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First published online 21 April 2004
doi: 10.1242/dev.01115
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1 Laboratory of Pathology, Center for Cancer Research, NCI, NIH, Bethesda, MD
20892, USA
2 Cell and Cancer Biology Branch, Center for Cancer Research, NCI, NIH,
Bethesda, MD 20892, USA
Author for correspondence (e-mail:
smack{at}helix.nih.gov)
Accepted 9 February 2004
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SUMMARY |
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 TOP SUMMARY IntroductionMaterials and methodsResultsDiscussionREFERENCES |
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Key words: AP pattern, Digit formation, Limb development, Hoxd genes, Gli3, Sonic hedgehog, Mouse
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Introduction |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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) (see reviews by
Mariani and Martin, 2003;
Tickle, 2003). The pattern of
different digits (I to V) that form from anterior (A; digit I, e.g. thumb) to
posterior (P; digit V, e.g. little finger) is controlled by secreted Shh
signals produced in the posterior limb bud mesoderm (reviewed by
Ingham and McMahon, 2001;
Mariani and Martin, 2003;
Tickle, 2003). Shh regulates
both digit number and identity in a dose-dependent manner; increasing levels
of Shh expand digit-forming capacity and specify more posterior digit
identities (Yang et al., 1997;
Lewis et al., 2001). The zinc
finger transcription factor Gli3 is the direct intracellular mediator of Shh
(Altaba, 1999;
Dai et al., 1999;
Shin et al., 1999) (reviewed
by Ingham and McMahon, 2001)
and Shh signaling protects Gli3 from cleavage to a repressor form
(Wang et al., 2000). Without
Shh, Gli3 repressor predominates, Shh/Gli3 target genes are repressed
(Altaba, 1999;
Dai et al., 1999;
Shin et al., 1999) and digit
formation largely fails (Chiang et al.,
2001; Kraus et al.,
2001). Eliminating Gli3 renders Shh dispensable for digit
formation, but normal digit identity is lost and polydactyly occurs
(Litingtung et al., 2002;
te Welscher et al., 2002). In
addition to functioning antagonistically, Gli3 also represses Shh
expression and, in Gli3-/- embryos, Shh is
expressed ectopically in the anterior limb bud
(Masuya et al., 1995).
Although de-regulated Shh expression is a consequence of altered Gli3
function, it is not the principle cause for polydactyly, because
Gli3-/-;Shh-/- embryos are likewise
polydactylous (Litingtung et al.,
2002; te Welscher et al.,
2002). In both Gli3-/- and
Gli3-/-;Shh-/- limbs, the digits are
indistinguishable, dysmorphic and of indeterminate identity
(Litingtung et al., 2002).
Thus, other factors conferring normal digit identity, previously presumed to
be mainly full-length Gli3 activator, are lacking or rendered nonfunctional in
these mutants.
Several 5'Hoxd genes are expressed in posterior-distal domains in the
early limb bud mesoderm, and play roles in regulating digit number and pattern
downstream of Shh (Dolle et al.,
1989; Nelson et al.,
1996) (reviewed by Zakany and
Duboule, 1999). Analysis of single- and compound-null mutants has
revealed extensive functional overlap between different 5'Hox members,
and indicates that they act in an additive, dose-dependent fashion
(Fromental-Ramain et al.,
1996; Zakany et al.,
1997; Wellik and Capecchi,
2003) (reviewed by Zakany and
Duboule, 1999). By contrast, forced expression of individual
5'Hoxd genes in the limb bud has more dramatic consequences; elevated
Hoxd11, Hoxd12 or Hoxd13 levels each cause duplications and
transformations of anterior digits to posterior identities
(Morgan et al., 1992;
Knezevic et al., 1997)
(Hoxd13) (J. Innis, personal communication). We previously showed
that a Hoxd12 transgene (Tg-Hoxd12), expressed throughout
the limb bud, causes polydactyly and also ectopic anterior Shh
expression (Knezevic et al.,
1997). 5'Hoxd genes are downstream targets of Gli3, and
their expression is broadly activated across the early limb bud in
Gli3-/- embryos
(Zuniga and Zeller, 1999). Yet
in the Gli3-/- background, extended high-level Hoxd
expression is not associated with the production of distinct digit identities,
as is seen in a wild-type background. The assumption is that the presence of
the full-length Gli3 activator is entirely responsible for such phenotypic
differences. We present evidence that Gli3 and Hoxd12 interact genetically and
physically, and that this interaction modulates Gli3 repressor function. By
extension to other 5'Hoxd members, this finding provides a foundation
for understanding how Hoxd proteins might function semi-quantitatively to
regulate digit pattern and identity, and also has implications for how
polydactyly may arise in certain human syndromes caused by mutations expected
to produce a constitutive repressor form of Gli3.
