Regulatory Changes of N-Acetylgalactosamine

Original Article
Regulatory Changes of N-Acetylgalactosamine Terminal Sugar in Early Mouse Embryonic Paraxial Mesenchyme
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* Corresponding Address: P.O.Box: 379, School of Health, Birjand University of Medical Sciences, Birjand, Iran
Email: [email protected]
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Abstract
2EMHFWLYH The development of vertebrae is a complex phenomenon that is correlated
with distinct morphological and biochemical alterations in the paraxial mesenchyme and
glycoconjugates. The purpose of this study is to investigate the glycosylation pattern in
paraxial mesenchyme-forming vertebrae by using the lectin histochemical technique.
0DWHULDOVDQG0HWKRGV,QWKLVGHVFULSWLYHDQDO\WLFVWXG\%*¿[HGSDUDI¿QVHFWLRQVRI
9 to 15 day Balb/c mouse embryos were processed for histochemical studies using seven
GLIIHUHQW+53ODEHOOHGOHFWLQV*O\FLQPD[6%$0DFOXUDSRPLIHUD03$:LVWDULDÀRULEXQGD:)$9LFLDYLOORVD99$ZKLFKDOORIWKHPDUHVSHFL¿FIRU1DFHW\OJDODFWRVDPLQH
(GalNAc), 8OH[HXURSLXV8($ELQGVWRĮ/IXFRVHZKHDWJHUPDJJOXWLQLQ:*$ELQGV
WRVLDOLFDFLGDQG*ULIIRQLDVLPSOLFLIROLD*6$%ELQGVWRJDODFWRVHWHUPLQDOVXJDUV
The sections were observed separately by three examiners who were blinded to the lectins. Grading was done according to the intensity of the tested lectins’ reactions with the
VSHFLPHQIURPQHJDWLYHWRVHYHUH'DWDZDVDQDO\VHGZLWK6366VRIWZDUHYHUVLRQDQGWKHQRQSDUDPHWULF.UXVNDO:DOOLVWHVWSZDVFRQVLGHUHGVLJQL¿FDQW
5HVXOWV2XU¿QGLQJVVKRZHGWKDWDPRQJWKHWHVWHGOHFWLQVRQO\*DO1$FUHVLGXHVHQVLtive lectins showed regulated changes in paraxial mesenchyme. Reactions of WFA and
MPA lectins with paraxial mesenchyme were severe on GD9. Reactions of WFA continXHG WR *' FRQVWDQWO\ ZKLOH 03$ UHDFWLRQV FRQWLQXHG VWURQJO\ WR *' VLJQL¿FDQWO\
GHFUHDVHGWKHUHDIWHUSDQGWKHQGLVDSSHDUHG99$DQG6%$ELQGLQJVLQLWLDWHG
ZHDNO\ RQ *' DQG FRQWLQXHG WR *' ZLWKRXW FKDQJLQJ7KHVH UHDFWLRQV LQFUHDVHG
VLJQL¿FDQWO\ S WKHUHDIWHU EHFDPH VHYHUH WR *' DQG ODWHU GLVDSSHDUHG7KH
other tested lectins did not reveal regulated changes.
&RQFOXVLRQ$FFRUGLQJWRWKHVH¿QGLQJVLWFDQEHFRQFOXGHGWKDWRQO\WKH*DO1$FWHUPLnal sugar showed temporally regulated changes during the early embryonic development
of vertebrae in mice. Therefore it most likely plays a key role (s) in the development of vertebrae, especially in the conversion of mesenchymal cells into chondroblasts. The other
tested terminal sugars may have no role in this phenomenon.
