1 Department of Cell Biology, Harvard Medical School, Boston, MA 02115,
USA
2 Department of Oral Biology, Harvard School of Dental Medicine, Boston, MA
02115, USA
* Present address: The Burnham Institute, Stem Cell and Regeneration Program,
10901 N. Torrey Pines Road, La Jolla, CA 92037, USA
Present address: Department of Craniofacial Biology, University of Colorado
Health Sciences Center, Denver, CO, USA
Author for correspondence (e-mail:
mmercola{at}burnham.org)
Accepted 10 October 2002
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SUMMARY |
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Key words: Dlx, Neural crest, Neural induction, Xenopus, hairy2a, slug, snail, msx
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INTRODUCTION |
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Diffusible proteins such as BMP, Wnt and FGF isoforms play an important
role in patterning neural and non-neural ectoderm along the mediolateral axis
in Xenopus and zebrafish. BMP antagonists, such as chordin and
noggin, initiate neural induction in the dorsal ectoderm while BMP signaling
in the ventral ectoderm represses neural fates and promotes epidermal
differentiation (Brewster et al.,
1998; Hemmati-Brivanlou and
Melton, 1997
; Kuo et al.,
1998
; Mizuseki et al.,
1998
; Nakata et al.,
1997
; Sasai et al.,
1994
; Smith et al.,
1993
; Wilson and
Hemmati-Brivanlou, 1995
). One model for neural plate border
formation suggests that the ectoderm is differentially patterned by threshold
levels of BMP signaling: high levels induce epidermal fates, low or absent
signaling permits neural differentiation while intermediate levels induce
border fates (Marchant et al.,
1998
; Morgan and Sargent,
1997
; Nguyen et al.,
1998
; Wilson et al.,
1997
). However, studies showing that not all aspects of neural and
neural crest induction are recapitulated by modulating BMP levels in
non-neural tissues (e.g. LaBonne and
Bronner-Fraser, 1998
) have prompted an examination of additional
factors that might synergize with BMP to control cell fate at the border
region of late blastula embryos (Bachiller
et al., 2000
; Klingensmith et
al., 1999
; Streit and Stern,
1999
). In particular, Wnt and FGF isoforms appear critical for
induction of neural crest and cranial placodal cells
(Adamska et al., 2000
;
Chang and Hemmati-Brivanlou,
1998
; LaBonne and
Bronner-Fraser, 1998
; Mayor et
al., 1997
; Phillips et al.,
2001
; Saint-Jeannet et al.,
1997
; Streit and Stern,
1999
; Vallin et al.,
2001
; Wilson et al.,
2001
).
Several transcription factors have been proposed to influence
neural/non-neural ectodermal patterning. For example, Xiro, Xash-3 and Zic
family members are induced by pre- to early gastrula stage dorsalizing and
neuralizing signals, such as noggin, and with time their expression becomes
localized to the future neural plate
(Gomez-Skarmeta et al., 1998;
Kuo et al., 1998
;
Mizuseki et al., 1998
;
Nakata et al., 1997
;
Turner and Weintraub, 1994
).
Overexpression of these factors in whole embryos expands the neural plate.
Where examined, however, either an abnormally broad domain of neural crest
marker expression overlaps the ectopic neural plate region or the neural crest
markers are lost (Gomez-Skarmeta et al.,
1998
; Kuo et al.,
1998
; Mizuseki et al.,
1998
; Nakata et al.,
1997
; Turner and Weintraub,
1994
), indicating that normal border signaling has not occurred.
