Tbx2 Terminates Shh/Fgf Signaling in the Developing Mouse Limb Bud by Direct Repression of Gremlin1
et al. (2013) Tbx2 Terminates Shh/Fgf Signaling in the Developing Mouse Limb Bud
by Direct Repression of Gremlin1. PLoS Genet 9(4): e1003467. doi:10.1371/journal.pgen.1003467
Tbx2 Terminates Shh/Fgf Signaling in the Developing Mouse Limb Bud by Direct Repression of Gremlin1
Henner F. Farin 0
Timo H.-W. Lu dtke 0
Martina K. Schmidt 0
Susann Placzko 0
Karin Schuster-Gossler 0
Marianne Petry 0
Vincent M. Christoffels 0
Andreas Kispert 0
Rolf Zeller, University of Basel, Switzerland
0 1 Institute for Molecular Biology, Medizinische Hochschule Hannover, Hannover, Germany, 2 Department of Anatomy, Embryology, and Physiology, Heart Failure Research Center, Academic Medical Center, University of Amsterdam , Amsterdam , The Netherlands
Vertebrate limb outgrowth is driven by a positive feedback loop that involves Sonic hedgehog (Shh) and Gremlin1 (Grem1) in the posterior limb bud mesenchyme and Fibroblast growth factors (Fgfs) in the overlying epithelium. Proper spatiotemporal control of these signaling activities is required to avoid limb malformations such as polydactyly. Here we show that, in Tbx2-deficient hindlimbs, Shh/Fgf4 signaling is prolonged, resulting in increased limb bud size and duplication of digit 4. In turn, limb-specific Tbx2 overexpression leads to premature termination of this signaling loop with smaller limbs and reduced digit number as phenotypic manifestation. We show that Tbx2 directly represses Grem1 in distal regions of the posterior limb mesenchyme allowing Bone morphogenetic protein (Bmp) signaling to abrogate Fgf4/9/17 expression in the overlying epithelium. Since Tbx2 itself is a target of Bmp signaling, our data identify a growth-inhibiting positive feedback loop (Bmp/Tbx2/Grem1). We propose that proliferative expansion of Tbx2-expressing cells mediates self-termination of limb bud outgrowth due to their refractoriness to Grem1 induction.
Funding: Work in the laboratory of VMC was supported by the European Communitys Seventh Framework Programme contract (CardioGeNet 223463). Work
in the laboratory of AK was supported by funding from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) for the grant KI 728/5-1 DFG
and for the Cluster of Excellence REBIRTH (from Regenerative Biology to Reconstructive Therapy) at Medizinische Hochschule Hannover. HFF was supported by an
EMBO long-term fellowship. Publication charges were supported by the Deutsche Forschungsgemeinschaft DFG in the framework of the program Open Access
Publishing at Medizinische Hochschule Hannover. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
Competing Interests: The authors have declared that no competing interests exist.
Polydactyly, the condition of having more than the normal
number of toes and/or fingers is the most frequent form of limb
malformation in humans with an incidence of 1:500. It most
commonly occurs as post-axial polydactyly (extra digit(s) towards
5th finger in limb), less common is pre-axial polydactyly (extra
digit(s) towards thumb or toe), very rare is central (mesoaxial)
polydactyly (with extra digits within the three middle digits). In all
cases, the excess digits can be undeveloped and only attached by a
little stalk mostly on the small finger side of the hand or fully
formed and working. Polydactyly can occur by itself, or more
commonly, as one feature of a syndrome of congenital anomalies
as e.g. in Pallister-Hall syndrome, Smith-Lemli-Opitz syndrome or
Bardet-Biedl syndrome (OMIM 146510, 270400, 209900).
