We report the first extended culture system for analysing zebrafish (Danio rerio) embryogenesis with which we demonstrate neural induction and anteroposterior patterning. Explants from the animal pole region of blastula embryos ('animal caps') survived for at least two days and increased in cell number. Mesodermal and neural-specific genes were not expressed in cultured animal caps, although low levels of the dorsoanterior marker otx2 were seen. In contrast, we observed strong expression of gta3, a ventral marker and cyt1, a novel type I cytokeratin expressed in the outer enveloping layer. Isolated ‘embryonic shield’, that corresponds to the amphibian organizer and amniote node, went on to express the mesodermal genes gsc and ntl, otx2, the anterior neural marker pax6, and posterior neural markers eng3 and krx20. The expression of these genes defined a precise anteroposterior axis in shield explants. When conjugated to animal caps, the shield frequently induced expression of anterior neural markers. More posterior markers were rarely induced, suggesting that anterior and posterior neural induction are separable events. Mesodermal genes were also seldom activated in animal caps by the shield, demonstrating that neural induction did not require co-induction of mesoderm in the caps. Strikingly, ventral marginal zone explants suppressed the low levels of otx2 in animal caps, indicating that ventral tissues may play an active role in axial patterning. These data suggest that anteroposterior patterning in the zebrafish is a multi-step process.
Reference
Abdelilah
S.
, Solnica-Krezel
L.
, Stainier
D. Y.
, Driever
W.
(1994
) Implications for dorsoventral axis determination from the zebrafish mutation janus.
Nature
370
, 468
–471
Allende
M. L.
, Weinberg
E. S.
(1994
) The expression of two zebrafish achaete-scute homolog (ash) genes is altered in the embryonic brain of the cyclops mutant.
Dev. Biol
166
, 509
–530
Ang
S.-L.
, Conlon
R. A.
, Jin
O.
, Rossant
J.
(1994
) Positive and negative signals from mesoderm regulate the expression of mouse Otx2 in ectoderm explants.
Development
120
, 2979
–2989
Barnes
J. D.
, Crosby
J. L.
, Jones
C. M.
, Wright
C. V.
, Hogan
B. L.
(1994
) Embryonic expression of Lim-1, the mouse homolog of Xenopus Xlim-1, suggests a role in lateral mesoderm differentiation and neurogenesis.
Dev. Biol
161
, 168
–178
Bierkamp
C.
, Campos-Ortega
J. A.
(1993
) A zebrafish homologue of the Drosophila neurogenic gene Notch and its pattern of transcription during early embryogenesis.
Mech. Dev
43
, 87
–100
Blum
M.
, Gaunt
S. J.
, Cho
K. W.
, Steinbeisser
H.
, Blumberg
B.
, Bittner
D.
, De Robertis
E. M.
(1992
) Gastrulation in the mouse: the role of the homeobox gene goosecoid.
Cell
69
, 1096
–1107
Cho
K. W.
, Blumberg
B.
, Steinbeisser
H.
, De Robertis
E. M.
(1991
) Molecular nature of Spemann's organizer: the role of the Xenopus homeobox gene goosecoid.
Cell
67
, 1111
–1120
Chomczynski
P.
, Sacchi
N.
(1987
) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Analyt. Biochem
162
, 156
–159
Christian
J. L.
, Moon
R. T.
(1993
) Interactions between Xwnt-8 and Spemann organizer signaling pathways generate dorsoventral pattern in the embryonic mesoderm of Xenopus.
Genes Dev
7
, 13
–28
Dale
L.
, Howes
G.
, Price
B. M. J.
, Smith
J. M. C.
(1992
) Bone morphogenetic protein 4: a ventralizing factor in early Xenopus development.
Development
115
, 573
–585
Doniach
T.
(1993
) Planar and vertical induction of anteroposterior pattern during the development of the central nervous system.
J. Neurobiol
24
, 1256
–1275
Driever
W.
, Stemple
D.
, Schier
A.
, Solnica-Krezel
L.
(1994
) Zebrafish: genetic tools for studying vertebrate development.
Trends Genet
10
, 152
–159
Ekker
M.
, Wegner
J.
, Akimenko
M. A.
, Westerfield
M.
(1992
) Coordinate expression of three zebrafish engrailed genes.
Development
116
, 1001
–1010
Fainsod
A.
, Steinbeisser
H.
, De Robertis
E. M.
(1994
) On the function of BMP-4 in patterning the marginal zone of the Xenopus embryo.
EMBO J
13
, 5015
–5025
Godsave
S. F.
, Slack
J. M. W.
(1989
) Clonal analysis of mesoderm induction in Xenopus laevis.
Dev. Biol
134
, 486
–490
Griffin
K.
, Patient
R.
, Holder
N.
(1995
) Analysis of Fgf function in normal and notail zebrafish embryos reveals separate mechanisms for formation of the trunk and the tail.
Development
121
, 2983
–2994
Halpern
M. E.
, Ho
R. K.
, Walker
C.
, Kimmel
C. B.
(1993
) Induction of muscle pioneers and floor plate is distinguished by the zebrafish notail mutation.