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Materials and methods |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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). The
Gli3-XtJ line was obtained from Jackson
Laboratories and genotyped as described
(Litingtung et al., 2002;
Buscher et al., 1998). Compound
hemizygous Tg-Hoxd12;Gli3+/- embryos were
generated in crosses between Tg-Hoxd12 (inbred FVB/N) and
Xt+/- (inbred C3H) mice.
Tg-Hoxd12;Gli3-/- embryos were generated by crossing live
born F1 Tg-Hoxd12;Gli3+/- mice (FVB/N-C3H mix)
with Xt+/- mice (inbred C3H). Embryos from a large series
of test crosses were first analyzed, to rule out effects due to genetic
variation from the mixed F1 background on the phenotype of Tg-Hoxd12
and of Xt+/-. In primary crosses between wild-type FVB/N
and Xt+/- (inbred C3H), the typical Xt-phenotype
(single extra digit 1, all limbs) was consistently observed (23/45 total
progeny). In primary crosses between wild-type C3H and Tg-Hoxd12
(inbred FVB/N), all Tg-Hoxd12 progeny displayed wild-type limb
phenotypes, except for a single embryo with a triphalangeal digit 1 in one
hind limb (1/52 Tg positives). The Gli3-/- limb phenotype,
evaluated on mixed background, was also found to be indistinguishable from the
inbred C3H background [15 Gli3-/- embryos from crosses of
F1 Xt+/- (FVB/N-C3H mix)].
Expression plasmids and antibodies
Hoxd12/Gst fusion proteins contained chick Hoxd12 sequences C-terminal to
Gst as follows: FL (full length), amino acids (aa) 9-266; 
HD
(homeodomain deleted), aa 9-151; and HD (homeodomain), aa 167-266. In all
transfection experiments, the full-length protein constructs included aa 1-266
expressed in pSG5. Full-length Hoxa1 and Hoxb1 were also expressed from pSG5
(DiRocco et al., 1997). Hoxd12
mutated in the homeodomain (HD) to inactivate DNA binding capacity (mtHD)
contained a two residue conservative substitution of WF to AA (aa 245-246) in
helix 3 of the HD, generated using Quick Change mutagenesis (Stratagene). The
resulting protein was expressed at the same level as wild type, but was
non-functional in gel shift and transfection assays using Hoxd12-consensus
element-driven reporters (data not shown). Hoxd13/Gst included chick sequences
encoding aa 112-309, and this fusion protein was also used as an immunogen to
generate the Hoxd13 antibody. Gst-fusion proteins were checked on gels to
normalize the amounts of all fusions used in pull-down experiments (data not
shown). Full-length and truncated (TR, aa 1-674) Gli3-expressing constructs in
pcDNA3.1 (Shin et al., 1999)
were used as described. Gli3 N-ZnF was generated by cleavage of Gli3 TR with
BstEII to produce a 426 aa run-off protein, in vitro, with all zinc
fingers (ZnF) deleted. Hoxd12 polyclonal rabbit antibody was generated by
immunization with the Hoxd12-HD/Gst fusion protein and
affinity-purified. The polyclonal affinity-purified Gli3 antibody used for
some experiments was a gift from C. Chiang, or was generated using a Human
GLI3/Gst (aa 1-497) fusion protein as an immunogen.