Keywords: Embryonic, Glycoconjugate, Terminal Sugar, Mesenchyme
Cell Journal(Yakhteh), Vol 14, No 2, Summer 2012, Pages: 130-141
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Introduction
'XULQJHDUO\GHYHORSPHQWRIWKHYHUWHEUDWHHPbryo, the primary segmented tissue of the body axis
is the paraxial mesoderm that lies bilaterally to the
D[LDORUJDQVQHXUDOWXEHDQGQRWRFKRUG7KH
term for the segmented aggregates cell is somites
1H[WWKHVHFHOOVGLIIHUHQWLDWHDQGWKHYHQWUR
medial portion changes into its mesenchymal form
CELL JOURNAL(Yakhteh), Vol 14, No 2, Summer 2012
130
Regulatory Changes of GalNAc in Paraxial Mesenchyme
(sclerotome), which are the precursor cells for verWHEUDHULEVDQGLQWHUYHUWHEUDOGLVFV
The molecular control of somite differentiation and
sclerotome proliferation and differentiation has only
VRPHZKDWEHHQHOXFLDWHGLQERWKFKLFNHQDQGPRXVH
PRGHOV7KHVRQLFKHGJHKRJVKKVLJQDOLQJPROHFXOHDQGVHYHUDOWUDQVFULSWLRQIDFWRUVLQFOXGing members of the pax and soxIDPLOLHVDUHUHTXLUHG
IRUWKHVHSKHQRPHQDWRRFFXU,WLVZHOONQRZQ
that the notochord induces sclerotomal cells by secretion of inductive factors such as Shh and Noggin,
among others, to migrate, proliferate, differentiate,
and adhere to one another for creating vertebrae (7,
7KHGHYHORSPHQWRIWKHYHUWHEUDOFROXPQ
in vertebrates is a complex phenomenon that is correlated with distinct morphological and biochemical
alterations of the mesenchymal component, such as:
induction, cell migration, differentiation, recognition,
adhesion, aggregation, condensation, and transformation which is regulated by different subsets of morSKRJHQHV,WFDQEHDVVXPHGWKDWVWUXFWXUDO
PRGL¿FDWLRQRIJO\FRFRQMXJDWHVDUHLQYROYHGLQWKHVH
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Glycoconjugates are present at animal cell surfaces
DQGLQWKHH[WUDFHOOXODUPDWUL[(&07HUPLQDO
sugars of these macromolecules correlate with essential functions of cellular interactions such as cell recognition, receptor function, cell adhesion, and migraWLRQ7KH\FDQEHGHWHFWHGKLVWRFKHPLFDOO\XVLQJ
natural polypeptides (lectins), which are obtained
IURPSODQWRUDQLPDOVRXUFHV
'HVSLWHH[WHQVLYHVWXGLHVRQWKHXVHRIOHFWLQKLVWRFKHPLVWU\LQWKHGHYHORSPHQWRIFHUWDLQRUJDQV>VPDOO
DQGODUJHLQWHVWLQHWHVWLVSURVWDWHSODcenta (20), and differentiation of chondrocytes (21,
22), few studies have focused on the development of
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Studies focusing on the expression of glycoconjugates and their role (s) in vertebral column developPHQWDUHOLPLWHG,QDVWXG\FRQGXFWHGE\*|W]DQG
colleagues in 1991, the lectin binding pattern in the
embryonal and early fetal human vertebral column
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binding pattern in the human paraxial mesenchyme
DQGLQWKH\VWXGLHGWKHGLVWULEXWLRQRIJO\coconjugates in the notochord and axial mesenchyme
RIKXPDQHPEU\RV%DJQDOODQG6DQGHUV
VWXGLHGWKHELQGLQJSDWWHUQRI31$OHFWLQDVVRFLDWHG
with sclerotome migration during the formation of
CELL JOURNAL(Yakhteh), Vol 14, No 2, Summer 2012
131
YHUWHEUDOD[LVLQFKLFNHPEU\RV4XRQGDPDWteo et al. noted that in undulated mouse mutants
(substitution in the Pax1 gene), a malformation
not only occurred in the vertebral column, but the
glycosylation pattern was also altered in normal
DQG PDOIRUPHG RUJDQV 0RLLQ HW DO VWXGLHG
changes in the terminal sugars of glycoconjugates
in precursor cartilage that formed the vertebrae in
rat embryos (27). Expression and changes of some
terminal carbohydrate residues of glycoconjugates were studied during the conversion of mesenchyme to cartilage forming vertebrae in mouse
HPEU\RVYLD:)$DQG2)$OHFWLQVE\1LNUDYHVK
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The aim of the present study was to investigate
the lectin binding pattern of paraxial mesenchyme
forming vertebrae and their possible alterations durLQJHDUO\PRUSKRJHQHVLVLQ%DOEFPRXVHHPEU\RV
by means of the lectin histochemistry method.
