonregional signal in all tissues, and 18% were not detected. Figure 1
shows examples of these three categories. All images and their
annotation are available and searchable at http://www.eurexpress.
org.
The Eurexpress database allows basic and advanced queries by
annotated anatomy, gene name, symbol, template, and gene
sequence. The search interface provides both a thumbnail view of
a representative section and the annotation summary (Figure 2A).
The expression data can be visualized in the form of either a
montage viewer (Figure 2B) or a zoom/panning viewer (virtual
microscope, Figure 2C). All expression patterns are linked to
expression databases, such as the ABA [8], EMAGE [12,17], and
the Gene Expression Nervous System Altas [11], and to
bioinformatics resources such as Entrez Gene, ENSEMBL, and
MGI. Additional features of Eurexpress include a standard
anatomy reference atlas based on a set of eight sagittal histology
sections that have been graphically annotated. These section views
have a user-controlled overlay capability as well as the standard
zoom viewer and can be used in conjunction with the assay image
views to enable convenient comparison (http://www.eurexpress.
org/eAtlasViewer/php/eurexpressAnatomyAtlas.php).
Introduction
Genomic research has significantly advanced our understanding
of physiological and pathophysiological processes, ranging from
infectious diseases to cancer. Two fundamental aspects of this
approach are the generation of large datasets and the systematic
integration of the information contained therein. Transcriptome
analysis has been in the forefront of this research field.
Ascertaining when and where genes are expressed is of crucial
importance to understanding or predicting the physiological role
of genes and proteins and how they interact to form the complex
networks that underlie organ development and function. Progress
in understanding gene networks is driven by massive parallel
approaches [1–4] that capture the complexity of a gene network as
a whole. However, genome-scale approaches capable of unraveling events occurring in single cells or small groups of cells still pose
a major challenge. In recent years, high-throughput methods that
collect such information at cellular resolution on a gene-by-gene
basis have been developed. Of particular relevance was the
development of high-throughput technology for RNA in situ
hybridization (ISH) to map gene expression patterns on tissue
sections [5–7]. A widely used resource based on this technology is
the Allen Brain Atlas (ABA) [8], a digital genome-wide atlas of
gene expression in the adult mouse brain. Additional valuable
resources documenting organ-specific gene expression using
similar approaches include the Gene Expression Nervous System
Altas (GENSAT), the GenitoUrinary Development Molecular
Anatomy Project (GUDMAP), and the St. Jude Brain Gene
Expression Map (BGEM) [9–11]. Efforts to integrate expression
data that bring together information from diverse sources are the
Edinburgh Mouse Atlas of Gene Expression (EMAGE) [12] and
the Mouse Genome Informatics (MGI) Gene Expression Database
(GXD) [13]. These databases use published gene expression data
descriptions to provide expression annotations that follow standard
anatomy ontology. The next challenge, partially addressed in
Drosophila melanogaster [14,15], is the generation of a transcriptome
map of an entire organism at cellular resolution.
Here we report the generation of the Eurexpress transcriptome
atlas, which delivers the expression patterns of almost all Mus
musculus protein-coding genes (more than 18,000 genes) in the
developing mouse at embryonic day 14.5 (E14.5) by RNA ISH.
These data were organized and annotated to build a Web-based
gene expression atlas freely available to the scientific community
(http://www.eurexpress.org). This atlas is to our knowledge the
first resource generated in a mammalian organism that provides a
simultaneous visualization of thoroughly annotated gene expression patterns at cellular resolution at one developmental stage.
Results
Validation
The Transcriptome Atlas
A quality control study on 250 solute carrier genes (Slc)
characterized with the same ISH protocol [18] but using probes
generated by PCR amplification with specific primers revealed
over 90% concordance, indicating that our template resource was
reliable (see Table S1). We also compared 1,089 expression
patterns (including genes with tissue-restricted expression and a
subset of disease genes) to previously published data, collected at
We analyzed the expression patterns of over 18,000 transcripts
(18,264), mostly corresponding to protein-coding genes, by RNA
ISH in the developing wild-type laboratory mouse. The colorimetric ISH was performed on frozen sagittal sections of C57BL/6J
wild-type mice at E14.5. At this developmental stage, organogenesis is largely complete, making it an adequate model to study
2
Author Summary
In situ hybridization (ISH) can be used to visualize gene
expression in cells and tissues in their native context. Highthroughput ISH using nonradioactive RNA probes allowed
the Eurexpress consortium to generate a comprehensive,
interactive, and freely accessible digital gene expression
atlas, the Eurexpress transcriptome atlas (http://www.
eurexpress.org), of the E14.5 mouse embryo. Expression
data for over 15,000 genes were annotated for hundreds of
anatomical structures, thus allowing us to systematically
identify tissue-specific and tissue-overlapping gene networks. We illustrate the value of the Eurexpress atlas by
finding novel regional subdivisions in the developing
brain. We also use the transcriptome atlas to allocate
specific components of the complex Wnt signaling
pathway to kidney development, and we identify regionally expressed genes in liver that may be markers of
hematopoietic stem cell differentiation.
the same stage and using the same methodology, by using the
literature query form of the MGI Gene Expression Database
( http://www.informatics.jax.org/searches/gxdindex_form.shtml ).
We found data in the literature for 14% of these, and the analysis
revealed 84% overall concordance between the two datasets. The
comparison was done by visual inspection, and concordance/
partial concordance was scored when the sites of expression were
the same or overlapping in the two datasets. Table S2 includes the
results and the appropriate literature references. Interestingly, if
we restrict the same analysis to a subset of more characterized
genes, namely, 100 disease genes, for which we found published
expression data in 72% of cases, the concordance reaches 97%,
giving a clear indication of the equivalence between datasets when
studying well-characterized genes. Overall, these results underscore the reliability of our data as tested against published data.
We compared our expression data to those obtained from
microarrays using RNA from whole E14.5 embryos [19]. This
comparison revealed that 30% of the genes determined as regional
by ISH could not be detected by microarray (GSE-6081) (e.g.,
Titf1; Figure 1). In addition, we also compared Eurexpress data to
the results of a microarray experiment carried out using RNA
from the E14.5 mouse heart (E-GEOD-1479 in the Gene
Expression Omnibus database). The comparative analysis revealed
that of the 397 regional genes annotated to be expressed in the
heart in Eurexpress, 20% (78 genes) were not detected by the
microarray experiment described above. These data underline the
value of ISH for revealing the expression of genes with very
specific or restricted patterns.
