1 Molecular and Cellular Biology Program, University of Washington, Seattle, WA
98195-7420 USA
2 Department of Biological Structure, University of Washington, Seattle, WA
98195-7420 USA
3 Department of Biology, University of Massachusetts, Amherst, MA 01003,
USA
* Author for correspondence (e-mail: draible{at}u.washington.edu)
Accepted 10 July 2003
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SUMMARY |
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Key words: Dorsal root ganglia, Hedgehog, Cyclopamine, Neurogenin, Neural crest, Zebrafish
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Introduction |
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Neurogenin (Ngn) transcription factors are key regulators of DRG
development. ngn1 and ngn2 are both expressed in the dorsal
root ganglia (Ma et al., 1998;
Ma et al., 1999
).
ngn2 is expressed in a subset of migrating neural crest precursors,
whereas ngn1 expression appears later in the nascent DRG
(Ma et al., 1999
;
Perez et al., 1999
).
Inactivation of ngn1 and ngn2 completely blocks development
of DRG neurons (Ma et al.,
1999
), and overexpression of ngn1 in neural crest cells
biases them to contribute to the DRG
(Perez et al., 1999
). In
zebrafish, a single ngn gene, ngn1, performs many of the
functions controlled by the two separate mammalian neurogenins
(Andermann et al., 2002
;
Cornell and Eisen, 2002
).
Blocking Ngn1 function by injection of antisense morpholino oligonucleotides
disrupts development of zebrafish DRG neurons. Understanding the signals that
regulate neurogenin gene expression in neural crest cells should shed light on
the early steps of DRG development.
In zebrafish, neural crest cells that give rise to the DRG are amongst the
earliest cells to migrate from the dorsal neural tube on a ventral pathway
between neural tube and somite (Raible et
al., 1992). Observation of labeled neural crest cells in living
zebrafish embryos revealed that DRG precursors subsequently undergo a
characteristic dorsal migration from a position lateral to the notochord to
their final position lateral to the neural tube
(Raible and Eisen, 1994
).
ngn1 is first expressed in DRG precursors once they reach this final
position (Andermann et al.,
2002
; Cornell and Eisen,
2002
). Together, these results suggest that DRG precursors may be
specified by local signals from notochord, somite or neural tube.
Several studies in avian embryos support the idea that signals released
from nearby tissues are involved in DRG specification. A role for somitic
mesenchyme in producing the segmental arrangement of the DRG is
well-established (Kalcheim and Teillet,
1989). Manipulation of rostrocaudal polarity of somites alters DRG
spacing and size, and can influence proliferation of DRG precursors
(Goldstein et al., 1990
;
Goldstein and Kalcheim, 1991
).
Axial structures have also been implicated in the development of the DRG.
Removal of the neural tube results in loss of DRG and produces unsegmented
sympathetic ganglia (Teillet and Le
Douarin, 1983
). Similarly, placement of an impermeable membrane
between the neural tube and migrating neural crest eliminates the development
of the DRG (Kalcheim and Le Douarin,
1986
). Consistent with these results, loss of either axial signals
in zebrafish floating head (flh) mutant embryos
(Halpern et al., 1995
;
Talbot et al., 1995
) or loss
of paraxial tissues in the zebrafish spadetail (spt) mutant
(Kimmel et al., 1989
;
Griffin et al., 1998
) severely
disrupts DRG development (J.U. and D.W.R., unpublished). All of these results
indicate a role for diffusible signals, either from axial or paraxial tissues,
in the development and patterning of the DRG.
Sonic hedgehog (Shh) is a secreted protein that signals from the notochord
to locally pattern adjacent tissues
(Krauss et al., 1993;
Roelink et al., 1994
). Shh
specifies the differentiation of both sclerotome and epaxial muscle from
paraxial mesoderm (Fan and
Tessier-Lavigne, 1994
; Johnson
et al., 1994
). In zebrafish, Shh signaling is required for the
development of slow muscle fibers and muscle pioneers
(Barresi et al., 2000
). Shh
also induces differentiation of several cell types in the ventral neural tube
through graded Shh signaling (Ericson et
al., 1997
). Similarly, midline Shh signaling is required for the
induction of both primary and secondary motor neurons in zebrafish
(Beattie et al., 1997
;
Lewis and Eisen, 2001
).
