Limb anterior–posterior polarity integrates activator and repressor
functions of GLI2 as well as GLI3
Megan Bowersa,b, Liane Enga, Zhimin Laoa, Rowena K. Turnbulla, Xiaozhong Baoc,
Elyn Riedeld, Susan Mackemc, Alexandra L. Joynera,n
aDevelopmental Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA
bDevelopmental Genetics Program, Skirball Institute of Biomolecular Medicine, New York, NY 10016, USA
cCancer and Developmental Biology Laboratory, NCI-Frederick, Frederick, MD 21702, USA
dDepartment of Epidemiology and Biostatistics, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA
a r t i c l e i n f o
Received 16 May 2012
Received in revised form
13 July 2012
Accepted 17 July 2012
Available online 25 July 2012
a b s t r a c t
Anterior–posterior (AP) limb patterning is directed by sonic hedgehog (SHH) signaling from the
posteriorly located zone of polarizing activity (ZPA). GLI3 and GLI2 are the transcriptional mediators
generally utilized in SHH signaling, and each can function as an activator (A) and repressor (R).
Although GLI3R has been suggested to be the primary effector of SHH signaling during limb AP
patterning, a role for GLI3A or GLI2 has not been fully ruled out, nor has it been determined whether
Gli3 plays distinct roles in limb development at different stages. By conditionally removing Gli3 in the
limb at multiple different time points, we uncovered four Gli3-mediated functions in limb development
that occur at distinct but partially over-lapping time windows: AP patterning of the proximal limb, AP
patterning of the distal limb, regulation of digit number and bone differentiation. Furthermore, by
removing Gli2 in Gli3 temporal conditional knock-outs, we uncovered an essential role for Gli2 in
providing the remaining posterior limb patterning seen in Gli3 single mutants. To test whether GLIAs or
GLIRs regulate different aspects of AP limb patterning and/or digit number, we utilized a knock-in allele
in which GLI1, which functions solely as an activator, is expressed in place of the bifunctional GLI2
protein. Interestingly, we found that GLIAs contribute to AP patterning specifically in the posterior limb,
whereas GLIRs predominantly regulate anterior patterning and digit number. Since GLI3 is a more
effective repressor, our results explain why GLI3 is required only for anterior limb patterning and why
GLI2 can compensate for GLI3A in posterior limb patterning. Taken together, our data suggest that
establishment of a complete range of AP positional identities in the limb requires integration of the
spatial distribution, timing, and dosage of GLI2 and GLI3 activators and repressors.
& 2012 Elsevier Inc. All rights reserved.
A long-standing question in developmental biology is how the
vertebrate limb acquires a stereotyped polarity and number of
skeletal elements along the anterior–posterior (AP) axis within
each segment of the proximal–distal axis (stylopod, zeugopod,
autopod). The secreted factor sonic hedgehog (SHH) is responsible
for mediating the AP organizing function of the zone of polarizing
activity (ZPA), a posterior cell-population that promotes digit
formation and AP polarity (Bastida and Ros, 2008). The mechanism
by which SHH signaling determines digit number in the autopod
(distal to the wrist) appears to involve regulating proliferation and
survival of digit precursor cells (Towers et al., 2008; Zhu et al.,
2008). However, studies focused on Shh have not fully delineated
the mechanism by which SHH signaling specifies AP pattern.
Whereas gain-of-function studies have provided evidence for a
classical morphogen gradient model in which AP positional iden-
tities are defined by the concentration of SHH protein (Riddle et al.,
1993; Tickle, 1981; Tickle et al., 1975), fate-mapping studies and
experimental manipulations have suggested that integration of
temporal SHH exposure and responsiveness influence AP identity
(Ahn and Joyner, 2004; Harfe et al., 2004; Scherz et al., 2007). In
addition, experiments addressing whether regulation of digit
number and specification of digit identity by SHH signaling are
temporally separable can be interpreted to mean either that
proliferation of limb mesenchymal cells is coincident with AP
patterning (Towers et al., 2008), or that Shh is required only
transiently for AP specification, after which it is required only for
mesenchymal expansion (Zhu et al., 2008). We reasoned that
additional insight into the mechanisms of AP patterning could be
gained through better understanding how the key intracellular
mediators of SHH signaling temporally regulate this process.
