1 McLaughlin Research Institute for Biomedical Sciences, 1520 23rd Street South,
Great Falls, MT 59405, USA
2 Department of Biomedical Sciences, Creighton University, 2500 California
Plaza, Omaha, NE 68178, USA
* Author for correspondence (e-mail: pxu{at}po.mri.montana.edu)
Accepted 9 September 2004
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SUMMARY |
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Key words: Eya1, Six1, Otic, Epibranchial, Placode, Sensory neurons, Neurogenesis, Neurogenins, bHLH protein, Phox2a, Phox2b, Cranial nerve patterning
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Introduction |
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Targeted mutagenesis in mice has shown that the basic helix-loop-helix
(bHLH) transcription factor neurogenins (Neurogs) play a critical role in
early development of the placodally derived cranial sensory neurons. Neurog1
and Neurog2 are expressed in the cranial placodes as early as E8.5 and act as
critical determination factors for cell fate commitment
(Fode et al., 1998;
Ma et al., 1998
;
Ma et al., 1999
;
Ma et al., 2000
). While
Neurog1 is required for the formation of the V and VIII sensory neurons
(Ma et al., 1998
), Neurog2 is
necessary for normal development of the epibranchial placode-derived sensory
neurons of VII, IX and X (Fode et al.,
1998
). Recent studies have found that Neurogs may activate a
cascade of downstream bHLH factors, including Math3, Neurod and Nscl1, to
promote the development of the placode-derived sensory neurons
(Fode et al., 1998
;
Ma et al., 1998
). Neurogs are
necessary for normal expression of these factors, but it is unclear whether
all these genes act in a linear pathway and none of them has been proven to be
a direct target of Neurog1 or Neurog2.
The epibranchial placode-derived sensory neurons of the distal VII, IX, and
X ganglia also depend on the paired homeodomain transcription factors Phox2a
and Phox2b for their differentiation and survival. Phox2a has been
shown to function upstream of Phox2b
(Pattyn et al., 1999) and in
Phox2a/ and
Phox2b/ mice, epibranchial placodes give
rise to the normal number of neuroblasts, but these neuroblasts fail to
activate their more specific differentiation program
(Morin et al., 1997
;
Pattyn et al., 1999
;
Pattyn et al., 2000
).
Epibranchial placodal sensory precursors require Phox2a for a subprogram of
neuron-subtype-specific gene expression that can be genetically independent
from a subprogram for pan-neuronal gene expression, which requires Neurog2
function (Fode et al., 1998
).
However, details of the Neurogs and Phox2 genes' mode of
action in neuronal cells have not been elucidated.
The murine Eya gene family, homologous to eyes absent
(eya), which is required for normal eye development in
Drosophila (Bonini et al.,
1993), is composed of four members (Eya1-4) and encodes a
transcriptional co-activator containing a conserved C-terminal Eya domain
involved in protein-protein interaction, and a divergent N-terminal
transactivation domain (Xu et al.,
1997a
; Xu et al.,
1997b
; Borsani et al.,
1999
). The conserved Eya domain of Eya proteins interacts with
Sine oculis (So) or Dachshund (Dach), other regulators for Drosophila
eye development (Chen et al.,
1997
; Pignoni et al.,
1997
). Eya1 genes are expressed in multiple domains
including many ectodermal placodes and this expression pattern appears to be
conserved from Xenopus and zebrafish to mammals
(Xu et al., 1997a
;
Sahly et al., 1999
;
David et al., 2001
;
Schlosser and Ahrens, 2004
).
Among the cranial placodes, Eya1 has been shown to play an essential
role in otic placode development (Xu et
al., 1999
; Zheng et al.,
2003
). Six1, a member of the Six gene family homologous
to Drosophila so, encodes a homeodomain protein and its gene product
physically interacts with Eya1 (Buller et
al., 2001
). During otic placodal development, Six1
functions downstream of and genetically interacts with Eya1
(Zheng et al., 2003
).
