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First published online October 12, 2007
doi: 10.1242/10.1242/dev.02885
Division of Biology, 139-74, California Institute of Technology, Pasadena, CA 91125, USA.
* Author for correspondence (e-mail: mbronner{at}caltech.edu)
Accepted 24 July 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONConclusionsREFERENCES |
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Key words: SALL4, Sox10, Electroporation, Placode formation
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONConclusionsREFERENCES |
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;
Webb and Noden, 1993).
Initially, all placodes arise from a horse-shoe shaped `preplacodal domain' at
the border of the rostral neural plate prior to neurulation
(Baker and Bronner-Fraser,
2001; Brugmann and Moody,
2005; Streit,
2004). They subsequently segregate into separate domains
designated, from rostral to caudal, as olfactory, lens, trigeminal, otic and
epibranchial, each with a distinctive gene expression profile. For example,
all placodes express characteristic Pax genes, with the most rostral (nose,
lens) expressing Pax6, intermediate level (trigeminal) expressing
Pax3 and most caudal (ear, epibranchial) expressing Pax2
(Baker and Bronner-Fraser,
2001). After thickening of the ectoderm, placodal cells either
invaginate or ingress to internalize as the first step in their conversion
from epithelia to sensory structures.
Although a number of genes have been implicated in specification of
placodal identity, little is known about what imbues placodal ectoderm with
the ability to internalize, migrate and contribute to sensory organs and/or
ganglia and thus distinguishes it from non-placodal ectoderm. At early stages,
ectoderm from other axial levels is competent to form particular placodes when
heterotopically grafted in place of the endogenous placode. However, this
broad potential to respond to placode-inducing signals becomes limited with
time (Groves and Bronner-Fraser,
2000). One intriguing possibility is that a factor(s), initially
expressed throughout the cranial ectoderm is critical for invagination of
ectodermal cells in response to placode inducing signals and that this becomes
restricted with time to individual placodes.
Here, we show that a zinc finger transcription factor of the spalt gene
family (Sweetman and Munsterberg,
2006), chick Spalt4 (also known as Sall4),
fulfills these criteria in the cranial ectoderm. Although its early expression
is uniform throughout the head ectoderm, its localization later becomes
restricted to the otic, lens and olfactory placodes. In Drosophila,
mutations in members of the spalt gene family cause defects in both migration
and cell fate (de Celis et al.,
1999; Elstob et al.,
2001; Kuhnlein and Schuh,
1996; Rusten et al.,
2001). In Xenopus, spalt genes have been shown to be
involved in brain development (Onai et
al., 2004) and limb regeneration
(Neff et al., 2005). Here, we
show that expression of Spalt4 in non-placodal ectoderm is sufficient
to induce invagination or ingression of cranial ectodermal cells. Knockdown of
Spalt4 function in the otic placode results in deficient otic vesicle
development whereas overexpression causes ear abnormalities. These results
suggest that Spalt4 is important for otic vesicle formation, and may
be generally important for the invagination/ingression of placodal
ectoderm.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONConclusionsREFERENCES |
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).
It also contains GFP driven from an IRES sequence downstream of the coding
sequence, allowing for coexpression of the desired protein and GFP in the same
cell. The sequence coding the amino terminus of Spalt4 including the CCHC zinc
finger (amino acids 1-98) was cloned into pCS2+NLS in frame with the nuclear
localization signal. The fragment with the NLS and truncated Spalt4
was then cloned into pCIG. The Sox10 expression construct was
described previously (McKeown et al.,
2005). Control electroporations used either empty pCIG or empty
pMES (in the Sox10 overexpression experiments) plasmids.