Cell
75
, 99
–112
Ho
R. K.
, Kimmel
C. B.
(1993
) Commitment of cell fate in the early zebrafish embryo.
Science
261
, 109
–111
Izpisua-Belmonte
J. C. M. D. R. E.
, Storey
K. G.
, Stern
C. D.
(1993
) The homeobox gene goosecoid and the origin of organizer cells in the early chick blastoderm.
Cell
74
, 645
–659
Jamrich
M.
, Sargent
T. D.
, Dawid
I. B.
(1987
) Cell-type-specific expression of epidermal cytokeratin genes during gastrulation of Xenopus laevis.
Genes Dev
1
, 124
–132
Joly
J.-S.
, Joly
C.
, Schulte-Merker
S.
, Boulekbache
H.
, Condamine
H.
(1993
) The ventral and posterior expression of the zebrafish homeobox gene eve1 is perturbed in dorsalized and mutant embryos.
Development
119
, 1261
–1275
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
Kelly
G. M.
, Greenstein
P.
, Erezyilmaz
D. F.
, Moon
R. T.
(1995
) Zebrafish wnt8 and wnt8b share a common activity but are involved in distinct developmental pathways.
Development
121
, 1787
–1799
Kengaku
M.
, Okamoto
H.
(1995
) bFGF as a possible morphogen for the anteroposterior axis of the central nervous system in Xenopus.
Development
121
, 3121
–3130
Kimmel
C. B.
(1989
) Genetics and early development of zebrafish.
Trends Genet
5
, 283
–288
Kimmel
C. B.
, Ballard
W. W.
, Kimmel
S. R.
, Ullman
B.
, Schilling
T. F.
(1995
) Stages of embryonic development of the zebrafish.
Dev. Dynamics
203
, 253
–310
Kimmel
C. B.
, Warga
R. M.
(1987
) Indeterminate cell lineage of the zebrafish embryo.
Dev. Biol
124
, 269
–280
Kimmel
C. B.
, Warga
R. M.
, Schilling
T. F.
(1990
) Origin and organization of the zebrafish fatemap.
Development
108
, 581
–594
Krauss
S.
, Concordet
J.-P.
, Ingham
P. W.
(1993
) A functionally conserved homolog of the Drosophila segment polarity gene hh is expressed in tissues with polarizing activity in zebrafish embryos.
Cell
75
, 1431
–1444
Krauss
S.
, Johansen
T.
, Korzh
V.
, Moens
U.
, Ericson
J. U.
, Fjose
A.
(1991
) Zebrafish pax[zf-a]: a paired box-containing gene expressed in the neural tube.
EMBO J
10
, 3609
–3619
Lamb
T. M.
, Harland
R. M.
(1995
) Fibroblast growth factor is a direct neural inducer, which combined with noggin generates anterior-posterior neural pattern.
Development
121
, 3627
–3636
Li
Y.
, Allende
M. L.
, Finkelstein
R.
, Weinberg
E. S.
(1994
) Expression of two zebrafish orthodenticle -related genes in the embryonic brain.
Mech. Dev
48
, 229
–244
Maeno
M.
, Ong
R. C.
, Suzuki
A.
, Ueno
N.
, Kung
H. F.
(1994
) A truncated bone morphogenetic protein 4 receptor alters the fate of ventral mesoderm to dorsal mesoderm: roles of animal pole tissue in the development of ventral mesoderm.
Proc. Natl. Acad. Sci. USA
91
, 10260
–10264
Molven
A.
, Njolstad
P. R.
, Fjose
A.
(1991
) Genomic structure and restricted neural expression of the zebrafish wnt-1 (int-1) gene.
EMBO J
10
, 799
–807
Mullins
M. C.
, Nusslein-Volhard
C.
(1993
) Mutational approaches to studying embryonic pattern formation in the zebrafish.
Curr. Opin. Genet Dev
3
, 648
–654
Neave
B.
, Rodaway
A.
, Wilson
S. W.
, Patient
R.
, Holder
N.
(1995
) Expression of zebrafish GATA3 (gta3) during gastrulation and neurulation suggests a role in the specification of cell fate.
Mech. Dev
51
, 169
–182
Oppenheimer
J. M.
(1936
) Transplantation experiments on developing teleosts (Fundulus and Perca).
J. Exp. Zool
72
, 409
–437
Oppenheimer
J. M.
(1959
) Extraembryonic transplantations of sections of the Fundulus embryonic shield.
J. Exp. Zool
140
, 247
–268
Oxtoby
E.
, Jowett
T.
(1993
) Cloning of the zebrafish krox-20 gene (krx20) and its expression during development.
Nucl. Acids Res
21
, 1087
–1095
Pannese
M.
, Polo
C.
, Andreazzoli
M.
, Vignali
R.
, Kablar
B.
, Barsacchi
B.
, Boncinelli
B.
(1995
) The Xenopus homologue of Otx2 is a maternal homeobox gene that demarcates and specifies anterior body regions.
