Neural crest forms four major categories of derivatives: pigment cells, peripheral neurons, peripheral glia, and ectomesenchymal cells. Some early neural crest cells generate progeny of several fates. How specific cell fates become specified is still poorly understood. Here we show that zebrafish embryos with mutations in the colourless gene have severe defects in most crest-derived cell types, including pigment cells, neurons and specific glia. In contrast, craniofacial skeleton and medial fin mesenchyme are normal. These observations suggest that colourless has a key role in development of non-ectomesenchymal neural crest fates, but not in development of ectomesenchymal fates. Thus, the cls mutant phenotype reveals a segregation of ectomesenchymal and non-ectomesenchymal fates during zebrafish neural crest development. The combination of pigmentation and enteric nervous system defects makes colourless mutations a model for two human neurocristopathies, Waardenburg-Shah syndrome and Hirschsprung's disease.

Akimenko
M. A.
,
Ekker
M.
,
Wegner
J.
,
Lin
W.
,
Westerfield
M.
(
1994
)
Combinatorial expression of three zebrafish genes related to distal-less: part of a homeobox gene code for the head.
J. Neurosci
14
,
3475
86
Anderson
D.
,
Axel
R.
(
1986
)
A bipotential neuroendocrine precursor whose choice of cell fate is determined by NGF and glucocorticoids.
Cell
47
,
1079
1090
Anderson
D. J.
(
1993
)
Molecular control of cell fate in the neural crest—the sympathoadrenal lineage.
Ann. Rev. Neurosci
16
,
129
158
Anderson
D. J.
(
1997
)
Cellular and molecular biology of neural crest cell lineage determination.
Trends Genet
13
,
276
280
Attie
T.
,
Till
M.
,
Pelet
A.
,
Amiel
J.
,
Edery
P.
,
Boutrand
L.
,
Munnich
A.
,
Lyonnet
S.
(
1995
)
Mutation of the endothelin-receptor B gene in Waardenburg-Hirschsprung disease.
Hum. Mol. Genet
4
,
2407
2409
Badner
J. A.
,
Chakravarti
A.
(
1990
)
Waardenburg syndrome and Hirschsprung disease: evidence for pleiotropic effects of a single dominant gene.
Am. J. Med. Genet
35
,
100
104
Baroffio
A.
,
Dupin
E.
,
Le Douarin
N.
(
1988
)
Clone-forming ability and differentiation potential of migratory neural crest cells.
Proc. Natl. Acad. Sci. USA
85
,
5325
5329
Baroffio
A.
,
Dupin
E.
,
Le Douarin
N.
(
1990
)
Cell lineage studies in avian neural crest ontogeny.
Adv. Exp. Med. Biol
265
,
53
62
Baroffio
A.
,
Dupin
E.
,
Le Douarin
N. M.
(
1991
)
Common precursors for neural and mesectodermal derivatives in the cephalic neural crest.
Development
112
,
301
305
Baynash
A. G.
,
Hosoda
K.
,
Giaid
A.
,
Richardson
J. A.
,
Emoto
N.
,
Hammer
R. E.
,
Yanagisawa
M.
(
1994
)
Interaction of endothelin-3 with endothelin-B receptor is essential for development of epidermal melanocytes and enteric neurons.
Cell
79
,
1277
1285
Bisgrove
B.
,
Raible
D.
,
Walter
V.
,
Eisen
J.
,
Grunwald
D.
(
1997
)
Expression of c-ret in the zebrafish embryo: potential roles in motoneuronal development.
J. Neurobiol
33
,
749
68
Bjorklund
H.
,
Dahl
D.
,
Seiger
A.
(
1984
)
Neurofilament and glial fibrillary acid protein-related immunoreactivity in rodent enteric nervous system.
Neuroscience
12
,
277
287
Bronner-Fraser
M.
(
1986
)
Analysis of the early stages of trunk neural crest migration in avian embryos using monoclonal antibody HNK-1.
Dev. Biol
115
,
44
55
Bronner-Fraser
M.
,
Fraser
S. E.
(
1991
)
Cell lineage analysis of the avian neural crest.
Development
2
,
17
22
Chakrabarti
S.
,
Streisinger
G.
,
Singer
F.