In contrast, the Dlx family of transcription factors represent a class of
proteins that, along with Msx-1 and PV.1, inhibit neural plate differentiation
when overexpressed in Xenopus
(Ault et al., 1997
;
Feledy et al., 1999
;
Pera and Kessel, 1999
;
Suzuki et al., 1997
). Prior to
gastrulation, transcripts encoding these factors are expressed diffusely in
the ectoderm but subsequently become excluded from the developing neural plate
(Luo et al., 2001a
). At least
one Dlx family member, Dlx3, has been shown to be regulated by BMP and
canonical Wnt/ß-catenin signaling that provide ventralizing signals in
pre-gastrula stage Xenopus embryos
(Beanan et al., 2000
). These
data suggest that Dlx genes might reinforce or refine the early
ectodermal pattern established by BMP and Wnt signaling. Interestingly, the
medial expression borders, adjacent to the neural plate, differ among Dlx
family members, leading Luo et al., to propose that distinct cell fates might
arise through differential action of Dlx family members
(Luo et al., 2001b
). These
studies suggest a model in which Dlx activity may regulate the position of the
neural plate border. A direct test of this model by loss-of-function has not
been done nor have the consequences of shifting the endogenous spatial
expression pattern of Dlx expression on adjacent cell fates been examined.
Moreover, it is not certain whether the normal role of Dlx proteins is solely
inhibitory. We originally postulated a positive role based on our observation
that Dlx3 transcripts were downregulated in narrowminded, a zebrafish
mutant that is deficient in Rohon-Beard neurons and that exhibits delayed
appearance of neural crest cells (Artinger
et al., 1999
).
In this paper, we describe loss- and gain-of-function studies to investigate the role of Dlx genes in positioning the lateral edge of the neural plate and specifying adjacent cell fates. Dlx3 or Dlx5 homeodomains, which are highly conserved among Dlx family members, were fused to the Engrailed transcriptional repressor or the VP16 transcriptional activator domains to modulate transcription of genes regulated by Dlx. These fusion proteins were misexpressed in localized regions within Xenopus ectoderm to modify endogenous Dlx function and subsequent alterations in cell fates were analyzed. Whereas we envisaged that pre-gastrula stage BMP, Wnt and FGF signaling biases ectodermal cells towards neural or epidermal fates, our results indicate that Dlx-dependent transcription positions of the lateral border of the neural plate and, importantly, is required in non-neural ectoderm to induce signals that specify the stereotypic pattern of neural crest cells and cranial placodes.
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MATERIALS AND METHODS |
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Embryos and microinjections
Xenopus laevis embryos were fertilized in vitro, dejellied in 2%
cysteine-HCl (pH 7.8), and maintained in 0.1xMMR. Embryos were reared at
14-22°C and staged according to Nieuwkoop and Faber
(Nieuwkoop and Faber, 1994).
Capped mRNA was synthesized using the mMessage Machine kit (Ambion). mRNA was
injected into one dorsoanimal blastomere of four to eight cell stage embryos
in 3% Ficoll in 1x MMR. For all VP16 constructs, 100-200 pg of mRNA was
injected while for the EnR constructs, 50-80 pg of mRNA was injected. All
injections included 250-300 pg of ß-galactosidase mRNA to provide a
lineage tracer. Dexamethasone (10 µM) was added at either stage 5/6, stage
8/9, stage 10 or stage 11.5-12 to induce the GR fusion proteins.
Transplants and explants
EnR-Dlx3hd mRNA along with ß-galactosidase mRNA and GFP mRNA as
lineage tracers were injected into two ventral animal blastomeres of four to
eight cell stage host embryos. Control host embryos were uninjected or were
injected with the lineage tracers alone. Donor embryos were first injected
with rhodamine dextran (10x103 Mr;
Molecular Probes) into both blastomeres of a two-cell stage embryos. Embryos
were then cultured until stage 12 when a portion of the neural plate and
subjacent mesoderm was transplanted into the ventral ectoderm of recipient
stage 12 embryos. The fluorescent signal was acquired in monochrome and
pseudo-colored green for clarity.
In situ hybridization and probes
Embryos that were co-stained for ß-galactosidase were fixed for 40
minutes at room temperature in MEMFA (0.1 M Mops pH 7.4, 2 mM EGTA, 1 mM
MgSO4, 3.7% formaldehyde), rinsed in 1x PBS with 2 mM
MgCl2 and incubated in staining solution at 37°C with Magenta
Gal (Biosynth) before processing for in situ hybridization as described
previously (Harland, 1991).
