Hoxd and Gli3 interactions modulate digit number in the

Developmental Biology 310 (2007) 430 – 441
www.elsevier.com/developmentalbiology
Genomes & Developmental Control
Hoxd and Gli3 interactions modulate digit number in the amniote limb
Rushikesh Sheth a , M. Félix Bastida a , Marian Ros b,⁎
a
Departamento de Anatomía y Biología Celular, Universidad de Cantabria, 39011 Santander, Spain
b
Instituto de Biomedicina y Biotecnología de Cantabria, CSIC-UC-IDICAN, Santander, Spain
Received for publication 18 June 2007; revised 18 July 2007; accepted 23 July 2007
Available online 27 July 2007
Abstract
During limb development, Sonic hedgehog (SHH) and HOX proteins are considered among the most important factors regulating digit number
and identity. SHH signaling prevents the processing of GLI3 into a short form that functions as a strong transcriptional repressor. Gli3 mutant
limbs are characterized by a severe polydactyly and associated ectopic anterior expression of 5′Hoxd genes. To genetically determine the
involvement of 5′Hoxd genes in the polydactyly of Gli3 mutants, we have generated a compound mutant that simultaneously removes the three
most 5′-located Hoxd genes and Gli3. Remarkably, the limbs that form in the absence of all four of these genes show the most severe polydactyly
so far reported in the mouse. The analysis of gene expression performed in compound mutants allows us to propose that the increase in the number
of digits is mediated by the gain in function of Hoxd10 and Hoxd9. Our results also support the notion that an adequate balance between positive
and negative effects of different Hoxd genes is required for pentadactyly.
© 2007 Elsevier Inc. All rights reserved.
Keywords: Gli3; Hoxd; Hoxa; Shh; Limb development; Extra-toes; Polydactyly; Posterior prevalence
Introduction
Pentadactyly is the ancestral amniote digit formula. All extant
tetrapods descend from an ancestor with a pentadactyl limb,
although many species have reduced the number of digits or
even lost the limb (Cohn and Tickle, 1999; Cohn, 2001). Despite
the multiple variations on the basic pentadactylous pattern, a
limit of 5 in the number of digits appears to be constant.
The detailed way in which the number and identity of the
digits is established and controlled during limb development is
not completely understood and remains a subject of active
investigation. In humans, alterations in the number of digits,
preferentially polydactyly, are among the most frequent congenital malformations (Lamb et al., 1982; Graham and Ress,
1998).
It is known that the zone of polarizing activity (ZPA),
through its production of Sonic hedgehog (SHH), controls distal
Abbreviations: FL, forelimb; HL, hindlimb; P3, third phalanx; P?, undefined
phalanx.
⁎ Corresponding author. Departamento de Anatomía y Biología Celular,
Universidad de Cantabria, Herrera Oria s/n, 39011 Santander, Spain. Fax: +34
942 201903.
E-mail address: rosm@unican.es (M. Ros).
0012-1606/$ - see front matter © 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.ydbio.2007.07.023
limb patterning including the number and identity of the digits
(Saunders and Gasseling, 1968; Tickle et al., 1975; Riddle et al.,
1993). SHH signaling in vertebrates is mediated by the three Gli
genes (Gli1, Gli2 and Gli3). Of these, Gli3 is the one that is
essential for limb development as evidenced by the polydactylous phenotype of the Extra-toes (Xt) mutation, which
represents the complete loss of function of Gli3 (Schimmang
et al., 1992; Hui and Joyner, 1993; Maynard et al., 2002). In the
absence of Shh, GLI3 is processed to a short form that acts as
a strong transcriptional repressor (GLI3R) (Dai et al., 1999;
Aza-Blanc et al., 2000; Wang et al., 2000). Therefore, as a
consequence of the posterior secretion of SHH during normal
limb development, a gradient of GLI3R is established along the
anteroposterior axis of the bud with maximum levels at the
anterior border (Wang et al., 2000; Litingtung et al., 2002;
Bastida et al., 2004). The molecular study of Xt homozygous
limbs and several other polydactylous mutations in mice
revealed an ectopic spot of Shh expression at the anterior
border, which was considered responsible for the polydactylous
phenotype (Chan et al., 1995; Masuya et al., 1995; Buscher
et al., 1997; Masuya et al., 1997). However, the subsequent
study of Shh;Gli3 double mutant embryos, phenotypically
indistinguishable from Gli3 mutants, demonstrated that the
R. Sheth et al. / Developmental Biology 310 (2007) 430–441
polydactyly in the absence of Gli3 was independent of Shh
(Litingtung et al., 2002; te Welscher et al., 2002b). Consequently, two types of polydactylous phenotypes can be distinguished with different underlying molecular mechanisms.
Type 1 depends on the ectopic anterior activation of Shh, with
intact Gli3 function, and preaxial mirror-image duplication of
digits with clear identities. Type 2 polydactyly disrupts Gli3
function, is Shh independent and shows unidentifiable digits
(Litingtung et al., 2002; te Welscher et al., 2002b).
It has also been clearly demonstrated that the morphogenesis
of the vertebrate digits requires the function of genes of the
HoxA and HoxD clusters (Davis et al., 1995; reviewed by
Zakany and Duboule, 1999). During limb development Hoxa
and Hoxd genes are activated sequentially in time and space,
following their genomic topography, a phenomenon known as
temporal and spatial collinearity (Lewis, 1978; Gaunt et al.,
1988). Genes located more 3′ in the chromosome (also referred
to as anterior or proximal) are expressed earlier and at more
anterior locations than genes located more 5′ (also referred to as
posterior or distal). The division between anterior and posterior
Hoxd genes has not been clearly defined although a separation
between Hoxd genes expressed throughout the limb bud and
those excluded from anterior cells and capable of inducing Shh
has been precisely mapped between Hoxd9 and Hoxd10
(Tarchini et al., 2006). Interestingly, the forced expression of
a posterior gene earlier and/or more anteriorly than normal
results in a posteriorization phenotype (Duboule, 1991;
Duboule and Morata, 1994), an observation that led to the
model of “posterior prevalence” meaning that the product of a
posterior gene can inactivate the function of a more anterior one,
likely at the posttranscriptional level (van der Hoeven et al.,
1996; Herault et al., 1997; Williams et al., 2006).