Ectoderm electroporations
The plasmid was injected into embryos containing three to seven somites
(stages 8-9), as well as at later stages (stages 12-13) where indicated. The
concentration of DNA was between 2 and 2.5 µg/µl. Eggs were windowed and
inked using standard procedures. The needle was placed through the vitelline
membrane at the caudal end of the embryo nearly parallel to the embryo, and
moved rostrally between the vitelline membrane and the embryo until it lay
above the midbrain. The DNA was then injected using a glass needle until the
embryo was completely covered with the DNA solution, and then withdrawn. The
electrodes were constructed in the lab and were made of two pieces of platinum
wire (19 gauge) bent at 45° from the horizontal, about 5 mm from the end
and 4 mm apart. The positive platinum electrode was inserted through a hole at
the edge of the blastoderm and under the embryo. The negative electrode, 4 mm
away from the positive electrode, was placed on top of the vitelline membrane
above the embryo at the level of the hindbrain and submerged in albumin but
without touching the membrane. Three pulses of 8-10 V for 30 mseconds duration
were applied 100 mseconds apart. The electrodes were carefully removed and the
egg was sealed and incubated at 38°C for up to 72 hours.
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) and
incubated in modified New culture (Chapman
et al., 2001) for 5-7 hours and then collected and fixed overnight
in 4% paraformaldehyde.
Analysis of embryos
Embryos were collected in Ringer's solution and fixed in 4%
paraformaldehyde overnight. Embryos were washed in PBT and embedded in gelatin
for histochemical analysis or dehydrated in methanol for in situ
hybridization. In situ hybridization was performed as described previously
(Wilkinson, 1992). In situ
hybridization on sections was performed using embryos fixed in Carony's
fixative, embedded in paraffin and sectioned at 10 µm. In situ
hybridization was performed using published procedures
(Etchevers et al., 2001).
Antibodies
The anti-GFP (Abcam), anti-Pax2 (Zymed), anti-Pax6 (Covance), HuC/D
(Molecular Probes) and TUJ1 (Covance) antibodies were obtained commercially.
The Spalt4 antibody was generated using a GST fusion construct that included
the region encoding amino acids 654-835 of chicken Spalt4. This region lies
between two zinc finger regions and has low sequence homology to other spalt
genes. The antibody recognizes nuclei in tissues that express Spalt4
RNA but does not recognize cells electroporated with
Spalt1-expressing constructs. The pan-Dlx antibody was a kind gift
from Jhumuku Kohtz, which was made from a construct from Grace Panganiban
(Dong et al., 2000). The
polyclonal antibody to Dlx3 (Bailey et al.,
2006) gives specific nuclear staining in tissues that normally
express Dlx3, such as the otic vesicle and the olfactory epithelium.
However, it does not stain other CNS structures that express other Dlx genes
(e.g. Dlx1, 2, 5 and 6). Primary
antibodies were visualized with Alexa Fluor 488-conjugated donkey anti-goat or
Alexa Fluor 594-conjugated donkey anti-rabbit secondary antibodies (Molecular
Probes). TUNEL analysis was done using the In Situ Cell Death Detector, TMR
kit from Roche according to the manufacturer's instructions.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONConclusionsREFERENCES |
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). Spalt4 can also be detected in the
lens (Fig. 1J) and weakly in
the olfactory epithelium (Fig.
1K). It is also present in limb mesoderm
(Fig. 1L), but not the ectoderm
or apical ectodermal ridge (Fig.
1M).
Fgf signaling has been implicated in placode formation
(Litsiou et al., 2005;
Maroon et al., 2002;
Martin and Groves, 2006) as
well as induction of the otic vesicle
(Ladher et al., 2000;
Ladher et al., 2005;
Liu et al., 2003;
Maroon et al., 2002;
Vendrell et al., 2000).
Furthermore, insertion of Fgf8-coated beads in the area opaca results in the
induction of early placode markers such as Dlx5, Sox3 and
Eya2 (Litsiou et al.,
2005). To analyze the ability of Fgf signaling to induce
Spalt4, we implanted an Fgf2-soaked bead
(Litsiou et al., 2005) under
the area opaca of stage 4 embryos, as this region of ectoderm is competent to
respond to placode-inducing signals. Embryos were analyzed for Spalt4
expression 5 to 7 hours thereafter. Spalt4 was detected in the
ectoderm surrounding the bead in nine out of ten embryos
(Fig. 2A,B,C), while none was
detected in control embryos (0/8 embryos) in which a BSA-soaked bead was
implanted (Fig. 2D-F). In
addition to Spalt4, the preplacodal marker Eya2 was induced
by Fgf2 (3/5) (data not shown).