Interestingly, Six1-deficient mice show defects in all three parts of
the ear similar to that observed in Eya1 mutants
(Xu et al., 1999
;
Zheng et al., 2003
). However,
it is unknown whether Eya1 and Six1 play a role in the generation of otic and
other placode-derived sensory neurons.
Placodal and neural crest-derived ganglia appear to be necessary for
trigeminal branchial motoneurons to exit the brain
(Moody and Heaton, 1983;
Ma et al., 2000
). Absence or
lack of differentiation of all VII and VIII ganglia
(Zheng et al., 2003
) may not
only alter the trajectory of inner ear efferents into the facial nerve
(Ma et al., 2000
), but may
cause redirection of facial branchial motoneuron fibers into still existing
cranial nerves. Eya1- and Six1-null mutants could therefore
provide a test for the hypothesis that all branchial motoneurons require
sensory neuron fibers to grow along to the peripheral nerves
(Fritzsch and Northcutt,
1993
).
In this study, we address whether Eya1 and Six1 are required for specific placodal precursor specification, survival and differentiation, and for branchial motoneurons exiting from the brainstem. We found that in addition to affecting VIII ganglion formation, these genes control epibranchial placode neurogenesis. During inner ear neurogenesis, Eya1 and Six1 appear to be dispensable for the initiation of neurogenesis, but both may regulate the progressive differentiation of neuroblast precursor cells. Strikingly, in contrast, the mutant epibranchial placodal progenitor cells fail to express both neuronal fate and neuronal subtype identity of the epibranchial placode-derived sensory lineage due to defective expression of Neurog2 and Phox2a. Shortly thereafter, increased cell death is detected in the mutant placodal ectoderm, suggesting that in the absence of their normal developmental program, the placodal progenitor cells undergo apoptosis. Moreover, whole-mount staining with the neuroglial marker Sox10 and neurofilament antibody at E10.5, and retrograde as well as anterograde labeling of motor neurons at E13.5 indicated that both Eya1 and Six1 regulate the proper routing of facial branchial motoneurons. Together, our analyses establish that Eya1 functions upstream of Six1 and both act as critical determination factors for epibranchial placodal progenitor cells to acquire both neural fate and subtype sensory identity.
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Materials and methods |
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Phenotype analyses and in situ hybridization
Embryos for histology and in situ hybridization were dissected out in PBS
and fixed with 4% paraformaldehyde (PFA) at 4°C overnight. Embryonic
membranes were saved in DNA isolation buffer for genotyping. For whole-mount
and section in situ hybridization, we used 4-6 wild-type or mutant embryos at
each stage for each probe as described (Xu
et al., 1997a).
Antibody staining and neuronal tract tracing
Whole-mount immunostaining using the anti-neurofilament monoclonal antibody
2H3 (Developmental Studies Hybridoma Bank) was performed as described
(Mark et al., 1993).
For neuronal tracing, E13.5 embryos were dissected, fixed in 4% PFA and
labeled using fast diffusion of lipophilic dyes as described previously
(Maklad and Fritzsch, 2003a).
Embryos were dissected and the dye-filled profiles were viewed with a BioRad
2000 confocal microscope.
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Results |
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|
|
Degeneration of neuroblasts in the developing VIIIth ganglion in Eya1 and Six1 mutants
Because we failed to detect the VIIIth ganglion in older
Eya1/ and
Six1/ embryos at its normal location on
histological sections, it is possible that neuroblast cells observed in the
younger mutant embryos degenerate and thus fail to form a morphologically
detectable ganglion. We therefore sought to determine whether the neuroblasts
in the developing VIIIth ganglion undergo abnormal cell death in the mutants.
Transverse sections of E8.5 to 9.5 normal and mutant embryos were processed
for the TUNEL detection method of apoptotic nuclei. Cell death in the VIIIth
ganglion anlage in Eya1/ and
Six1/ embryos was first observed at around
E9.25 and became apparent by E9.5 in both mutants (arrow,
Fig. 1Q,R), whereas very few
apoptotic cells were seen in the controls
(Fig. 1P). Thus, the defective
formation of the VIIIth ganglion can be attributed, at least in part, to
increased cell death.
