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First published online 25 October 2006
doi: 10.1242/dev.02668
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1 Centre for Regenerative Medicine, Developmental Biology Programme, Department
of Biology and Biochemistry, University of Bath, Bath BA2 7AY, UK.
2 Centre de Biologie du Développement, UMR5547, Université Paul
Sabatier, bât. 4R3, 118, route de Narbonne, 31062 Toulouse,
France.
* Author for correspondence (e-mail: bssrnk{at}bath.ac.uk)
Accepted 21 September 2006
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Sox10, Neural crest, Fate specification, Determination, Dorsal root ganglion, Neurogenin, Zebrafish, Transgene, Waardenburg-Shah syndrome
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Sensory afferent axons are myelinated by Schwann cells,
whereas DRG neuron cell bodies are surrounded by satellite glia. Both DRG
neurons and associated glia are generated from an ectodermally derived stem
cell population, the neural crest (NC).
The transcription factor Sox10 is pivotal to NC ontogeny
(Kelsh, 2006). In humans,
Sox10 mutations are associated with Waardenburg-Shah syndrome,
characterised by defects in enteric nervous system and pigmentation
(Pingault et al., 1998;
Southard-Smith et al., 1998),
and with severe dysmyelination syndromes
(Inoue et al., 1999;
Pingault et al., 2000;
Touraine et al., 2000).
Similarly, mouse Sox10 mutations exhibit similar dominant defects, as
well as an embryonic lethal recessive phenotype, primarily characterised by
widespread defects in NC derivatives, including enteric and sympathetic
ganglia, melanocytes, glia and DRG sensory neurons
(Herbarth et al., 1998;
Kapur, 1999;
Southard-Smith et al., 1998).
Subsequent studies have highlighted the importance of Sox10 for generation of
certain derivatives from the NC and, in particular, for development of glia
(Britsch et al., 2001;
Herbarth et al., 1998;
Paratore et al., 2001;
Southard-Smith et al., 1998).
Thus, homozygous Sox10 mutants lack early markers of PNS glial
progenitors (e.g. Erbb3 and Notch1), as well as glial
differentiation markers (Britsch et al.,
2001; Sonnenberg-Riethmacher
et al., 2001). Complete lack of Sox10 function results in cell
death before overt fate acquisition, and any surviving cells fail to
subsequently differentiate as glia (Britsch
et al., 2001; Kapur,
1999; Paratore et al.,
2001). Consistent with these defects, in mammals and also in
chick, Sox10 is expressed in undifferentiated NC and persistently in
mature glia, but not in mature DRG neurons
(Bondurand et al., 1998;
Britsch et al., 2001;
Cheng et al., 2000;
Herbarth et al., 1998;
Southard-Smith et al.,
1998).
Zebrafish sox10, also known as colourless (cls),
shows a strong conservation of gene expression pattern and function, and
homozygous sox10 mutants display a phenotype strikingly similar to
that of mouse Sox10 null homozygotes
(Dutton et al., 2001a;
Dutton et al., 2001b;
Kelsh et al., 1996;
Kelsh et al., 2000;
Kelsh and Eisen, 2000). As in
mouse, premigratory NC forms in normal numbers in zebrafish sox10
mutants (Dutton et al.,
2001b). Counts of NC cells (NCCs) on the medial pathway
demonstrated that DRG precursor cells migrate in normal numbers in
sox10 mutants. We recently proposed, based in part on single-cell
analysis of NCCs in sox10 mutants, that zebrafish sox10 has
a primary role in NCC fate specification, specifically in all neuronal, glial
and pigment cell lineages (Dutton et al.,
2001b; Kelsh and Raible,
2002). We have subsequently provided strong support for this model
for the melanocyte and enteric neuron lineages, showing that critical genes
encoding transcription factors required for specification of these lineages
(namely mitfa and phox2b) fail to be transcribed in the NC
of sox10 mutants (Dutton et al.,
2001b; Elworthy et al.,
2003; Elworthy et al.,
2005). Using both single-cell labelling studies and TUNEL, we have
shown that NCCs die, but this occurs in a narrow window between 35 and 45
hours post fertilisation (hpf), after they fail to become fate specified
(Dutton et al., 2001b;
Kelsh and Raible, 2002).