Protein interaction assays
Gst-fusion proteins loaded onto glutathione-sepharose beads were blocked
with 2% BSA, and bound to 35S-labeled in vitro translated proteins
(Promega TNT) as indicated. For co-immunoprecipitation (co-IP) assays from
transfected cells, cells were lysed [lysis buffer: 10 mM HEPES (pH 7.5), 1mM
EDTA, 250 mM NaCl, 0.5% NP-40, protease inhibitors] on ice for 25 minutes with
trituration, lysates were centrifuged at 5000 rpm for 5 minutes, and the
supernatants bound to Protein G Agarose loaded with affinity-purified
anti-Hoxd12 or affinity-purified anti-N peptide-Gli3
(Dai et al., 1999) antibodies.
Bound proteins were detected on western blots with anti-Xpress-tag
(Invitrogen), anti-Hoxd12, or anti-Hoxb1 (Covance) antibodies. For co-IP of
endogenous embryonic proteins, limb bud tissues (see below) were dissected in
PBS, pooled and lysed as above (
150 limb buds/ml), except that the lysis
buffer salt concentration was increased to 420 mM NaCl. The Hoxd12 antibody
used for co-IP was covalently cross-linked to beads (Pierce co-IP kit) and
bound proteins were detected with affinity-purified anti-Gli3. Chick embryo
protein lysates were made from separated anterior- or posterior-third early
(stage 22) limb buds, or from later distal digital arch region, including
condensations and interdigit mesenchyme (stage 27/28), that was used either
intact or separated into anterior and posterior halves. In control experiments
(not shown), Gli3 protein expression profiles in posterior limb bud halves
from stage 21-24 were very similar to those in posterior thirds, and clearly
displayed a very high ratio of full-length to repressor forms. All tissue
lysates were prepared and analyzed on western blots as previously described
(Wang et al., 2000;
Litingtung et al., 2002),
using polyclonal affinity-purified anti-Hoxd12, anti-Hoxd13 or anti-Gli3
antibodies.
Transfection assays
DF-1 cells (chick embryo fibroblast line, ATCC) were transfected (Qiagen
Superfect) as indicated. For reporter assays, Ptc/luc
(Shin et al., 1999) or
8xGli/luc (Sasaki et al.,
1997) values were normalized to pSV/RL (Promega dual reporter
system). All reporter assays were performed in duplicate, and at least three
independent experiments were performed to verify reproducibility.
Immunofluoresence co-localization
Co-transfected DF-1 cells were fixed in 4% paraformaldehyde, then
co-incubated with Anti-Xpress (for tagged Gli3) and affinity-purified
anti-Hoxd12 antibodies, followed by anti-rabbit-FITC and anti-mouse-Alexa Red
secondary antibodies. Immunofluoresence was detected using a Zeiss Axiovert
microscope (100x/1.4 oil immersion), and compared with control cultures
of cells transfected singly with either the full-length Hoxd12 or the Gli3-TR
expression vector; no differences in cellular localization were observed (data
not shown). Confocal images were generated using a Zeiss LSM 510.
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Results |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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) that,
when hemizygous, has no abnormal phenotype alone
(Fig. 1A). The Gli3
null mutant used is haplo-insufficient
(Hui and Joyner, 1993;
Buscher et al., 1998) and
Gli3+/- hemizygotes have a consistent, mild phenotype
(single extra digit 1, all limbs; Fig.
1B). [The phenotypes of both these alleles were invariant in all
backgrounds generated by the crosses used (described in Materials and
methods).] By contrast, the compound hemizygous
Tg-Hoxd12;Gli3+/- embryos had much more severe
digit phenotypes than the single hemizygotes for either allele, including
multiple digit duplications and posterior transformations (e.g.