The following plasmids were used to generate digoxigenin-labeled probes (RNA
polymerase was used for linearization): pSp72-Xslug (BglII,
SP6) (Mayor et al., 1995
),
pSp72-XSnail (BglII, Sp6) (Essex
et al., 1993
), pKS-XSox2 (XbaI, T7)
(Mizuseki et al., 1998
),
pKS-Msx-1 (Hox7.1) (EcoRI, T7)
(Su et al., 1991
),
pKS-Dll2 (Dlx3) (BglII, T7)
(Papalopulu and Kintner,
1993
), pKS-XHairy2a (BamHI, T7) (J. W.),
pGEM3-keratin (BamHI, SP6)
(Jonas et al., 1989
),
pBSII-Xsix1 (NotI, T7)
(Pandur and Moody, 2000
),
sp70-NCAM (EcoRV, SP6) and N-tubulin (BamHI, T3)
(Oschwald et al., 1991
;
Richter et al., 1988
). Stained
embryos were postfixed in MEMFA and embedded in JB4 according to the
manufacturer's directions (Polysciences) for histological examination.
Immunohistochemistry
Embryos were fixed in MEMFA and processed for immunohistochemistry
(Hemmati Brivanlou and Harland,
1989) using an EpA antibody
(Jones, 1985
) detected with an
alkaline-phosphatase-conjugated secondary antibody.
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RESULTS |
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Ectopic activation of target genes by injection of VP16-Dlx3hd mRNA
resulted in a loss of the early neural plate marker, Xsox2
(Kishi et al., 2000) (91% of
embryos showed a loss, n=69; Fig.
2B). This confirms that the anti-neural plate potential of the
intact Dlx3 protein (Feledy et al.,
1999
) (Fig. 3C)
depends on transcriptional activator function. In this and all subsequent
experiments, ß-galactosidase mRNA was co-injected to mark the progeny of
injected blastomeres (magenta stain). We then investigated whether repression
of downstream targets of endogenous Dlx proteins would have a reciprocal
effect on neural plate formation. As shown in
Fig. 2C, injection of
EnR-Dlx3hd mRNA expanded Xsox2 expression laterally on the injected
side of the embryo (92% expanded, n=79). Control embryos injected
with ß-galactosidase mRNA alone showed no change in Xsox2
expression (0% expanded, n=136;
Fig. 2A). Similar results were
seen when embryos were injected with VP16-Dlx5hd-GR (83% showed a loss,
n=30) (not shown) and EnR-Dlx5hd-Gr (61% expanded Xsox2,
n=31) (Fig. 2D) when
dexamethasone was added immediately (stage 5/6).
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|
To ensure that the effects on the neural plate were not limited to Xsox2 or to early stages of neural induction, we examined the effects of the Dlx constructs on NCAM expression. NCAM was lost after VP16-Dlx3hd injection (93% lost, n=42) but was expanded laterally by EnR-Dlx3hd (89% expanded, n=57; Fig. 2E-G). Transverse sections of older stage (stage 27-30) embryos revealed that the neural tube had grossly normal morphology although the region marked by ß-galacotosidase had expanded (Fig. 2I) as compared to the contralateral side or comparable region in Fig. 2H.
Co-injection with full-length Dlx3 mRNA rescued the EnR-Dlx3hd effects in a
dose-dependent manner (Fig. 3)
indicating that the EnR-Dlx3hd phenotype reflects repression of normal targets
of Dlx proteins. As would be epxected, the highest doses of full-length Dlx3
caused the overexpression phenotype (Fig.
3C). Thus, we conclude that the phenotypic effects of the Dlx
fusion proteins reflect a modulation of natural Dlx target genes. Taken
together, these data suggests that Dlx activity delimits the neural plate and
prevents its expansion, consistent with conclusions drawn from overexpression
studies (Feledy et al., 1999;
Luo et al., 2001b
).