Distinct phenotype in the distal cranial ganglia between Eya1 and Six1 mutant mice
To determine whether the formation of other cranial sensory ganglia is also
affected in the mutants, we performed whole-mount staining with
SCG10, an early marker of differentiating neurons, to label the
cranial sensory ganglia. At E10.5, SCG10 expression in the Vth,
VIIIth and distal VIIth, IXth, and Xth ganglia was readily visible in normal
embryos (Fig. 2A). However,
Eya1/ and
Six1/ mutant precursors in the forming
VIIIth ganglion anlage failed to express SCG10
(Fig. 2B,C). Strikingly,
Eya1/ embryos also lacked SCG10
expression in the epibranchial placode-derived ganglia
(Fig. 2B), whereas its
expression was present in the Eya1/ Vth
ganglion with slight reduction. Similarly, SCG10 staining was visible
in the Six1/ Vth ganglion, but was
undetectable in the Six1/ VIIth, and reduced
in the IXth and Xth ganglia of all six embryos analyzed
(Fig. 2C). These results
indicate that in addition to the defective formation of the VIIIth ganglion in
Eya1 and Six1 mutants, the formation of epibranchial
placode-derived ganglia, which are specified by the bHLH transcription factor
Neurog2, also requires Eya1 and Six1 function. The lack of expression of the
early neural differentiation marker SCG10 in the mutant embryos
suggests that Eya1 and Six1 are required for overt neural
differentiation of the precursors of cranial sensory ganglia in E9.5 to 10.5
embryos. These data demonstrate that Eya1 is essential for the formation of
all epibranchial placode-derived distal ganglia, while Six1 may regulate the
formation of subsets of the sensory neurons.
|
As all Eya1/ embryos at E9.5-10.0 lacked Neurog2 expression, we sought to determine the onset of the developmental arrest by examining Neurog2 expression at earlier stages, from E8.5 to 9.5 using section in situ hybridization. Neurog2 expression was strongly detected in the epibranchial placodes (Fig. 2G,J,M). Strikingly, no Neurog2 expression was detected in these structures of Eya1/ embryos at these stages (Fig. 2H,K,N), differing from the obvious Neurog1 expression in the otic ectoderm. In all three Six1/ embryos, only residual Neurog2 expression was observed in the VIIth precursors (Fig. 2I), and its expression was also reduced in the IXth but was relatively normal in Xth precursors (Fig. 2I,L,O), similar to that observed by whole-mount staining (Fig. 2F). This result suggests that the activation of Neurog2 expression in epibranchial placodes requires Eya1 function.
Since deletion of Neurog2 results in a transient loss of neural
fate in distal VII and IX ganglia at early stages, and the development of
these ganglia appears to recover at later stages in
Neurog2/ animals, probably due to functional
compensation by Neurog1 (Fode et
al., 1998; Ma et al.,
1998
; Ma et al.,
1999
), we therefore analyzed the other neuronal bHLH genes in the
mutant embryos at E9.5 to 10.5 to further define the developmental failure in
Eya1 and Six1 mutant epibranchial placodes. Transcripts for
Neurog1, which is weakly coexpressed with Neurog2 in
placodal cells and strongly in the migratory neuronal precursors
(Fig. 3A,D), were not detected
in Eya1/ distal VII, IX or X precursors
(Fig. 3B,E and data not shown).
Consistent with the observation of variable phenotype in the VIIth ganglion of
Six1/ embryos detected with the
Neurog2 probe, two of six Six1/
embryos showed a few Neurog1-positive cells in the VIIth ganglion
anlage bilaterally (Fig. 3C)
and the other four embryos lacked Neurog1 expression (data not
shown). Similarly, Neurog1 expression was markedly reduced in the
IXth (Fig. 3F) and present at
lower levels in the Xth precursors in all six
Six1/ embryos (data not shown). In sections
through E9.5 wild-type epibranchial placodes, we found that Neurod is
also expressed in cells within the placodal ectoderm prior to delamination, as
well as in delaminated precursors undergoing migration toward the condensing
VIIth, IXth and Xth ganglia (Fig.