Fig. 1C, and
Table 1). In the hindlimb,
where the transgenic promoter used drives uniform expression of
Tg-Hoxd12 throughout the limb bud, 85% (22/26) of compound hemizygous
Tg-Hoxd12;Gli3+/- embryos had severe phenotypes,
whereas the remainder had a simple Gli3+/- phenotype (only
extra digit 1). Compound hemizygous
Tg-Hoxd12;Gli3+/- embryos also frequently
displayed long bone shortening (especially tibia) in hindlimbs. In the
forelimb, where the promoter driving Tg-Hoxd12 expression is more
postero-distally restricted and variable
(Knezevic et al., 1997), 58%
(15/26) of the compound hemizygotes had strong digit phenotypes.
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),
ectopic Shh expression occurred relatively late (only seen after E12)
and thus may be a downstream consequence of altered Gli3 function,
rather than a cause of polydactyly. Furthermore, the frequency of Shh
misexpression in compound hemizygous
Tg-Hoxd12;Gli3+/- embryos was considerably lower
than the incidence of subsequent severe digit phenotypes observed [for
hindlimbs: 5/20 (25%) limb buds versus 38/52 (75%) skeletons]. In forelimbs,
ectopic Shh expression was never detected even though severe digit
skeletal phenotypes were sometimes seen (50%,
Table 1). This again suggests
that although Shh deregulation was a consequence of Gli3-Hoxd
interaction, it was not the primary cause for the skeletal abnormalities.
Although not causative of digit phenotypes, the Shh misexpression
occasionally seen in compound hemizygous Tg-Hoxd12;Gli3
+/- embryos, but never in single hemizygotes, did suggest a genetic
interaction (synergistic effect).
Gli3-Hoxd12 genetic interaction in digit formation requires functional Gli3
Unlike the compound hemizygous
Tg-Hoxd12;Gli3+/-, Gli3-/-
(or likewise Tg-Hoxd12;Gli3-/-, see below)
embryos displayed polydactylous digits that were largely indistinguishable,
short and highly dysmorphic (Fig.
1D,E; Fig. 2). At
E18.5, all Gli3-/- digits had only a single ossification
center, and an overall appearance suggesting two malformed phalangeal segments
(rather than the normal three present in all digits posterior to digit I, see
Fig. 2D-F). Gdf5
expression, a marker for inter-phalangeal segmentation
(Storm and Kingsley, 1996),
was evaluated at E14.5 to confirm this impression. Gdf5 staining
revealed only one discrete, well-formed inter-phalangeal segment in
Gli3-/- digits; a second more proximal zone of incomplete
Gdf5 staining suggested an abortive attempt at segmentation more
proximally (Fig. 2A-C). Thus,
Gli3-/- digits appear to have only two completely formed
phalanges, although the high degree of dysmorphology makes this difficult to
ascertain (see, for example, the variable, middle phalanx rudiment in some
digits in Fig. 1D,E) (see also
Litingtung et al., 2002). By
contrast, the polydactylous digits in compound hemizygous
Tg-Hoxd12;Gli3+/- embryos always had distinct
identities and very well-defined phalanges at both E14.5 and E18.5 (compare
Fig. 2B,E with C,F).
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;
Litingtung et al., 2002;
te Welscher et al., 2002).
Likewise, ectopic Shh expression in
Tg-Hoxd12;Gli3-/- embryos was very similar to
that seen in Gli3-/- embryos and was restricted to a small
anterior focus in the limb bud (12/12 embryos;
Fig. 1D,E). The broad
Shh misexpression apparent in a subset of compound hemizygous
Tg-Hoxd12;Gli3+/- limb buds was not observed in
Tg-Hoxd12;Gli3-/- embryos. Together, these
results indicate that Tg-Hoxd12 requires the presence of functional
Gli3 protein (albeit at a reduced level) to exert effects on digit morphology
and on Shh expression.