Contents lists available at SciVerse ScienceDirect
journal homepage: www.elsevier.com/locate/developmentalbiology
0012-1606/$-see front matter & 2012 Elsevier Inc. All rights reserved.
Abbreviations: AP, anterior–posterior; ZPA, zone of polarizing activity; A,
activator; R, repressor; p, phalanx; AER, apical ectodermal ridge; IDM, interdigi-
nCorresponding author. Fax: þ1 212 639 3962.
E-mail address: email@example.com (A.L. Joyner).
Developmental Biology 370 (2012) 110–124
The downstream effectors of SHH signaling include the GLI
family of transcription factors, each with a distinct biochemical
activity. Whereas Gli2 and Gli3 are required for patterning most
organs and survival past birth, Gli1 is dispensable in the mouse
(Jiang and Hui, 2008). GLI2 and GLI3 are bifunctional transcription
factors that have an activator function (GLI2A/GLI3A) only in the
presence of SHH, and otherwise undergo proteolytic cleavage to
produce repressors (GLI2/GLI3R) (Pan et al., 2006; Wang et al.,
2000). In contrast, Gli1 is a direct transcriptional target of GLI2A/
3A (Bai et al., 2004) that functions only as an activator (Dai et al.,
1999; Park et al., 2000; Sasaki et al., 1997). Although both GLI2
and GLI3 can form activators and repressors, genetic experiments
in mice have demonstrated that GLI3 predominates in its repres-
sor form, whereas GLI2 is the main activator induced by SHH
(Fuccillo et al., 2006). Since Gli1 can rescue all the Gli2?/?
phenotypes when expressed from the Gli2 locus (Bai and Joyner,
2001; Bai et al., 2004), it is possible that any repressor function of
GLI2 is masked by GLI3R. Conversely, as Gli3 cannot rescue most
Gli2?/?phenotypes (Bai et al., 2004), any GLI3A function would
appear to be weak. Given the distinct activities of GLI2 and
GLI3, and their overlapping expression throughout most of limb
development (Buscher and Ruther, 1998; Zulch et al., 2001), it
was surprising to find that only Gli3 is required for limb AP
patterning (Hui and Joyner, 1993; Mo et al., 1997; Park et al.,
2000; Bai et al., 2002). Since the posteriorly restricted expression
of Shh in the limb results in a gradient of GLI3 transcriptional
activity along the AP limb axis (Wang et al., 2000), it is possible
that AP positional identities are specified by different levels of
GLI3R. Consistent with this, the autopods of Shh?/?limbs, in
which the only form of GLI3 (and GLI2) present is the repressor,
develop only a single digit thought to retain digit 1 identity
(anterior-most digit) (Kraus et al., 2001; Litingtung et al., 2002),
whereas Gli3?/?limbs develop polydactyly and lack digit 1 iden-
tity (Johnson, 1967). Furthermore, removal of Gli3 rescues the
digit loss observed in Shh?/?autopods and results in polydactyly
(Litingtung et al., 2002; te Welscher et al., 2002b). However, since
some pattern remains in the posterior autopod of Gli3 mutants
(Hill et al., 2009; Panman et al., 2005; Zuniga and Zeller, 1999),
and Gli2?/?; Gli3?/?embryos die before the onset of skeletogen-
esis (Mo et al., 1997), it remains an open question whether indeed
GLI3R alone is sufficient to pattern the entire AP axis of the limb,
or whether instead Gli2 contributes to limb patterning.
Fig. 1. Gli3?/?hindlimbs develop polydactyly and severe AP patterning defects only in the anterior zeugopod and autopod. (A) Schematic of AP patterning mediated by
Shh signaling. Grey shading represents a gradient of Shh signaling. (B) and (C) Schematic representations of wild type and Gli3?/?hindlimbs with each digit identity
represented by a different color. Dotted lines denote bones that are variably lost. (D) Experimental design for CKO study showing when Tm was administered relative to
Gli3 expression (green) and Shh expression (blue) in the hindlimb. (E) and (F) Wild type E17.5 hindlimb. (G) and (H) Gli3?/?hindlimbs develop digit polydactyly, with
variable phalangeal bone duplications, variable abnormal p1 differentiation (red arrow-head), tibial agenesis (black arrow) and posteriorization of structures anterior to
digit 3, including loss of digit 1 metatarsal morphology and ossification center, loss of digit 1 tarsal bone (black arrowhead) and anterior elongation of the navicular bone.