Expression Analysis and Expression Clustering
We performed data mining on genes annotated as regional to
gain insight into the transcriptome complexity of the main organs
and anatomical structures at E14.5. This analysis revealed that the
tissues displaying the highest expression complexity belong to the
central nervous system (CNS), accounting for 60% (n = 3,902) of
regionally expressed genes, followed by the alimentary system
(45%, n = 2,912) and the sensory organs (43%, n = 2,730) (Figure
S2). We identified approximately 1,000 genes that display
exclusive expression in a specific anatomical structure (Table
S3), 16% of which have unknown function. For example, we
identified 106 markers for specific structures of the CNS (e.g.,
cerebral cortex, thalamus, hypothalamus), 218 for specific
structures of the alimentary system (147 of which are exclusively
expressed in the liver), and 127 for the thymus. This collection
Figure 1. Representative examples of RNA ISH data of E14.5
embryos. The expression categories defined by the annotation
summary are illustrated by the following examples. (1) Expression not
detected: Rassf1 messenger RNA is not detected at this stage. (2)
Homogeneous (non-regional) signal: Wdr68 shows hybridization signal
in all tissues and structures. (3) Regionally expressed genes: Crmp1,
Mir124, Titf1, and 1300010A20Rik. Crmp1 signal is evident in the brain,
the V trigeminal ganglion, the spinal cord, and the neural retina. miR124
is restricted to the nervous system. Titf1 expression is detected in the
diencephalon, hypothalamus, telencephalon, thyroid, and lung.
1300010A20Rik is an example of a tissue-specific gene with expression
limited to the liver. Complete sets of images for 19,411 genes are
available at http://www.eurexpress.org.
doi:10.1371/journal.pbio.1000582.g001
3
biological process. At a threshold value of the Pearson coefficient
of r$0.7, we found 496 clusters, 90 of which included at least ten
genes (additional information available at http://www.eurexpress.
org/ee/project/publication/cluster.jsp). We determined the expression occupancy of these clusters, which provides a measure of
how many of the genes in a cluster are expressed in a specific
anatomical structure. This approach allowed us to group
clusters expressed in the same sets of tissues (Figure 4A), thus
facilitating the identification of complex synexpression groups.
Figure 4B shows an example of a cluster with a complex
expression pattern (cluster 83). We found that genes in this cluster
continue to be synexpressed in the adult (Figure 4C), as assessed by
analysis of publicly available microarray data. This case raises the
possibility that embryonic expression patterns have predictive
value for adult mice. The clusters can be browsed online at http://
www.eurexpress.org/ee/project/publication/cluster.jsp, a Web
link that also provides interactive access to the gene lists and
associated assays, and the results of the functional enrichment
analysis with respect to Gene Ontology (GO), InterPro domains,
Mammalian Phenotype Ontology, and cytogenetic band mappings.
The individual cluster Web pages are also accessible directly
from each assay view via the ‘‘Syn-Expression’’ link on the assay
Web page (e.g., http://www.eurexpress.org/ee/databases/assay.
jsp?assayID = euxassay_009028). The identification of these expression clusters will facilitate the dissection of transcriptional
networks by integrating the high-resolution power of RNA ISH with
the currently available high-throughput—but generally lowresolution—procedures such as microarray and next generation
sequencing.
To gain insight into the dynamics of gene expression in the
embryo versus the adult, we took advantage of the ABA dataset
[8]. We compared gene expression patterns of 80 genes we found
to be confined to the following CNS structures: cerebral cortex,
striatum, thalamus, hypothalamus, midbrain, cerebellum, pons,
medulla, and spinal cord (taken from Table S3). We found that
26% of the genes had a conserved expression pattern, 43% had
extended their expression pattern into new domains of the adult
brain, and 30% were divergent (Table S5). Figure S3 shows two
examples for partial (Figure S3A and S3B) and full conservation
(Figure S3C and S3D) of expression sites. A similar comparison
was done for a subset of the solute carrier family of genes (Slc) for
which a cognate ABA dataset was available (99 genes in total).
Concordance for this data set was 89% (Table S6). Figure S4
illustrates examples where a particular Slc was expressed in
progenitor (E14.5) and differentiated (adult) cells. In the future,
gene expression at cellular resolution, refined by double-labeling
experiments with specific cell type markers, will uncover to what
extent gene expression networks are conserved across stages.
The Eurexpress atlas is highly informative with regard to
expression patterns of disease-causing genes. We selected 100
disease genes that are representative examples of genes responsible
for either diseases targeting specific tissues (e.g., eye, skeletal
muscle, heart, skeleton, immune system) or syndromic conditions
affecting multiple tissues. This analysis was carried out by
comparing the information present, for each disease, in the
clinical synopsis section of the Online Mendelian Inheritance of
Man (OMIM) database with the gene expression annotation data
present in Eurexpress. In all cases the expression pattern observed
was predictive for the phenotypes seen in human (Table S7; Figure
S5).
The above-described comparative analyses between embryonic
and adult brain and the foray into expression of human disease
genes emphasize that the reach of Eurexpress is well beyond the
mid-gestation mouse embryo.
Figure 2. Snapshot view of the Web-based transcriptome atlas.
(A) Keyword search results showing a table format including a
thumbnail view of an image, and visualizing each embryonic section
and associated anatomical annotation, color-coded according to
expression strength. (B) Clicking on a particular image allows viewing
the annotation associated with the particular image (left panel). Top
tabs give additional details and links to other gene expression Web sites
and genomic resources. (C) Zoom viewer. The image viewer provides
full resolution images with standard zoom and pan capability. In
addition, the viewed section can be selected using the 3-D embryo
view. The left-hand panel shows the annotation in the context of the
anatomy ontology, and the tabs provide additional detail and links to
other gene expression and genomic resources.
represents an extraordinary source of novel histological markers
for 37 different anatomical structures (see Figure 3 for specific
examples and Table S4 for a complete summary). This novel
catalog of genes with restricted expression patterns constitutes an
invaluable tool for the identification of sequence control elements
driving gene expression in specific tissues and organs and will be
useful for the design of tissue-specific mouse CRE driver lines [20].
Hierarchical clustering of expression data is a powerful tool to
assess synexpression, with the ultimate goals of elucidating
transcriptional pathways and dissecting gene co-regulation mechanisms. We decided to apply this methodology to our expression
atlas. Towards this goal, a subset of 5,933 regionally expressed genes
was clustered according to the tissue annotations across 831
anatomical terms. For each gene, an expression value was set
according to the expression strength. For hierarchical clustering we
then used the Pearson correlation coefficient, which means the
actual selected values are normalized and only relative expression
strength across the tissues is used. Clustering by annotation
identified numerous synexpression groups, i.e., genes with coordinated expression and that are potentially involved in the same
4
Wnt Signaling in the Developing Kidney
Wnt signaling in embryogenesis is characterized by an extensive
crosstalk between ligands, receptors and co-receptors, regulators,
and downstream messengers [21]. Surprisingly, the expression
patterns for many of the newly identified Wnt pathway
components are largely elusive, a gap in knowledge Eurexpress
begins to close. Table S8 summarizes the expression patterns of
117 Wnt signaling components for the major organ systems.
Collectively these data illustrate which components are expressed
in a given tissue and thus are an entryway into the identification of
organ-relevant pathways. In the developing kidney, 58 genes of the
Wnt signaling pathway show regional expression. Figure 5A
displays the expression strength of these genes in ten renal
structures that are recognizable at E14.5. The scheme in Figure 5B
illustrates that the different steps of nephron formation occur
concurrently at this stage. An early event is the induction of the
condensing mesenchyme (Figure 5B, image 3), which subsequently
undergoes a mesenchyme-to-epithelium transition leading to the
development of the renal vesicle (Figure 5B, image 4). This process
involves WNT9B and its downstream target WNT4 [22].