The sox10 mutant sensory neuron phenotype is weaker than that of
other derivatives, in both mouse and in zebrafish
(Kapur, 1999;
Kelsh and Eisen, 2000). In
mice, DRG neurons are absent posteriorly whereas anterior DRGs are reduced in
size, and initially contain apparently normal sensory neurons, yet these
eventually die (Britsch et al.,
2001; Kapur,
1999). In zebrafish, sensory neuron number is strongly reduced in
the tail, but less affected in the trunk
(Kelsh and Eisen, 2000). In
mouse, based partly on comparison to mouse Erbb3 mutants
(Riethmacher et al., 1997),
loss of trunk motor and sensory neurons in Sox10 mutants was proposed
to be a secondary consequence of the failure of differentiation of DRG
satellite glia and Schwann cells, with neuronal death due to loss of glial
trophic support (Britsch et al.,
2001). Thus, in contrast to glial fates, Sox10 was
attributed no direct role in sensory neuron development. Specification of DRG
sensory neuron fate is critically dependent upon the Neurogenin (Ngn) gene
family; encoding transcription factors, these are key regulatory genes for the
sensory neuron lineage in both mouse and zebrafish
(Blader et al., 1997;
Perez et al., 1999). Ngn genes
are expressed in a subset of NCCs early during migration, but are rapidly
downregulated in the nascent DRGs
(Greenwood et al., 1999;
Perez et al., 1999). DRG
neuron specification was not examined in mouse Sox10 mutants
(Britsch et al., 2001;
Sonnenberg-Riethmacher et al.,
2001). Hence, whether Sox10 mutants show an early
reduction in nascent sensory neurons, in addition to later neuronal death due
to absence of glial support remains untested. The DRG sensory neuron phenotype
therefore provides a critical test of the model that Sox10 is
required for fate specification of all nonskeletal NC derivatives.
Here, we analyse in detail the DRG phenotype in zebrafish sox10 mutants. We generate a sox10:egfp line to allow in vivo observation of PNS glia, and show defects in both DRG-associated Schwann cells and satellite glia in sox10 mutants. We show that neither motorneuron nor residual DRG neuron survival depends upon proximity to differentiated glia. Importantly, we show quantitatively that DRG neuron specification is defective in sox10 mutants, and that Sox10 is able to induce ngn1 (neurog1 - Zebrafish Information Network) robustly and cell autonomously. Furthermore, we present evidence for early, but only transient, expression of Sox10 in DRG precursors. Finally we introduce a new sox10 allele that shows a neurogenic DRG phenotype, underscored by an excess of ngn1-positive cells, but again without glial support. Thus, Sox10 is required for specification of DRG neurons and hence plays an active role in the generation of all cell types of the zebrafish DRG.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). sox10 alleles clst3 or
clsm618 were used interchangeably; they show equally
strong DRG phenotypes (Dutton et al.,
2001b) (G. Ford, K.A.D. and R.N.K., unpublished). The
-3.1ngn1:gfp transgenic line has been described previously
(Blader et al., 2003).
Constructs
Constructs used in this study include pCSHSP, hs>sox10 and
hs>sox10m618(L142Q)
(Dutton et al., 2001b). A
sox10 reporter construct, p-4.9sox10:egfp, was generated
using 4.9kb of zebrafish sox10 promoter sequence upstream of the
start of translation, subcloned as a BamHI-SpeI fragment
from a sox10 containing PAC (RZPD, Germany). This fragment was
inserted in front of the egfp gene in the vector
XLT.GFPLT.CS2+ (kind gift of R. Moon), using
conventional cloning methods (Sambrook,
1989).
Morpholino and DNA injections
Morpholinos obtained from Gene Tools (Corvallis, OR) were used as described
previously (Dutton et al.,
2001a; Andermann et al.,
2002). Sox9b morpholinos were as follows:
Sox9b.MO1, 5'-TGTGTGTGTGTGTGTGTGTGAGCAC-3';
Sox9b.MO2: 5'-AGCTGCTGAAACACACACAGATCCT-3'; and 4MM
Sox9b.MO2, 5'-AGCTcCTGAtACACAgACAGtTCCT-3' (mismatched
bases shown in lower case). Only embryos injected with the Sox10 morpholino
showing a clear melanophore phenotype were subsequently analysed. Plasmid DNA
was prepared using the Wizard Midi system (Promega, Madison, WI) and diluted
to a concentration of 25 ng/µl. Both DNA and morpholino solutions were
supplemented with 0.1% phenol red.