Given its clear roles in patterning both the neural tube and somites, and
its expression adjacent to the site where DRG develop, Shh is a candidate for
mediating local signaling required for specification of DRG precursors. To
examine the role of Shh signaling in the development of the zebrafish DRG, we
have taken advantage of the availability of several zebrafish mutations that
disrupt different components of the Hh signaling pathway: sonic you
(syu), which encodes sonic hedgehog; smooth muscle omitted
(smu), which encodes the smoothened (smo) receptor;
detour (dtr), which encodes the transcriptional effector
gli1, and you-too (yot), which encodes
gli2 (Brand et al.,
1996; Schauerte et al.,
1998
; Karlstrom et al.,
1999
; Chen et al.,
2001
; Varga et al.,
2001
; Karlstrom et al.,
2003
). These mutants generally share several morphological
defects, including: ventral cyclopia, curved-down tails, U-shaped somites
lacking the horizontal myoseptum and circulation defects associated with
disrupted dorsal aorta formation. Furthermore, we use the steroidal alkaloid,
cyclopamine, to vary the timing of loss of Hh signaling during neural crest
development. It has been previously demonstrated that cyclopamine inhibits
activation of the Hh pathway through direct binding to Smo
(Cooper et al., 1998
;
Incardona et al., 1998
;
Chen et al., 2002
), and has
been used previously to disrupt Hh signaling in zebrafish
(Neumann et al., 1999
;
Chen et al., 2001
;
Sbrogna et al., 2003
;
Stenkamp and Frey, 2003
). Our
analysis of the timing and tissue requirements for Shh signaling reveals a
cell-autonomous requirement for Hh signal transduction in DRG precursors and
shows that Shh signaling functions upstream of Ngn1 to promote sensory
neurogenesis.
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Materials and Methods |
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Cyclopamine treatment of embryos
Cyclopamine (gift from Henk Roelink) was diluted in embryo medium to 0.5
µg/ml-18 µg/ml from a stock of 4 mg/ml dissolved in 45% (w/v)
2-hydroxypropyl-ß-cyclodextrin (Sigma) in phosphate buffered saline
(PBS). Wild-type embryos were soaked in embryo medium containing cyclopamine
and allowed to develop at 28°C. Unless otherwise specified, embryos were
left in cyclopaminecontaining media until they were processed for
immunohistochemistry or whole-mount in situ hybridization. Embryos treated in
embryo medium containing 2-hydroxypropyl-ß-cyclodextrin alone were
indistinguishable from untreated wild-type embryos.
Immunohistochemistry
Embryos were anesthetized in tricaine (10 mg/ml; Sigma) in embryo medium,
fixed in 4% formalin in fix buffer for 2 hours at room temperature (RT)
(Westerfield, 1994).
Seventy-two hours postfertilization (hpf) and older embryos were permeabilized
by washing 3x30 minutes in distilled water. Embryos were incubated for 1 hour
in blocking solution (2% goat serum, 1% bovine serum albumin, 1%
dimethylsulfoxide, 0.1% Triton X-100 in PBS), then overnight at RT in primary
antibody diluted in blocking solution. Primary antibodies used were anti-Hu
(1:700; mAB 16A11) (Marusich et al.,
1994
), anti-zn-5 (1:400)
(Fashena and Westerfield,
1999
), anti-acetylated tubulin (1:1000; Sigma), anti-Islet 1 4D5
[1:200; gift from H. Roelink and Developmental Studies Hybridoma Bank (DSHB),
www.uiowa.edu/~dshbwww/],
anti-Lim-1/2 4F2 (1:500; gift from H. Roelink and DSHB) and anti-GFP (1:200;
Molecular Probes, Eugene, OR, USA). Embryos were rinsed extensively in PBS
with Triton X-100 (PBTx) and incubated overnight at RT in Alexa488- or
Alexa568-conjugated secondary antibodies diluted in blocking solution (1:750;
Molecular Probes). After rinsing in PBTx, embryos were transferred to 50%
glycerol in PBS and mounted on bridged coverslips.
Whole-mount in situ hybridization and RNA probe synthesis
Embryos were collected from timed matings, raised at 28.5°C and
carefully staged before fixing overnight at 4°C in 4% paraformaldehyde in
PBS. RNA in situ hybridization was performed following Thisse et al.
(Thisse et al., 1993), except
embryos were hybridized at 65°C. Digoxigenin or fluorescein-labeled
antisense RNA probes were generated for ngn1 and neurod
(Blader et al., 1997
),
patched1 (ptc1)
(Concordet et al., 1996
) and
crestin (Rubinstein et al.,
2000
; Luo et al.,
2001
) by digesting DNA with restriction enzyme and synthesizing
with RNA polymerase as follows: ngn1, XhoI/T7; neurod,
NotI/T3; ptc1, BamHI/T3; crestin, SacI/T7. Probes were
detected using anti-digoxigenin or anti-fluorescein antibodies conjugated to
alkaline phosphatase (Roche), followed by incubation with 5-bromo 4-chloro
3-indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT). For cryostat
sectioning, embryos were cryopreserved in 30% sucrose and mounted in OCT. For
plastic sectioning, embryos were processed for in situ hybridization,
dehydrated in a graded ethanol series and embedded in Araldite resin
(Polysciences, Warrington, PA, USA). All images were captured on a Nikon
Microphot-SA microscope (Nikon, Melville, NJ, USA) using a Spot Digital Camera
and software (Diagnostic Instruments, Sterling Heights, MI, USA). Images were
processed using Photoshop 6.0 (Adobe, San Jose, CA, USA).