3G,J). These data differ slightly from previously reported
expression only in migrating precursors in the mouse
(Fode et al., 1998
).
Expression of Neurod in Eya1/
epibranchial precursors was absent (Fig.
3H,K), lacking in the Six1/
VIIth ganglion in four of six embryos (Fig.
3I) and reduced in IXth and Xth ganglia in all six embryos
analyzed (Fig. 3L). Only a few
Neurod-positive cells were observed in the VIIth precursors
bilaterally in two of six Six1/ embryos
(data not shown). Similarly, a few Math3- or Nscl1-positive
cells were observed in the VIIth anlage in two of six
Six1/ embryos bilaterally for both probes
(data not shown and Fig. 3R),
while their expression was largely reduced in the IXth and slightly reduced in
the Xth anlage in all six Six1/ embryos
(Fig. 3O,R). In
Eya1/ embryos, a few Math3- and
Nscl1-positve cells were also observed in
Eya1/ Xth anlage unilaterally or bilaterally
in all six embryos analyzed (Fig.
3N,Q). Together, the lack of expression of these markers further
suggests that Eya1 functions upstream of the
Neurog2-regulatory pathway, and loss of Eya1 leads to the
inactivation of the genetic program controlling sensory neuronal fate that
normally expresses these bHLH genes.
|
To further define the consequences of the early block in delamination of
placodal precursors and test whether the cranial neural crest cells might
migrate to populate the distal ganglia, we examined the development of the
distal ganglia at later stages. Histological analysis at E11.5 to birth and
marker staining with neurofilament at E11.5-13.5 revealed that
Eya1/ embryos completely lacked the
epibranchial placode-derived distal VIIth and IXth cranial ganglia
(Fig. 5). However, partial
structure of the distal Xth ganglion was observed in
Eya1/ embryos
(Fig. 5E). In
Six1/ embryos, while the VIIth ganglion was
completely absent in all four embryos at each stage, the IXth ganglion was
present but reduced in size and the Xth ganglion was relatively normal in all
embryos (Fig. 5C,F,I,L). Taken
together, these analyses further confirmed an early arrest of neurogenesis in
the mutants and that in the absence of Eya1 or Six1, the cranial neural crest
cells are unable to populate the distal ganglia, differing from
Neurog2 mutants (Fode et al.,
1998).
|
|
Six1 expression in the epibranchial placode is Eya1 dependent
We have previously reported that murine Eya1 is expressed in the
cranial placodes including the epibranchial placodes at E9.5
(Xu et al., 1997a). However,
the Six1 expression in the epibranchial placodes during mouse
development and the detailed relationship between the expression of these two
genes in the generation of epibranchial placode-derived sensory neurons have
not been studied. We therefore set out to establish how the expression of
these genes related to the region and period of expression of Neurog2.
Eya1 expression was observed in all three epibranchial placodes as early
as E8.5 (before turning of the embryos). In transverse sections of E8.5 to
10.5 embryos, Eya1 expression was detected in the placodal cells,
migrating precursors and developing ganglia
(Fig. 7A,B and data not shown).