Materials and Methods
Preparation of mouse embryos
)RU WKLV GHVFULSWLYHDQDO\WLF VWXG\ VH[XDOO\
PDWXUH DGXOW YLUJLQ IHPDOH %DOE& PLFH DYHUDJH
DJHZHHNVDJHWKDWZHLJKHGJDQGIHUtile males of the same strain were obtained from the
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RI0HGLFDO6FLHQFHV0DVKKDG,UDQ
Animals were fed standard laboratory chow and
water ad libitum, under controlled conditions (12
KRXUOLJKWGDUNF\FOH“Û&UHODWLYH
humidity).
$QLPDOV ZHUH PDWHG RYHUQLJKW IHPDOH PDOH LQ VHSDUDWH FDJHV DQG VXEVHTXHQWO\ H[DPined the next morning for the presence of a vaginal
SOXJWKDWFRQ¿UPHGSUHJQDQF\WKHVDPHGD\ZDV
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WR*'IRXUSUHJQDQWPLFHZHUHUDQGRPO\VDFUL¿FHGE\FHUYLFDOGLVORFDWLRQIRUHDFKVWDJH(Pbryos were dissected and freed carefully from the
uterus and extraembryonic membranes (mean numEHUVRIHPEU\RVZHUHIRUHDFKSUHJQDQWPRXVH
All protocols for the animal experiments were apSURYHGE\WKH,QVWLWXWLRQ¶V$QLPDO&DUH&RPPLWWHH
RIWKH0DVKKDG8QLYHUVLW\RI0HGLFDO6FLHQFHV
Hassanzadeh Taheri et al.
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LQ WKH GDUN $OO VHFWLRQV ZHUH LQFXEDWHG
with chosen lectins in a humid chamber at room
temperature for 2 hours. After extensive rinising in
3%6VHFWLRQVZHUHLQFXEDWHGLQGLDPLQREHQ]LGLQH
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All sections were counterstained with a 1% soluWLRQ RI DOFLDQ EOXH DW S+ IRU PLQXWHV )LQDOO\WKHVHFWLRQVZHUHGHK\GUDWHGFOHDUHG
and mounted on glass slides. Controls for lectin
staining were carried out as such: substitution of
XQFRQMXJDWHGOHFWLQIRUOHFWLQ+53FRQMXJDWHVDQG
H[SRVXUHRIVHFWLRQVWR+53DQGVXEVWUDWHPHGLXP
without lectin.
Preparation of tissue sections
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VXEVHTXHQWO\¿[HGLQ%*¿[DWLYHVRGLXPDFHWDWH PHUFXULF FKORULGH JOXWDUDOGHK\GH
overnight at room temperature. After specimen dehydration by passing through a series of successive
graded ethanols, the embryos were cleared with xyOHQHDQGHPEHGGHGLQSDUDI¿QEORFNV$URWDU\PLcrotome were used to cut transverse serial sections
RIPPWKLFNQHVVHV
Lectin histochemistry
Seven horseradish peroxidase-conjugated lectins (Table 1) were purchased from Sigma ChemiFDO &RPSDQ\ 86$ DQG GLOXWHG ZLWK SKRVSKDWH
EXIIHUHGVDOLQH3%6WR—JRIOHFWLQLQ
03%6/HFWLQVFRQVLVWHGRIGlycin max
6%$ 0DFOXUD SRPLIHUD 03$ :LVWDULD ÀRULEXQGD :)$ DQG9LFLD YLOORVD 99$ ZKLFK DOO
RI WKHP DUH VSHFL¿F IRU 1DFHW\OJDODFWRVDPLQH
(GalNAc), 8OH[ HXURSLXV 8($ ELQGV WR Į/
IXFRVH ZKHDW JHUP DJJOXWLQLQ :*$ ELQGV WR
VLDOLFDFLGDQG*ULIIRQLDVLPSOLFLIROLD*6$%
binds to galactose terminal sugars).