The generation of an extensive series of mutations in the
HoxA and HoxD clusters including inversions, deletions,
duplications and compound mutations has shed light on the
regulation and function of Hox genes during limb development
(Kmita et al., 2002; Spitz et al., 2003; Kmita et al., 2005;
Tarchini and Duboule, 2006). Hoxd genes are activated in two
consecutive waves under different transcriptional control. The
first phase of Hoxd gene expression, essential for the
development of the limb up to the forearm, relies on two
opposite regulations: one, the early limb control region (ELCR),
located at the telomeric end of the complex, and the other
located at the centromeric end of the complex (Zakany et al.,
2004; Tarchini and Duboule, 2006). The ELCR acts as a timer
activator relying on relative distance to the promoter while the
centromeric enhancer acts by preventing expression in anterior
cells (Tarchini and Duboule, 2006). The second phase of Hoxd
activation, regulated by a global control region (GCR) located
centromeric to the cluster, is essential for digit development
(Spitz et al., 2001; Spitz et al., 2003).
The HoxA and the HoxD gene clusters are also required for
the initiation of Shh expression in the ZPA (Kmita et al., 2005).
Furthermore, the study of a series of Hoxa/Hoxd double mutant
mice showed that correct Shh expression depends on
quantitative and qualitative combinations of Hoxa and Hoxd
genes of the paralogous groups 10 to 13 (Tarchini et al., 2006).
431
Therefore, the notion that anterior-posterior patterning of the
limb bud is a consequence of the intrinsic colinearity of Hox
gene expression has emerged (Tarchini et al., 2006).
Interestingly, the 5′-located Hoxd genes are broadly and
ectopically expressed in the anterior limb bud mesoderm of Xt
mutants, indicating that GLI3R represses their transcription
(Zuniga and Zeller, 1999; Litingtung et al., 2002; te Welscher
et al., 2002b). Since overexpression of these genes cause
preaxial polydactyly (Morgan et al., 1992; Goff and Tabin,
1997; Knezevic et al., 1997; Kmita et al., 2002), it has been
proposed that the polydactyly in Xt mutants is mediated by the
ectopic anterior expression of posterior Hoxd genes. Interestingly, it has been recently shown that GLI3 and HOXD12
interact genetically and physically, and that this interaction
modulates GLI3R function (Chen et al., 2004). This finding
indicates that HOXD proteins may function semiquantitatively
to regulate digit pattern and identity through the interaction with
GLI3 (Chen et al., 2004). The graded posteroanterior level of 5′
HOXD proteins during their second phase of expression in the
developing limb can be envisaged as functioning to counteract
the transcriptional function of GLI3R, therefore potentiating
SHH function (Chen et al., 2004).
To directly determine the involvement of 5′Hoxd genes in
the polydactyly of Gli3 mutants, we have generated a
compound mutant that simultaneously removes the three most
5′-located Hoxd genes and Gli3. SHH prevents the processing
of GLI3 while HOXD12 and HOXD13 convert its function
from a transcriptional repressor to a transcriptional activator
(Wang et al., 2000; Chen et al., 2004). Therefore, removal of the
5′ Hoxd genes in the absence of Gli3 may also help to
determine other functions of 5′ Hoxd genes not related with
their interaction with GLI3.
Remarkably, the limbs deficient in all four of these genes are
extremely polydactylous, arguing against most 5′-located Hoxd
genes mediating the polydactyly characteristic of the Xt
phenotype. Compound mutants differ from Gli3 mutants in
the number, length and chondrogenic differentiation of the
digits, indicating that posterior Hoxd genes have functions
independent of GLI3. Based on the molecular study of the
compound mutant, we propose that the polydactylous phenotype is mediated by the gain of function of HOXD10 and
HOXD9. Our results also support the notion that an adequate
balance between positive and negative effects of different Hoxd
genes is required for pentadactyly.
Materials and methods
Mice mutant genotyping
The Extra-toes (Gli3XtJ Jackson allele; Hui and Joyner, 1993), the
HoxdDel(11–13) (Zakany and Duboule, 1996) and the Shh (Chiang et al., 1996)
mutant lines were maintained in a mixed background. The HoxdDel(11–13) allele
is the deletion of the Hoxd13 and Hoxd12 locus plus the insertion of the lacZ
reporter gene within the Hoxd11 gene, thus representing the loss-of-function of
Hoxd13, 12 and 11 (Zakany and Duboule, 1996).
The embryos were obtained by caesarean from pregnant mice and dissected
in cold phosphate buffered saline (PBS). Genotyping was performed by PCR as
described (Hui and Joyner, 1993; Chiang et al., 1996; Zakany and Duboule,
1996). Noon of the day the vaginal plug was observed was considered as
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R. Sheth et al. / Developmental Biology 310 (2007) 430–441
embryonic day 0.5 (E0.5). Besides the gestational age, the external
characteristics of the limbs were also used to stage the embryos (FernandezTeran et al., 2006).
In situ hybridization
Digoxigenin-labeled antisense riboprobes were prepared, and whole mount
in situ hybridization analysis performed according to standard procedures (Nieto
et al., 1996). The probes used were Shh, Fgf4, Fgf8, Grem1, Jag1, Hoxd9,
Hoxd10, Hoxa13, Gli1 and Hand2 (kindly provided by P. Beachy, D. Duboule,
G. Martin and A. Joyner). For each gene analyzed, the complete genotypic series
of limbs were processed jointly in the same hybridization tube to minimize
possible differences in the staining due to the procedure.
Skeletal analysis
Skeletal staining was performed according to standard protocols. Briefly,
embryos were skinned, eviscerated and fixed in 95% ethanol. Cartilages were
stained with Alcian blue and bones with Alizarin red. The skeletal analysis was
performed on newborns except for litters including the Shh mutant allele, which
were collected at E16.5 to avoid the late fetal lethality of the Shh mutation.