,
Walker
C.
(
1983
)
Frequency of-ray induced specific locus and recessive lthal mutations in mature germ cells of the zebrafish, Brachydanio rerio.
Genetics
103
,
109
123
Chitnis
A. B.
,
Kuwada
J. Y.
(
1990
)
Axonogenesis in the brain of zebrafish embryos.
J. Neurosci
10
,
1892
905
Collazo
A.
,
Bronner-Fraser
M.
,
Fraser
S. E.
(
1993
)
Vital dye labelling of Xenopus laevis trunk neural crest reveals multipotency and novel pathways of migration.
Development
118
,
363
376
Cretekos
C. J.
,
Grunwald
D. J.
(
1999
)
alyron, an insertional mutation affecting early neural crest development in zebrafish.
Dev. Biol
210
,
322
338
Dorsky
R. I.
,
Moon
R. T.
,
Raible
D. W.
(
1998
)
Control of neural crest fate by the Wnt signalling pathway.
Nature
396
,
370
373
Dow
E.
,
Cross
S.
,
Wolgemuth
D. J.
,
Lyonnet
S.
,
Mulligan
L. M.
,
Mascari
M.
,
Ladda
R.
,
Williamson
R.
(
1994
)
Second locus for Hirschsprung disease/Waardenburg syndrome in a large Mennonite kindred.
Am. J. Med. Genet
53
,
75
80
Driever
W.
,
Solnica-Krezel
L.
,
Schier
A. F.
,
Neuhauss
S. C.
,
Malicki
J.
,
Stemple
D. L.
,
Stainier
D. Y.
,
Zwartkruis
F.
,
Abdelilah
S.
,
Rangini
Z.
, et al. 
(
1996
)
A genetic screen for mutations affecting embryogenesis in zebrafish.
Development
123
,
37
46
Dupin
E.
,
Baroffio
A.
,
Dulac
C.
,
Cameron-Curry
P.
,
Le Douarin
N.
(
1990
)
Schwann-cell differentiation in clonal cultures of the neural crest, as evidenced by the anti-Schwann cell myelin protein monoclonal antibody.
Proc. Natl. Acad. Sci. USA
87
,
1119
23
Durbec
P. L.
,
Larsson-Blomberg
L. B.
,
Schuchardt
A.
,
Costantini
F.
,
Pachnis
V.
(
1996
)
Common origin and developmental dependence on c-ret of subsets of enteric and sympathetic neuroblasts.
Development
122
,
349
358
Edery
P.
,
Atti'e
T.
,
Amiel
J.
,
Pelet
A.
,
Eng
C.
,
Hofstra
R. M.
,
Martelli
H.
,
Bidaud
C.
,
Munnich
A.
,
Lyonnet
S.
(
1996
)
Mutation of the endothelin-3 gene in the Waardenburg-Hirschsprung disease (Shah-Waardenburg syndrome).
Nat. Genet
12
,
442
4
Eisen
J. S.
,
Weston
J. A.
(
1993
)
Development of the neural crest in the zebrafish.
Dev. Biol
159
,
50
59
Ekker
M.
,
Wegner
J.
,
Akimenko
M.
,
Westerfield
M.
(
1992
)
Coordinate embryonic expression of three zebrafish engrailed genes.
Development
116
,
1001
1010
Frank
E.
,
Sanes
J. R.
(
1991
)
Lineage of neurons and glia in chick dorsal-root ganglia—analysis invivo with a recombinant retrovirus.
Development
111
,
895
908
Fraser
S. E.
,
Bronner-Fraser
M.
(
1991
)
Migrating neural crest cells in the trunk of the avian embryo are multipotent.
Development
112
,
913
920
Haffter
P.
,
Granato
M.
,
Brand
M.
,
Mullins
M.
,
Hammerschmidt
M.
,
Kane
D.
,
Odenthal
J.
,
van Eeden
F.
,
Jiang
Y.
,
Heisenberg
C.
(
1996
)
The identification of genes with unique and essential functions in the development of the zebrafish, Danio rerio.
Development
123
,
1
36
Henion
P.
,
Weston
J.
(
1997
)
Timing and pattern of cell fate restrictions in the neural crest lineage.
