1 Department of Hematology/Oncology, St Jude Children's Research Hospital,
Memphis, TN 38105, USA
2 Department of Molecular Sciences, University of Tennessee Health Science
Center, Memphis, TN 38105, USA
3 Department of Pathology, St Jude Children's Research Hospital, Memphis, TN
38105, USA
4 Rotary Bone Marrow Laboratory, Melbourne, Australia
5 Department of Pediatrics, University of Tennessee Health Science Center,
Memphis, TN 38105, USA
* Authors for correspondence (e-mail: john.cunningham{at}stjude.org, paul.mead{at}stjude.org)
Accepted 30 November 2004
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SUMMARY |
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Key words: Xenopus, Grainyhead-like 1, BMP
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Introduction |
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The importance of the BMP4 signaling has resulted in the identification of
two major types of effector molecules, downstream of the BMP4 receptor, that
modulate epidermal-specific gene expression. Activation of Smad signal
transducers, and expression of the `immediate early response' (IER)
transacting factors Msx1 and XVent2, occurs with BMP4
binding to its cognate receptor
(Onichtchouk et al., 1996;
Suzuki et al., 1997b
;
Wilson et al., 1997
). In
naïve ectodermal cells, ectopic expression of these factors (like BMP4)
induces an epidermal fate with concomitant repression of neurogenesis. IER
factors, in turn, modulate transcription of a second set of widely expressed
trans-acting factor genes, including XAP-2 and
Dlx-3, which have a narrower range of action in ectodermal cells,
activating epidermal gene expression with concomitant repression of the neural
gene expression (Feledy et al.,
1999b
; Luo et al.,
2001b
; Luo et al.,
2002
; Snape et al.,
1991
).
Although a plethora of genes activated by this deceptively simple signaling
cascade have been identified, it remains unclear how epidermal-specific gene
expression is achieved. Examination of the regulatory sequences of several
mammalian epidermal-specific structural genes implicate a combinatorial
network of ubiquitous factors, such as Ap1 or Sp1, and
tissue-restricted proteins, such as Ap2 or Dlx3, that bind
promoter/enhancer elements in a context-specific manner to facilitate
appropriate high-level expression (Byrne,
1997; Crish et al.,
2002
; Kaufman et al.,
2003
; Ng et al.,
2000
; Presland et al.,
2001
; Sinha et al.,
2000
; Sinha and Fuchs,
2001
). However, several tissue-specific binding activities have
been reported, suggesting that, as yet, unidentified regulators of vertebrate
epidermal differentiation await characterization.
This hypothesis is supported by Drosophila mutagenesis screens,
numerous tissue-specific gene loci being identified that influence ectodermal
differentiation (Juergens et al.,
1984; Nusslein-Volhard et al.,
1994
; Wieschaus et al.,
1984
). One of these, grainyhead or grh (also known as
NTF-1 or Elf1), has an ectodermal-restricted pattern of
expression (Bray et al., 1989
;
Dynlacht et al., 1989
;
Uv et al., 1997
). Loss of
grh function results in ectodermal defects including flimsy cuticles,
abnormal head structures and a `blimp' phenotype
(Attardi et al., 1993
;
Bray and Kafatos, 1991
;
Ostrowski et al., 2002
).
Recent studies suggest an evolutionary conservation of grh-like
function. Cuticular defects occur with disruption of ceGrh, an
ectodermal-restricted, C. elegans ortholog of grh
(Venkatesan et al., 2003
).
Moreover, we, and others, have identified three mammalian orthologs of
grh, grainyhead-like factors 1, 2 and 3 (Grhl1-3), which share an
ectodermally restricted pattern of expression and a high degree of identity in
the DNA binding and dimerization domains
(Huang and Miller, 2000
;
Kudryavtseva et al., 2003
;
Ting et al., 2003b
;
Wilanowski et al., 2002
).
In this report, we provide the first evidence for a critical role of a
vertebrate grainyhead-like factor, Xenopus grainyhead-like 1
(XGrhl1) in epidermal ontogeny. This factor is regulated in an
epidermal-specific BMP4-dependent manner, modulating expression of
epidermal-specific gene expression. Concordant with these observations,
disruption of XGrhl1 activity results in defective epidermal
differentiation. Moreover, we identify a XGRHL1-dependent target gene,
epidermal keratin (XK81A1) (Dawid
et al., 1985; Jonas et al.,
1985
), and show that a crucial 5' regulatory motif of the
XK81A1 gene, which is necessary for high level promoter activity,
binds XGRHL1 directly. Together, these observations provide key mechanistic
insights into the role of an epidermal-specific transacting factor in
vertebrate development.