We then asked if suppression or expansion of the neural plate is
accompanied by compensatory changes in epidermal specification, which is
visualized by expression of epidermal keratin and the
epidermal-specific antibody, EpA (Jones,
1985). VP16-Dlx3hd did not affect keratin or EpA levels
when expressed in the neural plate region (keratin: 76% of embryos
exhibited no change, n=84, Fig.
2K; EpA: 86% no change, n=59
Fig. 2O) despite its marked
ability to inhibit neural plate differentiation. In contrast, EnR-Dlx3hd
suppressed keratin and EpA expression where injected
(keratin: 95% suppressed, n=138
Fig. 2L,M; EpA: 85%,
n=73, Fig. 2P),
consistent with expansion of the neural plate
(Fig. 2C,G). Control embryos
injected with ß-galactosidase mRNA showed no change in keratin
or EpA expression (keratin: 0% affected, n=129
Fig. 2J; EpA: 4%,
n=81, Fig. 2N). We
conclude that although Dlx activity inhibits neural plate differentiation, it
is not sufficient to redirect presumptive neural plate cells to adopt an
epidermal cell fate.
The neural plate-epidermal border region is patterned normally but
displaced laterally in EnR-Dlx3hd-expressing tissue
The preceding experiments show that EnR-Dlx3hd can expand the neural plate.
Expansion of the neural plate is also seen upon overexpression of Zic, Xash3
and Xiro family members, but the neural crest domain is either expanded
diffusely or missing and lateral neurons occur ectopically
(Gomez-Skarmeta et al., 1998;
Kuo et al., 1998
;
Mizuseki et al., 1998
;
Nakata et al., 1997
;
Turner and Weintraub, 1994
),
most likely reflecting a disturbance of the normal patterning mechanism. We
therefore examined a panel of markers to ask whether patterning of these cell
lineages were similarly disordered or occurred normally in EnR-Dlx3hd-injected
tissues. Xh2a and Xmsx-1 mark the neural plate border at the
end of gastrulation (stage 12). Xsnail and Xslug are
expressed in the neural crest, which appears during late gastrula/early neural
plate stages (stages 12-14). N-tubulin marks the medial, intermediate
and lateral rows of primary neurons in neurula and neural tube stage (stages
13-16) embryos (Chitnis et al.,
1995
). Cranial placodes arise from thickenings in the ectoderm
immediately lateral to the neural plate and give rise to a wide variety of
derivatives, including paired sense organs (reviewed by
Baker and Bronner-Fraser,
2001
). Xsix1, a homeobox-containing transcription factor,
is expressed in the cranial placodal thickenings present in an anterior and
lateral band of early neurulae, marking the prospective olfactory anlagen and
persists in the late neurulae, where it marks the olfactory, otic, trigeminal
and dorsolateral placodes (Pandur and
Moody, 2000
).
We frequently observed that each marker was displaced to the lateral margin of the areas that contained the injected EnR-Dlx3hd and ß-galactosidase mRNAs (Fig. 4; Table 1). Notably, the size of the Xh2a and Xmsx expression domains were not changed when displaced laterally (Fig. 4C,F) nor was the premigratory neural crest field, marked by Xslug and Xsnail, altered (compare the injected and uninjected sides in Fig. 4I,N,P). Similarly, the characteristic three rows of primary neurons, marked by N-tubulin, arose in the expanded neural plate in EnR-Dlx3hd-injected embryos, but their pattern was shifted outwards such that the lateral neurons were now positioned along the new neural plate border (Fig. 4S). An identical result was observed in EnR-Dlx3hd-injected zebrafish as well (Fig. 4T,U) suggesting that a Dlx-dependent mechanism positions lateral neurons in both species. The neurons remained tightly organized in rows and ectopic neurons were not observed. The placodal marker Xsix1 was also shifted outwards in embryos expressing EnR-Dlx3hd (compare Fig. 4V with 4X). Interestingly, Xsix1 was displaced to a region just lateral to the domain of EnR-Dlx3 expression, consistent with expression outside the expanded neural plate. Lateral displacement of trigeminal placodes was also observed in embryos stained with N-tubulin, which also marks these placodes (not shown). The ability of EnR-Dlx3hd to displace the stereotypic pattern of marker expression laterally by late gastrula stages suggests that endogenous Dlx activity is upstream of neural crest, lateral primary neuron and cranial placode precursor specification.