Development
124
,
4351
4359
Henion
P. D.
,
Raible
D. W.
,
Beattie
C. E.
,
Stoesser
K. L.
,
Weston
J. A.
,
Eisen
J. S.
(
1995
)
A screen for mutations affecting development of zebrafish neural crest.
Dev. Genetics
18
,
11
17
Henion
P. D.
,
Raible
D. W.
,
Beattie
C. E.
,
Stoesser
K. L.
,
Weston
J. A.
,
Eisen
J. S.
(
1995
)
A screen for mutations affecting development of zebrafish neural crest.
Dev. Genet
18
,
11
17
Herbarth
B.
,
Pingault
V.
,
Bondurand
N.
,
Kuhlbrodt
K.
,
Hermans-Borgmeyer
I.
,
Puliti
A.
,
Lemort
N.
,
Goossens
M.
,
Wegner
M.
(
1998
)
Mutation of the Sry-related Sox10 gene in Dominant megacolon, a mouse model for human Hirschsprung disease.
Proc. Natl. Acad. Sci. USA
95
,
5161
5
Hofstra
R. M.
,
Osinga
J.
,
Tan Sindhunata
G.
,
Wu
Y.
,
Kamsteeg
E. J.
,
Stulp
R. P.
,
van Ravenswaaij Arts
C.
,
Majoor Krakauer
D.
,
Angrist
M.
,
Chakravarti
A.
, et al. 
(
1996
)
A homozygous mutation in the endothelin-3 gene associated with a combined Waardenburg type 2 and Hirschsprung phenotype (Shah-Waardenburg syndrome).
Nat. Genet
12
,
445
447
Hosoda
K.
,
Hammer
R. E.
,
Richardson
J. A.
,
Baynash
A. G.
,
Cheung
J. C.
,
Giaid
A.
,
Yanagisawa
M.
(
1994
)
Targeted and natural (piebald-lethal) mutations of endothelin-B receptor gene produce megacolon associated with spotted coat color in mice.
Cell
79
,
1267
1276
Hughes
A.
(
1957
)
The development of the primary sensory system in Xenopus laevis (Daudin).
J. Anat
91
,
323
338
Jessen
K.
,
Mirsky
R.
(
1980
)
Glial cells in the enteric nervous system contain glial fibrillary acidic protein.
Nature
286
,
736
737
Jessen
K.
,
Mirsky
R.
(
1983
)
Astrocyte-like glia in the peripheral nervous system: an immunohistochemical study of enteric glia.
J. Neurosci
3
,
2206
2218
Jessen
K.
,
Mirsky
R.
(
1985
)
Glial fibrillary acidic polypeptides in peripheral glia. Molecular weight, heterogeneity and distribution.
J. Neuroimmunol
8
,
377
393
Kapur
R. P.
,
Sweetser
D. A.
,
Doggett
B.
,
Siebert
J. R.
,
Palmiter
R. D.
(
1995
)
Intercellular signals downstream of endothelin receptor-B mediate colonization of the large intestine by enteric neuroblasts.
Development
121
,
3787
3795
Kelsh
R. N.
,
Brand
M.
,
Jiang
Y.-J.
,
Heisenberg
C. P.
,
Lin
S.
,
Haffter
P.
,
Odenthal
J.
,
Mullins
M. C.
,
van Eeden
F. J.
,
Furutani-Seiki
M.
, et al. 
(
1996
)
Zebrafish pigmentation mutations and the processes of neural crest development.
Development
123
,
369
389
Kimmel
C. B.
,
Ballard
W. W.
,
Kimmel
S. R.
,
Ullmann
B.
,
Schilling
T. F.
(
1995
)
Stages of embryonic development of the zebrafish.
Dev. Dynam
203
,
253
310
Kimmel
C. B.
,
Miller
C. T.
,
Kruze
G.
,
Ullman
B.
,
Bremiller
R. A.
,
Larison
K. D.
,
Snyder
H. C.
(
1998
)
The shaping of pharyngeal cartilages during early development of the zebrafish.
Dev. Biol
203
,
245
263
Krauss
S.
,
Concordet
J.
,
Ingham
P.
(
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
Krotoski
D. M.
,
Fraser
S. E.
,
Bronner Fraser
M.