DRG counts
Embryos were processed for anti-Hu immunoreactivity at 60 hpf, mounted in
50% glycerol/PBS between bridged coverslips and oriented on their side to view
DRG neurons. Each embryo was scored on one side for the presence or absence of
DRG neurons and the presence of large neuronal clusters. Analysis of both
sides of many embryos revealed no obligate bilateral symmetry in appearance of
large clusters of neuronal cells (data not shown). Ganglia were scored as
`normal' if the number of neurons was in the appropriate range given the age
of the embryo and rostrocaudal level of the ganglion. However, given that the
abnormal clusters can vary significantly in cell number, some of the ganglia
that were scored as `normal DRG' may have been abnormal clusters in the early
stages of development.
Mosaic analysis
Donors were labeled by injecting 1 nl of a 3% solution of rhodaminedextran
(10,000 MW dextran, tetramethylrhodamine lysine fixable, D-3312; Molecular
Probes) in 0.2 M KCl into the yolk cytoplasm of 1- to 8-cell stage embryos
using an ASI pressure injection apparatus (ASI, Eugene, OR, USA). Mosaic
embryos were generated by transplanting cells from blastula-stage donors into
shield-stage hosts. Embryos were mounted in 3% methylcellulose (Sigma) in
embryo medium containing 1% penicillin-streptomycin (Sigma). To confirm
contribution of donor cells to host neural crest, in some experiments, cells
from embryos of mutant heterozygote crosses were transplanted into
nacre homozygotes that lack melanophores
(Lister et al., 1999).
nacre hosts could then be scored for the presence of neural
crest-derived melanophores. All embryos were fixed at 48-72 hpf and stained
with anti-Hu and anti-mouse Alexa488 to visualize DRG neurons. Stained embryos
were examined on a Zeiss LSM Pascal confocal microscope.
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Results |
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Hh signaling mutants lose expression of ngn1 and
neurod in DRG precursors
The ngn1 gene is critical for zebrafish DRG development and is the
earliest known marker for sensory neuroblasts
(Cornell and Eisen, 2002;
Andermann et al., 2002
). To
determine when the development of DRG precursors is first affected by loss of
Hh signaling, we examined expression of ngn1 in Shh/midline class
mutants (Fig. 5). yot
embryos and wild-type siblings were fixed at 36 hpf and processed for in situ
hybridization. Although spinal cord expression of ngn1 appears normal
in mutant embryos, expression in DRG precursors is completely eliminated in
yot homozygotes (Fig.
5C). Similarly, expression of ngn1 is absent in DRG
precursors in smu mutant embryos (data not shown). Expression of the
bHLH gene neurod normally follows expression of ngn1 in DRG
precursors. This expression is also abolished in smu mutants
(Fig. 5). Together, these
results demonstrate that Hh signaling is required prior to neurogenic bHLH
expression in DRG precursors.
Neural crest develops normally in the absence of Hh signaling
In other organisms, Shh signaling influences the dorsoventral pattern of
the neural tube, suggesting that the observed DRG phenotypes might reflect a
disruption of neural crest induction or may result from defects in neural
crest migration. Consistent with these possibilities, in addition to the DRG
defects, many of the Hh pathway mutants also have defects in neural
crest-derived cartilage formation in the developing jaw
(Brand et al., 1996;
Barresi et al., 2000
;
Varga et al., 2001
;
Kimmel et al., 2001
). However,
the other trunk neural crest derivatives, such as pigment cells and fin
ectomesenchyme, develop normally following attenuation of Hh signaling (data
not shown). To further explore the possibility that Hh signaling is required
for early events in neural crest development, we examined expression of the
neural crest marker crestin (Rubenstein et al., 2000;
Luo et al., 2001
) in
smu mutant embryos (Fig.
6). The level of crestin expression appears comparable
between wild-type and smu mutant embryos
(Fig. 6A,B), suggesting that
blocking Hh signaling does not significantly alter neural crest formation.
Although crestin expression reveals that migration of neural crest is
abnormal in smu mutant embryos, neural crest cells do still migrate
ventrally (Fig. 6D). Moreover,
although treatment of embryos with high doses of cyclopamine during
somitogenesis stages has severe effects on DRG development
(Fig. 6G), there is no obvious
effect on the pattern of neural crest migration
(Fig. 6F). Taken together,
these observations suggest that Hh signaling is required in the trunk
specifically for the formation of DRG neurons, and the observed effects on DRG
development are probably not the result of affecting earlier stages of trunk
neural crest development.
|
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Significant contributions of wild-type cells to both spinal cord and somitic tissue never rescued adjacent DRG neurons in smu or in yot mutant embryos (Table 1). Similarly, in reciprocal transplants of smu or yot cells into wild-type hosts, significant contributions of mutant cells to spinal cord or somitic tissue never disrupted development of adjacent DRG neurons (Table 1). In contrast, wild-type cells were capable of producing normal DRG neurons in both smu and yot mutant backgrounds (Fig. 8; Table 1). Although smu mutant cells were capable of producing neural crest-derived melanophores in wild-type embryos, they never formed DRG neurons (Table 1). Together, these results suggest that Hh signal transduction must occur directly within DRG precursors for normal DRG development to proceed.