In vivo physical interaction between Gli3-Hoxd12
The strong genetic interaction and synergistic Shh activation seen
only in compound hemizygous Tg-Hoxd12;Gli3+/-
embryos but not in Tg-Hoxd12;Gli3-/- embryos,
suggested a possible physical interaction between Hoxd12 and Gli3. When could
Gli3-Hoxd interactions be physiologically relevant in the developing limb? At
early patterning stages, Hoxd transcripts are expressed in nested posterior
domains that overlap anteriorly with Gli3, which is expressed in the
anterior three-quarters of the limb bud and is excluded from the
posterior-most mesoderm (Dolle et al.,
1989; Nelson et al.,
1996; Mo et al.,
1997; Schweitzer et al.,
2000) (see also Fig.
3A). Later, when digit condensations just begin to form, Hoxd
transcripts are expressed in overlapping distal domains in the interdigit
regions, and Gli3 RNA is also strongly expressed, uniformly
throughout all of the interdigit zones (see
Fig. 3A) (see also
Dolle et al., 1989;
Nelson et al., 1996;
Mo et al., 1997). This late
expression overlap is potentially relevant to digit patterning because digit
identity/morphology can still be regulated by interdigital mesenchymal signals
at late stages (Dahn and Fallon,
2000). Full-length Gli3 is protected from cleavage to repressor by
Hedgehog signaling (Wang et al.,
2000) (reviewed by Ingham and
McMahon, 2001); however, in late interdigit zones the extent of
such signaling is unclear, as Shh expression in the posterior limb bud has
declined and expression of Indian hedgehog (Ihh) produced in chondrogenic
mesenchyme is just initiating. Therefore, interdigit Gli3 protein was
evaluated to determine which form prevails at this stage. In early limb buds,
the ratio of repressor to full-length Gli3 protein is dramatically regulated:
high in the anterior and low in the posterior limb bud (see
Fig. 3B) (see also
Wang et al., 2000). The
profile of Gli3 proteins present in interdigit mesenchyme
(Fig. 3B) was both
qualitatively and quantitatively very similar to Gli3 present in early
anterior limb bud, even when the posterior interdigit region was analyzed
separately. Thus, it is primarily the Gli3 repressor form that is likely to be
active in interdigit zones at these later stages. As representatives of
5'Hoxd members, Hoxd12 and Hoxd13 protein levels were also checked, and
expression was evident at both early (posterior limb bud) and late
(interdigit) stages (Fig.
3B).
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Domain requirements for Gli3-Hoxd protein interaction
To determine which protein domains in Gli3 and Hoxd12 were necessary for
interaction, various in vitro translated Gli3 and Hoxd12/Gst-fusion protein
domains (Fig. 4A) were tested
in pull-down assays (Fig. 4B). The N-terminal, Zn-finger-containing region of Gli3 (Gli3 TR) and the
C-terminal homeodomain region of Hoxd12 (HD) interacted
(Fig. 4B). DNA-bridging did not
explain this interaction, as a Gli3 N-terminal region lacking its zinc-finger
DNA-binding domain still bound to a mutated Hoxd12 with inactivated
DNA-binding (mtHD). Gli3 interacted preferentially with certain classes of
homeodomains (Fig. 4C). Gli3
bound to Hoxd11/12/13, but only bound minimally to Hoxb1 or Hoxa1 when
challenged in assays containing both 5'Hoxd (AbdB type) and
3'Hoxa/b (Lab type) proteins
(Fig. 4C). Hoxd12 and Gli3 also
co-immunoprecipitated specifically and selectively when co-expressed in
transfected cells (Fig. 4D). Co-immunoprecipitation did not require Hoxd12 DNA-binding activity, and, when
challenged, Hoxd12 was again selectively co-immunoprecipitated, even in the
presence of co-transfected Hoxb1 protein
(Fig. 4D). Gli3 and Hoxd12 also
co-localized within the nucleus, but not the cytoplasm, of transfected cells
(Fig. 4E). Hence the
interaction with Hoxd12 did not act to sequester truncated Gli3-repressor
protein in the cytoplasm, because the bulk of Gli3 protein was still nuclear
and displayed a distribution comparable to Gli3 TR transfected alone (not
shown).