Colored bar at the top of panel (G) indicates that digit and tarsal bones anterior to digit 3 adopt progressively more anterior pattern, but exclude digit 1 pattern. For all
panels anterior is left, posterior right. m: medial cuneiform, i: intermediate cuneiform, l: lateral cuneiform, c: cuboid, and n: navicular.
M. Bowers et al. / Developmental Biology 370 (2012) 110–124
Efforts at determining the mechanism by which GLI3 patterns
the AP limb axis downstream of SHH have been confounded by
the expression of Gli3 during multiple stages of limb develop-
ment. Prior to expression of Shh in the ZPA at embryonic day (E)
?10.5 (or ?32 somites) in the hindlimbs, GLI3R (expressed in the
anterior limb) posteriorly restricts the expression of several AP
patterning genes, setting up a ‘‘pre-pattern’’ that could be required
for normal AP limb morphology (te Welscher et al., 2002a; Zuniga
and Zeller, 1999). Subsequently, during the time Shh is expressed
from the ZPA (E10.5-E12.5 in the hindlimbs), the extracellular SHH
gradient present along the AP limb axis (highest posterior) gen-
erates an opposing intracellular gradient of GLI3R (highest ante-
rior) that could provide a mechanism for specifying AP positional
values (Wang et al., 2000) (Fig. 1). Furthermore, Gli3 persists after
Shh is downregulated (after ?E12.5) and acts downstream of
indian hedgehog (IHH) in chondrocyte differentiation of the digital
rays (Hilton et al., 2005; Koziel et al., 2005). However, since Gli3 is
also expressed in the interdigital mesenchyme (IDM) (Buscher and
Ruther, 1998) and digit identity can be manipulated by signals
emanating from the IDM late in chick limb development (Dahn and
Fallon, 2000), it is possible that GLI3 plays a role in regulating digit
identity during this late developmental stage. Given the multiple
roles Gli3 could play during AP limb patterning and its apparent
status within the GLI family as the principle mediator of SHH
signaling in the limb, resolving the temporal specific roles of Gli3
during limb development would provide new understanding of the
mechanisms by which SHH signaling patterns the limb.
Using a Hoxb6-CreER transgenic line and Tamoxifen (Tm) to
conditionally inactivate Gli3 in the hindlimb mesenchyme before,
during, and after Shh expression, we have uncovered a temporal
sequence of Gli3 requirements, first in specifying AP pattern
sequentially along the proximal–distal axis, then in regulating digit
number, and finally in sustaining normal bone morphogenesis.
Importantly, by removing Gli2 in Gli3 conditional limb mutants, we
have uncovered a role for Gli2 in providing AP pattern to Gli3
mutant limbs throughout the AP axis. Furthermore, we show that
although regulation of AP patterning and digit number are tempo-
rally uncoupled after ?E12.0 in Gli3 conditional mutants (our
results and (Lopez-Rios et al.)), the two processes remain coupled
in Gli2/3 double mutants. Finally, by replacing the bifunctional GLI2
protein with the GLI1 activator, we show that GLI repressors
pattern the anterior autopod, whereas posterior autopod pattern-
ing involves a combination of activators and repressors or a weak
activator. Thus, the mechanism of GLI-mediated limb patterning is
complex, and depends on the integration of timing and dosage of
total Gli expression, as well as the distribution of GLI2R/3R and
GLI2A/3A functions within spatially distinct domains.
Materials and methods
Hoxb6-CreERT1transgenic mice (Nguyen et al., 2009), R26lox-STOP-lacZ
(R26R) (Soriano, 1999) and Gli3XtJ(Maynard et al., 2002), Gli2zfd(Mo
et al., 1997), and Gli21ki(Bai and Joyner, 2001) alleles were genotyped
as previously described. Gli3recand Gli3fl(Blaess et al., 2008) alleles
were detected using the following primers: 50-GGAAAGTCCTCTA-
CAGTCTG-30and 50-CAGTAGTAGCCTGGTTACAG-30. Gli3 CKO embryos
were generated by mating Hoxb6-CreERT1; Gli3XtJ/þcompound het-
erozygous males to Gli3fl/flhomozygous females. Gli3 CKO; Gli2zfd/zxfd
embryos were generated by mating Hoxb6-CreERT1; Gli3XtJ/þ; Gli2zfd/þ
compound heterozygous males to Gli2zfd/þ; Gli3fl/flfemales. Gli3 CKO;
Gli21ki/zfdembryos were generated by mating Hoxb6-CreERT1; Gli3XtJ/þ;
Gli2zfd/þcompound heterozygous males to Gli21ki/þ; Gli3fl/flfemales.