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Discussion |
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In addition to the prevalent loss of DRG in midline/Hh mutants and in
cyclopamine-treated embryos, in some segments we observed the appearance of
abnormal neuronal clusters ventrolateral to the spinal cord. The expression of
the spinal cord neuron marker, Lim-1/2, in these cells in smu mutant
embryos suggests that these may be cells that have inappropriately exited the
spinal cord. It is possible that normal Hh signaling is required for
structural integrity of the neural tube. Alternatively, given the specific
ventrolateral appearance of these clusters, Hh signaling may be more
specifically required for impeding migration of cells out of the motor exit
points. Neural crest-derived cells are required to `cap' the motor exit points
and act as a barrier to movement of cell bodies between the CNS and PNS
compartments in both mouse and chick embryos
(Vermeren et al., 2003). It is
possible that Hh signaling, in addition to its requirement for development of
DRG precursors, is also required for development of these neural crest-derived
border cells. The location of border cap cells at the ventrolateral edge of
the spinal cord, adjacent to the sources of Shh, is consistent with a role for
Hh in their development. Identifying specific markers for border cap cells
would allow testing of this hypothesis.
Previous studies examining the effects of Shh on neural crest suggest
several possible cellular mechanisms underlying the DRG phenotypes we observe
in zebrafish. Shh signaling has been shown to play an important role in the
survival of both neural tube and neural crest cell populations
(Dunn et al., 1995;
Miao et al., 1997
;
Ahlgren and Bronner-Fraser,
1999
; Charrier et al.,
2001
). Injection of function-blocking anti-Shh antibody into chick
cranial mesenchyme results in a loss of branchial arch structures that is
associated with significant cell death in both the neural tube and neural
crest (Ahlgren and Bronner-Fraser,
1999
). Similarly, cyclopamine treatment of Xenopus
embryos results in a reduction of craniofacial cartilages and promotes cell
death in explants of cranial neural crest
(Dunn et al., 1995
). In our
experiments, zebrafish migratory trunk neural crest appears normal in embryos
in which Hh signaling is blocked. In addition, we do not observe any obvious
cell death associated with the DRG in zebrafish Hh signaling mutants (data not
shown). Moreover, in both the chick and Xenopus studies, loss of Shh
signaling had no effect on the survival of trunk neural crest, suggesting that
the anti-apoptotic role of Shh appears restricted to cranial neural crest
populations. Similarly, although Shh promotes survival of specific CNS neurons
following toxic insult, Shh fails to show any neuroprotective effects on DRG
neurons in culture (Miao et al.,
1997
).
Hh signaling may alternatively be involved in regulating the migration of
neural crest cells. Dorsally expressed bone morphogenetic proteins (BMPs) have
been shown to promote neural crest dispersion through effects on integrins and
cadherins (Sela-Donenfeld and Kalcheim,
1999). The work of Testaz et al. suggests that ventrally expressed
Hh may play an opposing role in neural crest development by limiting the
migration of neural crest cells: in neural tube explants cultured on
fibronectin and immobilized N-terminal Shh, neural crest cell dispersion is
severely restricted when compared with migration of neural crest cells on
fibronectin alone (Testaz et al.,
2001
). Based on these results, we might predict that loss of DRG
neurons in zebrafish Hh signaling mutants reflects the failure to localize DRG
precursors to their appropriate positions. Consistent with this idea we see
disorganization of trunk neural crest streams in Hh signaling mutants.
However, although we observe significant loss of DRG neurons in embryos
treated with cyclopamine at 18 hpf, we do not see any obvious effects on
neural crest migration in these embryos. Moreover, other trunk neural crest
derivatives, such as melanophores, still differentiate in their normal
positions in the absence of Hh signaling (data not shown). Together, these
results argue against the idea that the effects of Hh signaling on DRG
development are mediated through general effects on neural crest migration.
Interestingly, Testaz et al. also suggest that the effects of Shh on neural
crest migration and adhesion probably involve mechanisms independent of the
canonical Patched/Smoothened/Gli signaling cascade because neither forskolin,
an activator of protein kinase A (PKA) and known antagonist of Hh signaling,
nor cyclopamine block the Shhmediated inhibition of neural crest cell
migration (Testaz et al.,
2001
). In contrast, zebrafish smoothened and gli
mutations have the same effect on development of DRG neurons as loss of
shh, indicating that the canonical Hh signaling pathway is required
for normal DRG development.
Another possibility is that Hh signaling is required to directly promote
the differentiation of DRG neurons. It is well-established that Shh signaling
is both necessary and sufficient for motor neuron differentiation
(Roelink et al., 1995;
Chiang et al., 1996
;
Ericson et al., 1996
) and
promotes differentiation of other neuronal cell-types according to the
appropriate axial position (Ericson et
al., 1995
; Hynes et al.,
1995
). All of our results are consistent with this role of Shh
signaling in neuronal development. Others and ourselves have shown previously
that Ngn1 is required for zebrafish sensory neuron specification
(Andermann et al., 2002
;
Cornell and Eisen, 2002
), and
expression of ngn1 is specifically absent from DRG precursors in
midline/Hh mutants and cyclopamine-treated embryos. Both cell transplantation
experiments and ptc1 expression suggest that DRG precursors receive
the Hh signal directly. Timed cyclopamine addition experiments further suggest
that the window of requirement for the Hh signal ends at approximately the
time ngn1 is first expressed in DRG precursors. Taken together, these
observations suggest the possibility that Hh promotes the determination of DRG
precursors by activating ngn1 expression, a role for Hh that has been
suggested previously for other neuronal cell-types in zebrafish
(Blader et al., 1997
). In these
studies, overexpression of Shh expanded the endogenous expression of zebrafish
ngn1 in the neural plate. Conversely, downregulation of Hh signaling
by injecting a constitutively active form of PKA abolished neural plate
expression of ngn1. Together, these results showed that Shh is
capable of driving expression of ngn1, either directly or indirectly.