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Ectopic vesicles at the level of the hindbrain express otic marker genes
We next tested whether the ectopic vesicles and pits resembled otic
vesicles using a number of molecular markers for placodes in general [e.g. the
Six-Eya-Dach gene network
(Streit, 2004)], early otic
markers such as Pax2 (Groves and
Bronner-Fraser, 2000), Dlx3
(Pera and Kessel, 1999),
Nkx5.1 (Herbrand et al.,
1998) as well as other transcription factors such as Sox8
(Bell et al., 2000),
Sox10 (Cheng et al.,
2000), Dlx5 (Streit,
2002), Tbx1 and Tbx3
(Chapman et al., 1996) and
signaling molecules such as Bmp4, Notch1 and Lunatic fringe
(Adam et al., 1998;
Cole et al., 2000) thought to
be involved in otic pit/vesicle or later ear development. By using a
combination of immunocytochemistry and in situ hybridization, we examined the
extent to which these ectopic vesicles mimicked the normal otic vesicle
program of gene expression.
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Overexpression of Spalt4 in the otic placode causes severe ear abnormalities
The above results show that expression of Spalt4 in naïve
ectoderm causes ectopic vesicle formation. We next investigated whether excess
Spalt4 would alter development if overexpressed in the endogenous
placode region. To this end, we targeted electroporations to the otic placode
itself. This resulted in profound morphological malformations in the
developing ear. The otic epithelium appeared enlarged and failed to close
properly. Frequently, electroporation of Spalt4 into the otic placode
resulted in a flat or opened otic pit (29/42) rather than closed otic vesicles
as in control embryos (0/31 with abnormal vesicles). The morphological
alterations were variable, perhaps due to differences in amount or
distribution of construct, but our data cumulatively suggest that excess
Spalt4 causes severe patterning defects in the developing ear.
Molecular markers that selectively localize in different domains within the
developing ear confirmed that cells overexpressing Spalt4 assumed
gene expression characteristic of the ear, though the spatial distribution was
sometimes altered. After electroporation of Spalt4, Sox10, which is
usually spatially restricted to the dorsal-lateral half of the otocyst
(Fig. 5A,C,D), was expressed
throughout the thickened ectoderm (Fig.
5B,E,F). Sox10 expression was not limited to the cells
expressing GFP (compare Fig. 5E with
F), suggesting the effects may be non cell-autonomous, perhaps as
a result of mispatterning of the otic ectoderm and failure to downregulate
endogenous Sox10 in the ventral-medial half as happens in control
embryos. Similarly, Pax2, normally expressed medially in control
electroporated otocysts, was observed in lateral regions after Spalt4
overexpression (Fig. 5G,H).
However the expression of Six1, which is not induced by
Spalt4 overexpression in the ectopic vesicles
(Fig. 4H), remains expressed in
a spatially restricted manner in the thickened otic ectoderm
(Fig. 5K,L). In rare cases
(<5%), multiple smaller vesicles were observed (data not shown). In
contrast to electroporation at early stages, those performed at later times
(stage 12 and 13) show normal otic vesicle formation (0/5 abnormal).
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-crystallin, which are
characteristic of the lens (data not shown).
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).