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Materials and methods |
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Xenopus embryo manipulations
Wild-type and albino Xenopus (NASCO) eggs were fertilized in
vitro, dejellied, cultured using standard methods
(Sive et al., 2000) and staged
according to Nieuwkoop's normal table of development
(Nieuwkoop and Farber, 1994
).
Microinjection, lineage tracing and animal cap assays were performed as
described (Luo et al., 2002
;
Parvin et al., 1995
;
Sargent et al., 1986
;
Smith and Harland, 1991
;
Wilson and Hemmati-Brivanlou,
1995
). Isolation of superficial and deep ectodermal layers was
performed as described with minor modification
(Chalmers et al., 2002
).
Expression analysis and probes
Whole-mount in situ hybridization was performed using albino
Xenopus embryos (Harland,
1991; Hemmati-Brivanlou et
al., 1990
). Digoxigenin-labeled antisense RNA probes were
generated from XGrhl1 full-length cDNA. Other probes used were
XK81A1, BMP4, XAP2 and Dlx3
(Feledy et al., 1999a
;
Jonas et al., 1985
;
Luo et al., 2002
;
Mariani and Harland, 1998
).
ß-Galactosidase activity was used as an injection tracer
(Sive et al., 2000
) using the
substrates XGal (blue) or Red-Gal (Research Organics, Cleveland, OH). For
histological analysis, embryos were embedded in paraffin. RNA extractions,
first-strand cDNA synthesis and PCR were carried out as previously described
(Kelley et al., 1994
;
Mead et al., 1996
). All assays
were performed in the linear range.
RT-PCR and primers
RNA extractions, first-strand cDNA synthesis and PCR were carried out as
previously described (Kelley et al.,
1994; Mead et al.,
1996
). Primers for ODC, XK81A1 keratin,
-actin, NCAM, geminin, XBra, GATA2, Sox3, BMP4, Msx1, XOtx2, XAG1,
XZic3, XNrp1, XVent-2, HIS4 and EDD have been reported
previously (Kroll et al.,
1998
; Sasai and de Robertis,
1997
; Wilson et al.,
1997
)
(http://www.xenbase.org/methods/RT-PCR.html).
Other primers for PCR included: XGrhl1
(5'-TGACCACCGCCTTCAGTGCT-3' and
5'-CCTTGGCTGCCCTGACATTG-3' amplify a 444 bp fragment at 56°C,
25 cycles), PCNA (5'-CGATCAGACGGCTTTGACAC-3' and
5'-CTCCGCTCGCAGAGAACTTT-3' amplify a 364 bp fragment at 56°C,
25 cycles), P63 (5'-CATGCCCAATCCAAATCAAA-3' and
5'-CATCTGCCTTGCGGTCTCT-3' amplify a 444 bp fragment at 56°C,
25 cycles), ESR-1 (5'-GGATTACAAGCAAGGGTTC-3' and
5'-TCCCATAGGATAACGTTCAT-3' amplify a 378 bp fragment at 54°C,
28 cycles), XNotch1 (5'-TGCCTTCCAATCTTACGC-3' and
5'-AGGGCAGTGTTTTAGGTCAA-3' amplify a 428 bp fragment at 54°C,
27 cycles), XDelta1 (5'-CTGTCCCCCTGGCTACATT-3' and
5'-CCCTCACACAGACAACCACA-3' amplify a 305 bp fragment at 56°C
25 cycles), Dlx3 (5'-GCTTGTGGGCAACGAG-3' and
5'-CTGCGTCTGAGTGAGTCCTA-3' amplify a 292 bp fragment at 56°C,
25 cycles), Dlx5 (5'-ATTCTCCCCAGTCTCCAGTG-3' and
5'-GATAGTGTCCCCAGTTGCGC-3' amplify a 425 bp fragment at 55°C,
25 cycles), XAP2 (5'-CGGGTATGTGTGCGAAACAG-3' and
5'-GGCGGGAGACCAATAGAGAA-3' amplify a 445 bp fragment at 56°C,
25 cycles) and ESR6e (5'-GGCACAGGGCAATACTGGT-3' and
5'-CCCCACTTGGCATTATGTTC-3' amplify a 400 bp fragment at 55°C,
27 cycles). PCR conditions were determined for each primer set to ensure that
amplification was within a linear range.