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While the cell fate markers were often displaced laterally to the border of the EnR-Dlx3hd region, we also observed a loss of markers in some embryos (Table 1, Fig. 4Y and see below). Loss was correlated with large domains of injected EnR-Dlx3hd that extended to the ventral side of the embryo. Thus, depletion of endogenous Dlx activity permits the neural plate to expand maximally to about twice its normal size, regardless of whether or not a larger area expresses EnR-Dlx3hd. The loss of markers, however, raised the possibility that Dlx proteins are required in the non-neural ectoderm to provide neural crest inducing signals. This hypothesis is tested by the transplant experiments below.
In contrast to the loss-of-function studies, injection of VP16-Dlx3hd or wild-type Dlx3 did not shift marker expression medially, but rather abolished expression of both border and neural crest markers (Table 1 and Fig. 4B,E,H,M,R). The sole exception was Xsix1, which lies outside the neural plate normally (Fig. 4W). Medial displacement occurred when only a small area of the neural plate contained the injected VP16-Dlx3hd mRNA; large patches of VP16-Dlx3hd-injected tissue resulted in a loss of Xsix1. The general inability to shift these markers medially suggests that ectopic Dlx activity in presumptive neural plate cannot induce factors needed for correct patterning of the cell lineages that arise at the neural plate border. However, the medial displacement of Xsix1 that occurs when only a minimal gap separates the neural plate from non-neural ectoderm suggests that short-range communication between neural and non-neural ectoderm is critical for placode formation.
Dlx proteins regulate neural plate border formation during
gastrulation
To investigate the temporal requirement for Dlx function in neural plate
border formation, embryos were injected as above but with mRNAs encoding the
conditional proteins EnR-Dlxh3-GR or VP16-Dlx3hd-GR. Dexamethasone was added
at either stage 6, 8, 10 or stage 11.5-12 and embryos were examined for
Xsox2 and Xslug expression. Injected embryos cultured in the
absence of dexamethasone exhibited no change in any of the markers tested (not
shown). When dexamethasone was added at stage 6, embryos injected with
EnR-Dlx3hd-GR (70% expanded, n=34) or VP16-Dlx3hd-GR (80% repressed,
n=25) showed the expansion or repression of Xsox2,
respectively (Fig. 5A,B),
typical of the preceding results with the constitutive constructs. Similarly,
the neural crest marker Xslug was either lost (48% lost,
n=39) or shifted laterally (52% shifted laterally, n=39;
Fig. 5C) when
EnR-Dlx3hd-GR-injected embryos were treated with dexamethasone at stage 6. The
incidence of expanded Xsox2 expression and laterally shifted
Xslug (Fig. 5I) decreased as dexamethsone was added at later stages. Addition at stage 11.5-12
did not affect Xsox2 expression (wild-type pattern was observed in
86% of EnR-Dlx3-GR embryos, n=66 and in 90% of the
VP16-Dlx3hd-GR-injected embryos, n=93;
Fig. 5E,F). Similarly,
EnR-Dlx3-GR did not alter Xslug expression when dexamethasone was
added at stage 11.5-12 (65% were unaffected, n=126;
Fig. 5G). Therefore,
specification of the neural plate border and induction of neural crest
requires Dlx function before the end of gastrulation. We did notice, however,
that VP16-Dlx3hd-GR eliminated or reduced Xslug expression regardless
of whether dexamethasone was added at stage 6 (88% eliminated or reduced,
n=20; Fig. 5D) or
stage 11.5-12 (72% eliminated or reduced, n=114;
Fig. 5H). Thus, neural crest,
but not neural plate, remains sensitive to Dlx inhibition after late
gastrulation.