Constructs were introduced into the presumptive otic epithelium by
electroporation in a similar manner to that used for the full-length
construct. The majority of embryos examined 2 days after electroporation with the dominant-negative construct (19/22) had significant reduction in the size of one or both otic vesicles after efficient levels of electroporation (as judged by high levels of GFP expression; Fig. 7C,D). By comparison, few embryos (4/25) electroporated with GFP-vector alone had smaller ears and none of these were as small as the experimental ears (Fig. 7A,B). GFP staining revealed uniform distribution of the electroporated construct throughout the ectoderm of both control and experimental embryos, including the miniature otic vesicles (data not shown). The reduced size of the vesicle in dominant-negative embryos was particularly dramatic in the rostrocaudal dimension, sometimes giving it a tightly squeezed look. There was a reduction of 30% (P<0.0001) in the length of the normal otocyst, with dominant-negative electroporated embryos averaging 212±41 µm (±s.e.m.; n=12) in length along the rostrocaudal axis compared with 301±30 µm (n=8) in controls. In some embryos, the vesicle lost the endolymphatic duct. In a few cases, we achieved unilateral electroporation. In these embryos, the electroporated side had a markedly smaller vesicle compared with a normal vesicle on the contralateral side (data not shown). In situ hybridization revealed that Sox10 (Fig. 7C,D) retained its normal pattern (compare with Fig. 7A,B). Notch and Lunatic fringe also retained their normal pattern, though Lunatic fringe expression was lost in the smallest vesicles (data not shown).
We next examined whether the changes in vesicle size were due to increased cell death and/or decreased proliferation. To this end, we performed TUNEL staining (Fig. 7E,F,G,H). At 8 hours after electroporation, we observed approximately twice as many TUNEL-positive cells in the ectoderm of dominant-negative electroporated embryos as in control electroporated embryos (9.4±5.4; ±s.e.m., n=8 TUNEL-positive cells per section compared with 5.25±2.4, n=8 in controls, P<0.05). By contrast, no significant alterations in phosphohistone H3 levels (P<0.4) were noted between dominant-negative [n=5; 1.94±0.4 (±s.e.m.) positive cells in the placode per section] and control (n=6; 1.71±0.18 positive cells) embryos. These data suggest that the decrease in otic vesicle size caused by electroporation of truncated Spalt4 may be due to an early increase in cell death but not to changes in cell proliferation.
In addition to the otic level, the truncated construct was electroporated at midbrain-rostral hindbrain level ectoderm, which does not normally express Spalt4. In these embryos, GFP-labeled cells migrated into the trigeminal ganglia in a manner similar to normal embryos (data not shown). This contrasts with embryos electroporated with full-length Spalt4, where GFP-positive cells remained in the ectoderm and contributed few or no cells to the trigeminal ganglion.
Sox10 overexpression can induce ectopic vesicles
Spalt4-induced vesicles have ectopic Sox10 expression. In
a recent study in Xenopus, Sox10 overexpression induced some
vesicle-like structures in the vicinity of the ear
(Taylor and Labonne, 2005). To
test if a similar function was present in birds and if Sox10 would
phenocopy Spalt4, we electroporated a construct encoding
Sox10 (McKeown et al.,
2005) into the cranial ectoderm. Similar to Spalt4
overexpression, this generated multiple ectopic vesicles that expressed
Notch1 (Fig. 8C,D) and
EphA4 (Fig. 8E,F)
suggesting that Sox10 is epistatic to Spalt4. However, we
did detect some weakly Spalt4-positive ectopic vesicles
(Fig. 7G,H), suggesting a more
complicated gene regulation. One difference between Spalt4 and
Sox10 is that the latter generated ectopic vesicles adjacent to the
trigeminal ganglia whereas Spalt4 did not (data not shown).
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONConclusionsREFERENCES |
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). Members of the Six-Eya-Dach pathway are expressed
in a crescent-shaped domain of anterior ectoderm that is postulated to be of
general placodal character (Brugmann and
Moody, 2005; Streit,
2004). Later, individual placodes become distinguishable in the
cranial ectoderm by thickening of the epithelium and expression of
characteristic genes. For example, Pax6 is expressed in lens and
olfactory placodes (Bhattacharyya et al.,
2004), Pax3 in trigeminal placodes
(Baker et al., 1999) and
Pax2 in otic and epibranchial placodes
(Baker and Bronner-Fraser,
2001; Groves and
Bronner-Fraser, 2000). No single gene imbues placodes with a
specific identity; instead, it is probable that co-expression of several genes
is required for placode formation and differentiation.