Plasmid construction
A 3.4 kb XGrhl1 cDNA was subcloned into pRN3 and pCS2+ (kind gifts
from P. Lemaire and D. Turner, respectively) to create RN3-XGrhl1 and
pCS2+XGrhl1. The plasmid RN3-227XGrhl1 or
RN3-EGFP
227XGrhl1 were generated from RN3-XGrhl1 by
replacing BglII/EcoRI fragment with an adaptor (annealing
5'-GATCTGAGAGCATCATGGCG-3' and
5'-AATTCGCCATGATGCTCTCA-3') or PCR amplified fragment of the EGFP
cDNA.
Synthetic RNA and antisense morpholino (MO) oligonucleotides
Synthetic RNA was made from linearized plasmid DNA with the mMessage
mMachine in vitro transcription kits (Ambion, TX). The RNA yield was
quantitated by spectrophotometer and its integrity checked by gel
electrophoresis. The following plasmids were digested and incubated with the
appropriate RNA polymerase: RN3-XGrhl1 (SfiI, T3),
RN3-227XGrhl1 (SfiI, T3),
RN3-EGFP1
227XGrhl1 (SfiI, T3), pSP64T-nucßGal
(XhoI, SP6), pSP64T-BMP4 (BamHI, SP6),
pSP64T-activin ßB (EcoRI, SP6), pCS2+XMAD
(NotI, SP6), pSP64T-tBR (EcoRI, SP6),
pCS2MT-Ngem (NotI, SP6) and pCS2+noggin
(NotI, SP6), RN3-XVent2 (PstI, T3)
(Huber et al., 1998
;
Huber et al., 2001
;
Kroll et al., 1998
;
Maeno et al., 1996
;
Onichtchouk et al., 1996
;
Smith and Harland, 1992
). MOs
were obtained from GeneTools, XGrhl1-MO2 having the sequence
5'-GTCGTAGTCTTGTGTCATGATGCTC-3', and a company-supplied control
morpholino (CMO) serving as a control.
GST chromatography, electrophoretic mobility shift analysis and luciferase reporter assays
Protein:protein interaction assays using GST chimeric factors and
electrophoretic mobility shift assays (EMSA) were performed as previously
described (Jane et al.,
1995).
Blastomeres at the four-cell stage were co-injected with 10 nl of RNA
encoding 227Grhl1 mutant and the KP487 reporter construct.
pRL-TK, a construct in which the thymidine kinase promoter was linked to the
renilla luciferase cDNA (30 pg/embryo), was injected to control for injection
efficiency. After injection, the embryos were cultured to stage 11. Ten
embryos were collected and lysed in 1 x lysis buffer (20 µl/embryo).
Luciferase activity was determined in 10 µl of the supernatant, according
to the manufacturer's instructions and quantified with a Model TD-20/20
luminometer (Turner Designs). Relative firefly luciferase activity (RLU) was
normalized with Renilla luciferase activity in cellular lysates.
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Results |
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Consistent with Drosophila grh expression
(Bray et al., 1989;
Dynlacht et al., 1989
;
Uv et al., 1997
),
XGrhl1 is expressed throughout ontogeny. Maternally derived
transcripts (stage 2-8) are replaced by zygotic expression after the
mid-blastula transition (stage 9+), which persists throughout embryogenesis
(Fig. 1A). Dissection of the
germ layers at stage 11 revealed that XGrhl1 transcripts were
restricted, like other epidermal-restricted genes, ESR6e and
XK81A1 keratin, to the superficial (non-neuronal) layer of the
ectoderm at mid-gastrulation (Fig.
1B) (Chalmers et al.,
2002
; Deblandre et al.,
1999
; Jonas et al.,
1985
). By contrast, unlike Drosophila grh, or the
neuronal-specific xNotch, xDelta and ESR1 genes,
XGrhl1 was neither expressed in the neuroectodermal layer, nor the
mesoderm or endoderm at any time point
(Fig. 1B; data not shown).