Similarly, our results, together with previous descriptions of ngn1
expression and DRG precursor migration patterns, suggest that Hh signaling may
normally help regulate expression of ngn1 in DRG sensory
precursors.
Neural crest cells that ultimately populate the DRG migrate ventrally on
the medial pathway along with sympathetic and pigment cell precursors.
However, at a point adjacent to the notochord, sensory precursors stop and
return dorsally to the position of the DRG
(Raible and Eisen, 1994) where
they begin to express ngn1
(Cornell and Eisen, 2002
;
Andermann et al., 2002
). The
timing of onset of ngn1 expression suggests that Hh signals emanating
from the notochord and/or neural tube may be involved in initiation of
ngn1 expression in DRG precursors. Ngns are known to be sufficient
for conferring neuronal identity on uncommitted precursors
(Farah et al., 2000
;
Sun et al., 2001
;
Nieto et al., 2001
).
Furthermore, Ngns are thought to reinforce the neuronal program by inhibiting
genes necessary for gliogenesis (Sun et
al., 2001
). This difference in migration behavior between sensory
precursors and autonomic and pigment cell precursors further suggests that DRG
precursors are already predisposed to respond to Hh signals early in their
migration. Rather than biasing neural crest cells toward a sensory fate, Hh
signaling may be influencing DRG precursors to adopt a neuronal cell fate by
promoting ngn1 expression.
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ACKNOWLEDGMENTS |
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REFERENCES |
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---|
Ahlgren, S. C. and Bronner-Fraser, M. (1999). Inhibition of sonic hedgehog signaling in vivo results in craniofacial neural crest cell death. Curr. Biol. 9,1304 -1314.[CrossRef][Medline]
Ahlgren, S. C., Thakur, V. and Bronner-Fraser, M.
(2002). Sonic hedgehog rescues cranial neural crest from cell
death induced by ethanol exposure. Proc. Natl. Acad. Sci.
USA 99,10476
-10481.
Andermann, P., Ungos, J. M. and Raible, D. W. (2002). Neurogenin1 defines zebrafish cranial sensory ganglia precursors. Dev. Biol. 125, 45-58.
Barresi, M. J., Stickney, H. L. and Devoto, S. H.
(2000). The zebrafish slow-muscle-omitted gene product
is required for Hedgehog signal transduction and the development of slow
muscle identity. Development
127,2189
-2199.
Beattie, C. E., Hatta, K., Halpern, M. E., Liu, H., Eisen, J. S. and Kimmel, C. B. (1997). Temporal separation in the specification of primary and secondary motoneurons in zebrafish. Dev. Biol. 187,171 -182.[CrossRef][Medline]
Blader, P., Fischer, N., Gradwohl, G., Guillemont, F. and
Strahle, U. (1997). The activity of neurogenin1 is
controlled by local cues in the zebrafish embryo.
Development 124,4557
-4569.
Brand, M., Heisenberg, C. P., Warga, R. M., Pelegri, F.,
Karlstrom, R. O., Beuchle, D., Picker, A., Jiang, Y. J.,
Furutani-Seiki, M., van Eeden, F. J. et al. (1996). Mutations
affecting development of the midline and general body shape during zebrafish
embryogenesis. Development
123,129
-142.
Charrier, J. B., Lapointe, F., Le Douarin, N. M. and Teillet, M. A. (2001). Anti-apoptotic role of Sonic hedgehog protein at the early stages of nervous system organogenesis. Development 128,4011 -4020.[Medline]
Chen, J. K., Taipale, J., Cooper, M. K. and Beachy, P. A.
(2002). Inhibition of hedgehog signaling by direct binding of
cyclopamine to smoothened. Genes Dev.
16,2743
-2748.
Chen, W., Burgess, S. and Hopkins, N. (2001). Analysis of the zebrafish smoothened mutant reveals conserved and divergent functions of hedgehog activity. Development 128,2385 -2396.[Medline]
Chiang, C., Litingtung, Y., Lee, E., Young, K. E., Corden, J. L., Westphal, H. and Beachy, P. A. (1996). Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function. Nature 383,407 -413.[CrossRef][Medline]
Concordet, J. P., Lewis, K. E., Moore, J. W., Goodrich, L. V.,
Johnson, R. L., Scott, M. P. and Ingham, P. W. (1996).
Spatial regulation of a zebrafish patched homologue reflects the
roles of sonic hedgehog and protein kinase A in neural tube and somite
patterning. Development
122,2835
-2846.
Cooper, M. K., Porter, J. A., Young, K. E. and Beachy, P. A.