Results
Analysis of limb phenotypes
It has been suggested that the polydactyly characteristic of
Gli3XtJ/XtJ mutants depends on the anterior upregulation of 5′
Hoxd genes (Zuniga and Zeller, 1999; Litingtung et al., 2002;
te Welscher et al., 2002b). To test this hypothesis directly, we
have generated the compound mutation for Gli3 and the three
most 5′-located Hoxd genes. We also reasoned that this
combined mutation would allow the analysis of 5′Hoxd
function in digit patterning, independent of GLI3.
For this genetic approach, we analyzed progeny from
crosses between Gli3XtJ/+ ;HoxdDel(11–13)/+ double heterozygous mice (Fig. 1). At birth all genotypes were recovered at
the expected Mendelian ratios but homozygous Gli3XtJ/XtJ;
HoxdDel(11–13)/Del(11–13) mutants died immediately after birth.
The limb skeletons of newborn homozygous for the two mutations were analyzed and all of them showed an identical phenotype characterized by an extreme polydactyly (Figs. 1Q–T).
As described, Gli3XtJ homozygous limbs exhibit a severe
polysyndactyly with 7–9 digits in the forelimb and 6–7 in the
hindlimb (Figs. 1I, J; Hui and Joyner, 1993; Mo et al., 1997),
whereas heterozygous mutants show a variable degree of
duplication of digit 1 (Figs. 1E, F). Gli3XtJ/XtJ digits have three
phalanges but the chondrogenesis of the proximal phalanx is
very defective (Chen et al., 2004; Hilton et al., 2005). The digits
in the Gli3XtJ/XtJ limb lack distinct anteroposterior identities
(Figs. 1I, J).
Homozygous HoxdDel(11–13) mice also show polysyndactyly
but of a very different morphology from that of Gli3XtJ/XtJ
mutants (Figs. 1K, L; Zakany and Duboule, 1996). The autopod
is characterized by brachydactyly and central/postaxial polydactyly of 6–7 digits mostly biphalangeal and with disorganized
cartilage pattern (Figs. 1K, L). In our background, we only
observed 6 digits in forelimbs while hindlimbs were pentadactylous. A prominent excrescence in the first metatarsal, a trait of
the Hoxd13 deficiency, is always present (arrowhead in Fig. 1L;
Dolle et al., 1993). Heterozygous Hoxd Del(11–13) mutants
frequently show a slight reduction in the second phalanx of
digits II and V (Figs. 1G, H).
Remarkably, mice double homozygous for Gli3 XtJ and
HoxdDel(11–13) had a more severe polysyndactyly phenotype
than in any individual mutation (Figs. 1Q–T). Compound
mutants developed 10 to 11 short and syndactylous digits in the
forelimb and 8 to 9 in the hindlimb (Figs. 1S, T). To our
knowledge this is the most severe polydactyly reported so far in
the mouse. All digits in the compound mutant were biphalangeal
and looked identical with undefined identity, except for the
posterior slender extradigit typical of the HoxdDel(11–13)/Del(11–13)
deficiency, which was also found in the compound mutant
(Figs. 1Q, R and Fig. S1). Distinct anteroposterior identities
were only retained at the level of the hindlimb proximal autopod
but not in the digits proper, neither in the whole forelimb
autopod (Figs. 1Q, R and Fig. S1). The terminal phalanx was
always clearly identifiable and normally ossified (marked as P3
in Fig. S1) but which of the two proximal phalanges had been
eliminated was difficult to discern (therefore marked as P? in
Fig. S1). The absence of one phalanx is the main cause of the
characteristic brachydactyly but a pronounced ventral bending
of the whole autopod also contributed to making the digits
appear even shorter than they were (the ventral bending is best
noted in the sections in Fig. S1). Remarkably, while the
hindlimb autopod showed a conspicuous primary ossification
center in each metatarsal, forelimb metacarpals showed no sign
of ossification at birth except for some occasional and small and
randomly located foci (Fig. 1Q and Fig. S1).
Interestingly, the syndactylous phenotype typical of the
Gli3XtJ/XtJ limbs was increased by the additional removal of the
three posterior Hoxd genes. In Gli3XtJ/XtJ limbs the soft tissue
syndactyly does not affect the distal (ungueal) phalanx.
However, in the compound mutation the syndactyly also
affected the distal phalanx (Fig. 1 and not shown). The extreme
polysyndactyly, together with the marked ventral flexion of the
digits, resulted in a cup-shape autopod, particularly in forelimbs
(Fig. 1Q and Fig. S1), reminiscent of the cup-shaped hands
described in human syndactyly type IV (Sato et al., 2007).
An increase in the potential to form digits was already
observed when one copy of the three posterior Hoxd genes was
deleted in the absence of Gli3 (Gli3XtJ/XtJ;HoxdDel(11–13)/+),
suggesting a quantitative dose-dependent effect. This was more
clearly observed in the hindlimb that showed a range of 6–9
digits, but with most specimens showing 7 or 8 digits, a
number intermediate between the 6 and 7 typical of the
Gli3XtJ/XtJ mutant and the 8–9 typical of the compound
homozygous mutant (Figs. 1M, N). Equally interesting was
the observation that the additional inactivation of one Gli3 allele
in the absence of Hoxd11-13 (Gli3XtJ/+;HoxdDel(11–13)/Del(11–13))
resulted in the variable duplication of digit 1, typical of Gli3XtJ
heterozygous, added to the phenotype of HoxdDel(11–13) homozygous (Figs. 1O, P).