Consistent with published data [22], Wnt9b and Wnt4 are
expressed in the ureteric bud and the condensing mesenchyme
(white and black arrows in Figure 5C). In addition to WNT4, we
identified seven Wnt signaling components that were markedly
expressed in the condensing mesenchyme (Figure 5A, column 3)
and in cells involved in the mesenchyme-to-epithelium transition.
Among them are Fzd3 and Fzd4 (Figure 5C, black arrows), which
are both expressed in the appropriate place and time to potentially
mediate downstream effects of paracrine WNT9B and autocrine
WNT4 signals. The condensing mesenchyme expresses essential
components of the canonical b-catenin-dependent pathway such
as the Wnt co-receptor Lrp5 and the transcription factor Tcf7
(Figure 5A). Additionally, regulators of canonical signaling such as
DKK1 and its receptor, KREMEN1, as well as AES, a repressor
competing with b-catenin for binding to transcription factors, are
expressed (Figure 5A). We noticed that Fzd3 is prominently
expressed in structures of early nephrogenesis (Figure 5A, columns
3–5), while Fzd4 expression is more pronounced in the renal
vesicle and in structures derived from it, such as the proximal
tubules (Figure 5A, columns 5–7). This observation could support
the idea of a receptor-mediated switch from canonical to
noncanonical signaling thought to occur at the beginning of
tubulogenesis [23]. We conclude that the comprehensive nature of
the Eurexpress database allows one to select those components of
signaling pathways that are expressed at the right time and
location.
Hematopoietic Stem Cell Lineages in Liver
Many of the regulators that control hepatocyte and cholangiocyte differentiation [24] are represented in the Eurexpress
database. In total, 147 genes were largely confined to liver (Table
S3), and these will provide markers to investigate liver development, especially at later stages. In the embryo, hepatocytes are
closely associated with hematopoietic stem cells (HSCs). During
fetal development, HSCs change anatomical localization several
times and are abundant in liver between E10 and E18, with HSC
cell number peaking at ,5,100 around E14.5 [25,26]. At E14.5,
HSC markers such as Itgab2 (CD41), Ptprc (CD45), Ly6a (Sca1), Kit
(CD117), Runx1, and Gata2 are strongly expressed in single,
discrete cells scattered throughout the liver. Cells expressing these
bona fide markers can be classified into three categories (Table
S9): (1) in the case of Gata2, Itgab2, and Runx1, intercellular
distance (d) is much larger than the cell diameter (cd) (d&cd); (2)
Ly6a-positive cells also obey this rule but in addition tend to form
Figure 3. Representative examples of RNA ISH data that show
gene expression patterns restricted to specific anatomical
structures. (A) 0610009A07Rik is expressed in the thyroid; (B)
9030227G01Rik in the salivary glands; (C) Tle6 in the pancreas; (D)
E130119H09Rik in the eye; (E) 6330406I15Rik in the cerebellum; and (F)
Gpr151 in the thalamus. Insets are higher magnification views of
expression shown in main panels and show in greater detail the sites of
expression. crb, cerebellum; pan, pancreas; sgl, salivary glands; thl,
thalamus; thy, thyroid.
5
Figure 4. Hierarchical clustering of regionally expressed genes. (A) Graphical representation of clusters (listed on the right) with more than
eight genes in terms of expression occupancy. The occupancy is calculated as the number of genes in each cluster that are expressed in the
anatomical structures (listed at the top) divided by the number of genes in that cluster (normalization). The matrix of occupancy values for each tissue
group clusters with tissue distribution. More information on clustering can be found at http://www.eurexpress.org/ee/project/publication/
PlosBiol2010.html. (B) Cluster 83, with a Pearson coefficient of 0.73, is composed of eight different genes showing expression in epithelia (oral and
nasal cavities, respiratory tract, and middle and internal auditory cavities), choroid plexus, and middle-gut mucosa. (C) Genes in Cluster 83 are also
synexpressed in adult tissues. Publicly available microarray data (http://symatlas.gnf.org) were clustered using the MeV program (http://www.tm4.
org/mev.html). The figure shows synexpression in intestine, stomach, lacrimal gland, salivary gland, uterus, prostate, mammary gland, placenta, and
bladder. Note that some tissues listed on the top of the diagram are duplicated because they represent two independent datasets. Gene symbols are
on the right.
small clusters and intercluster distances are much larger than cd;
and (3) cells expressing Kit or Ptprc are in proximity to each other
(d<cd). We mined the transcriptome atlas for genes whose
expression patterns in liver fall into the above groups. Table S9
lists the members of these groups and, in addition, defines a fourth
group of scattered cells where d#cd. Collectively, these groups
contain many genes that are implicated in immune functions
encoding membrane-bound cell surface receptors, extracellular
proteins, transcription factors, extracellular cytokines, protease
inhibitors, focal adhesion proteins, and proteins generally involved
in cell adhesion. Many of our markers tag a few thousand cells per
liver, corresponding to the HSC number estimates for fetal liver
6
[27], which raises the possibility that they identify HSCs.
However, double-labeling analyses will be required to resolve
which markers (or marker combinations) actually identify HSCs
and which their descendants.
expression in the medial pallium epithelium (prospective hippocampus), Dct and Zic3 were expressed in progressively more
anterior neuroepithelial domains (the prospective progenitors for
lateral pallium and ventral pallium, respectively) (Figure S6C–
S6F).
Moreover, hierarchical clustering of brain-specific transcription
factors (using the approach described in Figure 4) revealed a group
of ten transcription factor genes that show co-localized or
complementary expression patterns in the telencephalic pallium
and subpallium (Nfe213, Hivep2, Klf7, Fos12, Satb2, Zfhx1b, Zfp184,
Foxp4, Phf13, and Dmrtal). Therefore, the intricate organization of
molecular markers identified allowed us to develop combinatorial
maps that represent the molecular organization of the telencephalon
(Figure S6B, S6D, and S6F). At the same time these markers will
provide an entryway into future genetic fate mapping strategies.
Given that this combinatorial analysis of expression patterns in
the developing diencephalon mainly agrees with previously
proposed molecular maps [31–33], we were interested to explore
the efficiency of this approach for studying regionalization and
topology in the hypothalamus, where controversial models have
been postulated [31,32,34] (see [35] for a review). Using the digital
atlas we selected expression patterns of genes encoding DNA
binding proteins that showed ‘‘regional expression’’ (1,395 genes)
and analyzed in detail the expression of 126 of them expressed in
brain. This analysis revealed that genes mainly expressed in the
basal plate domains of the diencephalon, including the hypothalamus, were exclusively expressed in the caudal hypothalamic
regions: mammillar region and retromammillar areas (13 genes
were identified with this pattern: Foxa1, Mx1a, Lmx1b, Barhl1, Dbx1,
Pax7, Olig2, Rarb, Dfp3, Lhx1, Lhx5, Irx1, and Irx3; Figure 7A and 7D).