Elucidation of the genetic, molecular and cellular changes that
underlie polydactyly (as well as other limb defects) in humans has
greatly benefitted from the analysis of normal limb development,
and of the consequences of altered gene functions in suitable
animal models such as the chicken and the mouse . All of these
studies unraveled that proper establishment and elaboration of the
two main limb axes during development is crucial for setting up a
correct number and identity of digits. In the limb primordium two
signaling centers control the morphogenesis along these limb axes.
The apical ectodermal ridge (AER), a distal thickening of the
ectodermal jacket of the limb bud, controls proximal-distal (from
shoulder to finger tips), whereas the zone of polarizing activity
(ZPA) in the posterior region of the mesenchymal core mediates
anterior-posterior (A-P, from thumb to the small finger)
development. Fibroblast growth factors (Fgf4, Fgf8, Fgf9 and Fgf17) and
Shh secreted from the AER and ZPA, respectively, specify distal
and posterior positional values in the early limb bud mesenchyme
(in the mouse until E9.5) [2,3]. Removal of the signaling centers or
the signals, results in time-dependent distal truncations  and loss
of posterior limb positions (ulna and digits 25) , respectively.
However, both AER-FGFs and ZPA-Shh do not only provide
patterning functions, they also account for the massive outgrowth
of the limb bud at subsequent stages (from E9.5 to 11.5 in the
mouse) by promoting cell survival and proliferative expansion of
the distal and posterior limb bud mesenchyme, respectively [6,7].
During this phase the three primordia of the stylopod (the future
upper arm/leg), the zeugopod (lower arm/leg) and the autopod
(hand/foot) are laid down and expanded and digit formation is
initiated. It has long been noted that AER and ZPA are mutually
dependent on each other, thus linking the two signaling centers by
an epithelial-mesenchymal (e-m) signaling loop [8,9,10]. Fgf
signaling is likely to maintain Shh directly, whereas Shh signaling
to the overlying AER is relayed in the posterior limb bud
Developmental defects of the limb skeleton, such as
variations from the normal number of digits, can result
from an abnormal size of the early limb bud. The
mechanisms that restrict limb bud growth to avoid
polydactyly, i.e. the formation of extra digits, are unclear.
Gremlin 1 (Grem1) has been identified as a key regulator in
this process via its role as secreted antagonist of Bone
morphogenetic protein (Bmp) signaling. But it remains
unknown how Grem1 expression is switched off
appropriately to achieve normal limb bud size. Here we show in the
mouse embryo that T-box transcription factor 2 (Tbx2)
directly represses Grem1. We show that Tbx2-positive
mesenchymal cells at the posterior margin of the limb bud
create a Grem1-negative zone that expands concomitantly
with limb bud growth. Progressive displacement of the
source of Grem1 and its target region, the apical
ectodermal ridge, eventually disrupts
epithelial-mesenchymal signaling that is crucial for further proliferative
expansion. Our data show how local control of signaling
activities is translated into the architecture of the adult
skeleton, i.e. the number or digits, which helps us to
understand the molecular bases of human polydactyly.
mesenchyme by Gremlin 1 (Grem1), a secreted antagonist of Bone
morphogenetic protein (Bmp) signaling [11,12,13]. Inhibition of
Bmp signaling allows Fgf4 expression in the posterior AER
enabling the further propagation of the loop. Limb bud outgrowth
comes to a halt (around E11.5 to E12.0 in the mouse) when Fgf4
and Shh expression is shut-off due to a rise of Bmp signaling,
resulting in cell-cycle exit and initiation of chondrogenic
differentiation [10,14]. Although the precise mechanisms are unclear,
refractoriness of ZPA descendants to induce Grem1 may be of
critical importance to self-terminate the e-m signaling loop .