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Neural crest and cranial placode induction requires Dlx function in
the non-neural ectoderm
The preceding results showed that local depletion of Dlx function during
gastrulation causes an expansion of the neural plate and the lateral
displacement of the normal pattern of lateral primary neuron, neural crest and
cranial placode cell fates. This could occur by a repressive mechanism whereby
Dlx genes simply prevent neural plate formation. However, when the area of
EnR-Dlx3hd expression extended to the most ventral ectoderm, we noticed that
the neural plate expanded maximally to only about twice its normal distance
from the dorsal midline and that all of the cell lineage markers examined were
absent (Fig. 6A-D; identical
results obtained with EnR-Dlx5hd, not shown). This suggests that Dlx proteins
not only repress cell fates, but might also be necessary in non-neural
ectoderm for the production of factors that are critical for specification of
lateral primary neuron, neural crest, and cranial placode cells.
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To determine if Dlx function is required in non-neural ectoderm, we took
advantage of previous studies showing that grafting neural plate tissue to the
non-neural ectoderm causes neural crest cells to be induced in both host and
donor tissues where juxtaposed (Mancilla
and Mayor, 1996; Moury and
Jacobson, 1990
; Selleck and
Bronner-Fraser, 2000
). Regions of neural plate from fluorescent
dextran-injected donors were grafted to the ventral ectoderm of host embryos
that had been injected with nuclear-localized ß-galactosidase mRNA,
either alone or with EnR-Dlx3hd mRNA (Fig.
6E). We then assayed the graft region for the induction of
Xslug and Xsix1 to determine whether the induction of neural
crest and cranial placodes is inhibited when Dlx activity is downregulated. As
expected, control grafts showed Xslug expression induced at the
graft-host tissue interface (blue stain,
Fig. 6E inset). Higher
magnification views (Fig. 6J-N)
show induction of both Xslug and Xsix1 (blue stain) at the
interface of the graft (green fluorescent label) and host (magenta
ß-galactosidase stain) tissues, confirming that juxtaposition of
competent neural plate and non-neural ectoderm induces neural crest and
cranial placodes. In contrast, Xslug and Xsix1 were not
induced when neural plate was transplanted into ventral ectoderm expressing
EnR-Dlx3hd (Fig. 6O-S;
Table 2). In cases where donor
neural ectoderm spanned EnR-Dlx3hd-expressing and -non-expressing host tissue
(Fig. 6P), Xslug was
induced only at the site where the host tissue lacked injected mRNAs (black
arrowhead) but not in or near cells with the injected mRNA (red arrowhead). No
or minimal Xslug and Xsix1 transcripts were detected in
donor neural ectoderm cultured alone (Fig.
6F,G) whereas similar explants taken from a more lateral position
that spanned the border region expressed Xslug and Xsix1
(Fig. 6H,I), indicating that
expression in the grafts reflects induction and not contamination with donor
border region tissue. Taken together, these studies demonstrate that
Dlx-dependent transcription is required in non-neural ectoderm to produce
factors that act at over short range to induce neural crest and cranial
placode fates.
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DISCUSSION |
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A central finding of our study is that local inhibition of Dlx function shifts the stereotypic pattern of lateral primary neurons, neural crest and cranial placodes laterally (Figs 2,3,4). Thus, Dlx family members act upstream of the specification of cell fates that arise near the lateral border of the neural plate. Furthermore, Dlx genes are required for the induction of these cell fates, as revealed by the lack of marker expression when neural plate is grafted to EnR-Dlx3hd-expressing ectoderm (Fig. 6). Fig. 7 shows a schematic model of Dlx function at the lateral margin of the neural plate. Normally, Dlx activity in non-neural ectoderm (yellow) delimits the mediolateral position of the neural plate/non-neural ectoderm border and lateral neurons, neural crest and cranial placode cells (Fig. 7B). Inhibition of Dlx function in a localized region results in both an expansion of the neural plate and a lateral displacement of border region cell fates to a position abutting Dlx-positive ectoderm (Fig. 7C). Inhibition of a broader region of Dlx activity, however, causes the neural plate to expand maximally to about twice its normal distance from the dorsal midline, but normal border region cell lineages do not arise (Fig. 7D). The absence of stereotypic border region markers in these cases suggests that Dlx activity functions in the prospective epidermis to induce short range signals that specify border cell fates. In this model, the short-range signals are unable to traverse the interposed tissue (Fig. 7D). Ectopic Dlx activity generally fails to displace border cell fates medially (Fig. 7E). In these cases, as with broad regions of Dlx underexpression, we presume that signals needed to induce and pattern lateral neurons, neural crest and cranial placode cells cannot traverse the interposing tissue, which does not express neural or epidermal markers (Fig. 2).