However, no Eya1 expression was observed in the V placodal cells at
these stages (data not shown). X-gal staining of heterozygous
Six1lacZ embryos at E8.5 to 10.0, which recapitulated the
Six1 mRNA expression pattern (Xu
et al., 2003
; Zheng et al.,
2003
), revealed that Six1 is strongly expressed in the
distal VIIth and IXth placodal precursor cells and weakly in the Xth placodal
precursors in E8.5 (before turning) embryos (data not shown). From E9.0 to
10.0, its expression is observed in placodal, migrating, and aggregating
neuronal precursors in ganglion anlagen
(Fig. 7C,D,H). Similar to
Eya1, no Six1 expression is observed in the V placodal cells
(data not shown). Therefore, Six1 appears to be expressed as early as
Eya1 in the distal epibranchial placodes, and both slightly precede
Neurog2 (Fode et al.,
1998
).
|
Bmp7 expression in the pharyngeal endoderm is preserved in the mutants
Bmp7 in the pharyngeal endoderm has been shown to act as a
signaling molecule for epibranchial placode induction and it can induce
Phox2a-positive neurons directly from nonepibranchial placode head
ectoderm (Begbie et al., 1999).
In addition to the pharyngeal ectoderm, Eya1 and Six1 are
expressed in the pharyngeal endoderm and neural crest mesenchyme
(Xu et al., 2002
). We
therefore examined Bmp7 expression in
Eya1/ and
Six1/ pharyngeal endoderm and found that its
expression was unaffected (Fig.
7E,J and data not shown). Thus, Eya1 and Six1 may function
cell-autonomously in early development of epibranchial sensory neurons.
Increased cell death in the mutant epibranchial placodes
To look into the absence of neuronal precursor cells in
Eya1/ embryos, we carried out TUNEL labeling
to determine whether the placodal ectodermal cells undergo abnormal apoptosis.
More cell death was apparent in E10.0 Eya1/
placodes (Fig. 8A-D). Apoptotic
cells were also increased in the VIIth precursors but were relatively normal
in the IXth and Xth precursors in all three
Six1/ embryos analyzed (data not shown).
This analysis suggests that upon the failure to activate their normal
developmental program, the progenitor cells undergo apoptosis in the mutant
embryos.
|
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Discussion |
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The role of Eya1 and Six1 during neurogenesis of the inner ear
The otic ectoderm is programmed for neurogenesis of the VIIIth ganglion
from as early as a stage before the otic placode even becomes morphologically
apparent. Recent studies have found that many genes are required for normal
neurogenesis, but little is known of the cellular mechanisms involved in their
development. In this study, we carefully assessed neurogenesis of the VIIIth
ganglion in Eya1 and Six1 mutants, using Neurog1
and Neurod as early molecular markers, and have now demonstrated that
both Eya1 and Six1 genes are dispensable for the initiation
of neurogenesis. However, our finding that lack of Eya1 or Six1 reduces the
population of neuroblast precursors within the otic ectoderm indicates their
requirement for maintenance of neurogenesis. Since Neurog1 expression
level is relatively normal in Neurog1-positive cells that are
detected in the mutant otic ectoderm, but the number of
Neurog1-positive cells was markedly reduced in the null mutant
embryos (Fig. 1), one likely
explanation for the reduction of neurogenesis within the otic ectoderm is that
Eya1 and Six1 are required for proliferation of neuroblast progenitors.
Consistent with this idea, pulse-chase BrdU incorporation studies revealed
fewer BrdU-labeled cells in the ventral region within which the neuroblasts
are normally specified in the mutant embryos from as early as E8.75
(Zheng et al., 2003). In
addition, abnormal apoptosis occurred in the mutant otic ectoderm and might
also contribute to the reduction of neuroblast progenitors, as detected by
TUNEL assay (Xu et al., 1999
;
Zheng et al., 2003
).
After specification, the neuroblast precursors normally migrate away from
the ectoderm and aggregate to form the VIIIth ganglion. This
epithelial-mesenchymal transition involves a number of changes in the
delaminating precursors and their overt differentiation can be induced and
enhanced by extracellular matrix (ECM)
(Hay, 1993). Our data also
demonstrated that both Eya1 and Six1 are not required for neuroblast
delamination (Fig. 1). As
abnormal apoptosis, which probably reflects the death of Eya1- or
Six1-dependent precursor cells, was observed in the mutant ganglion
anlage that has not yet expressed SCG10
(Fig. 2), they might regulate
the progressive differentiation of the precursor cells. It should be noted
that Eya1 and Six1 are also expressed in the periotic
mesenchyme. However, it is unclear whether loss of their mesenchymal
expression also contributes to the defect. Tissue-specific deletion of Eya1 or
Six1 would be required to address the relative contribution of their
expression in the mesenchyme to the development of the otic ganglion.