Embryo and limb staging
Embryonic day 0.5 (E0.5) was defined as noon the day a
vaginal plug was detected. For the Shh and dHand expression
analysis, limbs were stage-matched by somite number and limb
morphology, as described in Wanek et al. (1989).
Tamoxifen administration and dosage
Pregnant females were administered 200 mg/g Tm admixed
with 50 mg/g progesterone dissolved in corn oil via oral gavage.
E17.5 embryos were processed for alizarin red/alcian blue
skeletal staining as described (Lufkin et al., 1992).
Western blot analysis
Embryos were dissected and lysates prepared from individual
hindlimb buds, electrophoresed and probed with anti-GLI3 anti-
body as previously described (Chen et al., 2004; Wang et al., 2000).
Anti-vinculin was used as a control for total protein loading.
RNA in situ
Standard whole mount RNA in situ hybridization analysis was
performed as described on the Joyner Lab website: http://www.
mskcc.org/mskcc/html/75282.cfm. Shh (Echelard et al., 1993),
Hoxd11/12 (Herault et al., 1999), Pax9 (Neubuser et al., 1995), Tbx2
(Gibson-Brown et al., 1996), dHand (Fernandez-Teran et al., 2000).
Detection of b-gal activity
Embryos were fixed in 0.2% gluteraldehyde/4% formaldehyde,
processed for cryosectioning at 14 mm, stained for beta-galacto-
sidase (b-gal) activity at 371C in X-gal solution for ?12 h and
counterstained with Fast Red. Detailed protocols can be found on
the Joyner Lab website.
Autopod phenotype analysis
Digit number of E17.5 embryos was determined as the number of
complete metatarsal bones that developed. If phalanges were dupli-
cated but associated with the same metatarsal bone, it was counted
as one digit, as in the first digit of Fig. 3E. Cartilagenous structures
that developed in the position of a metatarsal bone, but did not
develop into complete metatarsal bones, were not counted as digits,
as in the structures that formed anterior to the first digit in Fig. 5E. In
the text, ‘‘digit’’ therefore refers to the entire digital ray, a proximal–
distal unit that includes a metatarsal bone and its associated
Autopod AP patterning
In the wild-type mouse autopod, the most anterior tarsal bone
(medial cuneiform) underlying digit 1 is distinguishable from the
tarsal bones underlying digits 2 and 3 due to its elongated morphol-
ogy. The two rectangular tarsal bones underlying digits 2 and 3
(intermediate and lateral cuneiform, respectively) have similar
shapes, but the tarsal bone underling digit 2 is smaller. The most
posterior tarsal bone (cuboid) is shared by digits 4 and 5, and has a
trapezoidal shape. The more proximal tarsal bone (navicular) is
positioned directly beneath the tarsal bones underlying digits
2 and 3. The lengths of the metatarsal bones were used to distinguish
M. Bowers et al. / Developmental Biology 370 (2012) 110–124
affecting autopod AP polarity independent of SHH. Second, a
comparison of Gli3D699/?
limbs (expressing GLI3R only), to
Gli3D699/?; Shh?/?limbs concluded that GLI3A is unlikely to play
a role in AP patterning, since removal of Shh was interpreted as
having no affect on AP asymmetry in Gli3D699/?mutants (Hill
et al., 2009). However, the morphology of digits and carpal bones
in the posterior Gli3D699/?; Shh?/?forelimb autopods compared
to Gli3D699/?suggests that the distinctions between digits 3, 4,
and 5 are retained in Gli3D699/?mutants, but are partially lost in
Gli3D699/?; Shh?/?double mutant limbs. As we have observed
in Gli2?/?; Gli3 CKOs, the posterior carpal bones in Gli3D699/?;
Shh?/?forelimbs (the forelimb-equivalent to tarsal bones in the
hindlimb autopod) appeared fused and homogenous in morpho-
logy, whereas their patterning was normal in Gli3D699/?limbs
(Fig. 6 of (Hill et al., 2009). The overall digit length also appeared
similar in the three posterior-most digits of Gli3D699/?; Shh?/?