Tbx2 encodes a transcriptional repressor of the T-box gene
family that has recently been implicated in digit development. In
the mouse, Tbx2 is expressed in the anterior and posterior
mesenchymal flanks of the early limb bud. From E11.5 on, the
posterior domain of Tbx2 extends more distally and is then found
in the interdigital mesenchyme (IDM), most prominently in IDM4,
and at the distal tips of the digit condensates at E12.5  (Figure
S1). Limbs of Tbx2-deficient mice exhibit a hindlimb-specific
duplication of digit 4 . The spatially restricted nature of this
phenotype may be due to redundancy with the closely related
Tbx3 gene. While both genes are coexpressed in the proximal
mesenchyme on either limb margin, Tbx3 is absent from the distal
mesenychme of the posterior flank  (Figure S1). Exclusive
expression of Tbx3 in the AER may relate to the variable distal
truncations and oligodactyly observed in Tbx32/2 embryos .
Retroviral mis- and overexpression experiments in the chick model
provided phenotypic outcomes that suggested additional or
alternative functions for Tbx2 (and Tbx3) in regulating digit
identity rather than digit number  and in anterior-posterior
positioning of the limb bud .
Here, we set out to gain further insight into the role of Tbx2 in
digit development by genetic loss- and gain-of-function
experiments in the mouse. We show that increased digit number in
Tbx2mutant mice and oligodactyly in embryos overexpressing Tbx2 (in
the limbs) relates to the maintenance of the e-m feedback loop in
the posterior limb bud mesenchyme. Tbx2 terminates this loop by
locally repressing Grem1. Our experiments identify a Bmp/Tbx2/
Grem1 loop that counteracts the Shh/Grem1/Fgf4 loop to
mediate self-termination of limb bud outgrowth.
Etiology of digit 4 duplication in Tbx2-mutant hindlimbs
Mice homozygous for a Tbx2 null allele (Tbx2cre) maintained on
a NMRI outbred background died shortly after birth due to
craniofacial defects . Mutant E18.5 embryos had normal
forelimbs but hindlimbs displayed six instead of the normal five
digits. Soft tissue webbing, i.e. the persistence of IDM tissue was
not observed (Figure 1B). Hindlimb polydactyly showed 70%
penetrance in homozygous embryos, heterozygous embryos were
not affected. Skeletal preparations of hindlimbs of E18.5 Tbx22/2
embryos revealed that the proximal segment of digit 4 was
broadened and split distally to connect to a duplicated pair of
second and third phalangeal segments (Figure 1D). Analysis of
chondrogenic elements at E15.5 and E13.5 showed that the
duplication of skeletal elements of digit 4 was established shortly
after onset of chondrogenesis in the mutant hindlimb (Figure 1H
and 1L). Comparative expression analysis for the
(pre-)chondrogenic marker gene Sox9  and Raldh2, which marks the IDM
, showed that the occurrence of duplicated distal cartilagenous
segments of digit 4 was preceded by a posterior expansion of the
prechondrogenic condensate at the expense of the adjacent IDM4
at E12.5 (Figure 1M1P).
Since Tbx2 expression in the developing limb is confined to
flank and ID mesenchyme that are removed by programmed cell
death , we determined the distribution of apoptotic cells by
Lysotracker Red staining. We noted a specific reduction of
apoptotic cells in IDM4 of Tbx22/2 hindlimbs at E13.5, whereas
other domains of programmed cell death (IDM1-3) were
unchanged (Figure 1Q and 1R). In E12.5 wild-type embryos,
apoptosis was confined to the mesenchyme underlying the AER at
the anterior and posterior margins of the hindlimb (Figure 1S).
Programmed cell death appeared unaffected in the anterior region
whereas it was largely reduced in the posterior mesenchymal
region of mutant hindlimbs at this stage (Figure 1T). The latter
may explain the increased size and the characteristic outward
curvature of the posterior edge of the mutant hindlimb. Together
with the strong expression of Tbx2 in the distal region of the
posterior flank mesenchyme at E11.5 and in IDM4 at E12.5
(Figure S1), this suggests that Tbx2 functions after E11.5 in the
development of the posterior autopod region to maintain IDM4
fate and to restrict the expansion of chondrogenic material at the
posterior limit of the digit 4 condensation.