Nonetheless, our results at earlier stages suggest that both Eya1 and Six1
regulate early neuronal differentiation and maintenance.
Conserved molecular mechanisms underlying epibranchial placodes
In addition to the otic placode, Eya1 and Six1 are
coexpressed in other neurogenic placodes and cranial sensory ganglia
(Xu et al., 1997a;
Sahly et al., 1999
;
David et al., 2001
). Studies
in Drosophila eye imaginal disc indicate that eya is
epistatic to so and both genes cross-regulate each other to maintain
their expression (Pignoni et al.,
1997
). Our finding that Six1 expression in the
epibranchial placodes is Eya1 dependent suggests evolutionary
conservation of the Drosophila Eya-Six regulatory cassette in the
epibranchial placodal development, similar to that observed in early otic
development (Zheng et al.,
2003
). In contrast to the common ear phenotype in both mutants,
the phenotype in the distal cranial ganglia appears less severe in
Six1 than in Eya1 mutants. As we found that Six1 is
almost expressed as early as Eya1, one classic explanation for this
finding is functional redundancy with another coexpressed molecule. The
closely related family member Six4 is also expressed in the cranial
sensory ganglion from early stages and shows an identical pattern with
Six1 (Oliver et al.,
1995
; Esteve and Bovolenta,
1999
; Kobayashi et al.,
2000
; Pandur and Moody,
2000
; Ghanbari et al.,
2001
; Ozaki et al.,
2001
). Although the onset of Six4 expression during mouse
embryonic development has not been described, in the chick Six4
transcripts were detected in the ectodermal placodes, including olfactory,
optic, otic, and all epibranchial placodes as early as they acquire their
identity (Esteve and Bovolenta,
1999
). Thus, Six1 and Six4 may compensate for
each other's role in cranial sensory ganglia. Such a compensatory mechanism
has been noted between different bHLH genes Neurog2 and Neurog1,
Mash1 and Math3, or Mash1 and Neurog2 in
neurogenesis (Ma et al., 1999
;
Tomita et al., 2000
;
Nieto et al., 2001
), and for
the paralogous Pax3 and Pax7, and Pax1 and
Pax9 genes in other developmental systems
(Borycki et al., 1999
;
Peters et al., 1999
). This
could explain why the Six1 mutant phenotype appears to be variable
and less severe than that in the Eya1 mutants and why the
Six4 mutant mice do not display an embryonic phenotype
(Ozaki et al., 2001
).
Our data show that while a few neuronal precursors form in the VIIth and
VIIIth ganglia in the mutants, no traces of sensory neurons are found at
E13.5. In the absence of any viable sensory neurons, VII motoneurons project
through the V ganglion system in both mutants. This is consistent with
previous findings of rerouting of V motor fibers into the VII nerve in the
absence of a V ganglion (Ma et al.,
2000) and supports the hypothesis that sensory neurons may attract
branchial motoneuron fibers (Fritzsch and
Northcutt, 1993
). Central expansion into vestibular nuclei by
somatosensory fibers has been noticed before
(Fritzsch, 1990
). However, the
massive expansion seen in Six1 and Eya1 mutant mice may be
related to both central and peripheral effects of these genes. It is of
interest to note that haploinsufficiency for the human EYA1 or
SIX1 results in branchio-oto-renal (BOR) syndrome, an autosomal
dominant developmental disorder characterized by branchial clefts, hearing
loss, and renal anomalies (Abdelhak et al.,
1997
; Ruf et al.,
2004
). Minor anomalies, which occur in less than 20% of affected
patients, including facial nerve paresis, lacrimal duct aplasia and gustatory
lacrimation, were also described (Smith
and Schwartz, 1998
) and these features reflect anomalies in the
cranial nerve trajectories.