double mutants, further suggesting loss of posterior polarity. Our
interpretation of the Gli3D699/?; Shh?/?morphological data pre-
sented in Hill et al. (2009) study is therefore consistent with a
model of GLI-mediated AP patterning in which GLI repressors
alone are not sufficient to pattern the posterior autopod.
An on-going debate about the role of SHH signaling during
vertebrate limb development concerns whether SHH-mediated
AP limb patterning and regulation of digit number are temporally
uncoupled, as indicated in the mouse (Zhu et al., 2008), or if these
two processes take place at the same time, as suggested in the
chick (Towers et al., 2008). In our study, depending on the type of
conditional mutant, we observed either uncoupled or coupled
patterning and digit number regulation. In Gli3-E10.5 CKOs digit
number and AP pattern was temporally uncoupled, consistent
with a biphasic model in which AP pattern is specified by SHH
signaling early in limb development, but regulation of digit
number occurs over an extended time (Zhu et al., 2008). However,
temporal uncoupling was not observed in Gli2?/?mutants lack-
ing Gli3 after ?E12. Given that only 6–12 h of Shh expression is
required to specify AP pattern (Zhu et al., 2008), yet Gli2 and Gli3
function is required to regulate AP polarity until between ?E12.0
and ?E12.75, it could be interpreted that a mechanism for
differentially regulating GLI2 and GLI3 activity across the limb
independent of SHH must take over after ?E11. One possibility is
that GLI3-binding partners (HOXD12, SMAD, beta-catenin) can
modify GLI3 transcriptional activity independent of late SHH
signaling (Chen et al., 2004; Liu et al., 1998; Ulloa et al., 2007),
as a high percentage of regulatory regions bound by GLI3R in the
limb do not contain GLI-recognition sites, and are therefore likely
to require GLI3 binding partners to regulate gene expression in
the limb or are inert (Vokes et al., 2008). Our results suggest that
whether or not temporal uncoupling of regulation of digit number
and identity by SHH signaling is observed depends, fundamen-
tally, on the assay employed.
Using a combination of temporal conditional knock-out and
knock-in mutant analyses we have demonstrated that the task of
translating SHH signaling into normal digit number and AP
pattern is distributed between GLI3 and GLI2, and requires both
GLIR and GLIA function. Spatially, the dependence of the anterior
autopod on GLIR, and the posterior autopod on both GLIA and
GLIR corresponds to the regional distributions of the proteins in
the limb (Fig. 9B). Interestingly, as the tarsal bone morphologies
become increasingly homogenous along the AP axis upon pro-
gressively decreasing the time and dose of GLI function, expres-
sion of AP polarity markers become progressively homogenous as
well. The tarsal bone morphologies appear to converge upon a
morphology resembling the bone normally located in the middle
of the autopod. Since the middle of the autopod is the region most
likely to experience a similar dose of GLIR and GLIA function, the
‘medial’ morphology of the tarsal bones in Gli2?/?; Gli3 CKO
hindlimbs might correspond to the AP limb polarity produced
when the ratio of GLI3R and GLI3A function is close to 1 (Fig. 9C).
The results of our genetic studies demonstrate that the mechan-
ism by which GLI-transcription factors provide a full range of
polarized positional identities along the autopod AP axis involves
integrating spatial, temporal, and dosage requirements for both
GLIR and GLIA functions.
We would like to thank Dr. Cindy Loomis for advice and
insightful comments throughout the studies and Dr. Sandra
Wilson for critical reading of the manuscript. We would like to
thank Dr. Virginia E Papaioannou and Dr. Cindy Loomis for
generously providing the Tbx2 and Pax9 in situ probes, respec-
tively. This work was funded by the NIH grants nos. R01
CA128158 and R01 DE016779 to ALJ and by the Center for Cancer
Research, National Cancer Institute to SM.
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