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To assess whether the specificity of these effects was related to
inhibition of BMP4 signaling directly, we examined the outcome of co-injection
of tBR and increasing amounts of XMad1 RNA. Enforced
expression of XMad1, a BMP4-specific signal transduction molecule, is
sufficient to activate the BMP4-dependent signaling cascade, reversing
tBR-induced neuroectodermal specification
(Wilson et al., 1997), as
demonstrated by downregulation of NCAM expression
(Fig. 3D). XAG
expression, a marker of the cement gland whose expression is modulated by the
relative concentration of BMP4, was also repressed
(Gammill and Sive, 2000
). By
contrast, XK81A1 and XGrhl1 expression was restored to
levels observed in caps derived from uninjected embryos. We observed a similar
outcome with co-injection of noggin and XMad1 (data not
shown). Thus, XGrhl1 expression is dependent on activation of the
epidermal differentiation program, is modulated by the BMP4-signaling pathway,
and is repressed by regulatory factors that induce neuralization.
These observations raised the possibility that XGrhl1 alone may specify epidermal fate and/or inhibit neural specification. To address this hypothesis, we co-injected increasing amounts of XGrhl- and ß-gal-encoding RNA into animal pole blastomeres, observing no effect on epidermal (top) or neural (bottom) specification at any stage of ontogeny (Fig. 3E; data not shown). Furthermore, co-injection of XGrhl1-expressing RNA with either tBR (Fig. 3F) or noggin (data not shown) at the one-cell stage failed to suppress neural gene expression (NCAM, Sox3 or Nrp1), or rescue known BMP4 epidermal-specific targets including XK81A1 in animal cap explants (Fig. 3F). Taken together, our studies demonstrate that XGrhl1 expression is activated by BMP4 signaling, but in the absence of such a cue, enforced XGrhl1 expression is insufficient to specify an epidermal fate.
XGrhl1 can modulate downstream components of the BMP4 signaling pathway
Establishment of the relative position of XGrhl1 in the BMP4
signaling cascade is complicated by the high concentrations of endogenous BMP4
protein present in ectodermal explants. To circumvent this problem, embryos
were isolated at stage 9, and dissociated in Ca2+- and
Mg2+-free buffer (Fig.
4A) (Sargent et al.,
1986; Wilson and
Hemmati-Brivanlou, 1995
). After washing, to remove endogenous
extracellular factors, re-aggregated cells were incubated with recombinant
BMP4 or media alone until control embryos reached the mid-neurula stage. In
the absence of BMP4, cap cells had a neural fate, expressing NCAM,
but failing to express either the XK81A1 and ESR6e
epidermal-specific markers, or XGrhl1 mRNA, as assayed by
semi-quantitative RT-PCR (Fig.
4B). By contrast, increasing amounts of BMP4 induced a significant
increase in XK81A1 and ESR6e expression, and a concomitant
decrease in NCAM expression. Moreover, epidermal marker expression
was associated with a BMP4-dependent induction of XGrhl1
transcription. Concomitantly, induction of the Dlx3, Dlx5 and
XAP2 transacting factors was observed in BMP4-treated cells. Given
that these factors are known downstream components of the BMP4 signaling
cascade, these results suggest strongly that XGrhl1 is modulated by
BMP4 directly.
|
To localize the relative position of XGrhl1 in the BMP4 signaling
pathway, we micro-injected fertilized eggs with XGrhl1 encoding RNA
and assessed its effect(s) in the dissociated cap assay. Induction of BMP4, or
of the IER genes, Msx1, xVent2 or p63 was not observed
(Fig. 4C), consistent with the
absence of BMP4 stimulus, and a lack of effect of XGrhl1 on
expression of these factors (Bakkers et
al., 2002; Wilson and
Hemmati-Brivanlou, 1995
). By contrast, not only was
XK81A1 induced by XGrhl1, but upregulation of Dlx3,
Dlx5, XAP2 and ESR6e expression was also observed. Our results
strongly support the conclusion that XGrhl1 is induced by BMP4
signaling, and is located downstream of the IER genes, but upstream of
structural genes, such as XK81A1, that are necessary for epidermal
differentiation.