(
1988
)
Mapping of neural crest pathways in Xenopus laevis using inter-and intra-specific cell markers.
Dev. Biol
127
,
119
132
Lahav
R.
,
Ziller
C.
,
Dupin
E.
,
Ledouarin
N. M.
(
1996
)
Endothelin-3 promotes neural crest cell-proliferation and mediates a vast increase in melanocyte number in culture.
Proc. Natl. Acad. Sci. USA
93
,
3892
3897
Lane
P. W.
,
Liu
H. M.
(
1984
)
Association of megacolon with a new dominant spotting gene (Dom) in the mouse.
J. Hered
75
,
435
439
Le Douarin
N.
,
Dulac
C.
,
Dupin
E.
,
Cameron Curry
P.
(
1991
)
Glial cell lineages in the neural crest.
Glia
4
,
175
184
Le Douarin
N. M.
(
1986
)
Cell line segregation during peripheral nervous system ontogeny.
Science
231
,
1515
1522
Le Douarin
N. M.
(
1990
)
Cell lineage segregation during neural crest ontogeny.
Ann. N. Y. Acad. Sci
599
,
131
40
Le Douarin
N. M.
,
Dupin
E.
,
Ziller
C.
(
1994
)
Genetic and epigenetic control in neural crest development.
Curr. Opin. Genet. Dev
4
,
685
695
Le Douarin
N. M.
,
Teillet
M. A.
(
1973
)
The migration of neural crest cells to the wall of the digestive tract in avian embryo.
J. Embryol. Exp. Morphol
30
,
31
48
Lo
L.
,
Guillemot
F.
,
Joyner
A. L.
,
Anderson
D. J.
(
1994
)
MASH-1: a marker and a mutation for mammalian neural crest development.
Perspect Dev. Neurobiol
2
,
191
201
Loring
J. F.
,
Erickson
C. A.
(
1987
)
Neural Crest Cell Migratory Pathways in the Trunk of the Chick Embryo.
Dev. Biol
121
,
220
236
Malicki
J.
,
Schier
A. F.
,
Solnica-Krezel
L.
,
Stemple
D. L.
,
Neuhauss
S. C. F.
,
Stainier
D. Y. R.
,
Abdelilah
S.
,
Rangini
Z.
,
Zwartkruis
F.
,
Driever
W.
(
1996
)
Mutations affecting development of the zebrafish ear.
Development
123
,
275
283
Marcos-Gutierrez
C.
,
Wilson
S.
,
Holder
N.
,
Pachnis
V.
(
1997
)
The zebrafish homologue of the ret receptor and its pattern of expression during embryogenesis.
Oncogene
14
,
879
889
Marusich
M. F.
,
Furneaux
H. M.
,
Henion
P. D.
,
Weston
J. A.
(
1994
)
Hu neuronal proteins are expressed in proliferating neurogenic cells.
J. Neurobiol
25
,
143
155
Mayer
T. C.
(
1965
)
The development of piebald spotting in mice.
Dev. Biol
11
,
319
334
Mayer
T. C.
,
Maltby
E. L.
(
1964
)
An experimental investigation of pattern development in lethal spotting and belted mouse embryos.
Dev. Biol
9
,
269
286
Milos
N.
,
Dingle
A. D.
(
1978
).
Dynamics of pigment pattern formation in the zebrafish, Brachydanio rerio. I. Establishment and regulation of the lateral line melanophore stripe during the first eight days of development.
J. Exp. Zool
205
,
205
216
Morrison
S. J.
,
Shah
N. M.
,
Anderson
D. J.
(
1997
)
Regulatory mechanisms in stem cell biology.
Cell
88
,
287
298
Myers
P. Z.
(
1985
)
Spinal motoneurons of the larval zebrafish.
J. Comp. Neurol
236
,
555
561
Neuhauss
S. C.
,
Solnica-Krezel
L.
,
Schier
A. F.
,
Zwartkruis
F.
,
Stemple
D. L.
,
Malicki
J.
,
Abdelilah
S.
,
Stainier
D. Y.
,
Driever
W.
(
1996
)
Mutations affecting craniofacial development in zebrafish.
Development
123
,
357
367
Oxtoby
E.
,
Jowett
T.