Conversely, genes mainly expressed in the diencephalic alar plate
and/or in the telencephalon extended their expression into the
tuberomammillar (TM) hypothalamus and/or anterior hypothalamic (AH) and suprachiasmatic nucleus (12 genes were identified
with this pattern: Lhx2, Lhx6, Lhx9, Dlx1, Dlx2, Dlx5, Unc4, Cited,
Rorb, Arx, Foxa2, and Otx2; Figure 7B–7D). Thus, this analysis
revealed that both mammillar and retromammillar regions express
genes of generic basal plate character, while the TM, AH, and
suprachiasmatic hypothalamic areas, although classified as basal
plate derivatives, express mainly ‘‘alar’’ genes. The expression
analysis of the developing hypothalamus strongly suggests that the
TM hypothalamus (including the neurohypophysis) and the anterior
hypothalamus have an alar plate character. The expression patterns
of Shh and Nkx2.1 in the tuberal and the AH areas could be used
against this new interpretation [31]. However, grafting data showed
different inductive properties of diencephalic and hypothalamic
SHH signals [36], suggesting that these differences in SHH
signaling could be attributed to its alar and basal nature. In
conclusion, our data suggest a novel regional map of the
hypothalamus (Figure 7E and 7F) that interprets the data more
appropriately than the previous model [31] (Figure 7G) and that
allows us to understand the different inductive effects of the anterior
axial mesoderm in the anterior neural plate [37] and its ability to
induce basal plate and alar plate derivatives. More interestingly, this
new interpretation that places primary sensorial hypothalamic areas
(i.e., AH and TM areas [38]) as alar plate derivatives agrees with the
hypothesis of ‘‘functional columns’’ in the vertebrate brain, where
sensorial information is primarily processed by alar derivatives
(extensively reviewed in [39]).
Molecular Organization of the CNS
In the E14.5 embryo, most neurons of the CNS have been
generated and have migrated from the germinative epithelium into
the mantle layer. However, important migratory processes that
shape the future CNS have not yet initiated. Thus, this atlas is a
rich source of additional gene markers that characterize diverse
neuronal populations. Figure 6A shows examples of expression
patterns of five genes collectively delineating the stratification of
the nascent neocortex. 2610306H15Rik and Hist1h1d are localized
at different apico-basal levels of the ventricular epithelium, Nhlh1 is
expressed in the subventricular and intermediate zones, and Nin
and Rorb are expressed in cells localized at different radial levels of
the mantle layer.
At E14.5, the complex cytoarchitecture of the mature spinal
cord is not evident, although most neurons have been generated
and have migrated into the mantle layer. To date, many molecular
markers for the motoneuron columns have been identified in the
ventral horn [28], but there are few markers for the central zone
and for the dorsal horn that do not show any internal subdivisions
and appear as homogeneous cellular fields. We found that
expression patterns of four genes revealed molecular differences
of neurons at different ventro-dorsal levels along the length of the
spinal cord (Figure 6D). Nhlh and Lrrtm1 are expressed at different
layers of the dorsal horn, Adcyap1 is expressed in the dorsal-most
cells of the dorsal horn and in motoneurons, and Zdhhc2 is mainly
expressed in visceral motoneurons. These cellular populations that
show different molecular expressions may belong to the primordium of Rexed’s lamina in the mature spinal cord [29].
The thalamus also appears as a homogeneous cellular field at
E14.5, except for the thalamo-cortical fiber confluence (Figure 6B).
Mining the transcriptome digital atlas allowed us to detect genes
marking an early molecular regionalization of the thalamic mantle
layer, where undifferentiated neurons accumulate. Figure 6C,
shows four examples of genes that show graded expression with
respect to putative diencephalic ‘‘secondary organizers’’ that are
the basal plate and zona limitans (as sources of SHH ventralizing
and rostralizing signals) and the dorsal midline (which produces
FGF8, BMPs, and Wnt dorsalizing signals) [30]. These intrathalamic regionalized genes may specify different cell fates in a
concentration-dependent manner and thus underlie the development of functional domains in the mature thalamus.
The developing mammalian CNS is characterized by complex
gene expression patterns, and the interpretation of these data has
led to the prosomeric model of the mammalian brain [31]. This
model predicts the existence of domains within the ventricular
zones that give rise to diverse segments and morphogenetic fields
[31]. We mined the digital expression atlas for genes that have a
restricted expression pattern within the ventricular zone along the
rostral–caudal axis and hence could be involved in early
specification of the pallial domains of the telencephalon [31].
Nissl staining showed a mainly homogeneous cellular organization
along the midline (Figure S6A) and progressive lateral sections of
the telencephalic pallium (Figure S6C and S6E). Gene expression
patterns clearly demonstrated a molecular heterogeneity among
different regions at the level of the ventricular epithelium and
mantle layer in the corresponding midline (Figure S6B) and lateral
sections (Figure S6D and S6F), thus mapping the predicted
molecular regions in the subpallium and pallium. For instance,
while a new marker gene (0610040j01Rik) showed a localized
Discussion
This is to our knowledge the first gene expression atlas of an
entire mammalian organism that is thoroughly annotated so as to
7
Figure 5. Expression sites of Wnt signaling components in the E14.5 mouse kidney. (A) The matrix shows the level of expression of all 58
regionally expressed genes in ten different renal structures that are defined in (B). Colors represent expression strength: strong (red), moderate (light
red), weak (pink), and not detected (white). The Wnt signaling components are grouped into seven blocks (ligands, receptors, extracellular inhibitors,
canonical signaling, Ca2+ signaling, PCP signaling, and GO Wnt receptor signaling pathway). (B) The scheme in the center illustrates the ten main
anatomical structures characterizing the developing kidney. The image gallery composed of low- and high-power (inset) images reveals that each of
the ten structures characteristically expresses a particular Wnt component. 1: Wnt7b; 2: Wnt11; 3: Dkk1; 4: Sfrp2; 5: Lrp6; 6: Slc9a3r1; 7: Tle4; 8: Tcf4; 9:
Wnt5a; 10: Rspo3. (C) Wnt signaling components involved in the mesenchyme-to-epithelium transition. Wnt9b is expressed in the ureteric bud (white
arrowhead) and acts upstream of WNT4, which is expressed in condensing mesenchyme (black arrowhead). The Wnt receptors FZD3 (black
arrowhead) and FZD4 (black arrowhead) are expressed in a way that allows them to function as candidate transducers for WNT9B/WNT4 signaling
and could possibly underlie a shift from canonical to noncanonical signaling.