Ectopic expression in zebrafish embryos by heat shock
Wild-type embryos were injected with 50-75 pg of pCSHSP,
hs>sox10 or hs>sox10m618(L142Q) and
incubated at 28.5°C until 2 hpf, heat-shocked by incubation at 37°C
for 2 hours and then fixed after a further 30 minutes at 28.5°C.
Generation of a transgenic line
To generate a stable transgenic line, the p-4.9sox10:egfp
construct was linearised and 50-80 pg of DNA injected per embryo. Offspring of
incrosses of raised injected fish were screened by fluorescent microscopy for
GFP and a stable line, designated Tg(-4.9sox10:egfp)ba2,
generated.
Mutagenesis and sox10 allele screening
Forty adult male Tübingen fish were mutagenised using ENU according to
the established protocol (Haffter et al.,
1996). Fifteen surviving F0 males were crossed to AB wild-type
females at weekly intervals and progeny raised as separate F1 families. New
sox10 alleles were isolated by non-complementation screen using carriers for
clst3, clstw2 or clsty22f.
The sox10baz1 mutation was identified by directly
sequencing RT-PCR products amplified from cDNA.
Whole-mount in situ hybridisation, antibody staining and TUNEL analysis
RNA in situ hybridisation was performed largely as previously described
(Kelsh and Eisen, 2000),
except that a tenfold concentration of Proteinase K was used on 5 days post
fertilisation (dpf) embryos. Probes used were ngn1
(Blader et al., 1997),
sox10 (Dutton et al.,
2001b) and mbp
(Brosamle and Halpern,
2002).
Single or double antibody staining was performed largely as previously
described (Ungos et al.,
2003). Primary antibodies used were anti-Hu (1:700 mAb 16A11)
(Marusich et al., 1994),
anti-DM-GRASP (1:400 mAb zn-5) (Fashena
and Westerfield, 1999), anti-Islet1 (1:200 mAb 4D5; Developmental
Studies Hybridoma Bank DSHB), anti-Sox10
(Park et al., 2005),
anti-phospho-histone H3 (1:1000; Upstate Biotechnology, NY), anti-Fluorescein
(1:400, Molecular Probes, Eugene, OR) and anti-GFP (1:200; Molecular Probes).
Fluorescent visualisation used Alexa488- or Alexa546-conjugated secondary
antibodies diluted in blocking solution (1:750; Molecular Probes).
TUNEL was performed as previously described, using fluorescein-11 dUTP,
imaged by fluorescent microscopy (Dutton
et al., 2001b).
Cell transplantation
For chimaera experiments, donor embryos were injected with 0.1% 10,000 MW
fluorescein dextran (Molecular Probes) at the one- to two-cell stage. At
approximately 30% epiboly, 15-20 cells were transplanted to shield stage hosts
into the presumptive NC domain. Donors derived from
sox10+/- incrosses were genotyped by PCR immediately
following transplantation. Host embryos were raised separately to 3 dpf,
pooled according to donor genotype where appropriate, fixed and
immunofluorescently stained for fluorescein and Hu. Donor-derived DRG neurons
were counted and compared with a Mann-Whitney test.
Microscopy and statistical analysis
Fluorescent images were taken with a Zeiss Confocal microscope (LSM510) or
on an Eclipse E800 (Nikon) microscope using appropriate filters and a SPOT
digital camera (Diagnostic Instruments). Screening for GFP transgenics was
performed on an MZ12-FL dissecting microscope (Leica) with fluorescent
attachment.
Statistical analysis was done with the Prism Statistical Package (GraphPad, San Diego, CA).
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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), being visible from around the one-somite stage in
two stripes lateral to the anterior neural plate (data not shown);
subsequently GFP expression was seen in both premigratory and migratory NCCs
(Fig. 1B-E), and reveals
details of cell morphology (Fig.
1C-E). At 24 hpf, expression mostly resulted from perdurance of
GFP from premigratory stages, but was strong in all cells, including in
branchial arch cartilage progenitors (Fig.
1E,F) (see also Wada et al.,
2005). GFP was later detected in NCCs on both the lateral (not
shown) and the medial (Fig. 1H)
NC migration pathways and in satellite cells of the cranial ganglia
(Fig. 1G). GFP was prominent in
all PNS glial precursors, including those of Schwann cells of the posterior
lateral line and spinal nerves (Fig.