(
1993
)
Cloning of the zebrafish krox-20 gene (krx-20) and its expression during hindbrain development.
Nucl. Acids Res
21
,
1087
1095
Passarge
E.
(
1973
)
Genetics of Hirschsprung's disease.
Clin. Gastroenterol
2
,
507
513
Pavan
W. J.
,
Tilghman
S. M.
(
1994
)
Piebald lethal (sl) acts early to disrupt the development of neural crest-derived melanocytes.
Proc. Natl Acad. Sci. USA
91
,
7159
7163
Piotrowski
T.
,
Schilling
T.
,
Brand
M.
,
Jiang
Y.
,
Heisenberg
C.
,
Beuchle
D.
,
Grandel
H.
,
van Eeden
F.
,
Furutani-Seiki
M.
,
Granato
M.
, et al. 
(
1996
)
Jaw and branchial arch mutants in zebrafish II: anterior arches and cartilage differentiation.
Development
123
,
345
356
Pomeranz
H. D.
,
Rothman
T. P.
,
Chalazonitis
A.
,
Tennyson
V. M.
,
Gershon
M. D.
(
1993
)
Neural crest-derived cells isolated from the gut by immunoselection develop neuronal and glial phenotypes when cultured on laminin.
Dev. Biol
156
,
341
361
Raible
D. W.
,
Eisen
J. S.
(
1994
)
Restriction of neural crest cell fate in the trunk of the embryonic zebrafish.
Development
120
,
495
503
Raible
D. W.
,
Eisen
J. S.
(
1996
)
Regulative interactions in zebrafish neural crest.
Development
122
,
501
507
Raible
D. W.
,
Wood
A.
,
Hodsdon
W.
,
Henion
P. D.
,
Weston
J. A.
,
Eisen
J. S.
(
1992
)
Segregation and early dispersal of neural crest cells in the embryonic zebrafish.
Dev. Dyn
195
,
29
42
Rao
M. S.
,
Anderson
D. J.
(
1997
)
Immortalization and controlled in vitro differentiation of murine multipotent neural crest stem cells.
J. Neurobiol
32
,
722
746
Reid
K.
,
Turnley
A.
,
Maxwell
G.
,
Kurihara
Y.
,
Kurihara
H.
,
Bartlett
P.
,
Murphy
M.
(
1996
)
Multiple roles for endothelin in melanocyte development: regulation of progenitor number and stimulation of differentiation.
Development
122
,
3911
3919
Schilling
T. F.
,
Kimmel
C. B.
(
1994
)
Segment and cell type lineage restrictions during pharyngeal arch development in the zebrafish embryo.
Development
120
,
483
494
Schilling
T.
,
Piotrowski
T.
,
Grandel
H.
,
Brand
M.
,
Heisenberg
C.
,
Jiang
Y.
,
Beuchle
D.
,
Hammerschmidt
M.
,
Kane
D.
,
Mullins
M.
, et al. 
(
1996
)
Jaw and branchial arch mutants in zebrafish I: branchial arches.
Development
123
,
329
344
Schilling
T. F.
,
Walker
C.
,
Kimmel
C. B.
(
1996
)
The chinless mutation and neural crest cell interactions in zebrafish jaw development.
Development
122
,
1417
1426
Schuchardt
A.
,
D'Agati
V.
,
Larsson Blomberg
L.
,
Costantini
F.
,
Pachnis
V.
(
1994
)
Defects in the kidney and enteric nervous system of mice lacking the tyrosine kinase receptor Ret.
Nature
367
,
380
383
Serbedzija
G. N.
,
Bronner-Fraser
M.
,
Fraser
S. E.
(
1989
)
A vital dye analysis of the timing and pathways of avian trunk neural crest cell migration.
Development
106
,
809
816
Serbedzija
G. N.
,
Bronner-Fraser
M.
,
Fraser
S. E.
(
1994
)
Developmental potential of trunk neural crest cells in the mouse.
Development
120
,
1709
1718
Serbedzija
G. N.
,
Fraser
S. E.
,
Bronner-Fraser
M.
(
1990
)
Pathways of trunk neural crest cell migration in the mouse embryo as revealed by vital dye labelling.