Eya1, Six1 and epibranchial placode-derived sensory neurons
Delamination of neuronal precursors is the first morphological sign of
differentiation during cranial placode neurogenesis. Among the transcription
factors involved in early sensory neurogenesis, Neurog2 appears to be required
for delamination of neuronal precursors, and this process is blocked in
Neurog2 mutant epibranchial placodes
(Fode et al., 1998). Although
Phox2a is also expressed in the epibranchial placodes, it does not control the
delamination of neuronal progenitors and their aggregation into the ganglion
anlagen (Morin et al., 1997
).
Our studies show that the initial expression of both Eya1 and Six1 occur in
the ectoderm of epibranchial placodes prior to delamination, and expression of
Neurog2 and Phox2a is lost in the mutant embryos (Figs
2,
3), suggesting that Eya1 and
Six1 function is required before delamination occurs. Therefore, Eya1 probably
acts in the initial selection of neuronal precursors within the epibranchial
placodal ectoderm and in the absence of Eya1, the initial Neurog2-
and Phox2a-expressing cells may not be specified. As Eya1 is
also expressed in the migratory precursors as well as in the forming ganglia,
it may regulate migration as well as aggregation during neuronal precursor
differentiation.
As the epibranchial placode cells that express Phox2a also express the
highest levels of Neurog2 transcripts, the same signal might be
involved in the induction of both genes, coupling the generic and neuronal
identity subprogram (Lo et al.,
1999). However, the nature of the inducing signal is currently
unclear. Our results indicate that Eya1 may induce Neurog2 and
Phox2a. When Neurog2 and Phox2a are activated, they will trigger
neuronal differentiation in the placodal ectodermal cells. Failure to activate
their normal differentiation programs will result in apoptosis of these
placodal ectodermal cells (Fig.
8). Consistent with the idea that Eya1 acts genetically upstream
of Neurog2 and Phox2a, the distal sensory ganglion formation appears to be
more severely affected in Eya1/ embryos than
in Neurog2/
(Fode et al., 1998
) or
Phox2a/ embryos
(Morin et al., 1997
). Our data
suggest the existence of differential mechanisms controlling epibranchial
neuronal differentiation between different placodes. Some epibranchial neurons
completely failed to develop but others, e.g. the Xth ganglion, were partially
present in Eya1/ embryos, or were mildly
affected in Six1/ embryos. The same ganglia
were also differentially affected in Neurog2 and Phox2a
mutant mice (Fode et al.,
1998
). Our results show that the Xth neurons appear to be more
severely affected in Eya1/ embryos than in
Neurog2/ embryos as judged by marker
analysis (Figs 4,
5), consistent with the idea
that Eya1 functions upstream of Neurog2. Thus, although it
is possible that different placodes respond to regional-specific signals, our
studies clearly show that Eya1 and Six1 are required for both generic and
subtype-specific gene expression in the epibranchial placodes by regulating
the expression of Neurog2 and Phox2a.
It is noteworthy that Eya and Six genes are also
co-expressed in myogenic cells and are involved in the molecular network
controlling muscle development (Heanue et
al., 1999; Fougerousse et al.,
2002
; Laclef et al.,
2003
). Similar to the nervous system, muscle differentiation is
crucially dependent on four bHLH factors, Myf5, MyoD, Myogenin and Mrf4
(Weintraub et al., 1991
). In
contrast to their early role in neurogenesis, recent data from muscle have
suggested that Eya and Six genes seem to be involved in
later steps of myogenic differentiation but do not activate the expression of
Myf5, which acts as a myogenic determination factor that activates other bHLH
myogenic factors (Fougerousse et al.,
2002
; Delfini and Duprez,
2004
). Thus, the Eya-Six regulatory hierarchy may operate
through a similar molecular network in nerves and muscles but acts at distinct
steps between sensory neuronal and muscle differentiation programs.
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ACKNOWLEDGMENTS |
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