1I,J,K) and satellite glia within the DRGs
(Fig. 1J,
Fig. 4C). At 5 dpf, GFP
expression clearly revealed the compacted morphology of the satellite glia in
the DRGs, but was absent from Hu+ cells within. Expression in
peripheral glia was strongly maintained to 10 dpf in posterior lateral line
nerve Schwann cells (data not shown). Similar GFP expression patterns have
been observed in multiple other sox10 transgenic lines (T.J.C., J.
Dutton and R.N.K., unpublished). Thus, this transgenic line (allele
designation Tg(-4.9sox10:egfp)ba2), which we here call
sox10:egfp, labels all DRG glia and NC precursors.
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). In
contrast, GFP+ cells were associated with spinal nerves in all
segments of sox10 mutants (Fig.
2B). Thus, even in sox10 mutants, NCCs migrate along the
medial pathway and remain in position along the spinal nerves. However, these
GFP+ cells were not normally differentiated. First, whereas in the
wild type these cells showed a consistent smooth morphology, with Schwann
cells organised along the middle of each somite segment and satellite glia
compacted around the sensory neurons (Fig.
2A), in sox10 mutants both the morphology and
organisation of cells associated with the nerve was disrupted, with
GFP+ branches diverging from the medial position. Further, no
morphologically normal satellite glia could be seen in any somite segment
(Fig. 2B). Finally, mRNA in
situ hybridisation detection of myelin basic protein (mbp),
currently the only zebrafish Schwann cell differentiation marker
(Brosamle and Halpern, 2002),
revealed expression in wild-type spinal nerve Schwann cells
(Fig. 2C), but was undetectable
in sox10 mutants (Fig.
2D). Thus, GFP+ NCCs near the spinal nerves failed to
differentiate as Schwann cells in sox10 mutants. We also observed an
absence of mbp expression in the oligodendrocytes of the CNS
(Fig. 2D). Thus, as in mouse
Sox10 mutants, differentiated PNS glia and CNS oligodendrocytes are
absent in zebrafish sox10 mutants.
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), sox10 siblings showed both abnormal
placement of and a strong reduction in numbers of DRG neurons and ganglia
(Fig. 3B,C). For example, at 5
dpf, sox10 mutants show only approximately 25% of the number of
neurons of their wild-type siblings. Individual segments showed different DRG
patterns, with Hu+ cell number entirely absent, reduced or normal;
we could detect no clear pattern to the distribution of each of these,
although fewer cells were detected posteriorly. A time-course study showed
that sensory neuron number increased with time in wild-type embryos, but
remained consistently reduced in mutants
(Fig. 3C). In mouse
Sox10 mutants, DRG sensory neurons degenerate, and this phenotype is
attributed to lack of Schwann cell-derived trophic support
(Britsch et al., 2001). To test
whether apoptosis of newly generated Hu+ sensory neurons made a major
contribution to the sensory neuron phenotype of zebrafish sox10
mutants, we used TUNEL combined with immunofluorescent detection of Hu to
label sensory neurons between 48 hpf and 6 dpf. All Hu+ DRG neurons
throughout the trunk and tail of each embryo were examined for TUNEL. Although
small numbers of apoptosing Hu+ neurons were seen in cranial
ganglia and in the brain of both wild-type and sox10 mutant embryos,
apoptosing DRG sensory neurons were seen only occasionally (0-2
TUNEL+;Hu+ DRG neuron per embryo) in wild type and not
at all in sox10 mutants at these stages
(Table 1). Thus, although a
strong DRG sensory neuron sox10 phenotype is conserved in zebrafish,
in contrast to mouse Sox10 mutants
(Britsch et al., 2001),
apoptotic loss of differentiated sensory neurons is unlikely to explain the
sensory neuron phenotype prior to 6 dpf.
|
). To
test whether changes in proliferation of DRG neurons might contribute to the
sox10 mutant phenotype, we used double immunofluorescent detection of
phosphoHistone H3 to detect proliferation combined with Hu to label neurons
(Table 2 and Fig. S2 in the
supplementary material). Intriguingly, we observed a significantly elevated
level of DRG neuron proliferation in sox10 mutant embryos at 60 hpf,
although rates were identical at 72 hpf. Thus, changes in proliferative rates
cannot explain the sox10 mutant DRG neuron phenotype.