Thus, the development of Gli3XtJ/XtJ;HoxdDel(11–13)/Del(11–13)
limbs indicates a genetic interaction between Gli3 and the
posterior Hoxd genes, which occurs in a dose-dependent manner
and results in an increase in digit number. The phenotype of this
R. Sheth et al. / Developmental Biology 310 (2007) 430–441
433
Fig. 1. Skeletal phenotypes of single and compound mutants of Gli3XtJ and HoxdDel(11–13). Genotypes are indicated at the top. For each genotype the autopod skeleton
of the forelimb (left) and the hindlimb (right) are shown. The complete fore and hindlimb skeleton is also shown for the control (C, D) and compound mutant (S, T). All
specimens are shown in dorsal views and with distal to the right and anterior to the top. Note that the number of digits increases as the dose of Hoxd11, Hoxd12 and
Hoxd13 is reduced in the absence of Gli3. The polydactyly is maximal when Gli3 and the three most 5′-located Hoxd genes are simultaneously removed. The arrow in
panels K and Q points to the posterior slender digit typical of HoxdDel(11–13) homozygous. The arrowhead in panel L indicates the protuberant first metatarsal typical of
the Hoxd13 deficiency.
combined mutation also proves that the polydactyly of the Gli3
mutant limb does not require the three most 5′ Hoxd genes and
suggests that posterior Hoxd genes may have some negative
effect in determining digit number since its additional removal,
in the absence of Gli3, leads to an increase in the number of
digits.
SHH signaling is irrelevant for the phenotype of the
compound Gli3XtJ/XtJ;HoxdDel(11–13)/Del(11–13)
It is known that Shh expression is ectopically activated in the
anterior mesoderm of Gli3XtJ/XtJ limbs (Masuya et al., 1995;
Buscher et al., 1997; Masuya et al., 1997). This ectopic
activation of Shh may depend on the ectopic anterior expression
of posterior Hoxd genes as overexpression of HOXD proteins in
mice and chicks results in anterior activation of Shh (Morgan
et al., 1992; Charite et al., 1994; Goff and Tabin, 1997;
Knezevic et al., 1997; Caronia et al., 2003; Chen et al., 2004;
Zakany et al., 2004). Thus, we asked whether Shh ectopic
activation would also occur when the three posterior Hoxd
genes are deleted from the Gli3XtJ/XtJ background. The analysis
of Shh expression in several compound mutants between E10.5
and E13.5 failed to detect any anterior ectopic domain, while the
posterior normal domain was spatially and temporally similar to
normal (Figs. 2A, B, see also Fig. 3D and data not shown). The
forelimb in Fig. 2A does not show the posterior domain of Shh
expression because it corresponds to the developmental time at
which Shh expression terminates.
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R. Sheth et al. / Developmental Biology 310 (2007) 430–441
However, since the ectopic activation of Shh at the anterior
border of Gli3XtJ/XtJ mutants is usually small, transitory and
sometimes difficult to detect, we decided to analyze the
expression of Gli1, a well-established target of SHH (Ahn and
Joyner, 2004). Interestingly, Gli1 transcripts were clearly
detected at the anterior border of E12 double mutant limbs
(Figs. 2C, D). The ectopic anterior expression was clearly
observed in the hindlimb (arrowhead in Fig. 2D) but not in the
forelimb, a difference that can be attributed to the normal
difference in developmental stage between fore and hindlimbs.
These results allow us to conclude that Shh is indeed activated
at the anterior border of the compound mutant limb,
disregarding the failure in detecting Shh transcripts. Therefore,
in the context of absence of Gli3, the three most 5′-located
Hoxd genes are dispensable for Shh anterior ectopic activation. It should be noted here that Hoxd10, capable of
triggering Shh expression (Tarchini et al., 2006), is upregulated at the anterior border of the compound mutant limb (see
below).
The double Shh;Gli3 mutant limb showed that SHH
signaling was irrelevant for the Gli3 XtJ/XtJ polydactyly,
disregarding its anterior ectopic activation (Litingtung et al.,
2002; te Welscher et al., 2002b). However, SHH signaling is
operative in Gli3XtJ/XtJ mutant limbs as it induces Gli1 and
Ptch1 (Litingtung et al., 2002; te Welscher et al., 2002b),
possibly through GLI2. In the neural tube and other systems,
GLI2 has been shown to play activator functions necessary for
SHH signaling (Sasaki et al., 1999; Mill et al., 2003). Also, the
reduction in bone length observed in Gli2−/− mutant limbs is
increased by the additional removal of one copy of Gli3 (Mo
et al., 1997). Therefore, to completely rule out the involvement
of SHH signaling in the polydactyly of the compound mutant,
we performed the triple mutation in which Gli3, the three most
5′-located Hoxd genes and Shh were simultaneously removed.
Embryos from crosses between triple heterozygotes (Gli3XtJ/+;
HoxdDel(11–13)/+;Shh−/+) were collected at E16.5 due to the late
fetal lethality of the Shh mutant allele. Although endochondral
ossification could not be completely evaluated because of the
relatively early developmental age of the embryos, we found
that the additional removal of Shh did not modify the polydactyly of double compound homozygous embryos (Figs.
2E, F). This result confirmed that Shh was irrelevant for the
phenotype of the compound Gli3XtJ/XtJ;HoxdDel(11–13)/Del(11–13)
mutant.
Gene expression analysis in mutant limbs
Compound mutant limbs developed relatively normally
during early stages, the phenotype not being distinguishable
by morphologic changes up to E11. Between E11 and E12 the
double mutant limb developed a dramatic expansion of the
autopod (please note the shape of the compound mutant limb in
Figs. 3 and 4).
Besides the posterior Hoxd genes, other factors know to
be downstream of GLI3 are Fgf8, Fgf4, Grem1, Hand2 and
Jag1. All of them are upregulated in the anterior mesoderm of
Fig. 2. Shh expression is irrelevant for the polydactylous phenotype of compound mutants. Panels A and B are E12 compound mutant forelimb (A) and hindlimb (B)
hybridized for Shh showing no detectable expression at the anterior border. Note that the normal Shh expression in the posterior mesoderm has already terminated in
the forelimb. Panels C and D are E12 compound mutant forelimb (C) and hindlimb (D) hybridized for Gli1 showing ectopic expression at the anterior border of the
hindlimb (arrowhead). Panels E and F are skeletal preparations of E16.5 Gli3XtJ/XtJ;HoxdDel(11–13)/Del(11–13);Shh−/− triple fore (E) and hindlimb (F) mutants.