systematically capture gene expression in hundreds of organs and
tissues. Because all this information is available in a searchable
database, users can retrieve information tailored to their own
needs. The present study provides a selection of examples
demonstrating how this resource can be applied to a broad range
of biomedical questions and drive scientific discovery. We showed
that we can correlate disease phenotypes to sites of expression of
underlying genes; we extracted information to demonstrate novel
8
Figure 6. High-resolution molecular regionalization in the central nervous system. (A) Genes expressed in cells at different radial levels in
the anterior pole of the dorsal pallium (presumptive frontal cortex). 2610306H15Rik and Hist1h1d are localized at different apico-basal levels of the
ventricular epithelium (VZ); Nhlh1 is expressed at the subventricular zone (SVZ) and intermedial zone (IZ); Nin and Rorb are expressed in cells localized
at different radial levels of the mantle layer (ML). Each transcript is depicted with a different color to show how the expression of each gene in pallial
cells is complementary to others, with some degree of overlap. MZ, marginal zone. (B) Picture of a mid-sagittal section of the brain from a section
series of a Eurexpress assay processed with Cresyl violet. The inserts show the area where the corresponding regions (arrows) have been localized. It is
important to note the homogeneity of cellular patterns in the mantle layer of the thalamus and spinal cord, as opposed to the complex molecular
patterns observed in (C) and (D). (C) Examples of three genes with a graded expression in the thalamic mantle layer (Th). BC055811 shows strong
expression in the caudal pole of the thalamus (close to the retroflexus tract [rf]), becoming weaker towards the anterior pole; Pde10a expression is
complementary to that of BC055811, with a strong signal at the anterior pole of the thalamus, showing a sharp edge of its expression domain at the
limit with the prethalamus (PTh). The expression of this gene becomes progressively weaker towards the caudal pole. Btbd3 transcripts have a dorsoventral decreasing gradient, strong at the dorsal thalamus and progressively weaker towards the ventral thalamus. The ventral pole of the thalamic
mantle layer is depicted by the expression of Calb1. The merged picture, using a color for each gene (right panel), shows how molecular
regionalization allows detection of differences in cell identities in the four areas of thalamic mantle layer: dorsal (DTh), anterior (ATh), ventral (VTh),
and posterior (PTh) thalamus. COM, commissural nuclei of pretectum; EPTh, eminentia thalami; ET, epithalamus; MP, medial pallium; PC,
precommisural nuclei of pretectum; PThTg, prehalamic tegmentum; PTTg, pretectal tegmentum; TTg, thalamic tegmentum; ZI, zona incerta. (D)
Sagittal section of the spinal cord, showing an overlay picture where the expression patterns of four genes have been combined. The picture
summarizes the localization of region-specific molecular codes in spinal cord cells. These molecular codes correspond to different structural levels of
the developing spinal cord: Adcyap1 is expressed in the gelatinous substance (SG, Rexed’s layer 2) and motoneurons (MN); Nhlh1 is expressed in the
spinal cord in the central nucleus of the dorsal horn (NP, Rexed’s layers 3 and 4); Lrrtm1 is located in the spinal reticular nucleus (Rt, Rexed’s layers 5
and 6); and Zdhhc2 is located in visceral motoneurons (vMN). Note that the expression patterns reported above, with the exception of Rorb and Calb,
are novel. The merged color composites are the product of alignment, superposition of sections, and editing using a computer program. A detailed
description of the methods used to obtain such figures is included in Text S1.
insights into the complex segmental organization of the mammalian brain; the cellular resolution provided by the Eurexpress atlas
enabled the discovery of gene markers that characterize the
molecular subdivision of organs, identified novel putative markers
of the hematopoietic lineage, and facilitated the comprehensive
organism-wide mapping of an important developmental signaling
pathway. Future applications of these data might include the
determination of elusive regional differences within structurally
complex organs, the identification of expression signatures for
specific cell populations, the search for regulatory elements that
9
Figure 7. Combinatorial analysis of several transcription factors’ patterns in the hypothalamus reveals a new model of mammalian
hypothalamic organization. (A) Foxa1 expression pattern in the basal plate of rhombencephalic, mesencephalic, diencephalic, and caudal
hypothalamic neuroepithelium. This pattern is representative of other transcription factors such as Lmx1a, Lmx1b, Barhl1, Dbx1, Pax7, Olig2, Rarb,
Dfp3, Lhx1, Lhx5, Irx1, and Irx3, expressed in the prosencephalic basal plate, including hypothalamus, where they were exclusively localized in the
caudal regions: mammillar (MM) and/or retromammillar (RM) areas. (B and C) Lhx2 and Dlx1 expression patterns are representative of transcription
factors expressed in alar prosencephalic derivatives (telencephalon, prethalamus, and thalamus) showing expression in TM and AH areas (currently
described as basal plate hypothalamic domains), as well as in alar hypothalamic regions such as the suprachiasmatic (SCH), paraventricular (PV), and
supraopto-paraventricular (SPV) areas. These patterns are representative of other genes expressed in alar derivatives including the TM and AH
regions: Lhx6, Lhx9, Dlx1, Dlx2, Dlx5, Unc4, Cited, Rorb, Arx, Foxa2, and Otx2. (D) Photoshop composition to illustrate the alar expression patterns of
Lhx2 and Dlx1 (green) and the Foxa1 basal expression (red). (E) Schematic representation of the analyzed patterns suggesting that the mammillar and
retromammillar areas show basal plate molecular characteristics, while the TM and AH regions showed alar plate molecular characteristics. (F and G)
Representation of the new revised topologic model that incorporates the TM and AH regions into the alar plate (F), compared to the currently
accepted prosomeric model (G).The merged color composites are the product of alignment, superposition of sections, and editing using a computer
program. A detailed description of the methods used to obtain such figures is included in Text S1. A, amygdale; ac, anterior commissure; Bst, bed
nucleus of stria terminalis; Cx, cortex; FF, forel fields; P1Tg, pretectal tegmentum; P2Tg, thalamic tegmentum; PH, posterior hypothalamic area; POA,
preoptic area; PT, pretectum; PTh, prethalamus; PV, paraventricular; SCH, suprachiasmatic; Se, septum; SPV, supraopto-paraventricular; ST/Pa,
striatum/pallidum; Th, thalamus.
confer tissue- or region-specific expression, the establishment of
gene networks that operate within and between organs, the
molecular characterization of genetic or otherwise modified mice,
and the design of new tissue-specific CRE driver lines and cell
lineage experiments. Finally, this atlas is ideal for the evaluation of
candidate genes for complex diseases and congenital disorders.
template generation, we used cDNA clones (IMAGE collection or
Mammalian Gene Collection) that were available and re-sequenced
at the German Resource Center for Genome Research (RZPD).
Approximately 10,000 clones could be used for template generation. The clones were used as direct templates for PCR and stored as
glycerol stock in 384-well plates at 280uC. This initial collection
was then enlarged to include about 8,000 PCR templates generated
from the ABA consortium [8]. The latter templates were dilutions of
first-round PCR products derived from EST clone, mouse brain
cDNA, or mouse genomic DNA (ABA templates).