Development
121
, 707
–720
Puschel
A. W.
, Gruss
P.
, Westerfield
M.
(1992
) Sequence and expression pattern of pax-6 are highly conserved between zebrafish and mice.
Development
114
, 643
–651
Rossant
J.
, Hopkins
N.
(1992
) Of fin and fur: mutational analysis of vertebrate embryonic development.
Genes Dev
6
, 1
–13
Schmidt
J. E.
, Suzuki
A.
, Ueno
N.
, Kimelman
D.
(1995
) Localized BMP-4 mediates dorsal/ventral patterning in the early Xenopus embryo.
Dev. Biol
169
, 37
–50
Schulte-Merker
S.
, Hammerschmidt
M.
, Beuchle
D.
, Cho
K. W.
, De Robertis
E. M.
, Nusslein-Volhard
C.
(1994
) Expression of zebrafish goosecoid and notail gene products in wild-type and mutant notail embryos.
Development
120
, 843
–852
Schulte-Merker
S.
, Ho
R. K.
, Herrmann
B. G.
, Nusslein-Volhard
C.
(1992
) The protein product of the zebrafish homologue of the mouse T geneis expressed in nuclei of the germ ring and the notochord of the early embryo.
Development
116
, 1021
–1032
Schulte-Merker
S.
, van Eeden
F.
, Halpern
M. E.
, Kimmel
C. B.
, Nusslein-Volhard
C.
(1994
) notail (ntl) is the zebrafish homologue of the mouse T (brachyury) gene.
Development
120
, 1009
–1015
Sharpe
C. R.
(1991
) Retinoic acid can mimic endogenous signals involved in transformation of the Xenopus nervous system.
Neuron
7
, 239
–247
Shih
J.
, Fraser
S. E.
(1995
) Distribution of tissue progenitors within the shield region of the zebrafish gastrula.
Development
121
, 2755
–2765
Simeone
A.
, Acompora
D.
, Mallamaci
A.
, Stornaiuolo
A.
, D'Apice
M. R.
, Nigro
V.
, Boncinelli
E.
(1993
) A vertebrate gene related to orthodenticle contains a homeodomain of the bicoid class and demarcates anterior neuroectoderm of the gastrulating mouse embryo.
EMBO J
12
, 2735
–2747
Sive
H.
, Draper
B.
, Harland
R.
, Weintraub
H.
(1990
) Identification of a retinoic acid sensitive period during primary axis formation in Xenopus laevis.
Genes Dev
4
, 932
–942
Slack
J. M. W.
, Tannahill
D.
(1992
) Mechanisms of anteroposterior axis specification in vertebrates.
Development
114
, 285
–302
Stachel
S. E.
, Grunwald
D. J.
, Myers
P. Z.
(1993
) Lithium perturbation and goosecoid expression identify a dorsal specification pathway in the pregastrula zebrafish.
Development
117
, 1261
–1274
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
Taira
M.
, Otani
H.
, Saint-Jeannet
J. P.
, Dawid
I. B.
(1993
) Role of the LIM class homeodomain protein Xlim-1 in neural and muscle induction by the Spemann organizer in Xenopus.
Nature
372
, 677
–679
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
Toyama
R.
, O'Connel
M. L.
, Wright
C. V. E.
, Kuehn
M. R.
, Dawid
I. B.
(1995
) Nodal induces ectopic goosecoid and lim1 expression and axis duplication in zebrafish.
Development
121
, 383
–391
Weinberg
E. S.
, Allende
M. L.
, Kelly
C. S.
, Abdelhamid
A.
, Murakami
T.
, Andermann
P.
, Doerre
O. G.
, Grunwald
D. J.
, Riggleman
B.
(1996
) Developmental regulation of zebrafish MyoD in wild-type, no tail and spadetail embryos.
Development
122
, 271
–280
Wittbrodt
J.
, Rosa
F. M.
(1994
) Disruption of mesoderm and axis formation in fish by ectopic expression of activin variants: the role of maternal activin.
Genes Dev
8
, 1448
–1462
Woo
K.
, Fraser
S. E.
(1995
) Order and coherence in the fate map of the zebrafish nervous system.
Development
121
, 2595
–2609
Zaraisky
A. G.
, Lukyanov
S. A.
, Vasiliev
O. L.
, Smirnov
Y. V.
, Belyavsky
A. V.
, Kazanskaya
O. V.
(1992
) A novel homeobox gene expressed in the anterior neural plate of the Xenopus embryo.
Dev. Biol
152
, 373
–382
Zhang
J.
, Jacobson
A. G.
(1993
) Evidence that the border of the neural plate may be positioned by the interaction between signals that induce ventral and dorsal mesoderm.
Dev. Dynam
196
, 79
–90
This content is only available via PDF.
© 1996 by Company of Biologists
1996
-JournalMeeting.png?versionId=3773)
(update)-ImmuneSI.png?versionId=3773)
-GSWorkshop.png?versionId=3773)
(update)-InPreprints.png?versionId=3773)
-FocalPlaneNetworkLaunch.png?versionId=3773)