R. Sheth et al. / Developmental Biology 310 (2007) 430–441
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Fig. 3. Expression of Fgf8, Shh, Fgf4, Grem1, Hand2 and Jag1 in mutant limbs. All the panels are dorsal pictures of forelimbs after hybridization with the specific
probes indicated on the left. Genotypes are marked at the top of each column. Panels A–H correspond to E10.5 to E10.75 forelimbs. Panels I–P correspond to E11.5 to
E12 forelimbs. The arrowhead in panels E–H points to the anterior limit of Fgf4 expression in the AER.
Gli3XtJ/XtJ limbs, and therefore possibly implicated in the
polydactylous phenotype (Zuniga and Zeller, 1999; Litingtung
et al., 2002; te Welscher et al., 2002b; McGlinn et al., 2005). In
situ hybridization for Fgf8, which is normally expressed along
the entire anteroposterior extension of the apical ectodermal
ridge (AER; Martin, 1998), revealed a marked anterior
extension of the AER in the compound mutant (Fig. 3D),
similar to Gli3XtJ/XtJ limbs (Fig. 3B) while Fgf8 expression in
HoxdDel(11–13) homozygous limbs was comparable to normal
(Figs. 3A, C). The limbs in Figs. 3A–D were hybridized
conjointly for Fgf8 and Shh; note that the expression domain of
Shh in the compound mutant looked similar to normal as
previously mentioned (compare Figs. 3A and D).
Expression of Fgf4 is normally restricted to the posterior
AER, parallel to the mesodermal expression of Grem1 (Martin,
1998; Zuniga et al., 1999; Fig. 3E). As reported, the expression
of these two genes was anteriorly expanded in Gli3XtJ/XtJ
mutant limbs (Litingtung et al., 2002; te Welscher et al., 2002b;
Fig. 3F). Compound mutants also showed a clear anterior
expansion of the domain of expression of Fgf4 and Grem1, but
curiously the level of expression was weaker than in Gli3XtJ/XtJ
limbs (Fig. 3H). Fgf4 expression appeared patched and irregular
all along the extension of the AER, whereas Grem1 domain of
expression appeared less well defined than in control and
Gli3XtJ/XtJ limbs. Expression of both Fgf4 and Grem1 was
within normal limits in HoxdDel(11–13)/Del(11–13) limbs (Fig. 3G).
The difference in pattern of expression between the limbs in
Figs. 3G and E was merely due to a slightly different developmental stage between both limbs.
Hand2 (formerly dHand) is a transcription factor involved in
the establishment of the anteroposterior patterning of the limb
bud prior to Shh through a reciprocal antagonism with Gli3
(Charite et al., 2000; Fernandez-Teran et al., 2000; te Welscher
et al., 2002a). Accordingly, Hand2 is, in fact, upregulated in
Gli3XtJ/XtJ anterior mesoderm (Fig. 3J; te Welscher et al., 2002a,
2002b; Litingtung et al., 2002). In the compound mutant Hand2
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R. Sheth et al. / Developmental Biology 310 (2007) 430–441
Fig. 4. Expression of Hoxd9, Hoxd10 and Hoxa13 in the different genotypes. All the panels show dorsal pictures of forelimbs hybridized with the specific probe
indicated on the left. The developmental stage of the limbs is also indicated on the left. Genotypes are marked at the top of each column. Note the strong upregulation of
Hoxd10 expression in Gli3 and compound mutants. The arrowheads in panels A and C delimit the anteroposterior extension of the Hoxd9 autopodial domain. The
arrowhead in panels I–L points to the anterior limit of Hoxa13 expression in the distal mesoderm.
expression also became upregulated in the anterior mesoderm of
E11.5 limb buds, but in a pattern more distally restricted than in
Gli3XtJ homozygous (Fig. 3L). At later stages, Hand2 upregulation occurred in the whole autopod of compound mutants,
in a pattern similar to Gli3XtJ/XtJ (not shown). Expression of
Hand2 in HoxdDel(11–13)/Del(11–13) limbs was similar to control
limbs (Figs. 3K and I).
Using microarray technology, the Notch ligand Jag1 was
identified as highly repressed by GLI3 (McGlinn et al., 2005).
Since Jag1 is also an excellent marker for the distal mesoderm,
the site of the phenotype in the double mutant, we decided to
analyze its expression. As reported, Jag1 was highly upregulated in the anterior mesoderm of Gli3XtJ/XtJ limb buds by
E11.5, in accordance with it being repressed by GLI3R (Fig.
3N; McGlinn et al., 2005; Panman et al., 2006). In the
compound mutant limb, Jag1 was also anteriorly upregulated
(Fig. 3P) while the pattern of expression in triple Hoxd deletants
was comparable to normal (Figs. 3O, M).
In short, our results are compatible with Fgf8, Hand2 and
Jag1 having a role in the polydactyly of Gli3XtJ/XtJ and
Gli3XtJ/XtJ;HoxdDel(11–13)/Del(11–13) mutants and indicate that
their anterior ectopic expansion, besides not requiring SHH
signaling (Litingtung et al., 2002; te Welscher et al., 2002b),
does not require the input of the three most 5′Hoxd genes
either. However, the similarity in the patterns of expression of
Fgf8, Hand2 and Jag1 between Gli3XtJ/XtJ and Gli3XtJ/XtJ;
HoxdDel(11–13)/Del(11–13) limbs does not provide an explanation
for the increase in the polydactylous potential exhibited by the
compound mutant. The fact that the expression of Grem1 and
Fgf4, although spatially expanded, is quite faint in the
compound mutant argues against these two genes playing a
major role in the polydactyly since a substantial decrease in
R. Sheth et al. / Developmental Biology 310 (2007) 430–441
their level of expression associates with an increase in the
polydactylous potential of the limb.