Development
108
,
605
612
Shah
N. M.
,
Anderson
D. J.
(
1997
)
Integration of multiple instructive cues by neural crest stem cells reveals cell-intrinsic biases in relative growth factor responsiveness.
Proc. Natl. Acad. Sci. USA
94
,
11369
11374
Shah
N. M.
,
Groves
A. K.
,
Anderson
D. J.
(
1996
)
Alternative neural crest cell fates are instructively promoted by TGFbeta superfamily members.
Cell
85
,
331
343
Shah
N. M.
,
Marchionni
M. A.
,
Isaacs
I.
,
Stroobant
P.
,
Anderson
D. J.
(
1994
)
Glial growth-factor restricts mammalian neural crest stem-cells to a glial fate.
Cell
77
,
349
360
Sieber-Blum
M.
,
Cohen
A. M.
(
1980
)
Clonal analysis of quail neural crest cells: they are pluripotent and differentiate in vitro inthe absence of noncrest cells.
Dev. Biol
80
,
96
106
Smith
M.
,
Hickman
A.
,
Amanze
D.
,
Lumsden
A.
,
Thorogood
P.
(
1994
)
Trunk neural crest origin of caudal fin mesenchyme in the zebrafish Brachydanio rerio.
Proc. R. Soc. Lond. B
256
,
137
145
Southard-Smith
E. M.
,
Kos
L.
,
Pavan
W. J.
(
1998
)
Sox10 mutation disrupts neural crest development in DOM Hirschsprung mouse model.
Nature Genet
18
,
60
64
Steel
K. P.
,
Davidson
D. R.
,
Jackson
I. J.
(
1992
)
TRP-2/DT, a new early melanoblast marker, shows that steel growth factor (c-kit ligand) is a survival factor.
Development
115
,
1111
1119
Stemple
D. L.
,
Anderson
D. J.
(
1992
)
Isolation of a stem-cell for neurons and glia from the mammalian neural crest.
Cell
71
,
973
985
Stone
J. G.
,
Spirling
L. I.
,
Richardson
M. K.
(
1997
)
The neural crest population responding to endothelin-3 in vitro includes multipotent cells.
J. Cell Sci
110
,
1673
1682
Streisinger
G.
,
Singer
F.
,
Walker
C.
,
Knauber
D.
,
Dower
N.
(
1986
)
Segregation analyses and gene-centromere distances in zebrafish.
Genetics
112
,
311
319
Thisse
C.
,
Thisse
B.
,
Postlethwait
J. H.
(
1995
)
Expression of snail2, a second member of the zebrafish snail family, in cephalic mesendoderm and presumptive neural crest of wild-type and spadetail mutant embryos.
Dev. Biol
172
,
86
99
Thisse
C.
,
Thisse
B.
,
Schilling
T. F.
,
Postlethwait
J. H.
(
1993
)
Structure of the zebrafish snail1 gene and its expression in wild-type, spadetail and no tail mutant embryos.
Development
119
,
1203
1215
Trevarrow
B.
,
Marks
D. L.
,
Kimmel
C. B.
(
1990
)
Organization of hindbrain segments in the zebrafish embryo.
Neuron
4
,
669
679
Webster
W.
(
1973
)
Embryogenesis of the enteric ganglia in normal mice and in mice that develop congenital aganglionic megacolon.
J. Embryology Exp. Morph
30
,
573
585
Weston
J. A.
(
1970
)
The migration and differentiation of neural crest cells.
Adv. Morph
8
,
41
114
Weston
J. A.
(
1991
)
Sequential segregation and fate of developmentally restricted intermediate cell populations in the neural crest lineage.
Curr. Topics Dev. Biol
25
,
133
153
Whitfield
T.
,
Granato
M.
,
van Eeden
F.
,
Schach
U.
,
Brand
M.
,
Furutani-Seiki
M.
,
Haffter
P.
,
Hammerschmidt
M.
,
Heisenberg
C.
,
Jiang
Y.
, et al. 
(
1996
)
Mutations affecting development of the zebrafish inner ear and lateral line.
Development
123
,
241
54
Yntema
C. L.
,
Hammond
W. S.
(
1954
)
The origin of intrinsic ganglia of trunk viscera from vagal neural crest in the chick embryo.
J. Comp. Neurol
101
,
515
541
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