7KH DI¿QLWLHV RI WKH WHVWHG OHFWLQV ZHUH VHSDrately observed by three examiners blinded to the
OHFWLQVXQGHUDOLJKWPLFURVFRSH2O\PSXV$+
and grading was done according to the intensity
of lectin reactions with the specimen, from negaWLYH WR VHYHUH 7KH PHGLXP RI
the suggested grades for each lectin on each day
was calculated.
Five sections of the thoracic region of each embryo
were chosen randomly. The specimens were deparDI¿QL]HGLQ[\OHQHDQGK\GUDWHGE\SDVVLQJLQDVHries of reduced grades ethanol. They were then treated
ZLWK/XJRO¶VVROXWLRQWRUHPRYHPHUFXULFVDOWVSULRU
to histochemical staining. Thereafter the sections
ZHUHULQVHGIRUPLQXWHVLQ03%6S+ Comparisons were then made among the various tested lectins and also across the different
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Lectin tested
Abbreviation
&DUERK\GUDWHELQGLQJVSHFL¿FLW\
Hairy winter vetch (Vicia villosa agglutinin)
VVA
ȕ*DOĺĮ*DO1DF!*DO
Glycine max (Soybean agglutinin)
6%$
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Maclura pomifera agglutinin
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Ulex europeus agglutinin
8($
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Triticum vulgaris (Wheat germ agglutinin)
:*$
>ȕĺ'*OF1DF@RU6LDOLFDFLG
Griffonia simplicifolia
*6$%
'*DO
Fuc; Fucose, Gal; Galactose, GalNac; N-Acetylgalactosamine and Glc; Glucose.
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Regulatory Changes of GalNAc in Paraxial Mesenchyme
Results
Somatic cell differentiation was noted in the
craniocaudal direction with time progression, their
histomorphological and histochemical changes
were evaluated by comparing similar sections in
GLIIHUHQW*'V
Histomorphology
8VLQJFRQYHQWLRQDOVWDLQLQJ+(WKHFKDUactristic pattern of somatic cell differentiation
and development of vertebrae occurred as follows:
2Q *' WKH HSLWKHOLDO FHOOV EHJDQ WR GHWDFK
from the ventro-medial portion of the somite and
transformed into mesenchymal cells, termed sclerotom (Fig 1).
2. The sclerotomal cells initiated migration to
the periphere of the axial structures, notochord,
and neural tube, and accumulated around them
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7KHVHPHVHQFK\PDOFHOOVZHUHFRQGHQVHGDQG
gradually a prechondroginous blastema formed
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Fig 1: Photomicrograph of GD9 mouse embryo, crosssection of paraxial mesenchyme, notochord, and ventral
portion of neural groove incubated with VVA lectin. Notochordal process (Small star), Neural groove (Big star), DA;
Dorsal aorta, PG; Primitive gut, IEC; Intera embryonic coelum Intra-embryonic coelom (×400).
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Fig 2: Photomicrograph of GD10 mouse embryo, cross-section of paraxial mesenchyme, notochord, and ventral portion of neural tube incubated with WFA lectin. Notochordal
plate (n), its sheath (small arrow), neural tube (star), DA;
Dorsal aorta. Severe reaction observed in paraxial mesenchyme (arrow; ×200).
Fig 3: Photomicrograph of GD10 mouse embryo, crosssection of paraxial mesenchyme, notochord, and ventral
portion of neural tube and primitive gut, incubated with
SBA lectin. Notochordal plate (n), its sheath (arrow),
neural tube (star), DA; Dorsal aorta, PG; Primitive gut
(×200) .
Hassanzadeh Taheri et al.
Fig 4: Photomicrograph of GD10 mouse embryo, crosssection of paraxial mesenchyme, notochord, and ventral
portion of neural tube, incubated with MPA lectin. Notochord (big arrow), its sheath (small arrow), neural tube
(star), C; Coelum Coelom. Severe reaction observed in
mesenchymal surfaces (head arrow) and in ECM (medium arrow) (×200).
Fig 6: Photomicrograph of GD11 mouse embryo, crosssection of paraxial mesenchyme, notochord, and ventral
portion of neural tube, incubated with MPA lectin. Notochord (n), its sheath (small arrow), neural tube (star), A;
Aorta. Severe reaction observed in paraxial mesenchyme
(big arrow) (×200).