XGrhl1 function/expression is required for epidermal differentiation in the developing embryo
Our results are consistent with a model in which XGrhl1 expression
modulates epidermal differentiation predominantly at a stage subsequent to
commitment of ectodermal cells to an epidermal fate. To elucidate further the
role of this factor in epidermal differentiation, we identified a specific
dominant-negative form of XGrhl1, guided in part by previous
observations of a Drosophila dominant-negative mutant. The fly
factor, 447grh, lacked a 447 amino acid N-terminal activation
domain, dimerized with the wild-type protein and blocked GRH function
(Attardi et al., 1993
). The
structurally homologous mutant of XGrhl1 tested here,
227XGRHL1, lacks a N-terminal activation domain encoding the first 227
amino acids, dimerizes with wild-type XGRHL1 and has comparable binding
affinity to XGRHLl for a consensus grh-binding motif (see Fig. S2 in
the supplementary material). In addition, both
447grh and
227XGrhl1 transcripts inhibited XGrhl1-induced
XK81A1, Dlx3, Dlx5 and XAP2 expression specifically in
dissociated cell explant assays (see Fig. S2 in the supplementary
material).
Microinjection of 227XGrhl1 encoding mRNA (1 ng) into
animal pole blastomeres at the two- to 16-cell stage resulted in normal
gastrulation and neurulation of
227XGrhl1/ß-gal injected
embryos (stages 1-25; data not shown). By contrast, the effect of
227XGrhl1 expression on non-neuronal ectoderm at later
timepoints was profound, with gross macroscopic distortion of pre-larval
epidermal differentiation (Fig.
5A-C; stages 35-40). We observed a consistent loss of specialized
surface structures and a failure of evolution of the normal pigmentation
pattern in injected ectoderm. Head and neck structures were defective, with
absence of eye and otic placodes, failure of formation of the stomatodeal
anlage and lack of melanization (Fig.
5A,B). Trunk/tailbud structures were also abnormal, with frequent
misshapen embryos and loss of appropriate fin formation
(Fig. 5B). Furthermore, there
was apparent persistence of embryonic pigment at stage 40 on the injected side
alone, which is more characteristic of earlier stages of pre-larval
development (Fig. 5A,C;
arrows). By contrast,
227XGrhl1 transcripts, even at high
concentrations, had no macroscopic effect when injected into blastomeres with
a neural or mesendodermal fate, specificity being confirmed by rescue of
227XGrhl1-mediated defects by co-injection of XGrhl1
transcripts (data not shown).
|
Additional features were evident from histological analysis of embryos in
which blastomeres with a non-neuronal ectodermal fate were injected with
227XGrhl1-encoding RNA transcripts. First, disorganization of
specialized epidermal structures including otic and optic placode formation
was observed with consequent failure of lens structure development in the
latter instance (Fig. 5C; data
not shown). Coincident with the latter changes, significant regression of the
neuroretinal structures was observed, with a reduction of the surrounding
pigment layer. Second, structures deep to the epidermis showed significant
changes, including disorganized head and trunk mesenchyme, and alteration in
the neural tube and the stomodeal anlage. By contrast, histology of embryos in
which
227XGrhl1 transcripts were injected into blastomeres
with a mesoendodermal fate was normal (data not shown). These results indicate
that loss of XGRHL1 function results in a specific primary alteration in
epidermal differentiation in the maturing Xenopus embryo, with
apparent loss of epidermal inductive signals to underlying structures.
XGrhl1 function/expression is required for appropriate epidermal keratin expression
Architectural changes in the OEL occurring with 227XGrhl1
expression, coupled with the modulation of expression of XK81A1,
ESR6e and other epidermal-restricted factors in explanted cells, support
the contention that XGrhl1 activity is necessary for appropriate
epidermal differentiation subsequent to commitment. To test this idea further,
we asked if XGrhl1 activity was required for epidermal structural
gene expression in vivo. We chose to focus our efforts on XK81A1
expression, given the central role of keratins in structural and morphogenetic
events in vertebrate epidermal cells
(Fuchs and Raghavan, 2002
;
Porter and Lane, 2003
).
Transcripts encoding
227XGrhl1, co-injected with ß-gal
into one blastomere at the four-cell stage, resulted in complete loss of
XK81A1 expression in the progeny of injected blastomeres when assayed
at stage 14 (Fig. 6A; data not
shown). This effect was reversed with co-injection of wild type
XGrhl1, confirming the specificity of
227XGrhl1
activity (Fig. 6A;
Table 1). By contrast,
227XGrhl1 had no effect on XAP2, Dlx3 or BMP4
expression (Fig. 6B). A green
fluorescent protein/
227XGrhl1 chimera gave a similar result
and confirmed that the dominant-negative factor, like the wild-type protein
was localized to the nucleus of injected blastomere progeny
(Fig. 6B; data not shown).