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Early markers of sensory neuron specification are reduced in sox10 mutants
Together, these observations argued against the proposal that DRG sensory
neurons were depleted in zebrafish sox10 mutants as a secondary
consequence of loss of peripheral glial cells. Consequently, we tested whether
sox10 might be required directly for specification of sensory
neurons, by examining ngn1 expression. In wild type, ngn1
expression was seen in a reiterated series of cells lying ventrolateral to the
spinal cord, whereas sox10 mutants show frequent gaps in this pattern
(Fig. 5A,B). Quantitation of
these cells at 36 hpf revealed significant deficiencies (52% reduction
compared with wild-type siblings) of ngn1+ cells in
sox10 embryos, even at this very early stage
(Fig. 5C). To rule out the
possibility that ngn1+ cells were simply obscured due to
delayed migration, we used a ngn1:GFP reporter line, which shows
strong GFP expression in nascent DRG neurons
(Fig. 5D)
(Blader et al., 2003). We used
a sox10 morpholino (Dutton et
al., 2001a) to knock down Sox10 in this transgenic reporter line.
As a positive control for successful Sox10 knock down, we noted the
characteristic strong reduction in pigment cell numbers, consistent with the
sox10 phenotype (compare left panels in
Fig. 5D,E). At 48 hpf,
uninjected embryos or embryos injected with a mismatch morpholino showed
normal pigmentation and a reiterated series of GFP+ cells alongside
the neural tube in a position consistent with forming DRG neurons
(Fig. 5D). In sox10
morpholino-injected embryos showing a strong pigment phenotype, we observed
significant loss (85% reduction) of GFP+ DRG neuron precursors at
all axial levels (Fig. 5F),
although the dorsal CNS GFP expression domain remained unaffected
(Fig. 5E). There was no sign of
misplaced cells, arguing against defects in sensory neuron migration. Instead,
we conclude that ngn1 transcription fails in the absence of Sox10
function.
|
). To test whether the remaining Hu+ DRG
neurons in sox10 mutants were also ngn1 dependent or somehow
formed independently of ngn1, we used morpholino-mediated Ngn1 knock
down. sox10 mutants injected with a ngn1 morpholino
consistently showed a significant reduction (by 91%) of Hu+ DRG
neurons at 5 dpf compared with uninjected sox10 mutants
(Fig. 5G-I). Thus, the
remaining Hu+ neurons in sox10 mutants were dependant on
ngn1 for their development.
Residual Hu+ DRG neurons in sox10 mutants are Sox9b dependent
We then asked whether functional redundancy between SoxE genes
might explain the relatively weak sensory neuron phenotype in sox10
mutants by knocking down Sox9b. sox9b is expressed transiently in
premigratory NCCs (Chiang et al.,
2001; Li et al.,
2002; Yan et al.,
2005). Injection of 9 ng of sox9b morpholino into
wild-type embryos gave the previously reported small head and down-curved tail
phenotype (Chiang et al.,
2001; Li et al.,
2002; Yan et al.,
2005). Interestingly, these embryos also showed a small reduction
of Hu+ DRG neurons and some disorganisation of the remaining cells
(Fig. 6A,B) compared with
uninjected siblings. When injected into sox10 mutant embryos, this
same dose of sox9b morpholino resulted in a dramatic phenotype,
showing the small head and curved tail, but also strongly accentuating the
sox10 mutant DRG neuron phenotype
(Fig. 6C,D). We saw similar DRG
neuron phenotypes with either of two non-overlapping sox9b
morpholinos, whereas an 18 ng dose of a 4 bp-mismatch morpholino had no effect
(Fig. 6E).