Fig 5: Photomicrograph of GD12 mouse embryo, cross-section of paraxial mesenchyme, notochord, and ventral portion of neural tube incubated with WFA lectin. Notochord
(n), reaction of its cells (head of arrow), neural tube (star),
A; Aorta. Severe reaction observed in paraxial mesenchyme
(arrow) (×200).
Fig 7: Photomicrograph of GD13 mouse embryo, crosssection of paraxial mesenchyme, notochord, and ventral
portion of neural tube, incubated with VVA lectin. Severe
reaction observed in paraxial mesenchyme around the notochord. Notochord (N), Neural tube (star) (×200).
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Regulatory Changes of GalNAc in Paraxial Mesenchyme
Lectin histochemistry
Reactions of glycoconjugates with tested lectins
RQ GLIIHUHQW *'V ZHUH VWXGLHG DQG ¿QGLQJV KDYH
EHHQVXPPDUL]HGLQWDEOH
WFA
Fig 8: Photomicrograph of GD13 mouse embryo, crosssection of paraxial mesenchyme, notochord, and ventral
portion of neural tube, incubated with SBA lectin. Notochord (n), neural tube (star). Severe reaction observed
in paraxial mesenchyme (white stars) (×200).
A severe reaction of paraxial mesenchyme was
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its sheath, vessel wall, and neuroepithelial cells
(especially at the apical plasma membrane of the
luminal cells) also showed strong staining.
MPA
Reactions of this lectin were similar to those of
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7KLV UHDFWLRQ GHFUHDVHG VLJQL¿FDQWO\ DJDLQ RQ
*' S DQG EHFDPH QHJDWLYH 5HDFtions of the notochord and its sheath were moderate, while neuroepithelial cells, especially at
the apical plasma membrane of the luminal cells
VKRZHGDVWURQJUHDFWLRQ)LJV
VVA
Fig 9: Photomicrograph of GD13 mouse embryo,
cross-section of paraxial mesenchyme, notochord, and
ventral portion of neural tube incubated with GSA1-B4
lectin. Notochord (arrow), neural tube (star), V; Vessel
(×200).
CELL JOURNAL(Yakhteh), Vol 14, No 2, Summer 2012
135
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these reactions changed significantly (p<0.001)
and became strong, especially around the notochord, while the notochord reaction was negaWLYHRQWKLVGD\*' (Fig 7). This reaction
FRQWLQXHG WR *' DQG FKDQJHG VLJQLILFDQWO\
WKHUHDIWHU RQ *' S DQG WKHQ GLVDSpeared, except in the periphere of the future
vertebral body (Fig 10). Reactions of the neural
tube, notochord, and its sheath were negative on
WKH*'VVWXGLHG
Hassanzadeh Taheri et al.
Table 2: Severity of reactions of tested lectins with paraxial mesenchyme on different Gestational dayss
Tested
GDs
Lectins
9
10
11
12
VVA
-
-
SBA
-
-
WFA
MPA
-
-
-
UEA1
-
-
-
-
-
-
-
WGA
-
GSA1-B4
-
-
-
-
-
-
-
Staining intensity based on estimated scale from 0 to +++, with: negative reaction;
(-), weak; (+), moderate; (++) and severe; (+++).
Fig 10: Photomicrograph of GD15 mouse embryo, cross section of paraxial mesenchyme, notochord and ventral portion of neural tube incubated with VVA lectin. Notochord (N), Neural tube (star), Reaction of this lectin restricted to
the peripher zone of future vertebral body. Magnification =×200.
UEA1
No reaction of this lectin was observed in the
paraxial mesenchyme (Fig 11).
SDUD[LDOPHVHQFK\PDOFHOOVGXULQJDOO*'VLQYHVtigated (Fig 12).
GSA1-B4
WGA
$ ZHDN XQLIRUP VWDLQLQJ ZDV REVHYHG LQ WKH
%LQGLQJ RI WKLV OHFWLQ ZLWK SDUD[LDO PHVHQFK\PH
was negative in the time period investigated (Fig 9).