All clones or PCR template sequences were compared to the
mouse gene reference databases (ENSEMBL and Entrez Gene) via
BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) prior to selection. For the probe generation we selected only templates with
sequences matching the reference with at least 95% identity across
at least 80% of the length. Templates were generated by PCR
using appropriate oligonucleotide primers. Full information on
templates, including the complete sequence of the product, the
sequences of the oligonucleotides used to generate them, and the
RNA polymerase promoters used for riboprobe synthesis, are
available on the Eurexpress Web site.
PCR reactions were performed in a 100- ml total volume with
final concentrations of 16 Taq buffer, 1.5 M Betaine, 0.2 mM
Materials and Methods
Template Selection and Generation
For gene selection, both the mouse ENSEMBL and the mouse
Entrez Gene databases were analyzed. Templates used for the
generation of the atlas were PCR products obtained from either
publicly available cDNA clones or reverse transcriptase PCR
reactions, a fraction of which was provided by the ABA consortium
[8]. Automated ISH was performed using previously described
protocols [7]. We set up semi-automated routines for designing one
appropriate probe per gene (Figure S1). Our approach was aimed at
covering most of the genes represented in public mouse databases
(ENSEMBL and Entrez Gene). Because of the high-throughput
nature of the project, we restricted our selection to one probe per
gene, capturing most of the isoforms generated by alternative
splicing, when possible. As an initial source of DNA for PCR
10
dNTPs, 5 U Taq polymerase, 10 U Pfu DNA polymerase, and
0.5 mM of each primer. As template material for the PCR, we used
clone glycerol stock, purified plasmid, or PCR product (ABA
collection).
The quality (size and quantity) of the PCR templates was
systematically assessed by standard gel electrophoresis (1% agarose
gel) and by spectrophotometry (Nanodrop). PCR products yielding
an unexpected size (6100 bp) or showing multiple bands were
excluded from riboprobe generation.
In vitro transcription was performed as previously described [5].
coefficient between genes in the cluster, the number of genes
within the cluster, and the IDs of the genes involved. Further
information on the enrichment of functional annotation within
each cluster is available to users by clicking on the cluster IDs.
This information includes the annotation terms and enrichment
p-values for the GO terms, the InterPro domains, the Mammalian Phenotype Ontology terms, and the cytogenetic band
mappings.
Data Annotation
Figure S1 Eurexpress template generation and riboprobe synthesis workflow.
Found at: doi:10.1371/journal.pbio.1000582.s001 (0.07 MB PDF)
Supporting Information
Approximately 360,000 images were viewed and annotated,
each of high resolution and typically 4K64K pixels. To allow the
annotators to rapidly pass through the data and assess each image,
we implemented a bespoke annotation Java-based interface
termed Fast Image Annotation Software (FIATAS). Key aspects
of the software are the fast interfaces for image viewing, focused
anatomy views with efficient menu and multi-select option
annotation, data ‘‘inbox’’ management, quality control and
multi-editor review, and automatic update to the tracking database
and publication to the Web site (Figure S8). FIATAS can be
installed for off-line operation or will start directly via Web-start
from links on the Eurexpress Web site.
For anatomy tissue annotation we adopted the standard mouse
ontology from EMAP. In the FIATAS interface, the full
anatomical tree of 1,420 terms at Theiler stage 23 is provided,
as well as a number of cut-down views, which can be used for
more detailed access. More information on data annotation can be
found in Text S1.
Figure S2 Transcriptome complexity of main organs
and anatomical structures. The bars represent the number of
genes displaying a regional expression pattern in selected organs
and structures.
Figure S3 Comparison of expression patterns for E14.5
CNS-specific genes between embryonic and adult brain.
This figure illustrates two examples of degrees of similarity
between fetal and adult brain. (A and B) show partial concordance
of the expression pattern of the RFamide-related peptide gene in
neurons of the dorsomedial hypothalamic nucleus (DM) at E14.5
(A) and adult (B). (C and D) show coincidence of expression of the
G-protein-coupled receptor 151 gene in the presumptive region of
the habenular nuclei (MHb) (C) and the habenular region (MHb
and LHb) (D).
Data Management
Figure S4 Comparison of expression patterns for E14.5
The link between the central database and each activity was
managed via a combination of Web services and ftp, with data
exchanged either in XLS, XML or JPEG formats. The
architecture is shown in Figure S7.
CNS-specific genes between embryonic and adult brain.
This figure illustrates typical cases of equivalent (A–F), partially
equivalent (G), and different (H) patterns. Images shown were
downloaded from either the Eurexpress database or the ABA. 4V,
fourth ventricle; bv, brain vasculature; cb, cerebellum; cp, choroid
plexus; cx, cortex; ep, ependyma; hy, hypothalamus; mb,
midbrain; md, medulla; pcp, Purkinje cell progenitors; pcl,
Purkinje cell layer; po, pons; sn, substantia nigra; st, striatum; th,
thalamus; vta, ventral tegmental area; vz, ventricular zone. (A)
The glutamate transporter SLC1A6 is expressed in Purkinje cell
progenitors of the developing cerebellum as well as in all adult
cerebellar Purkinje neurons. (B) Glucose transporter SLC2A1
expression persists in both embryonic and adult brain vasculature.
(C) SLC4A2, a chloride/bicarbonate transporter, is characteristically expressed in the epithelial lining of the choroid plexi. (D)
SLC6A3, a dopamine transporter, is highly expressed in the
substantia nigra and its progenitor region, the ventral tegmental
area. (E) Serotonin transporter SLC6A4 is strongly expressed in
raphe nuclei of the embryonic and adult brain. (F) SLC17A6
resides in synaptic vesicles and takes up glutamate for subsequent
release into the synaptic cleft. It is broadly expressed in neurons in
the adult brain, and this pattern is already seen in the E14.5 brain.
(G) The glial high-affinity glutamate transporter SLC1A3 is
strongly expressed in the ventricular lining of the developing brain.
Later, in the adult brain, expression is most prominent in astroglia
scattered throughout the brain and in the Purkinje cell layer of the
cerebellum (see overview article [40]). The characteristic cell shape
of SLC1A3-positive adult glia cells is already seen in embryonic
SLC1A3-positive cells, suggesting that these are glial progenitors
already expressing a typical adult brain Slc. (H) SLC4A4, a
sodium bicarbonate co-transporter, is highly expressed in
ependymal cells lining the ventricular floor from the midbrain to
Cluster Analysis
Functional inference using Eurexpress data employed hierarchical clustering with centered Pearson correlation coefficients and
the average linkage method. We employed a maximal propagation
strategy, where parent terms acquire the values of child terms
throughout the anatomical ontology. Four annotation types were
examined: GO terms, InterPro conserved domain identifiers,
Mammalian Phenotype Ontology terms, and cytogenetic band (as
a proxy for genomic position). Annotation enrichment was
calculated for each co-expressed cluster containing ten or more
genes (to ensure sufficient annotation to carry out tests), and the
significance of each test was measured using the hypergeometric
distribution according to the standard practice. The significance of
enrichment across all clusters in the dataset was determined using
a permutation strategy: 100,000 permuted datasets were produced
by permuting gene IDs with respect to their annotation, but
maintaining GO term interdependencies. The numbers of tests
passing given p-value thresholds, within each permuted dataset,
were then used to calculate the significance of tests passing those
thresholds in the observed dataset. This proportion provided us
with a permutation-derived p-value, which accounted for the large
number of tests performed while controlling for the interdependencies among the GO annotation terms.