GFP perdurance reveals activity of the sox10 promoter in DRG sensory neuron precursors
If the effect on DRG neurons seen in sox10 mutants does not
reflect a lack of glial trophic support, perhaps Sox10 acts autonomously in
the sensory neuron lineage. This requires that DRG neuron precursors express
Sox10 at some stage. Consequently, we used double immunofluorescent detection
of Hu antigen and GFP in sox10:egfp embryos to mark sox10
expression cell autonomously. Given the perdurance of GFP, we anticipated that
the strong GFP expression in premigratory NCCs would allow use of GFP as a
lineage tracer to determine if the sox10 promoter was active at some
stage in the development of DRG neurons. At 5 dpf, GFP signal is absent from
DRG neurons (Fig. 1J). However,
48 hpf embryos consistently showed double-labelled NCCs in nascent DRGs
(Fig. 7D). These Hu+
GFP+ DRG neurons were surrounded by Hu-GFP+
cells, which likely represent glial lineages
(Fig. 7E). We conclude that the
sox10 promoter was transiently active in DRG sensory neuron
progenitors, and thus infer that these cells expressed Sox10 transiently.
We then used a direct approach to confirm Sox10 expression in sensory
neuron precursors by using a polyclonal serum that recognises zebrafish Sox10
(Park et al., 2005) on the
ngn1:GFP reporter line, as ngn1 is the earliest available
marker for sensory neuron precursors. We assessed cells for co-expression of
GFP and Sox10 and noted that 7/26 (27%) and 10/52 (19%) GFP+
nascent DRG sensory neurons were expressing detectable Sox10 protein in 40 and
42 hpf embryos, respectively (Fig.
7F). Interestingly, the GFP+;Sox10- cells
tended to show rather stronger GFP expression, suggesting that they were more
mature, consistent with the suggestion that Sox10 is rapidly downregulated in
neuronal precursors.
Ectopic Sox10 expression induces ectopic ngn1
We then asked if Sox10 expression was sufficient to induce ngn1
expression. Embryos were injected with heat-shock constructs driving Sox10 or
various control constructs (Dutton et al.,
2001b), heat shocked at 37°C for 2 hours, then fixed and
examined for ectopic ngn1 transcripts by in situ hybridisation.
Embryos injected with the hs:sox10 construct reproducibly contained
many cells with strong ngn1 expression, whereas uninjected embryos or
embryos injected with an empty heat-shock vector showed none
(Fig. 7A,B). Interestingly,
embryos injected with a heat-shock construct driving expression of a mutant
Sox10 protein [hs>sox10m618(L142Q)]
(Dutton, et al., 2001b) only
weakly induced ngn1, presumably due to low level residual activity of
this protein. Injected, but not heatshocked, embryos showed greatly reduced
ngn1 induction (data not shown). Further, double in situ
hybridisation analysis showed that cells expressing ngn1 almost
always also contained detectable sox10 message
(Fig. 7C). Thus, Sox10
functioned cell autonomously to induce ngn1 expression.
|
). We used cell transplantation from sox10
mutants or their wild-type siblings into wild-type embryos to test whether the
wild-type environment is able to support sox10 mutant cells as well
as wildtype cells. sox10 mutant donor cells generated significantly
fewer sensory neurons compared with wild-type sibling donor cells in wild-type
host embryos, consistent with a cell-autonomous role in DRG sensory neurons
(see Table S1 in the supplementary material). In addition, experiments
transferring wild-type cells into sox10 mutant hosts provided two
clear cases of hosts containing a DRG with multiple wild-type glial cells, but
which failed to rescue sensory neuron survival, a result inconsistent with the
trophic support model, but consistent with our fate specification model (see
Fig. S1 in the supplementary material).
A new sox10 allele with a neurogenic sensory neuron phenotype
In a sox10 allele screen we recovered one allele,
sox10baz1, that showed a unique phenotype compared with
all reported mouse or zebrafish sox10 alleles. Like other zebrafish
sox10 alleles, sox10baz1 is fully recessive and
homozygous lethal. sox10baz1/baz1 embryos have a strong
melanophore phenotype similar to other reported sox10 alleles,
although other pigment cell types are less affected, with xanthophores and
iridophores mildly reduced (Fig.
8A,B; data not shown). In situ hybridisation using mbp
probe showed the complete absence of PNS Schwann cells in
sox10baz1/baz1 mutants, similar to the phenotype of strong
sox10 alleles (Fig.