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136
Regulatory Changes of GalNAc in Paraxial Mesenchyme
Fig 11: Photomicrograph of GD15 mouse embryo, cross-section of paraxial mesenchyme, notochord, and ventral portion of
neural tube incubated with UEA1 lectin. Notochord (N), neural tube (star) (×200).
Fig 12: Photomicrograph of GD11 mouse embryo, cross-section of paraxial mesenchyme, notochord, and ventral portion of neural tube incubated with WGA lectin. Notochord (arrow), its sheath (head of arrow), neural tube (star), V;
Vessel. Severe reaction only in the notochord (×200).
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Hassanzadeh Taheri et al.
Discussion
Lectins serve as useful histochemical probes
for studying the role of surface carbohydrate
moieties in glycoconjugates during normal development, including the vertebral column. In
this study seven different lectins were used to
determine whether differences could be detected
DPRQJYDULRXVFHOOVXUIDFHVDQG(&0LQWKHSDraxial mesenchyme during normal development
of the vertebral column in mouse embryos beWZHHQ*'DQG*'
,Q D SUHYLRXV VWXG\ ZH IRXQG 31$ SRVLWLYH
terminal sugars in paraxial mesenchyme that
changed regulatory and discussed LQ WKH
present study another subset of lectins were
tested. Among the tested lectins, reactions of
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their recognition of terminal sugars in the development of the vertebral column have been previously reported.
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the development of the vertebrae in chondroF\WHDQGSHULFKRGULXPPHVHQFK\PHLQUDWV
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sites probably detect glycotopes on the laminin
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bind GalNAc terminal sugar in spatial patterns,
ȕ *DO ĺ *DO1$F DQG Įȕ'*DO1$F UHspectively.
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*' DQG GLG QRW FKDQJH XQWLO *' 7KHUHafter, these reactions increased significantly
S DSSHDUHG VHYHUHO\ XQWLO *' DQG
ODWHU GLVDSSHDUHG S 3RVLWLYH PRGHUate reactions of human paraxial mesenchyme
ZLWK6%$OHFWLQKDYHEHHQUHSRUWHGSUHYLRXVO\
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binding specificity would be extremely useful
in detecting the initiation of 0-glycan biosynWKHVLV
8($ OHFWLQ ZDV XQUHDFWLYH RQ WKH SDUD[LDO
mesenchyme during the time period described.
This finding has indicated that the terminant
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by other sugars, such as neuraminic acid and
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fuc is a typical carbohydrate residue of the OOLQNHG JO\FRSURWHLQ ,Q DFFRUGDQFH ZLWK
RXU UHVXOWV =VFKlELW] HW DO GLG QRW ILQG HYLdence for the presence of µĮ/IXF residues in
WKHSDUD[LDOPHVHQFK\PH,QFRQWUDVWZLWK
our results, the expression of these moieties
ZDV UHSRUWHG E\ *|W] DQG 4XRQGXPDWWHR in human embryos.
$QRWKHUWHVWHGOHFWLQZDV:*$7KHUHDFWLRQ
RIWKLVOHFWLQZDVJHQHUDODQGLQLWLDWHGRQZHDNO\RQ*'DQGWKHQFRQWLQXHGWR*'6LQFH
this lectin binds GlcNAc and sialic acid terminal sugars, few glycocojugates with GlcNAc or
sialic acid are present in this region. The findLQJVRI=VFKlELW]HWDOIRUWKLVVXJDUZDVQHJDWLYH ZKLOH *|W] HW DO KDYH QRWHG ZHDN
staining in the axial mesenchyme (27), which
was similar to our findings. This latest invesWLJDWLRQ FRQFOXGHG WKDW DEVHQW RU ZHDN :*$
lectin binding in the axial mesenchyme could
be related to migration, because axial mesenchymal cells no longer migrate during the early