Effects of the compound mutation on the expression of other
Hox genes
One possible explanation for the exacerbation of the
polydactyly in compound mutants is that the deletion of the
three most 5′ Hoxd genes causes misexpression of other Hox
genes necessary for proper autopod formation. Since the pattern
of expression of a particular Hoxd gene changes following its
location in the cluster and relative distance to the enhancers
(Kmita et al., 2002; Tarchini and Duboule, 2006), we first
analyzed the expression of Hoxd10 and Hoxd9, the genes
nearest to the deletion point. We concentrated on the second
phase of expression since it is the one required for the
morphogenesis of the digits (Sordino et al., 1995; Tarchini and
Duboule, 2006). At E11.5, Hoxd9 expression mainly occurs in a
proximal forearm domain that corresponds to the early phase of
expression but in addition also shows a faint autopodial domain
corresponding to the late activation phase. The autopodial
domain appeared centrally located in the distal wild-type and
homozygous HoxdDel(11–13) autopods (delimited by arrowheads
in Figs. 4A and C). However, Gli3XtJ and Gli3XtJ;HoxdDel(11–13)
homozygous showed a marked expansion of Hoxd9 domain of
expression all along the anteroposterior distal mesoderm (Figs.
4B and D). At this stage, the early phase of Hoxd9 expression, as
reflected by the forearm domain, appeared much stronger in
HoxdDel(11–13) and compound mutants, probably reflecting a
stronger expression at earlier stages.
Normal Hoxd10 expression is posteriorly restricted and, at
E11, occurs in two bands, corresponding to the zeugopod and
autopod (early and late phases) domains of expression,
separated by a transversal strip devoid of transcripts (Fig. 4E).
This pattern is conserved in single HoxdDel(11–13)/Del(11–13)
mutants as previously reported (Fig. 4G; Zakany and Duboule,
1996). Remarkably, expression of Hoxd10 in both Gli3XtJ/XtJ
and double homozygous limbs lacked its characteristic posterior
restriction as well as the separation between the autopodal and
zeugopodal domains, except for a small area or lesser
expression at the very posterior margin of the bud (Figs. 4F
and H). Therefore, the compound mutant limb develops with a
strong and uniform expression of Hoxd10 across the whole
anteroposterior axis of the bud (Fig. 4H). It is worth noting here
that the upregulation of Hoxd10 provides an explanation for the
ectopic activation of Shh at the anterior border of the compound
mutant (Figs. 2A, B), as it is known that Hoxd10 can trigger
Shh expression (Tarchini et al., 2006).
Since several Hoxa and Hoxd genes have been shown to
function cooperatively in a redundant and quantitative manner
(Davis et al., 1995; Fromental-Ramain et al., 1996a; FromentalRamain et al., 1996b; Zakany et al., 1997), we also analyzed the
expression of Hoxa11 and Hoxa13 in the double mutant. In
E10.5 compound mutant limbs the expression of Hoxa13
appeared clearly expanded in the anterior mesoderm (Fig. 4L),
similarly to Gli3XtJ/XtJ limbs (Fig. 4J; te Welscher et al., 2002b).
The more distal restriction observed in compound (Fig. 4L)
437
versus Gli3XtJ/XtJ (Fig. 4J) mutant limbs at E10.5 is explained
by the slight developmental delay of the compound mutant
relative to the Gli3XtJ/XtJ limb and was not observed at later
stages. At E11.5 compound mutants were characterized by an
elevated and homogeneous expression of Hoxa13 throughout
the distal mesoderm, similarly to Gli3XtJ/XtJ (Figs. 4N, P).
HoxdDel(11–13) homozygous showed a pattern and level of
expression similar to wild type (Fig. 4O, compare with Fig.
4M). Finally, the analysis of expression of Hoxa11 in E11.5
double mutant limb buds showed restriction to the junction
between the zeugopod and autopod in a normal pattern (not
shown).
Altogether, the results of our gene expression analysis
revealed that the compound mutant limb developed with
uniform expression of Hoxd9, Hoxd10 and Hoxa13 across the
distal limb mesoderm in a pattern similar to Gli3 mutant limbs.
However, the expression of Hoxd10 and Hoxd9, although
similarly upregulated in these two genotypes, is predicted to
have a higher functional impact in compound mutants because
of the absence of the negative effect of posterior Hoxd genes
(posterior prevalence).
Discussion
Deletion of Hoxd11-13, in the absence of Gli3, increases digit
number
In this work, we have removed the three most 5′-located
Hoxd genes simultaneously with Gli3 in order to further
analyze the role these genes play during digit development. It is
currently accepted that the polydactyly of Gli3XtJ/XtJ mutants
depends on the ectopic anterior expression of 5′-located Hoxd
genes (Zuniga and Zeller, 1999; Litingtung et al., 2002; te
Welscher et al., 2002b). Therefore, we reasoned that the number
of digits would decrease if the most 5′Hoxd genes were deleted
from the Gli3XtJ/XtJ background. Unexpectedly, Gli3XtJ/XtJ;
HoxdDel(11–13)/Del(11–13) mutant limbs, although relatively normal at proximal level, were severely polydactylous; the number
of digits being larger than in the single Gli3 mutant. Indeed, to
our knowledge, these compound mutant limbs bear the largest
number of digits so far reported in the mouse, 10–11 in the
forelimb and 8–9 in the hindlimb. This phenotype shows that
the loss-of-function of Hoxd11 to 13, in the absence of Gli3,
increases the polydactylous potential of the limb and reveals a
negative effect of posterior Hoxd genes in determining digit
number.
The polydactyly in compound mutants can be classified as
type 2 polydactyly because of symmetric unidentifiable digits
and disruption of Gli3 function (Litingtung et al., 2002; te
Welscher et al., 2002b). As occurs in Gli3 mutants, ectopic Shh
expression, reported by Gli1 expression, is observed at the
anterior border of compound mutant limbs demonstrating that it
does not require HOXD11-13 inputs, although it likely relies on
the upregulation of Hoxd10, which is also capable of triggering
Shh expression (Tarchini and Duboule, 2006). To thoroughly
rule out a possible involvement of Shh in the polydactyly, we
performed the triple Gli3XtJ/XtJ;HoxdDel(11–13)/Del(11–13);Shh−/−
438
R. Sheth et al. / Developmental Biology 310 (2007) 430–441
mutant that showed identical limb phenotype as the double mutant
proving that the phenotype was independent of SHH signaling.