8C,D). Hence, glial and melanophore phenotypes were strongly
hypomorphic, and xanthophore and iridophore phenotypes more weakly so. In
striking contrast to any previously described sox10 phenotype, 5 dpf
sox10baz1/baz1 embryos were hypermorphic for DRG sensory
neurons. Thus, instead of the strong decrease in Hu+ DRG sensory
neurons seen in other strong sox10 mutant alleles,
sox10baz1/baz1 embryos showed a 98% increase in their
number compared with wild-type siblings
(Fig. 8E-H,K). This phenotype
was specific to sensory neurons, because as with the other sox10
alleles, sox10baz1/baz1 mutants also had no enteric
neurons (Fig. 8E, arrow). The
supernumerary DRG neurons survived until at least 5 dpf, despite the absence
of fully differentiated Schwann cells. To confirm its identity as a new
sox10 allele, we sequenced sox10 cDNA isolated from
sox10baz1/baz1 embryos. We found a G to A substitution at
position 724 (Fig. 8M), which
creates a Valine to Methionine substitution within the HMG domain of the Sox10
protein at amino acid position 117 (Fig.
8M). This position is fully conserved between human, mouse and
chicken Sox10 protein sequences, and is also within one of two nuclear
localisation sequences in the HMG box.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Sonnenberg-Riethmacher et al.,
2001). These authors noted the similar phenotype to that of
Erbb3 mutants and proposed that DRG neuron and motorneuron deficits
were likely secondary effects of loss of glial support. However, these same
studies did not assess neuronal number quantitatively, nor did they evaluate
the tail where the DRG deficit is more severe
(Kapur, 1999). Thus, a role
for Sox10 in fate specification of sensory neurons, as recently hypothesised
(Dutton et al., 2001b;
Kelsh and Raible, 2002), was
not eliminated by these studies.
The zebrafish DRG phenotype shows many similarities to that in mouse,
suggesting that an essential role for sox10 in DRG development is
conserved throughout vertebrates. Thus, here we have shown a similar absence
of glial differentiation, combined with expression of sensory neuron markers
in residual DRG neurons in zebrafish sox10 mutants. We have also
previously demonstrated that undifferentiated cells contributing to the
nascent DRG of sox10 mutants undergo apoptosis
(Dutton et al., 2001b).
However, although we have shown a quantitative reduction in DRG sensory
neurons, we have also shown that there is no elevated apoptotic loss of
Hu+ DRG sensory neurons up to 6 dpf in sox10 mutants.
Furthermore, using markers of motorneuron cell bodies and secondary
motorneuron axons we see normal numbers of motorneurons to at least 10 dpf. We
suggest that the timing of onset of glial dependency for trophic support of
sensory and motorneurons may be relatively later in zebrafish than in mice.
Consistent with this, residual sensory neurons were seen in the absence of
cells showing normal glial differentiation, and were often distant from even
undifferentiated GFP+ NCCs remaining on the medial pathway.
Reconstitution of a wild-type glial environment in chimaeras was insufficient
to rescue the sox10 mutant sensory neuron phenotype. Furthermore,
sox10 mutant NCCs generate sensory neurons with decreased efficiency
in a wild-type environment. In conclusion, although zebrafish sox10
mutants show equally dramatic sensory neuron defects to mouse Sox10
mutants, secondary effects due to absence of differentiated glial cells do not
explain the sensory neuron defects. Instead our data strongly indicate a
direct role for Sox10 in sensory neuron specification.
|
;
Lee et al., 2004;
Ma et al., 1999;
Perez et al., 1999). We tested
the mechanistic basis for the sensory neuron phenotype in sox10
mutants, especially the more severe phenotype in the tail and, in particular,
our hypothesis that sox10 might have a role in DRG sensory neuron
fate specification (Dutton et al.,
2001b; Kelsh and Raible,
2002). In contrast to the predictions of the trophic support
model, our fate specification model predicted that sox10 mutants
should show very early defects in sensory neuron markers, with fewer cells
expressing the sensory neuron specification gene ngn1 at its time of
onset. Furthermore, sensory neurons would be derived from
sox10-expressing NCCs, and sox10 expression should be
sufficient, at least in some cells, to drive ngn1 transcription cell
autonomously. We have tested all of these predictions and have shown here that
all are fulfilled. We conclude, therefore, that sox10 is required for
specification of sensory neurons from the NC, acting at the level of
initiation of ngn1 transcription. Thus, sox10 functions
directly in sensory neuron development, just as has been previously
demonstrated for the melanocyte lineage
(Bondurand et al., 2000;
Dutton et al., 2001b;
Elworthy et al., 2003;
Lee et al., 2000;
Potterf et al., 2000;
Verastegui et al., 2000). In
the case of melanocytes, Sox10 acts directly on the mitf/mitfa
promoter to activate expression (Bondurand
et al., 2000; Potterf et al.,
2000; Verastegui et al.,
2000; Elworthy et al.,
2003). Regulation of ngn1 has been investigated in the
CNS (Blader et al., 2004;
Blader et al., 2003), but not
in DRG sensory neuron precursors, so it remains to be tested whether Sox10
directly activates the ngn1 promoter, although we note that our
ngn1:gfp transgene studies localise a putative responsive region
within the ngn1 promoter and that in this fragment lie multiple
Sox-binding sites (P.D. and P.B., unpublished).