|
Stereotypic displacement of border fates was not observed in previous
studies in which the neural plate was expanded by overexpression of Zic family
members, XBF-2, and Xash-3 (Kuo et al.,
1998; Mariani and Harland,
1998
; Mizuseki et al.,
1998
; Nakata et al.,
1997
; Turner and Weintraub,
1994
). In these cases, the domains of neural crest and lateral
neurons were either expanded or eliminated, indicating that these genes
control differentiation of neural plate cells and their derivatives rather
than regulate formation of a normal border region per se.
We did not see any consistent effects on the forebrain marker Otx2
when embryos were injected with either the activating or inhibiting constructs
(not shown). We also did not see a shift in the anterior portion of the
Xsix1 domain (not shown). Previous studies have shown that
overexpression of Dlx3 does not affect Otx2 expression
(Feledy et al., 1999). These
results suggest that Dlx genes function primarily posterior to the forebrain.
Positioning the mediolateral and anteroposterior positions of forebrain
neurectoderm might rely on a separate mechanism, possibly involving Wnt, FGF
or retinoic acid signaling (Villanueva et
al., 2002
).
Dlx activity regulates cell fates during gastrulation
The modulation of late gastrula stage markers
(Fig. 4A-I) and the timecourse
experiment of EnR-Dlx3hd-GR activation by dexamethasone addition
(Fig. 5) both indicated that
Dlx activity patterns cell fates at the medial neural/non-neural ectodermal
interface by stages 11.5-12. Because this time precedes the morphological
appearance and expression of molecular markers of neural folds and lateral
primary neurons, neural crest cells and cranial placodes, Dlx activity is
needed upstream of the process that induces these cell fates. Classical graft
and extirpation experiments suggested that the anterior neural plate border is
no longer susceptible to signals that reposition the border by late blastula
stage (stage 9+) (Zhang and Jacobson,
1993). While the mediolateral border was not examined
specifically, it is possible that these studies revealed the action of
diffusible signals, such as BMP and Wnt, that bias ectodermal fates. Our data
indicates that endogenous Dlx activity is required somewhat later, through
stage 11.5-12 (Fig. 5),
consistent with Dlx genes being regulated by BMP and Wnt signaling.
Interestingly, the stages when Dlx activity is required corresponds to the
time when the transcripts encoding Dlx, Xmsx1 and Xiro become spatially
localized to respect the neural plate border. The potential involvement of Dlx
proteins with these and other proteins to regulate their own expression and
influence neural, epidermal and border region cell fates is discussed
below.
Complex interactions among proteins involved in patterning the neural
plate border and adjacent cell fates
Current models for partitioning ectoderm into neural plate and epidermis
involve initiating Wnt, BMP and FGF signals
(Baker et al., 1999;
Barth et al., 1999
;
Ikeya et al., 1997
;
Launay et al., 1996
;
Streit and Stern, 1999
;
Wilson and Hemmati-Brivanlou,
1995
; Wilson et al.,
2001
). Wnt signaling through the canonical ß-catenin pathway
provides dorsal cues during cleavage stages (e.g.
