The segregation of cells into germ layers is one of the earliest events in the establishment of cell fate in the embryo. In the zebrafish, endoderm and mesoderm are derived from cells that involute into an internal layer, the hypoblast, whereas ectoderm is derived from cells that remain in the outer layer, the epiblast. In this study, we examine the origin of the zebrafish endoderm and its separation from the mesoderm. By labeling individual cells located at the margin of the blastula, we demonstrate that all structures that are endodermal in origin are derived predominantly from the more dorsal and lateral cells of the blastoderm margin. Frequently marginal cells give rise to both endodermal and mesodermal derivatives, demonstrating that these two lineages have not yet separated. Cells located farther than 4 cell diameters from the margin give rise exclusively to mesoderm, and not to endoderm. Following involution, we see a variety of cellular changes indicating the differentiation of the two germ layers. Endodermal cells gradually flatten and extend filopodial processes forming a noncontiguous inner layer of cells against the yolk. At this time, they also begin to express Forkhead-domain 2 protein. Mesodermal cells form a coherent layer of round cells separating the endoderm and ectoderm. In cyclops-mutant embryos that have reduced mesodermal anlage, we demonstrate that by late gastrulation not only mesodermal but also endodermal cells are fewer in number. This suggests that a common pathway initially specifies germ layers together before a progressive sequence of determinative events segregate endoderm and mesoderm into morphologically distinct germ layers.

Reference

Ang
S. L.
,
Wierda
A.
,
Wong
D.
,
Stevens
K. A.
,
Cascio
S.
,
Rossant
J.
,
Zaret
K. S.
(
1993
)
The formation and maintenance of the definitive endoderm lineage in the mouse: involvement of HNF-3/forkhead proteins.
Development
119
,
1301
1315
Ang
S. L.
,
Conlon
R. A.
,
Jin
O.
,
Rossant
J.
(
1994
)
Negative signals from the mesoderm regulate the expression of mouse otx2 in ectoderm explants.
Development
120
,
2979
2989
Bolker
J. A.
(
1993
)
Gastrulation and mesoderm morphogenesis in the white sturgeon.
J. Exp. Zool
266
,
116
131
Cooper
M. S.
,
D'Amico
L. A.
(
1996
)
A cluster of noninvoluting endocytic cells at the margin of the zebrafish blastoderm marks the site of embryonic shield formation.
Dev. Biol
180
,
184
198
Delarue
M.
,
Sanchez
S.
,
Johnson
K. E.
,
Darribere
T.
,
Boucaut
J.-C.
(
1992
)
A fate map of superficial and deep circumblastoporal cells in the early gastrula of Pleurodeles waltl.
Development
114
,
135
146
Delarue
M.
,
Johnson
E. E.
,
Boucaut
J.-C.
(
1994
)
Superficial cells in the early gastrula of Rana pipiens contribute to mesodermal derivatives.
Dev. Biol
165
,
702
715
Dirksen
M. L.
,
Jamrich
M.
(
1995
)
Differential expression of fork head genes during early Xenopus and zebrafish development.
Dev. Genet
17
,
107
118
Dufort
D.
,
Schwartz
L.
,
Harpal
K.
,
Rossant
R.
(
1998
)
The transcription factor HNF3is required in visceral endoderm for normal primitive streak morphogenesis.
Development
125
,
3015
3025
Hammerschmidt
M.
,
Nusslein-Volhard
C.
(
1993
)
The expression of a zebrafish gene homologous to Drosophila snail suggests a conserved function in invertebrate and vertebrate gastrulation.
Development
119
,
1107
1118
Hatada
Y.
,
Stern
C. D.
(
1994
)
A fate map of the epiblast of the early chick embryo.
Development
120
,
2879
2889
Hatta
K.
,
Kimmel
C. B.
,
Ho
R. K.
,
Walker
C.
(
1991
)
The cyclops mutation blocks specification of the floor plate of the zebrafish central nervous system.
Nature
350
,
339
341
Henry
G. L.
,
Brivanlou
I. H.
,
Kessler
D. S.
,
Hemmati-Brivanlou
A.
,
Melton
D. A.
(
1996
)
TGF-signals and a prepattern in Xenopus laevis endodermal development.
Development
122
,
1007
1015
Henry
G. L.
,
Melton
D. A.
(
1998
)
Mixer, a homeobox gene required for endoderm development.