In this study, we show that Spalt4 expression overlaps that of
Six-Eya-Dach genes in the preplacodal ectoderm, and is detected as
early as stage 3 in the chick. Spalt4 subsequently resolves to the
presumptive otic, lens and olfactory placode regions by stage 10, concomitant
with the time during which non-placodal ectoderm loses competence to form otic
placode (Baker et al., 1999;
Groves and Bronner-Fraser,
2000).
Spalt4 expression causes the formation of ectopic pits
One of the best-studied placodes is the otic placode, which forms the inner
ear (Solomon et al., 2004;
Streit, 2001). It initiates as
a patch of thickened ectoderm on either side of the hindbrain that invaginates
to form otic vesicles (otocysts). Subsequently, the otocyst becomes
regionalized, giving rise to the complex inner ear including the cochlea, the
different parts of the vestibular system and the endolymphatic duct. A number
of different cell types originate from this epithelium, including
mechanosensory hair cells, support cells and various other specialized cell
types. Other cells delaminate from the placode and migrate next to the neural
tube to form the acoustic ganglion. Signals from neighboring tissue, such as
Fgf (Ladher et al., 2000;
Ladher et al., 2005;
Liu et al., 2003;
Maroon et al., 2002;
Vendrell et al., 2000) and
Bmp4 (Chang et al., 1999;
Gerlach et al., 2000;
Merlo et al., 2002), appear to
induce the otic placode and/or activate specific patterns of gene expression.
We have shown that Spalt4 is also induced in stage 4 ectoderm by
Fgf2. Recent results have also implicated Wnt signaling in otic vesicle
induction (Ladher et al.,
2000; Ohyama et al.,
2006).
Electroporation of Spalt4 results in formation of small ectodermal pits near the otic vesicle or even laterally in the branchial arch ectoderm. Adjacent to the hindbrain, Spalt4 induced ectopic vesicles that morphologically resemble otic vesicles and express otic markers. It is interesting to note that not all Spalt4-electroporated ectodermal cells invaginate. In almost all embryos, some remain in the ectoderm adjacent to the pits. Perhaps this reflects some intrinsic limits to ectopic vesicle size, density of electroporated cells, or requirement for additional signals in some cell populations. These results suggest that Spalt4 alone is not sufficient to induce invagination in all ectoderm.
Ectopic vesicles express a number of genes characteristic of the otic
vesicle and important for normal ear development; these include Notch
(Adam et al., 1998),
Lunatic fringe (Cole et al.,
2000), Bmp4 (Cole et
al., 2000), Dlx3
(Pera et al., 1999),
Dlx5 (Streit, 2002),
Sox8 (Bell et al.,
2000), Sox10 (Cheng
et al., 2000), Tbx1
(Chapman et al., 1996),
Tbx3 (Chapman et al.,
1996) and Nkx5.1
(Herbrand et al., 1998).
Spalt4 is sufficient to recapitulate some but not all of the
molecular events necessary for normal ear development. For example, we were
unable to detect the expression of many genes in the Six-Dach-Eya
pathway. One possibility is that the Six-Eya-Dach pathway is upstream
of Spalt4. Another possibility is that regulation of this pathway may
involve a gene network independent and perhaps parallel to that induced by
Spalt4. Six1 and Eya1 mutants have been shown to have poorly
developed auditory systems (Li et al.,
2003; Ozaki et al.,
2004; Xu et al.,
1999; Zheng et al.,
2003). Though the ears progress to the otic vesicle stage, they
fail to form middle and inner ear structures or neurons, as if stalled at the
vesicle stage rather than differentiating further
(Zheng et al., 2003).