Whereas knock down of ngn1 results in absence of DRG neuron
precursors, the strong sox10 mutant alleles in zebrafish consistently
show only a partial reduction. Other mechanisms specifying DRG sensory neurons
may also contribute to the weaker phenotype of these sox10 mutants.
In particular, we have shown here that functional redundancy with
sox9b, which is also expressed in premigratory NC, plays a role
(Chiang et al., 2001;
Li et al., 2002;
Yan et al., 2005). Regardless
of how they are formed, we have shown here that the residual sensory neurons
in sox10 mutants are ngn1-dependent. It is likely that the
residual ngn1+ cells seen on the medial migration pathway
in sox10 mutants generate the remaining Hu+ neurons seen
at later stages.
|
). This
led to the prediction that, at least under certain conditions, excess DRG
neurogenesis might be seen in mice with reduced Sox10 activity. The zebrafish
sox10baz1 mutant shows in vivo the excess neurogenesis
phenotype predicted from these in vitro studies. At a molecular level, this
mutation is a point mutant generating a single amino acid substitution in the
DNA binding domain. Phenotypically, it generally behaves as a hypomorphic
allele, having a somewhat weaker phenotype than the strong sox10
mutant alleles published before (Kelsh et
al., 1996; Malicki et al.,
1996). The mechanistic basis for the hypermorphic sensory neuron
phenotype remains unclear, but the neurogenic nature suggests an involvement
of the Notch-Delta signalling system, already implicated in sensory neuronal
and glial fate specification (Morrison et
al., 2000; Wakamatsu et al.,
2000), and may give insight into the interactions between the
Sox10-Ngn1 and Notch-Delta pathways. The soxbaz1 mutant
phenotype gives strong confirmation that Hu+ sensory neurons do not
require differentiated glia for survival and that levels of sox10
activity help regulate levels of sensory neuron specification.
This study extends the evidence for a general model of Sox10 function in NC
derivative specification (Kelsh,
2006). Key lineage specification transcription factors for each of
the sensory and enteric neuron and melanophore cell types have been shown to
require Sox10 for their expression in zebrafish (this work)
(Elworthy et al., 2003;
Elworthy et al., 2005), and in
mouse similar defects in sympathetic neuron and melanocyte fate specification
have been shown (Kim et al.,
2003; Lee et al.,
2000; Potterf et al.,
2000; Verastegui et al.,
2000). It will be of interest to use quantitative analysis of
mouse Sox10 mutants to test whether DRG sensory neuron specification
is affected.
Given that Sox10 is required for multiple diverse cell types, it is clear
that this transcription factor alone is insufficient to explain the logic of
NC fate specification. In general, fate specification of NC derivatives
requires both intrinsic factors and extrinsic signals
(Kelsh and Raible, 2002;
Le Douarin and Kalcheim,
1999). In the case of DRG sensory neurons, both Shh and Wnt
signalling also influence selection of that fate
(Lee et al., 2004;
Ungos et al., 2003), yet these
same signals are required for fate specification of melanocytes
(Bondurand et al., 2000;
Dorsky et al., 2000;
Elworthy et al., 2003;
Lee et al., 2000;
Potterf et al., 2000;
Takeda et al., 2000;
Verastegui et al., 2000;
Yasumoto et al., 2002). There
clearly remain further factors for both melanocyte and sensory neuron
specification to identify. As Sox proteins are known to interact with other
partner proteins (Wegner and Stolt,
2005), these are likely to have crucial influences on the response
of NCCs to intrinsic and extrinsic factors mediating fate choice.
Identification of the full complement of these factors will be necessary for a
comprehensive understanding of the logic of NCC fate specification.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/23/4619/DC1
|
ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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