Science
281
,
91
96
Ho
R. K.
,
Kimmel
C. B.
(
1993
)
Commitment of cell fate in the early zebrafish embryo.
Science
261
,
109
111
Hudson
C.
,
Clements
D.
,
Friday
R. V.
,
Stott
D.
,
Woodland
H. R.
(
1998
)
Xsox17 alpha and-beta mediate endoderm formation in Xenopus.
Cell
91
,
397
405
Kane
D. A.
,
Hammerschmidt
M.
,
Mullins
M. C.
,
Maischein
H.-M.
,
Brand
M.
,
van Eeden
F. J. M.
,
Furutani-Seiki
M.
,
Granato
M.
,
Haffter
P.
,
Heisenberg
C.-P.
,
Jiang
Y.-J.
,
Kelsh
R. N.
,
Odenthal
J.
,
Warga
R. M.
,
Nusslein-Volhard
C.
(
1996
)
The zebrafish epiboly mutants.
Development
123
,
47
55
Keller
R. E.
(
1975
)
Vital dye mapping of the gastrula and neurula of Xenopus laevis. I. Prospective areas and morphogenetic movements of the superficial layer.
Dev. Biol
42
,
222
241
Keller
R. E.
(
1976
)
Vital dye mapping of the gastrula and neurula of Xenopus laevis. II. Prospective areas and morphogenetic movements in the deep region.
Dev. Biol
51
,
118
137
Kimmel
C. B.
,
Warga
R. M.
,
Schilling
T. F.
(
1990
)
Origin and organization of the zebrafish fate map.
Development
108
,
581
594
Kimmel
C. B.
,
Ballard
W. W.
,
Kimmel
S. R.
,
Ullmann
B.
,
Schilling
T. F.
(
1995
)
Stages of embryonic development of the zebrafish.
Dev. Dyn
203
,
253
310
Lawson
K. A.
,
Meneses
J. J.
,
Pedersen
R. A.
(
1991
)
Clonal analysis of epiblast fate during germ layer formation in the mouse embryo.
Development
113
,
891
991
Lee
R. K.
,
Stainier
D. Y. R.
,
Fishman
M. C.
(
1994
)
Cardiovascular development in the zebrafish. II. Endocardial progenitors are sequestered within the heart field.
Development
120
,
3361
3366
Lemaire
P.
,
Darras
S.
,
Caillol
D.
,
Kodjabachian
L.
(
1998
).
A role for the vegetally expressed Xenopus gene Mix.1 in endoderm formation and in the restriction of mesoderm to the marginal zone.
Development
125
,
2371
2380
Long
W. L.
(
1983
)
The role of the yolk syncytial layer in determination of the plane of bilateral symmetry in the rainbow trout, Salmo gairdneri Richardson.
J. Exp. Zool
228
,
91
97
Lundmark
C.
(
1986
)
Role of bilateral zones of ingressing superficial cells during gastrulation of Ambystoma mexicanum.
J. Embryol. Exp. Morph
97
,
47
62
Melby
A. E.
,
Warga
R. M.
,
Kimmel
C. B.
(
1996
)
Specification of cell fates at the dorsal margin of the zebrafish gastrula.
Development
122
,
2225
2237
Minsuk
S. B.
,
Keller
R. E.
(
1996
)
Dorsal mesoderm has a dual origin and forms by a novel mechanism in Hymenochirus, a relative of Xenopus.
Dev. Biol
174
,
92
103
Monaghan
A. P.
,
Kaestner
K. H.
,
Grau
E.
,
Schutz
G.
(
1993
)
Postimplantation expression patterns indicate a role for the mouse forkhead / HNF- 3, andgenes in determination of the definitive endoderm, chordamesoderm and neuroectoderm.
Development
119
,
567
578
Nascone
N.
,
Marcola
M.
(
1995
)
An inductive role for the endoderm in Xenopus cardiogenesis.
Development
121
,
515
523
Pasteels
J.
(
1942
)
New observations concerning the maps of presumptive areas of the young amphibian gastrula (Amblystoma and Discoglossus).
J. Exp. Zool
89
,
255
281
Papaioannou
V. E.
,
Silver
L. M.
(
1998
)
The T-box family.
BioEssays
20
,
9
19
Purcell
S. M.
,
Keller
R. E.
(
1993
)
A different type of amphibian mesoderm morphogenesis in Ceratophrys ornata.