|
)
(reviewed by Ingham and McMahon,
2001). A previously characterized Ptc/luciferase reporter
(Shin et al., 1999), whose
basal expression is induced threefold by full-length Gli3 and repressed up to
10-fold by the truncated repressor form of Gli3 (Gli3 TR), was used to assess
the effect of Hoxd12 binding to Gli3 on Gli3 target promoters in transfection
assays. In our hands, activation of the reporter by full-length Gli3 was weak
and variable, and the effect of co-transfected Hoxd12 was likewise variable
and difficult to ascertain (Y.C. and S.M., unpublished). However, Gli3 TR
reproducibly repressed basal expression, allowing evaluation of Hoxd12 effects
on Gli3 transcriptional activity. Surprisingly, co-transfection of low levels
of Hoxd12 with low levels of Gli3 TR converted the Gli3 repressor into an
activator, with activity increasing according to the relative Gli3:Hoxd12
ratio (Fig. 5A). This
stoichiometry determines whether activator (Gli3TR-Hoxd12 complex) or
repressor (free Gli3 TR) function prevails. The DNA-binding activity of Hoxd12
was not required for this effect, as activation was comparable even when the
DNA-binding mutant Hoxd12 was co-transfected with Gli3 TR
(Fig. 5B). To further evaluate
whether recruitment of Gli3 TR-Hoxd12 complexes to Gli3-regulated promoters
requires only Gli3-binding sites or whether it might also depend on cryptic
Hoxd12-binding elements, a basal promoter driven solely by multimerized
Gli-consensus elements (Sasaki et al.,
1997) was assayed. Whereas Gli3 TR alone repressed basal reporter
expression, co-expressing low levels of the Hoxd12 DNA-binding mutant not only
prevented repression, but upregulated expression above the basal level in the
presence of Gli3 TR (Fig. 5C).
Experiments in which the level of Gli3 TR was held constant while
co-transfected Hoxd12 levels were increased likewise showed a decline in
repression activity; however, it was not possible to vary Hoxd12 levels over a
very broad range, owing to reporter activation by higher Hoxd12 levels that
complicates interpretation of the results (data not shown).
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Discussion |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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) (reviewed by Zakany
and Duboule, 1999), as cumulative recruitment of Hoxd proteins to
bound Gli3 repressor protein would modify the overall effect on Gli3 target
promoters. Rather than a combinatorial Hox code, a quantitative Hox-activity
gradient, determined by the total Hox protein relative to Gli3 protein at a
particular site (Fig. 3A; and
shown schematically in Fig. 6),
would modify `net' Gli3 function to regulate expression levels of Gli3 target
promoters differentially, and thereby potentially activate downstream Shh
pathway targets indirectly. The genetic evidence presented here suggests that
Gli3-Hoxd interaction pertains mainly to the regulation of digit
morphogenesis. This is not unexpected for an interaction with Gli3 shared
among several posterior Hox proteins, given that some of the 5'Hoxd
members normally only regulate digits physiologically (e.g. Hoxd13). In fact,
the long bone shortening observed may represent a distinct dominant-negative
effect independent of Gli3 (see Goff and
Tabin, 1997). Gli3-Hox interactions may represent a recent
evolutionary acquisition that, together with the distal recruitment of
5'Hox genes, enables the development of the distal autopod with its
multiple digits. As the distal autopod is probably a neomorphic structure of
tetrapod vertebrates (e.g. Sordino et al.,
1995), it is not surprising that an interaction between the
homologous Drosophila Ci and AbdB proteins has not been
described.