The Eurexpress Web site has implemented a link to visualize
clusters of co-expressed genes derived from hierarchical clustering
of Eurexpress anatomical expression patterns. In each case the
relevant cluster ID is given together with the average correlation
11
Table S3 List of genes that display exclusive expression
in selected structures.
the spinal cord, possibly regulating the electrolytic composition of
the cerebrospinal fluid. In the adult brain SLC4A is expressed
throughout the brain and co-localizes with glial cells. These rather
different patterns of expression raise the possibility of distinct
embryonic and adult functions for the proteins.
Distribution of genes with restricted spatial
expression in different anatomical structures.
Table S4
Figure S5 Tissue distribution at E14.5 of the murine
Table S5 Evaluation in the adult mouse brain of the
expression of the genes expressed exclusively in the CNS
at E14.5.
homologs of three human disease genes. The human
disease genes are SALL1, GDF5, and SLC26A2, responsible for
Townes-Brocks syndrome, brachydactyly type C, and achondrogenesis type 1B, respectively. The expression observed is consistent
with the phenotypic spectrum of the corresponding disease (see
Table S7 for further details and for additional examples).
Table S6 Comparison of Slc expression patterns between embryonic and adult mouse brain.
Genoarchitecture of developing mouse forebrain Nissl-stained sagittal sections. Midline (A) and
progressively more lateral sections (C and E) illustrating the
basic anatomy, with the pertinent anatomical structures labeled.
(B, D, and F) show the same planes as in (A, C, and E) with
expression patterns of several genes indicated by color. Names of
genes are provided in the same colors used to delineate their sites
of expression ([D] and [F] present the same genes). ac, anterior
commissure; AH, anterior hypothalamus; ch, choroidal plexus;
cp, commissural plate; DP, dorsal pallium; LGE, lateral
ganglionic eminence; LP, lateral pallium; LT, lamina terminals;
MGE, medial ganglionic eminence; ML, mantle layer; ML,
mantle layer; MM, mammillar region; MP, medial pallium; OB,
olfactory bulb; och, optic chiasm; POA, preoptic area; PTh,
prethalamus; SCH, suprachiasmatic nucleus; Se, septum; Th,
thalamus;VE, ventricular epithelium; VP, ventral pallium. The
merged colored composites are the product of alignment,
superposition of sections, and editing using a computer program.
A detailed description of the methods used to obtain such figures
is included in Text S1.
Figure S6
List of murine homologs of human disease
genes whose tissue distribution at E14.5 is consistent
with the corresponding human disease phenotype.
Table S7
Table S8 Expression of Wnt signaling components in
the E14.5 embryo.
Table S9 Classification of single cell expression patterns in the E14.5 liver.
Text S1 Supporting methods. This file gives an overview of
the methods used in this manuscript. Additional supplementary
data on clustering can be found at http://www.eurexpress.org/ee.
Found at: doi:10.1371/journal.pbio.1000582.s018 (0.20 MB
DOC)
Acknowledgments
We acknowledge the Allen Institute for Brain Science for providing us with
a set of templates for this study. We acknowledge C. Thaller for help with
the ISH set-up. Authors wish to acknowledge Sigmar Stricker, Julia Meier,
Bella Roßbach, Julia Repkow, and Clara Schäfer. We thank L. Borrelli for
editing the manuscript.
Figure S7 Eurexpress data management architecture.
Each process on the outer pipeline is tracked by data exchange
with the tracking database (TDB). The yellow arrows represent
data flow using protocols as described in the test.
PDF)
Author Contributions
The author(s) have made the following declarations about their
contributions: Conceived and designed the experiments: G Diez-Roux, S
Banfi, I Peluso, N Lin-Marq, M Koch, H Lehrach, P Sarmientos, A
Reymond, DR Davidson, P Dollé, SE Antonarakis, M-L Yaspo, S
Martinez, RA Baldock, G Eichele, A Ballabio. Performed the experiments:
M Sultan, L Geffers, E Canidio, M Pagani, I Peluso, N Lin-Marq, M
Koch, M Bilio, I Cantiello, R Verde, C De Masi, S Bianchi, E Perroud, S
Mehmeti, E Dagand, S Schrinner, A Nrnberger, K Schmidt, K Metz, K
Zwingmann, N Brieske, C Springer, A Martinez Hernandez, S Herzog, F
Grabbe, C Sieverding, B Fischer, K Schrader, M Brockmeyer, S Dettmer,
C Helbig, V Alunni, M-A Battaini, C Mura, CN Henrichsen, S Mundlos.
Analyzed the data: G Diez-Roux, S Banfi, M Sultan, L Geffers, R GarciaLopez, D Echevarria, E Puelles, E Garcia-Calero, CAM Semple, SE
Antonarakis. Contributed reagents/materials/analysis tools: S Anand, D
Rozado, A Magen, S Kruse, M Uhr, C Kauck, G Feng, N Milyaev, CK
Ong, L Kumar, M Lam, A Gyenesei, U Radelof. Wrote the paper: G DiezRoux, S Banfi, DR Davidson, P Dollé, SE Antonarakis, M-L Yaspo, S
Martinez, RA Baldock, G Eichele, A Ballabio.
Figure S8 Screen view of the FIATAS annotation interface. The image displayed in the left-hand view can be expanded
to full resolution and panned at will. The right-hand side image
selector also shows which images are annotated. The upper,
partially hidden dialog box shows the current ‘‘inbox’’ and which
user is currently annotating which assay, and provides the review
and quality control options. The small dialog box lower center
provides the annotation options for the selected anatomical terms.
Table S1 Comparison of independently produced ISH
data for the solute carrier superfamily.
Table S2 Validation of Eurexpress data against published data.
DOC)
References
2. Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B (2008) Mapping
and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods 5:
621–628.
1. Sultan M, Schulz MH, Richard H, Magen A, Klingenhoff A, et al. (2008) A
global view of gene activity and alternative splicing by deep sequencing of the
human transcriptome. Science 321: 956–960.
12
3. Kapranov P, Cawley SE, Drenkow J, Bekiranov S, Strausberg RL, et al. (2002)
Large-scale transcriptional activity in chromosomes 21 and 22. Science 296:
916–919.
4. Birney E, Stamatoyannopoulos JA, Dutta A, Guigo R, Gingeras TR, et al.
(2007) Identification and analysis of functional elements in 1% of the human
genome by the ENCODE pilot project. Nature 447: 799–816.