Similarly, we were unable to detect neurons in the ectopic vesicles 72 hours
after Spalt4 electroporation. An intriguing possible reason why
Spalt4-induced vesicles do not progress beyond the otic vesicle stage
and fail to generate neurons is because of their failure to upregulate
Six1 and Eya1. Alternatively, the Six-Eya-Dach
genes may require region-specific signals that are absent at the sites where
ectopic vesicles form. The latter possibility seems likely in the case of
Dach2, since it was expressed only in ectopic vesicles next to the
hindbrain. Tbx1 and Tbx3 are also influenced by other
factors since they were expressed only in ectopic vesicles caudal to the
endogenous otic vesicle.
Recent studies have highlighted interesting similarities between the
vertebrate inner ear and Johnston's organ in Drosophila
(Boekhoff-Falk, 2005). Many of
the genes necessary for specification or function of the auditory cells in
Drosophila are also required in the vertebrate inner ear. In
Drosophila spalt and spalt-related are required for the
formation of Johnston's organ (Dong et
al., 2003). However, the roles of spalt genes in vertebrate and
fly auditory development may not be completely analogous. In Drosophila,
spalt has been shown to be downstream of distalless
(Dong et al., 2002). By
contrast, we find that chick Dlx genes are upregulated by
misexpression of chick Spalt4. Furthermore, Dlx3 or
Dlx5 overexpression fails to induce Spalt4 expression (data
not shown). The Iroquois homologues Irx1 and Irx2 are
upregulated by Spalt4 (Table
1), though they are repressed by spalt in
Drosophila wing development (de
Celis and Barrio, 2000).
Ectopic Fgf2, Fgf3 or Fgf8 induce ectopic vesicles that express otic
markers such as Notch1, Pax2 and Nkx5.1
(Adamska et al., 2000;
Vendrell et al., 2000). In
addition to Fgf, other signals may be involved in regulation of
Spalt4 and/or other placodal determinants. Vitamin A-deficient chick
embryos lack posterior hindbrain, but develop ectopic Pax2-positive
vesicles (Kil et al., 2005).
Overexpression in Xenopus of the secreted phospholipase Rossy induces
ectopic olfactory vesicles, and, by microarray analysis, has been shown to
upregulate a member of the spalt family, Xspalt1
(Munoz-Sanjuan and Brivanlou,
2005). Overexpression of other transcription factors has been show
to result in ectopic vesicles in other species. Pax6 overexpression
generates ectopic lens vesicles (Altmann et
al., 1997) and Sox10 can generate ectopic otic vesicles
(Taylor and Labonne, 2005).
Ectopic Six3 expression in mice leads to ectopic vesicle formation
near the otocyst (Lagutin et al.,
2001), and injection of Sox3
(Koster et al., 2000) in
medaka gives rise to vesicles of either otic or lens character. However, we
failed to detect upregulation of either Six3 or Sox3 after
misexpression of Spalt4; this could reflect species differences or a
lack of epistatic interactions between these transcription factors. It is
currently unclear if any of these factors directly regulates Spalt4
in the chick. Interestingly, constitutively active Notch has been shown to
cause the formation of ectopic structures expressing ear-specific genes,
consistent with the possibility that Spalt4 is upstream of
Notch in this cascade (Daudet and
Lewis, 2005). We have found that ectopic Sox10 expression
can generate ectopic vesicles in the chick, similar to results previously
described in Xenopus (Taylor and
Labonne, 2005). These ectopic vesicles also express
Spalt4. The exact relationship between these two genes has yet to be
determined, but may involve a feedback loop. One difference is that
Spalt4 was unable to generate ectopic vesicles expressing
otic-specific genes at the level of the trigeminal ganglia, whereas
Sox10 was able to do so. This indicates that Spalt4 and
Sox10 respond differently to the signals in the ectoderm at midbrain
level and implies that Sox10 may act downstream of
Spalt4.