Heasman et al., 1994
;
Larabell et al., 1997
). By
pre-gastrula stages, ectopic activation of BMP and Wnt/ß-catenin
signaling pathways elicit a ventralizing effect on embryos suggesting that
these proteins might normally antagonize induction of the neural plate at
these stages (Baker et al.,
1999
; Christian and Moon,
1993
; Hawley et al.,
1995
; Sasai et al.,
1995
; Wilson and
Hemmati-Brivanlou, 1995
;
Wilson et al., 2001
). At least
Dlx3 is negatively regulated by the pre-MBT Wnt/ß-catenin
signaling (Beanan et al.,
2000
), and induced by pre-gastrula BMP signaling
(Feledy et al., 1999
); thus,
Dlx genes probably act downstream of these proteins. Whether Dlx activity
feeds back to influence gastrula-stage BMP and/or Wnt signaling is
unclear.
Dlx proteins might regulate border cell fates by cooperating with other
transcription factors that are induced by early BMP, Wnt and/or FGF signaling.
Injection of VP-Dlx3hd prevents induction of markers of lateral primary
neurons, neural crest and cranial placodes
(Fig. 4). Moreover, epidermal
markers were not induced (Fig.
2), suggesting that Dlx activity alone is insufficient to
respecify neural plate to non-neural ectoderm (see diagram in
Fig. 7E) and implicating an
additional factor. One candidate is Msx1, which is an immediate downstream
target of BMP signaling localized to the ventral ectoderm in early gastrula
stage Xenopus embryos (Feledy et
al., 1999; Suzuki et al.,
1997
). Overexpression of Msx1 represses neural plate and
border cell fates, but unlike Dlx3, also converts prospective neural
plate into epidermis (Suzuki et al.,
1997
). Neural inhibition by Msx-1, unlike Dlx factors, appears to
act via transcriptional repression
(Yamamoto et al., 2000
),
suggesting that Msx-1 and Dlx factors regulate distinct target genes. Since
Msx and Dlx proteins can heterodimerize
(Zhang et al., 1997
),
regulatory interaction between the factors is also likely. Msx-1
loss-of-function experiments are needed to address its role in cell fate
specification at the neural plate border. Defects of Msx1-deficient
mice are not known to involve an expanded neural plate, but potential
redundancy or compensation complicates interpretation
(Jumlongras et al., 2001
;
Satokata and Maas, 1994
).
Nonetheless, it seems possible that induction of neural crest, lateral neurons
and cranial placodes might require both Dlx activity and the
epidermal promoting activity of Msx1.
The lateral displacement of border cell fates by Dlx genes could indicate a
genetic interaction with prospective neural plate factors (see diagram in
Fig. 7A). One candidate is
Xiro1, which is initially expressed broadly in the dorsal ectoderm at
the onset of gastrulation but becomes restricted to the prospective anterior
neural plate as gastrulation proceeds
(Gomez-Skarmeta et al., 1998).
Overexpression of Xiro1 expands the neural plate and, in some
instances, can displace Xslug expression outward
(Gomez-Skarmeta et al., 1998
).
Furthermore, ectopic Xiro1 downregulates endogenous Bmp4
expression whereas ectopic Xmsx1 downregulates endogenous
Xiro1 (Gomez-Skarmeta et al.,
2001
). These data combined with our results raise the intriguing
possibility that Dlx genes, like Xmsx1, are involved in a mutual
repression circuit with Xiro1.
In summary, our results support a model in which diffusible BMP, Wnt, and FGF signaling establishes graded expression patterns of transcription factors involved in positioning the neural plate border and determining adjacent cell fates. The neural plate antagonizing activities of Dlx proteins probably function in a reciprocal (inhibitory) circuit with transcription factors that promote neural plate, such as the Xiro-1 protein. The consequence of this interaction is to sharpen the border between the neural and non-neural ectoderm. In addition, we showed that Dlx genes also play a positive role in non-neural ectoderm for the production of factors that are required for the induction and stereotypic mediolateral patterning of lateral neurons, neural crest and placode cells.
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ACKNOWLEDGMENTS |
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