Development
117
,
307
317
Rebagliati
M. R.
,
Toyama
R.
,
Haffter
P.
,
Dawid
I. B.
(
1998
)
cyclops encodes a nodal related factor involved in midline signalling.
Proc. Nat. Acad. Sci. USA
95
,
9932
9937
Sampath
K.
,
Rubinstein
A. L.
,
Cheng
A. H. S.
,
Liang
J. O.
,
Fekany
K.
,
Solnicakrezel
L.
,
Korzh
V.
,
Halpern
M. E.
,
Wright
C. V. E.
(
1998
)
Induction of the zebrafish ventral brain and floorplate requires cyclops/nodal signalling.
Nature
395
,
185
189
Sasai
Y.
,
Lu
B.
,
Piccolo
S.
,
De Robertis
E. D.
(
1996
)
Endoderm induction by the organizer-secreted factors chordin and noggin in Xenopus animal caps.
EMBOJ
15
,
4547
4555
Sasaki
H.
,
Hogan
B. L.
(
1993
)
Differential expression of multiple fork head related genes during gastrulation and axial pattern formation in the mouse embryo.
Development
118
,
47
59
Schulte-Merker
S.
,
Ho
R. K.
,
Herrmann
B. G.
,
Nusslein-Volhard
C.
(
1992
)
The protein product of the zebrafish homologue of the mouse T gene is expressed in nuclei of the germ ring and the notochord of the early embryo.
Development
116
,
1021
1032
Shih
J.
,
Fraser
S. E.
(
1995
)
Distribution of tissue progenitors within the shield region of the zebrafish.
Development
121
,
2755
2765
Shimamura
K.
,
Rubenstein
J. L. R.
(
1997
)
Inductive interactions direct early regionalization of the mouse forebrain.
Development
124
,
2709
2718
Stainier
D. Y. R.
,
Lee
R. K.
,
Fishman
M. C.
(
1993
)
Cardiovascular development in the zebrafish: I. Myocardial fate map and heart tube formation.
Development
119
,
31
40
Storey
K. G.
,
Selleck
M. A.
,
Stern
C. D.
(
1995
)
Neural induction and regionalization by different subpopulations of cells in Hensen's node.
Development
121
,
417
428
Strähle
U.
,
Blader
P.
,
Henrique
D.
,
Ingham
P. W.
(
1993
)
axial, a zebrafish gene expressed along the developing body axis, shows altered expression in cyclops mutant embryos.
Genes Dev
7
,
1436
1446
Sulik
K.
,
Dehart
D. B.
,
Inagaki
T.
,
Carson
J. L.
,
Vrablic
T.
,
Gesteland
K.
,
Schoenwolf
G. C.
(
1994
)
Morphogenesis of the murine node and notochordal plate.
Dev. Dyn
201
,
260
278
Thisse
C.
,
Thisse
B.
,
Halpern
M. E.
,
Postlethwait
J. H.
(
1994
)
goosecoid expression in neurectoderm and mesendoderm is disrupted in zebrafish cyclops gastrulas.
Dev. Biol
164
,
420
429
Trevarrow
B.
,
Marks
D. L.
,
Kimmel
C. B.
(
1990
)
Organization of hindbrain segments in the zebrafish embryo.
Neuron
4
,
669
679
Warga
R. M.
,
Kimmel
C. B.
(
1990
)
Cell movements during epiboly and gastrulation in zebrafish.
Development
108
,
569
580
Warga
R. M.
,
Nusslein-Volhard
C.
(
1998
)
spadetail -dependent cell compaction of the dorsal zebrafish blastula.
Dev. Biol
203
,
116
121
Weigel
D.
,
Jurgens
G.
,
Kuttner
F.
,
Seifert
E.
,
Jäckle
H.
(
1989
)
The homeotic gene fork head encodes a nuclear protein and is expressed in the terminal regions of the Drosophila embryo.
Cell
57
,
645
658
Woo
K.
,
Fraser
S. E.
(
1995
)
Order and coherence in the fate map of the zebrafish nervous system.
Development
121
,
2595
2609
Wylie
C. C.
,
Snape
A.
,
Heasman
J.
,
Smith
J. C.
(
1987
)
Vegetal pole cells and commitment to form endoderm in Xenopus-laevis.
Dev. Biol
119
,
496
502
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