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Discussion |
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Ectopic expression of BMP4 or of IER factors, such as Xmad1,
result in epidermal re-specification in cellular progeny of blastomeres with a
neural fate (Wilson et al.,
1997). Similarly, co-expression of IER factors and BMP
antagonists/inhibitors induces epidermal specification in injected ectodermal
cells, with coincident repression of neural gene expression
(Feledy et al., 1999a
;
Suzuki et al., 1997a
;
Suzuki et al., 1997b
). Given
the temporal pattern of endogenous expression, we expected a similar outcome
with enforced expression of XGrhl1. Differing sharply from the
effects of IER factors, ectopic expression of XGrhl1 failed to induce
epidermal specification. These observations suggest that XGrhl1
activity is dispensable for this process, a conclusion supported by the
inability of injection of
227XGrhl1-encoding transcripts or
XGrhl1-specific MOs to affect germ layer specification.
Our studies suggest an alternate model, XGrhl1 functioning
downstream of the IER factors in the BMP-signaling cascade
(Fig. 8). In this context,
AP2 and Dlx-like factors have been shown previously to be
essential for appropriate epidermal differentiation
(Fuchs and Raghavan, 2002;
Luo et al., 2002
;
Panganiban and Rubenstein,
2002
). However, it remains unclear how these factors achieve
tissue specificity given their wider pattern of gene expression
(Luo et al., 2001b
;
Luo et al., 2002
). We show
that induction of XK81A1 is dependent on appropriate XGrhl1
function. Like Dlx3, expression of XGrhl1 does not induce
expression of the epidermal structural gene XK81A1 in the absence of
a functional BMP4 pathway, suggesting that morphogen-induced expression of
other factors is necessary. One candidate may be AP2, given its
ability to rescue the epidermal defect induced by tBR expression in a similar
manner to IER regulatory factors (Luo et
al., 2001b
; Suzuki et al.,
1997b
; Wilson et al.,
1997
). Furthermore, like XGrhl1, AP2 fails to repress
expression of pan-neural gene markers, a divergence from the effects of IER
factor expression (Luo et al.,
2002
). These observations, together with our studies of the
XK81A1 promoter, indicate that XGrhl1 functions
predominantly downstream of the IER factors in the BMP4 signaling cascade.
Furthermore, our studies of the XK81A1 promoter demonstrate that both
AP2 and XGRHL1 are required for XK81A1 expression (see
below). Thus, we suggest that characterization of the expression of
XGrhl1 and its mechanism of action represents a significant new
insight into the regulation of BMP4-responsive epidermal-specific targets,
this tissue-specific factor modulating structural gene expression in concert
with the more widely expressed regulator AP2 during terminal
differentiation.
|
Our observations demonstrating a severe defect in end-stage epidermal
differentiation induced by perturbation of XGRHL1 function raise the issue of
whether this defect is related to loss of XK81A1 expression alone, or
to a more generalizable defect in expression of genes of the epidermal
program. The latter model is supported by several observations. Although the
effect of a knock down of XK81A1 expression on Xenopus
development has not been described, studies of dysregulation of keratin K14,
its murine homolog, suggest otherwise. Animals homozygous for K14 disruption
exhibit a similar increase in keratinocyte fragility that resembles the
227XGrhl1-induced phenotype
(Lloyd et al., 1995
).
Conversely, these mice are viable at parturition, have appropriate
differentiation of embryonic and adult epidermal keratinocytes, and have no
evidence of a secondary defect in underlying mesendodermal tissues. A second
line of evidence supporting a more generalized epidermal defect is provided by
the observation that enforced co-expression of XK81A1 in
227XGrhl1-expressing embryos (or in embryos injected with
XGrhl1-MOs) failed to reverse the phenotype (J.T., unpublished). Collectively,
these results suggest that XGrhl1 modulates transcription of a range
of epidermal-specific genes.
Additional lines of evidence support this conclusion. First, comparison of
Drosophila and Xenopus Grhl-defective phenotypes confirm an
evolutionary conservation of Grhl factor function during epidermal ontogeny.
Thus, the failure of appropriate epidermal differentiation, abnormal head
structures and defective specialized epidermal structures characteristic of
227XGrhl1 expression mirrors the defects observed with
expression of a structurally similar mutant,
447grh, in
Drosophila. The mutant fly phenotype includes cuticlar defects, a
`grainyhead' phenotype and deficits in hooks, mouth pieces and wing structures
(Attardi et al., 1993
;
Bray and Kafatos, 1991
;
Lee and Adler, 2004
).