5. Visel A, Thaller C, Eichele G (2004) GenePaint.org: an atlas of gene expression
patterns in the mouse embryo. Nucleic Acids Res 32: D552–D556.
6. Reymond A, Marigo V, Yaylaoglu MB, Leoni A, Ucla C, et al. (2002) Human
chromosome 21 gene expression atlas in the mouse. Nature 420: 582–586.
7. Gitton Y, Dahmane N, Baik S, Ruiz i Altaba A, Neidhardt L, et al. (2002) A
gene expression map of human chromosome 21 orthologues in the mouse.
Nature 420: 586–590.
8. Lein ES, Hawrylycz MJ, Ao N, Ayres M, Bensinger A, et al. (2007) Genomewide atlas of gene expression in the adult mouse brain. Nature 445: 168–176.
9. Brunskill EW, Aronow BJ, Georgas K, Rumballe B, Valerius MT, et al. (2008)
Atlas of gene expression in the developing kidney at microanatomic resolution.
Dev Cell 15: 781–791.
10. Magdaleno S, Jensen P, Brumwell CL, Seal A, Lehman K, et al. (2006) BGEM:
an in situ hybridization database of gene expression in the embryonic and adult
mouse nervous system. PLoS Biol 4: e86. doi:10.1371/journal.pbio.0040086.
11. Gong S, Zheng C, Doughty ML, Losos K, Didkovsky N, et al. (2003) A gene
expression atlas of the central nervous system based on bacterial artificial
chromosomes. Nature 425: 917–925.
12. Christiansen JH, Yang Y, Venkataraman S, Richardson L, Stevenson P, et al.
(2006) EMAGE: a spatial database of gene expression patterns during mouse
embryo development. Nucleic Acids Res 34: D637–D641.
13. Hill DP, Begley DA, Finger JH, Hayamizu TF, McCright IJ, et al. (2004) The
mouse Gene Expression Database (GXD): updates and enhancements. Nucleic
Acids Res 32: D568–D571.
14. Lecuyer E, Yoshida H, Parthasarathy N, Alm C, Babak T, et al. (2007) Global
analysis of mRNA localization reveals a prominent role in organizing cellular
architecture and function. Cell 131: 174–187.
15. Tomancak P, Berman BP, Beaton A, Weiszmann R, Kwan E, et al. (2007)
Global analysis of patterns of gene expression during Drosophila embryogenesis.
Genome Biol 8: R145.
16. Carson JP, Thaller C, Eichele G (2002) A transcriptome atlas of the mouse brain
at cellular resolution. Curr Opin Neurobiol 12: 562–565.
17. Ringwald M, Baldock R, Bard J, Kaufman M, Eppig JT, et al. (1994) A database
for mouse development. Science 265: 2033–2034.
18. Yaylaoglu MB, Titmus A, Visel A, Alvarez-Bolado G, Thaller C, et al. (2005)
Comprehensive expression atlas of fibroblast growth factors and their receptors
generated by a novel robotic in situ hybridization platform. Dev Dyn 234:
371–386.
19. Visel A, Carson J, Oldekamp J, Warnecke M, Jakubcakova V, et al. (2007)
Regulatory pathway analysis by high-throughput in situ hybridization. PLoS
Genet 3: e178. doi:10.1371/journal.pgen.0030178.
20. Madisen L, Zwingman TA, Sunkin SM, Oh SW, Zariwala HA, et al. (2010) A
robust and high-throughput Cre reporting and characterization system for the
whole mouse brain. Nat Neurosci 13: 133–140.
21. van Amerongen R, Nusse R (2009) Towards an integrated view of Wnt signaling
in development. Development 136: 3205–3214.
22. Schmidt-Ott KM, Barasch J (2008) WNT/beta-catenin signaling in nephron
progenitors and their epithelial progeny. Kidney Int 74: 1004–1008.
23. Merkel CE, Karner CM, Carroll TJ (2007) Molecular regulation of kidney
development: is the answer blowing in the Wnt? Pediatr Nephrol 22:
1825–1838.
24. Lemaigre FP (2009) Mechanisms of liver development: concepts for understanding liver disorders and design of novel therapies. Gastroenterology 137:
62–79.
25. Orkin SH, Zon LI (2008) Hematopoiesis: an evolving paradigm for stem cell
biology. Cell 132: 631–644.
26. Christensen JL, Wright DE, Wagers AJ, Weissman IL (2004) Circulation and
chemotaxis of fetal hematopoietic stem cells. PLoS Biol 2: e75. doi:10.1371/
journal.pbio.0020075.
27. Mikkola HK, Orkin SH (2006) The journey of developing hematopoietic stem
cells. Development 133: 3733–3744.
28. Dalla Torre di Sanguinetto SA, Dasen JS, Arber S (2008) Transcriptional
mechanisms controlling motor neuron diversity and connectivity. Curr Opin
Neurobiol 18: 36–43.
29. Rexed B (1952) The cytoarchitectonic organization of the spinal cord in the cat.
J Comp Neurol 96: 414–495.
30. Martinez-Ferre A, Martinez S (2009) The development of the thalamic motor
learning area is regulated by Fgf8 expression. J Neurosci 29: 13389–13400.
31. Puelles L, Rubenstein JL (2003) Forebrain gene expression domains and the
evolving prosomeric model. Trends Neurosci 26: 469–476.
32. Figdor MC, Stern CD (1993) Segmental organization of embryonic diencephalon. Nature 363: 630–634.
33. Rubenstein JL, Martinez S, Shimamura K, Puelles L (1994) The embryonic
vertebrate forebrain: the prosomeric model. Science 266: 578–580.
34. Shimogori T, Lee DA, Miranda-Angulo A, Yang Y, Wang H, et al. (2010) A
genomic atlas of mouse hypothalamic development. Nat Neurosci 13: 767–775.
35. Puelles L (2009) Contributions to neuroembryology of Santiago Ramon y Cajal
(1852–1934) and Jorge F. Tello (1880–1958). Int J Dev Biol 53: 1145–1160.
36. Vieira C, Martinez S (2006) Sonic hedgehog from the basal plate and the zona
limitans intrathalamica exhibits differential activity on diencephalic molecular
regionalization and nuclear structure. Neuroscience 143: 129–140.
37. Garcia-Calero E, Fernandez-Garre P, Martinez S, Puelles L (2008) Early
mammillary pouch specification in the course of prechordal ventralization of the
forebrain tegmentum. Dev Biol 320: 366–377.
38. Halford S, Pires SS, Turton M, Zheng L, Gonzalez-Menendez I, et al. (2009)
VA opsin-based photoreceptors in the hypothalamus of birds. Curr Biol 19:
1396–1402.
39. Nieuwenhuys R (1999) The morphological pattern of the vertebrate brain.
Eur J Morphol 37: 81–84.
40. Danbolt NC (2001) Glutamate uptake. Prog Neurobiol 65: 1–105.
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