(1998). Teratogen-mediated inhibition of target tissue response
to Shh signaling. Science
280,1603
-1607.
Cornell, R. A. and Eisen, J. S. (2002).
Delta/Notch signaling promotes formation of zebrafish neural crest by
repressing Neurogenin 1 function. Development
129,2639
-2648.
Dunn, M. K., Mercola, M. and Moore, D. D. (1995). Cyclopamine, a steroidal alkaloid, disrupts development of cranial neural crest cells in Xenopus. Dev. Dyn. 202,255 -270.[Medline]
Dutton, K. A., Pauliny, A., Lopes, S. S., Elworthy, S., Carney,
T. J., Rauch, J., Geisler, R., Haffter, P. and Kelsh, R. N.
(2001). Zebrafish colourless encodes sox10 and specifies
non-ectomesenchymal neural crest fates. Development
128,4113
-4125.
Ericson, J., Thor, S., Edlund, T., Jessell, T. M. and Yamada, T. (1992). Early stages of motor neuron differentiation revealed by expression of homeobox gene Islet-1. Science 256,1555 -1560.[Medline]
Ericson, J., Muhr, J., Placzek, M., Lints, T., Jessell, T. M. and Edlund, T. (1995). Sonic hedgehog induces the differentiation of ventral forebrain neurons: a common signal for ventral patterning within the neural tube. Cell 81,747 -756.[Medline]
Ericson, J., Morton, S., Kawakami, A., Roelink, H. and Jessell, T. M. (1996). Two critical periods of Sonic Hedgehog signaling required for the specification of motor neuron identity. Cell 87,661 -673.[Medline]
Ericson, J., Briscoe, J., Rashbass, P., van Heyningen, V. and Jessell, T. M. (1997). Graded sonic hedgehog signaling and the specification of cell fate in the ventral neural tube. Cold Spring Harbor Symp. Quant. Biol. 62,451 -466.[Medline]
Fan, C. M. and Tessier-Lavigne, M. (1994). Patterning of mammalian somites by surface ectoderm and notochord: evidence for sclerotome induction by a hedgehog homolog. Cell 79,1175 -1186.[Medline]
Farah, M. H., Olson, J. M., Sucic, H. B., Hume, R. I., Tapscott,
S. J. and Turner, D. L. (2000). Generation of neurons
by transient expression of neural bHLH proteins in mammalian cells.
Development 127,693
-702.
Fashena, D. and Westerfield, M. (1999). Secondary motoneuron axons localize DM-GRASP on their fasciculated segments. J. Comp. Neurol. 406,415 -424.[CrossRef][Medline]
Goldstein, R. S. and Kalcheim, C. (1991). Normal segmentation and size of the primary sympathetic ganglia depend upon the alternation of rostrocaudal properties of the somites. Development 112,327 -334.[Abstract]
Goldstein, R. S., Teillet, M. A. and Kalcheim, C. (1990). The microenvironment created by grafting rostral half-somites is mitogenic for neural crest cells. Proc. Natl. Acad. Sci. USA 87,4476 -4480.[Abstract]
Griffin, K. J., Amacher, S. L., Kimmel, C. B. and Kimelman,
D. (1998). Molecular identification of spadetail:
regulation of zebrafish trunk and tail mesoderm formation by T-box genes.
Development 125,3379
-3388.
Halpern, M. E., Thisse, C., Ho, R. K., Thisse, B., Riggleman,
B., Trevarrow, B., Weinberg, E. S., Postlethwait, J. H. and Kimmel, C.
B. (1995). Cell-autonomous shift from axial to paraxial
mesodermal development in zebrafish floating head mutants.
Development 121,4257
-4264.
Henion, P. D., Raible, D. W., Beattie, C. E., Stoesser, K. L., Weston, J. A. and Eisen, J. S. (1996). Screen for mutations affecting development of zebrafish neural crest. Dev. Genet. 18,11 -17.[CrossRef][Medline]
Hidalgo, A. and Ingham, P. (1990). Cell patterning in the Drosophila segment: spatial regulation of the segment polarity gene patched. Development 110,291 -302.[Abstract]
Hynes, M., Porter, J. A., Chiang, C., Chang, D., Tessier-Lavigne, M., Beachy, P. A. and Rosenthal, A. (1995). Induction of midbrain dopaminergic neurons by Sonic hedgehog. Neuron 15,35 -44.[Medline]
Incardona, J. P., Gaffield, W., Kapur, R. P. and Roelink, H.
(1998). The teratogenic Veratrum alkaloid cyclopamine inhibits
sonic hedgehog signal transduction. Development
125,3553
-3562.