Interestingly, injection of
447grh into Xenopus
blastomeres with an epidermal fate at the eight- to 16-cell stage results in a
similar outcome to that observed with
227XGrhl1 (data not
shown). Second, we observed XGrhl1-mediated expression of other
epidermal-restricted differentiation factors, including AP2, Dlx3 and
ESR6e in ectodermal cells (Fig.
4). As discussed above, this suggests a complex requirement for
XGrhl1 in the expression of these genes.
What are the identities and functions of other XGrhl1-dependent
genes? The failure to resorb cytoplasmic yolk platelets and assume the
flattened morphology of the mature peridermal cell suggests that
XGrhl1 may modulate repression of the primitive epidermal gene
program and/or modulate morphogenetic changes in cell shape. The latter
hypothesis is supported by observations demonstrating a role for
Drosophila grh in changes in tracheal cell shape and the
epidermal-specific failure of murine neural tube closure observed with mouse
Grhl3 deficiency (Hemphala et al.,
2003; Ting et al.,
2003a
). Given our demonstration of a conservation of Grhl
function, other Grhl targets may be provided by recent characterization of
orthologs of Drosophila blimp phenotypes similar to grh
(Ostrowski et al., 2002
).
Preliminary analysis has identified several genes epistatic to GRH,
including cadherins and adhesion molecules
(Lee and Adler, 2004
). Indeed,
some of these molecules are also involved in the development of specialized
epidermal appendages. Given the apparent evolutionary conservation of Grhl
function, it will be important to determine the functional role of vertebrate
orthologs of XGrhl1, particularly its role in the stratification of
the adult epidermis, as similar mechanisms are operative in the embryonic
epidermis and the basal layer of the adult skin
(Byrne et al., 1994
;
Koster et al., 2004
;
Nieuwkoop and Farber,
1994
).
Vertebrate promoter/enhancer regulatory elements of type I (and II)
keratins, and other structural genes contain functionally important motifs for
the non-epidermal specific AP2, AP1 and Sp1 DNA-binding factors, amongst
others (Byrne and Fuchs, 1993;
Jonas et al., 1989
;
Kaufman et al., 2002
;
Leask et al., 1990
;
Leask et al., 1991
;
Sinha et al., 2000
;
Sinha and Fuchs, 2001
;
Snape et al., 1990
;
Snape et al., 1991
). The
expression patterns of these factors suggest that non-epidermal specific
factors interact in a combinatorial manner, potentially recruiting
keratinocytic-specific co-regulators to modulate appropriate expression
(Fuchs and Raghavan, 2002
).
Several mammalian keratinocyte-specific promoter/enhancer-binding activities
have been identified recently, although detailed characterization is awaited
(Kaufman et al., 2002
;
Sinha et al., 2000
;
Sinha and Fuchs, 2001
). Our
studies alter this model significantly. We demonstrate clearly the molecular
basis by which binding of a novel epidermal-specific factor, XGRHL1, is
essential for high-level transcription in epidermal cells. Interestingly,
preliminary exploration of murine K14 sequences, as well as Xenopus
AP2 and Dlx3 promoters, identified similar Grhl binding motifs
(J.T., unpublished).
Adjacent to the XGRHL1 site is a previously defined AP2 motif crucial for
maximal promoter activity (Snape et al.,
1990; Snape et al.,
1991
). Our studies suggest a functional interaction between these
factors (Fig. 7D,E).
Drosophila GRH interacts with dTAFII110
(Attardi and Tjian, 1993
;
Dynlacht et al., 1989
;
Dynlacht et al., 1991
), a
component of the TFIID TATA-binding complex providing a structural basis for
the exploration of the molecular mechanism(s) of keratin gene transcription.
Interestingly, hTAFII130, the ortholog of dTAFII110,
interacts with the co-regulator CBP
(Nakajima et al., 1998
), the
latter co-factor being required for AP2-mediated gene activation
(Braganca et al., 2003
).
Together, these observations suggest the existence of a molecular `bridge'
between the DNA-bound transacting factors and the transcriptional initiation
complex which may explain the requirement for binding of both XGRHL1 and AP2
for maximal promoter function. It will be important to confirm these
relationships in future studies.
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Supplementary material |
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ACKNOWLEDGMENTS |
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