Also, the fact that the Gli3 XtJ/XtJ ;Hoxd Del(11–13)/Del(11–13)
mutant limbs are not identical to Gli3XtJ/XtJ limbs, as both
types of limbs differ in the number, morphology and chondrogenic differentiation of the digits, indicates that 5′ HOXD
proteins do, indeed, have functions independent of GLI3,
either acting as monomers or in association with multiple
partners (Svingen and Tonissen, 2006).
The phenotype of the compound mutants indicates that
Hoxd11-13 do not contribute to the polydactylous phenotype in
Gli3 XtJ/XtJ limb. This phenotype may appear difficult to
reconcile with multiple experiments demonstrating that overexpression of Hoxd12 and other posterior Hoxd genes results in
anterior polydactyly (Morgan et al., 1992; Goff and Tabin,
1997; Knezevic et al., 1997; Chen et al., 2004; Zakany et al.,
2004). However, the physical interaction between HOXD and
GLI3R proteins, which reverses the transcriptional activity of
the latter from a repressor into an activator, provides an
explanation for the preaxial polydactyly caused by 5′ Hoxd
overexpression, in the presence of Gli3 (Chen et al., 2004).
Indeed, overexpression of Hoxd12 requires Gli3 in order to
cause anterior polydactyly (Chen et al., 2004).
Chondrogenic defects in the mutants
Targeted disruption in mice as well as overexpression
experiments have shown that Hox genes are required for the
appropriate condensation, proliferation and differentiation of
skeletal elements (Morgan et al., 1992; Davis and Capecchi,
1996; Zakany and Duboule, 1996; Goff and Tabin, 1997; Yueh
et al., 1998). In particular, loss of function of posterior Hoxd
genes associates with a noticeable delay in the ossification
pattern of the autopodal bones revealing a strong deregulation
of bone formation (Davis and Capecchi, 1996; Zakany and
Duboule, 1996). Expression of Hoxd genes occurs in
chondrocytes but is normally switched off as differentiation
progresses to the prehypertrophic state (Zakany and Duboule,
1996; Suzuki and Kuroiwa, 2002). Interestingly, in the
HoxdDel(11–13)/Del(11–13) mutant, expression of Hoxd11/lacZ,
the marker gene present at the 5′ end of the HoxdDel(11–13)
allele (Zakany and Duboule, 1996) persists in chondrocytes
concomitantly with the delay in differentiation. Also, overexpression of Hoxc8 and Hoxd4 in the chondrogenic lineage
results in a marked chondrogenesis delay (Yueh et al., 1998).
These observations raise the possibility that the chondrogenic
phenotype in the absence of posterior Hoxd genes may be
mediated by abnormal persistent expression of remaining Hoxd
genes instead of a more direct requirement of 5′ HOXD
products in chondrogenesis. In this regard, it would be
interesting to analyze whether expression of Hoxd9 and
Hoxd10 abnormally persists in the growing skeleton of the
compound mutant.
Besides the delayed chondrogenic differentiation resulting
from the removal of posterior HOXD proteins, the defects in
ossification in the double mutant should also combine
modifications in IHH signaling due to the absence of GLI3
(Hilton et al., 2005; Koziel et al., 2005). It is worth noting the
puzzling difference between the ossification of metacarpals and
metartarsals in the newborn compound mutants. While
ossification is completely absent in metacarpals, it is prominent
in metatarsals. The reason for this variation remains unknown
but differences in gene expression between the fore and
hindlimb may account for this difference. In particular, several
Hox genes show differential patterns of expression between fore
and hindlimbs (Nelson et al., 1996). The chondrogenic
differentiation of the three genotypes used in this work is an
issue that deserves further investigation.
Functional upregulation of Hoxd10 and Hoxd9 is responsible
for the exacerbation of the polydactyly in compound mutants
The examination of the pattern of expression of Fgf8, Fgf4,
Grem1, Hand2 and Jag1, all of them previously associated with
the Gli3 mutant phenotype (Zuniga and Zeller, 1999; Litingtung
et al., 2002; te Welscher et al., 2002b; McGlinn et al., 2005),
failed to detect changes that could account for the increase in
digit number observed in the compound mutant relative to the
Gli3 mutant. Indeed, the expression of Fgf4 and Grem1 was
weaker in the double mutant suggesting that the modifications
in pattern of expression of these genes may not be involved in
the generation of the phenotype but rather associated with the
broadening of the mutant limb.
We also examined the expression of other Hox genes that
could play a role in the formation of the autopod. The
elimination of a group of Hoxd genes should impact on the
expression of remaining Hoxd genes because of their reallocation in the chromosome (Tarchini and Duboule, 2006).
However, the deletion we have used here contains a LacZ
reporter transgene positioned at the place of Hoxd11 and hence
it is the transcription of this reporter gene that results
upregulated rather than that of the next gene (Hoxd10) in the
chromosome (Zakany and Duboule, 1996). Accordingly, we
observed that the late transcriptional profile of Hoxd9 and 10
was not significantly modified in HoxdDel(11–13) homozygous,
confirming previous studies for Hoxd10 and Evx2 (Zakany and
Duboule, 1996). GLI3 is a firm candidate to interact with the
centromeric enhancer in the early phase of Hoxd expression and
with the digital enhancer embedded in the Global Control
Region implicated in the second phase of Hoxd expression
(Spitz et al., 2003; Tarchini and Duboule, 2006). The fact that
the spatial upregulation of the late Hoxd10 and Hoxd9
expression domains was similar in compound and Gli3XtJ/XtJ
mutants, but absent in Hoxd11-13 deletants, indicates that it was
mainly induced by the removal of Gli3.