Itoh, M., Kim, C. H., Palardy, G., Oda, T., Jiang, Y. J., Maust, D., Yeo, S. Y., Lorick, K., Wright, G. J., Ariza-McNaughton, L. et al. (2003). Mind bomb is a ubiquitin ligase that is essential for efficient activation of Notch signaling by Delta. Dev. Cell 4,67 -82.[Medline]
Johnson, R. L., Laufer, E., Riddle, R. D. and Tabin, C. (1994). Ectopic expression of sonic hedgehog alters dorsal-ventral patterning of somites. Cell 79,1165 -1173.[Medline]
Kalcheim, C. and Le Douarin, N. M. (1986). Requirement of a neural tube signal for the differentiation of neural crest cells into dorsal root ganglia. Dev. Biol. 116,451 -466.[Medline]
Kalcheim, C. and Teillet, M. A. (1989). Consequences of somite manipulation on the pattern of dorsal root ganglion development. Development 106, 85-93.[Abstract]
Karlstrom, R. O., Trowe, T., Klostermann, S., Baier, H., Brand,
M., Crawford, A. D., Grunewald, B., Haffter, P., Hoffmann, H., Meyer,
S. U. et al. (1996). Zebrafish mutations affecting
retinotectal axon pathfinding. Development
123,427
-438.
Karlstrom, R. O., Talbot, W. S. and Schier, A. F.
(1999). Comparative synteny cloning of zebrafish
you-too: mutations in the Hedgehog target gli2 affect
ventral forebrain patterning. Genes Dev.
13,388
-393.
Karlstrom, R. O., Tyurina, O., Kawakami, A., Talbot, W. S.,
Sasaki, H. and Schier, A. F. (2003). Genetic analysis of
zebrafish gli1 and gli2 reveals divergent requirements for
gli genes in vertebrate development.
Development 130,1549
-1564.
Kimmel, C. B., Kane, D. A., Walker, C., Warga, R. M. and Rothman, M. B. (1989). A mutation that changes cell movement and cell fate in the zebrafish embryo. Nature 337,358 -362.[CrossRef][Medline]
Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B. and Schilling, T. F. (1995). Stages of embryonic development of the zebrafish. Dev. Dyn. 203,253 -310.[Medline]
Kimmel, C. B., Miller, C. T. and Moens, C. B. (2001). Specification and morphogenesis of the zebrafish larval head skeleton. Dev. Biol. 233,239 -257.[CrossRef][Medline]
Korzh, V., Edlund, T. and Thor, S. (1993).
Zebrafish primary neurons initiate expression of the LIM homeodomain protein
Isl-1 at the end of gastrulation. Development
118,417
-425.
Krauss, S., Concordet, J. P. and 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.[Medline]
Lewis, K. E. and Eisen, J. S. (2001). Hedgehog signaling is required for primary motoneuron induction in zebrafish. Development 128,3485 -3495.[Medline]
Lister, J. A., Robertson, C. P., Lepage, T., Johnson, S. L. and
Raible, D. W. (1999). nacre encodes a
zebrafish microphthalmia-related protein that regulates neural-crest-derived
pigment cell fate. Development
126,3757
-3767.
Luo, R., An, M., Arduini, B. L. and Henion, P. D. (2001). Specific panneural crest expression of zebrafish Crestin throughout embryonic development. Dev. Dyn. 220,169 -174.[CrossRef][Medline]
Ma, Q., Chen, Z., del Barco Barrantes, I., de la Pompa, J. L. and Anderson, D. J. (1998). neurogenin1 is essential for the determination of neuronal precursors for proximal cranial sensory ganglia. Neuron 20,469 -482.[Medline]
Ma, Q., Fode, C., Guillemot, F. and Anderson, D. J.
(1999). Neurogenin1 and neurogenin2 control two distinct waves of
neurogenesis in developing dorsal root ganglia. Genes
Dev. 13,1717
-1728.
Marusich, M. F., Furneaux, H. M., Henion, P. D. and Weston, J. A. (1994). Hu neuronal proteins are expressed in proliferating neurogenic cells. J. Neurobiol. 25,143 -155.[Medline]
Miao, N., Wang, M., Ott, J. A., D'Alessandro, J. S., Woolf, T.
M., Bumcrot, D. A., Mahanthappa, N. K. and Pang, K.
(1997). Sonic hedgehog promotes the survival of specific CNS
neuron populations and protects these cells from toxic insult in vitro.
J. Neurosci. 17,5891
-5899.
Neumann, C. J., Grandel, H., Gaffield, W., Schulte-Merker, S.
and Nusslein-Volhard, C. (1999). Transient
establishment of anteroposterior polarity in the zebrafish pectoral fin bud in
the absence of sonic hedgehog activity. Development
126,4817
-4826.
Nieto, M., Schuurmans, C., Britz, O. and Guillemot, F. (2001). Neural bHLH genes control the neuronal versus glial fate decision in cortical progenitors. Neuron 29,401 -413.[Medline]
Park, H. C., Kim, C. H., Bae, Y. K., Yeo, S. Y., Kim, S. H., Hong, S. K., Shin, J., Yoo, K. W., Hibi, M., Hirano, T. et al. (2000). Analysis of upstream elements in the HuC promoter leads to the establishment of transgenic zebrafish with fluorescent neurons. Dev. Biol. 227,279 -293.[CrossRef][Medline]
Perez, S. E., Rebelo, S. and Anderson, D. J.
(1999). Early specification of sensory neuron fate revealed by
expression and function of neurogenins in the chick embryo.
Development 126,1715
-1728.