|
) where the
Gli3-Hoxd overlap occurs. The co-expression of Hoxd members along with the
Gli3 repressor in this border-zone could serve to modify (extend anteriorly)
the effective Shh activity gradient across the early limb bud anteroposterior
(AP) axis, and could thereby contribute to the specification of digit number
and/or pattern (Fig. 6). The
observed polydactyly ensuing from the enforced expression of any one of the
several 5'Hoxd members may then be the result of enhanced binding to
Gli3, and mitigation of repression in the early anterior limb bud. Enforced
expression of Hoxd12 does not result in any apparent change in the
expression pattern or levels of Gli3 transcripts (V.K. and S.M., unpublished).
By contrast, Gli3 does normally repress, and so restrict Hoxd expression to
the posterior limb bud at early stages
(Zuniga and Zeller, 1999);
this regulatory relationship will serve to limit the extent of overlap and
interaction between the Gli3 repressor and Hoxd proteins in the wild-type
early limb bud. However, the same simple regulatory hierarchy is clearly not
operational at later stages in the normal limb bud, because the interdigital
expression of the endogenous Gli3 repressor and Hoxd members overlaps quite
extensively in the wild-type embryo (e.g.
Fig. 3A,B).
Indeed, some features of digit identity/morphology are determined late.
Manipulation of interdigit mesenchyme in chick has revealed that adjacent
interdigit regions instruct digit anlage to develop different distinct
identitites (Dahn and Fallon,
2000), as judged by the number of phalangeal segments formed. The
responsible interdigit signaling factors remain to be elucidated, but any
regulation by Shh must be very indirect, as Shh expression has
subsided by this stage. Expression of several Hoxd genes persists late in the
interdigits, along with the Gli3 repressor
(Fig. 3A), and this interaction
could play a role in positively regulating the expression of late-secreted
interdigit signals that determine different digit identities
(Fig. 6). In this manner,
Gli3-Hoxd interactions could function either to indirectly sustain the Shh
pathway at these later stages, or, alternatively, to regulate novel (non-Shh
mediated) targets. A major focus for future work will be determining the
relative contributions and potential roles of early and later Gli3-Hoxd
interactions in regulating digit formation and morphogenesis. Considering the
redundancy of the posterior Hox genes, such approaches will entail a
mutational analysis of Gli3 residues mediating the interaction.
Gli3-Hoxd interactions may also have implications for digit abnormalities
in certain human syndromes arising from mutations in GLI3.
Pallister-Hall Syndrome (PHS) and Post-Axial Polydactyly (PAP) behave
semi-dominantly (reviewed by Biesecker,
1997), and arise from mutations expected to produce a truncated,
constitutive-repressor form of GLI3
(Altaba, 1999;
Dai et al., 1999;
Shin et al., 1999). Recent
mouse and chick models for PHS (Bose et
al., 2002; Meyer and Roelink,
2003) confirm the constitutive-repressor function of this mutated
Gli3 gene in many developmental processes, where the homozygous PHS
allele causes phenotypes resembling Shh-/- (Shh/Gli3
targets are repressed). However, surprisingly, the PHS allele does not block
the Shh pathway in the limb, but instead results in polydactyly with
non-identical digits (Bose et al.,
2002). Whether or not such Gli3 mutations behave as
dominant repressors during limb development may depend on their interactions
with Hoxd genes. In the limb, where Hoxd genes are uniquely expressed, the
function of an otherwise dominant-repressor Gli3 mutant could be modified by
an enhanced Hoxd-interaction affinity.
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ACKNOWLEDGMENTS |
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|
Footnotes |
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These authors contributed equally to this work 
* Present address: 20/20 Gene Systems Inc., Rockville, MD 20850, USA
Present address: US Deptartment of Commerce, USPTO, Washington, DC 20231,
USA
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