Considering the functional suppression exerted by posterior
HOXD products over more anterior products (Duboule and
Morata, 1994; van der Hoeven et al., 1996; Herault et al., 1997;
Williams et al., 2006), we propose that the expansion of the
distal mesoderm and subsequent generation of an elevated
number of short and identical digits typical of the Gli3XtJ/XtJ;
HoxdDel(11–13)/Del(11–13) compound mutation is mediated by the
gain in function of HOXD10 and HOXD9. This gain in function
relies on the derepression due to the absence of GLI3R and on
R. Sheth et al. / Developmental Biology 310 (2007) 430–441
the concomitant absence of the prevalent effect of posterior
HOXD products. Even if the transcriptional profile of Hoxd10
and Hoxd9 is similar in Gli3 and compound mutants, the
absence of posterior Hoxd genes in the later generates an
important functional difference that would explain the different
grade of polydactyly between both genotypes. The fact that, in
the absence of Gli3, removal of the whole HoxD cluster does
not modify the polydactyly (Zakany et al., 2007) while removal
of only Hoxd11 to 13 increases the polydactyly (our work),
strongly indicates that the gain in digit number is mediated by
the remaining anterior Hoxd genes. The results of the genetic
analysis performed by Zakany et al. (2007) along with our
results strongly support the notion that “anterior” Hox genes
have a positive effect on digit number, while “posterior” Hox
genes restrict digit number and that a correct balance between
anterior and posterior HOX products and GLI3 is required for
pentadactyly.
In the context of our proposal, it is also important to consider
why the upregulation of Hoxd10, which occurs in the absence
of Hoxd11-13 but in the presence of Gli3, does not induce
polydactyly (Kmita et al., 2002). Our interpretation is that the
presence of GLI3, known to interact with HOX products (Chen
et al., 2004), may interfere with HOXD10 function through a
presently unknown molecular mechanism. Interestingly, the
observation that a partial deletion of the HoxD cluster (Hoxd1 to
Hoxd10 included), which carries a gain in function of Hoxd12
and 13 results in symmetric and frequently oligodactylous
limbs (Zakany et al., 2004) but severely impairs limb
development when combined with loss of Gli3 (Zakany et al.,
2007), led Zakany et al. to suggest that GLI3 may exert a
protective effect against the deleterious effects of these HOX
proteins. Finally, the fact that Hoxd11 can trigger a spectacular
polydactyly upon deletion of Hoxd12 and 13 but in the
presence of Gli3 (Kmita et al., 2002) suggests specific GLI3/
HOXD11 interactions. The capacity of Hoxd11 to trigger
supernumerary digits indicates that the negative effect on digit
number is indeed exerted by Hoxd13 and Hoxd12, but not
Hoxd11.
Hoxa13 was also found to be upregulated in Gli3 and
compound mutants. Previous published results indicate that the
upregulation of Hoxa13 may mediate the polydactyly. For
example, misexpression experiments in chick limb buds
showed that Hoxa13 was involved in the adhesiveness of
mesodermal cells by controlling homophilic cell interactions
(Yokouchi et al., 1995). Hoxa13 was also found to be
responsible for controlling cartilage growth and differentiation
towards short bones characteristic of the autopod. The analysis
of Hoxa13 mutant limbs further supported this interpretation
and identified EphA7 as the molecule involved in this function
(Stadler et al., 2001). Furthermore, it has recently been shown
that Hoxa13 acts upstream of Sox9, since misexpression of
Sox9 is able to rescue the hypodactyly phenotype characteristic
of the Hoxa13 deficiency (Akiyama et al., 2007). Therefore, the
upregulation in Hoxa13 observed in Gli3 and compound
mutants may well contribute to the generation of an excessive
number of chondrogenic rays. It is worth mentioning here that,
on the basis of the phenotypes obtained after different
439
combinations of loss-of-function alleles of HoxD and HoxA
clusters, a predominant role for Hoxa13 in the generation of
polydactylous short-digited limbs was postulated (Zakany et al.,
1997). The findings reported here are in complete agreement
with this hypothetical role for Hoxa13.
Positive and negative effects of different Hoxd genes on
determining digit number
In summary, the phenotype of the compound Gli3XtJ/XtJ;
HoxdDel(11–13)/Del(11–13) mutant indicates that the posterior Hoxd
genes (probably only Hoxd13 and 12) play a negative role on
digit number, considering that the polydactyly is clearly more
severe than in Gli3XtJ/XtJ mutants. In accordance with the
“posterior prevalence” model, we propose that the negative
effect is exerted through the functional repression of HOXD10
and HOXD9, which have a positive effect on digit number.
Therefore, anterior and posterior HOXD products need to be
adequately balanced for the pentadactyl formula. The effects of
modifications in this balance are more easily appreciated in the
absence of Gli3, since GLI3, by directly binding HOXD
proteins (Chen et al., 2004), may prevent or modify their
function and interactions with other partners.
It has been speculated that the successive recruitment of the
HoxA and HoxD clusters in developing appendages may have,
at least partially, mediated the evolutionary transition from
polydactyly to pentadactyly in ancestral tetrapods (Zakany et
al., 1997). It is currently accepted that this recruitment may have
carried the implementation of Shh activation (Tarchini et al.,
2006). Our work suggests that the recruitment of the HoxD
cluster had to include a correct balance between anterior and
posterior products as well as with GLI3.
Acknowledgments
This work was supported by grant BFU2005-09309-CO2-01
from the Spanish Ministry of Education and Science. R.S. is a
recipient of an FPU grant from the Spanish Ministry of
Education and Science. We are very grateful to Denis Duboule
and Joseph Zakany for providing the HoxdDel(11–13) mutant
mice and for discussions and communicating results before
publication. We thank R. Dahn, S. Mackem, F. Meijlink, M.
Torres and A. Zuñiga for helpful discussions. We appreciate the
excellent technical assistance of Marisa Junco and the support
from the members of our group.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at doi:10.1016/j.ydbio.2007.07.023.
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