Overexpression of Spalt4 within the otic vesicle itself causes
alterations in morphology and patterns of gene expression in the developing
ear. Defects include formation of multiple vesicles resembling otic vesicles,
and the failure to form a closed otic vesicle. In both cases, Pax2,
Lunatic fringe and Notch1 are expressed in a non-regionalized
fashion. In the most extreme cases where the otic vesicle fails to close,
Sox10 is expressed throughout the otic ectoderm rather than being
confined to the lateral half as in control electroporated embryos. Normally,
the expression of Spalt4 in the closed vesicle begins to be
downregulated at stage 16. However, in electroporated embryos, Spalt4
expression is maintained. This may lead to altered expression of other genes,
the loss of regionalizing signals and the observed abnormalities in the otic
vesicle. Interestingly, activation of canonical Wnt signaling as well as Fgf
overexpression in the ear also leads to formation of open, oversized ears
(Ladher et al., 2000;
Ohyama et al., 2006;
Vendrell et al., 2000).
Expression of Spalt4 at the midbrain level interfered with the
normal ingression of placode cells into the trigeminal ganglia. Few GFP cells
contributed to the ganglia and the number of placode-derived cells was also
reduced, resulting in a malformed ganglia. A similar effect was seen in the
failure of Spalt4-expressing neural crest cells to contribute to the
trigeminal ganglia (Barembaum and
Bronner-Fraser, 2004). Furthermore, we were unable to detect
Pax3 in the GFP-expressing thickened ectoderm (data not shown). At
epibranchial placode levels, Spalt4-electroporated cells also formed
ectopic vesicles and failed to contribute to the ganglia derived from the
epibranchial placodes after 48 hours. Since ectopic vesicles were found in
ectoderm that would normally give rise to neurons, this probably reflects a
change in cell fate from neurogenic to sensory. The cell fate switch is
reminiscent of the activity of spalt in Drosophila where it
affects fate determination in a number of different lineages
(de Celis et al., 1996;
Elstob et al., 2001;
Rusten et al., 2001).
Reduction of Spalt4 activity causes abnormalities in ear development
Our results show that Spalt4 is not only sufficient for vesicle
formation but also necessary for proper otic development. Introduction of a
truncated, dominant-negative Spalt4 results in abnormal otic vesicles
that are drastically reduced in size. In general, otic gene expression remains
the same and vesicles retain a regionalized pattern. The reduction in vesicle
size appears to be caused by increased cell death, as assayed by TUNEL. That
otic vesicles do form in the presence of the dominant-negative
Spalt4, albeit reduced in size, may indicate that dominant-negative
Spalt4 may not fully abrogate endogenous Spalt4 activity.
Spalt4 is normally expressed well before the time that we introduce
the dominant-negative construct. Thus, it is likely that we do not achieve
full knockdown of transcription factor activity. Also we cannot rule out the
possibility that other genes may be acting on the ectoderm to partially
compensate for the loss of Spalt4. It is also worth noting that
whereas Sox10 can be induced in ectopic vesicles, it is not reduced
in otic vesicles electroporated with the dominant-negative construct. A
possible explanation is that Sox10 may not be directly induced by
Spalt4.
Consistent with our observations in chick, humans with Okihiro syndrome, in
which SALL4 is mutated, have hearing defects as well as abnormalities
of the heart, kidney and limbs (Kohlhase
et al., 2005). By contrast, no hearing defects have been reported
in heterozygous mutant mice (Sakaki-Yumoto
et al., 2006), though some hearing defects have been detected in
mice with a truncated Spalt4
(Warren et al., 2007).
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Conclusions |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONConclusionsREFERENCES |
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONConclusionsREFERENCES |
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Adamska, M., Leger, S., Brand, M., Hadrys, T., Braun, T. and Bober, E. (2000). Inner ear and lateral line expression of a zebrafish Nkx5-1 gene and its downregulation in the ears of FGF8 mutant, ace. Mech. Dev. 97,161 -165.[CrossRef][Medline]
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