Raible, D. W. and Eisen, J. S. (1994).
Restriction of neural crest cell fate in the trunk of the embryonic zebrafish.
Development 120,495
-503.
Raible, D. W., Wood, A., Hodsdon, W., Henion, P. D., Weston, J. A. and Eisen, J. S. (1992). Segregation and early dispersal of neural crest cells in the embryonic zebrafish. Dev. Dyn. 195,29 -42.[Medline]
Roelink, H., Augsburger, A., Heemskerk, J., Korzh, V., Norlin, S., Ruiz i Altaba, A., Tanabe, Y., Placzek, M., Edlund, T., Jessell, T. M. et al. (1994). Floor plate and motor neuron induction by vhh-1, a vertebrate homolog of hedgehog expressed by the notochord. Cell 76,761 -775.[Medline]
Roelink, H., Porter, J. A., Chiang, C., Tanabe, Y., Chang, D. T., Beachy, P. A. and Jessell, T. M. (1995). Floor plate and motor neuron induction by different concentrations of the amino-terminal cleavage product of sonic hedgehog autoproteolysis. Cell 81,445 -455.[Medline]
Rubinstein, A. L., Lee, D., Luo, R., Henion, P. D. and Halpern, M. E. (2000). Genes dependent on zebrafish cyclops function identified by AFLP differential gene expression screen. Genesis 26,86 -97.[CrossRef][Medline]
Sbrogna, J. L., Barresi, M. J. and Karlstrom, R. O. (2003). Multiple roles for Hedgehog signaling in zebrafish pituitary development. Dev. Biol. 254, 19-35.[CrossRef][Medline]
Schauerte, H. E., van Eeden, F. J., Fricke, C., Odenthal, J.,
Strahle, U. and Haffter, P. (1998). Sonic
hedgehog is not required for the induction of medial floor plate cells in
the zebrafish. Development
125,2983
-2993.
Sela-Donenfeld, D. and Kalcheim, C. (1999).
Regulation of the onset of neural crest migration by coordinated activity of
BMP4 and Noggin in the dorsal neural tube. Development
126,4749
-4762.
Stenkamp, D. L. and Frey, R. A. (2003). Extraretinal and retinal hedgehog signaling sequentially regulate retinal differentiation in zebrafish. Dev. Biol. 258,349 -363.[CrossRef][Medline]
Sun, Y., Nadal-Vicens, M., Misono, S., Lin, M. Z., Zubiaga, A., Hua, X., Fan, G. and Greenberg, M. E. (2001). Neurogenin promotes neurogenesis and inhibits glial differentiation by independent mechanisms. Cell 104,365 -376.[Medline]
Talbot, W. S., Trevarrow, B., Halpern, M. E., Melby, A. E., Farr, G., Postlethwait, J. H., Jowett, T., Kimmel, C. B. and Kimelman, D. (1995). A homeobox gene essential for zebrafish notochord development. Nature 378,150 -157.[CrossRef][Medline]
Teillet, M. A. and Le Douarin, N. M. (1983). Consequences of neural tube and notochord excision on the development of the peripheral nervous system in the chick embryo. Dev. Biol. 98,192 -211.[Medline]
Testaz, S., Jarov, A., Williams, K. P., Ling, L. E.,
Koteliansky, V. E., Fournier-Thibault, C. and Duband, J. L.
(2001). Sonic hedgehog restricts adhesion and migration of neural
crest cells independently of the Patched-Smoothened-Gli signaling pathway.
Proc. Natl. Acad. Sci. USA
98,12521
-12526.
Thisse, C., Thisse, B., Schilling, T. F. and 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.
Tsuchida, T., Ensini, M., Morton, S. B., Baldassare, M., Edlund, T., Jessell, T. M. and Pfaff, S. L. (1994). Topographic organization of embryonic motor neurons defined by expression of LIM homeobox genes. Cell 79,957 -970.[Medline]
van Eeden, F. J., Granato, M., Schach, U., Brand, M.,
Furutani-Seiki, M., Haffter, P., Hammerschmidt, M., Heisenberg, C. P.,
Jiang, Y. J., Kane, D. A. et al. (1996). Mutations affecting
somite formation and patterning in the zebrafish, Danio rerio.Development 123,153
-164.
Varga, Z. M., Amores, A., Lewis, K. E., Yan, Y. L., Postlethwait, J. H., Eisen, J. S. and Westerfield, M. (2001). Zebrafish smoothened functions in ventral neural tube specification and axon tract formation. Development 128,3497 -3509.[Medline]
Vermeren, M., Maro, G. S., Bron, R., McGonnell, I. M., Charnay, P., Topilko, P. and Cohen, J. (2003). Integrity of developing spinal motor columns is regulated by neural crest derivatives at motor exit points. Neuron 37,403 -415.[Medline]
Westerfield, M. (1994). The Zebrafish Book, University of Oregon Press.
Wolff, C., Roy, S. and Ingham, P. W. (2003). Multiple muscle cell identities induced by distinct levels and timing of hedgehog activity in the zebrafish embryo. Curr. Biol. 13,1169 -1181.[CrossRef][Medline]