GHYHORSPHQWRIWKHYHUWHEUDOFROXPQ6Xmida et al. reported that proteins binding Con A
DQG:*$OHFWLQVSURPRWHPLJUDWLRQRIFDUGLDF
PHVHQFK\PDOFHOOVLQWKHFKLFNHQ
*6$% OHFWLQ GLG QRW UHDFW ZLWK WKH SDraxial mesenchyme, therefore no galactose
terminal sugar was obtained in this region. In
FRQWUDVWWRRXUUHVXOWV=VFKlELW]HWDOUHSRUWHG
positive findings of paraxial mesenchyme in
UDWV
'XULQJ WKH *'V LQYHVWLJDWHG ILUVW DQ XQVHJmented paraxial mesenchyme was derived from
CELL JOURNAL(Yakhteh), Vol 14, No 2, Summer 2012
138
Regulatory Changes of GalNAc in Paraxial Mesenchyme
sclrotomal cells, then segmentation occurred
rapidly for the formation of vertebral bodies,
DUFKHVDQGLQWHUYHUWHEUDOGLVFV
$FFRUGLQJWR*|W]HWDOWKHD[LDOPHVHQFK\PH
of the early human embryo is rich in sulphated
glycosaminoglycans and hyaloronic acid and
FRQWDLQV JO\FRSURWHLQV OLNH ILEURQHFWLQ DQG
ODPLQLQEXWKDVQRNHUDWDQVXOSKDWH7RROH
has reported that during cell aggregation in the
paraxial mesenchyme, fibronectin, tenascin,
DQGW\SH,FROODJHQDFFXPXODWHLQWKH(&0E\
DORVVRIK\DOXURQDWH7KHSUHVHQFHDQGLQvolvement of aggrecans in the process of sclerotomal condensation around the notochord to
form the vertebral body has been reported by
9DVDQ 7KH WUDQVLWLRQ WR GHYHORSLQJ FDUWLODJH LV PDUNHG E\ WKH GHSRVLWLRQ RI FDUWLODJH
matrix protein, such as proteoglycan core protein, fibronectin, chonroitin sulphate biglycan,
and transients from type I and type II collagen
)LEURQHFWLQDQGWHQDVFLQFROODERUDWHLQILbroblasts in culture, and this may be an important feature for regulation of cell adhesion, cell
PLJUDWLRQDQGPRUSKRJHQHVLV
Although there are similarities between our
ILQGLQJV DQG SUHYLRXV VWXGLHV differences also exist, which are due to the different species examined. Species differences
DUH D ZHOO NQRZQ SKHQRPHQRQ LQ OHFWLQ KLVWRchemistry, and this can explain why our results
DUH GLIIHU IURPWKRVHRI *|W]DQG4XRQGXPDWWHRRQKXPDQHPEU\RVDQG=VFKlELW]HWDO
RQUDWHPEU\RV
Further studies are needed to understand
how these specific glycoconjugates and their
GalNAc terminal sugers cause morphological
changes during early development of the vertebrae.
As the contribution of Shh glycoprotein has been
estabilished in the development of vertebrae, furWKHU VWXG\ RI LPPXQRKLVWRFKHPLFDOV E\ VSHFL¿F
antibodies and lectin histochemistry by tested lectins are recommended.
Acknowlegments
This research was supported by grant number
IURP WKH RIILFH RI WKH 9LFHFKDQFHOORU IRU 5HVHDUFK $IIDLUV DW 0DVKKDG 8QLYHUVLW\ RI 0HGLFDO 6FLHQFHV 7KH DXWKRUV DUH
YHU\ JUDWHIXO WR 3URIHVVRUV )D]HO 1LNUDYHVK
DQG-DODOLIRUWKHLUJXLGDQFHDQGZRXOGOLNHWR
WKDQN0UV0RWDMDGGHGIRUKHUH[FHOOHQWWHFKQLFDODVVLVWDQFH:HDOVRGHFODUHWKDWWKHUHLVQR
conflict of interest for this article.
References
Conclusion
1.
According to our results, we have concluded
that specific glycoconjugate molecules with
GalNAc terminal sugar bind to all specific sensitive tested lectins and show temporally regulated changes, probably in the presence of components of prechondrogenic cells forming the
YHUWHEUDH RU LQ WKH (&0 7KHVH FDUERK\GUDWH
residues may be involved in phenomena such
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of these cells around axial organs, condensation
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CELL JOURNAL(Yakhteh), Vol 14, No 2, Summer 2012
cells to chondroblasts. Those GalNAc residues
ZKLFKELQG:)$6%$DQG99$OHFWLQVLQDGdition to the mentioned processes), may be correlated with the differentiation of chondrocytes
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139
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