The signaling loop between the AER and the underlying
mesenchyme is disturbed in the posterior region of
Proliferative expansion and apoptotic removal of the limb bud
mesenchyme is governed by reciprocal signaling with the overlying
AER . Given the increased posterior size and the decreased
apoptosis of E12.5 Tbx2-deficient hindlimbs, we studied the
expression of factors that are involved in e-m signaling before the
manifestation of morphological defects. In E10.5 hindlimb buds
expression of Fgf4, Fgf8, Fgf9, Fgf17 and Shh was unaffected
(Figure 2A2E). At E11.5, however, we found increased expression
of Fgf4 (in 7/8 embryos), Fgf9 (in 3/3 embryos) and Fgf17 (in 2/3
embryos) in the posterior AER of Tbx22/2 hindlimbs (Figure 2F
2H). At E12.5, Fgf4 expression was no longer detected in wild-type
hindlimbs, but residual AER expression of Fgf4 was observed in 3/
8 mutant embryos (Figure 2K). Shh was unchanged at E11.5
(Figure 2I), but expression was aberrantly maintained at E12.5 (in
5/5 embryos) (Figure 2N). Fgf8, which is continuously expressed in
the entire AER, was unaffected in Tbx2-deficient hindlimbs at all
analyzed stages (Figure 2E, 2J and 2O, and data not shown).
Together we conclude that Tbx2 is required to assure correct
termination of the Fgf4/9/17-Shh signaling loop in the posterior
mesenchyme of the hindlimb bud.
Polydactyly in Tbx2-deficient mice is ameliorated by
reduced mesenchymal Fgf-signaling
To explore if a gain in e-m signaling indeed causes polydactyly
in Tbx2-deficient hindlimbs, we genetically reduced the level of
mesenchymal Fgf-signaling. We took advantage of cre
recombinase driven from our Tbx2 mutant allele to delete a floxed allele of
the Fgf-receptor 1 gene in the posterior limb bud mesenchyme.
Double heterozygous animals (Tbx2cre/+;Fgfr1fl/+) were intercrossed
and the limb skeleton of compound mutants was analyzed.
Homozygous loss of Fgfr1 (Tbx2cre/+;Fgfr1fl/fl) caused a reduced
number of 4 digits in fore- and hindlimbs (Figure 3F). This result is
consistent with previous experiments using the Shh-cre line that
recombines in a domain very similar to that of Tbx2cre [7,25]
(Figure S2AS2D). At E16.5 Tbx2cre/cre;Fgfr1fl/+ embryos were
underrepresented and Tbx2cre/cre;Fgfr1fl/fl embryos were absent,
most likely due to a synthetic lethal cardiac defect. Double
heterozygous Tbx2cre/+;Fgfr1fl/+ embryos exhibited a normal
limb skeleton (Figure 3B) in agreement with previous results
using the Prrx1-cre line that deletes in the entire limb bud
mesenchyme [25,26]. In Tbx2-deficient embryos, however,
dose-reduction of Fgfr1 strongly reduced the penetrance of
polydactyly (3/7 compared to 7/10 in Tbx2cre/cre;Fgfr1wt/wt
embryos) or even caused oligodactyly (4 digits in 3/7 embryos
analyzed) (Figure 3D and 3E). In oligodactic limbs the
characteristic shape of the proximal end of the posterior
metacarpal indicated a digit 5 identity (white arrows). Absence
of Eomes expression in E12.5 Tbx2cre/cre;Fgfr1fl/fl hindlimbs
argued for a specific loss of digit 4 (Figure S2E, S2F) .
Our results suggest that polydactyly in Tbx2-deficient mice
critically depends on increased activity of the e-m signaling loop.
Figure 2. Prolonged posterior e-m signaling in Tbx2-deficient hindlimbs. Whole-mount in situ hybridization analysis of Fgf4, Fgf9, Fgf17, Shh
and Fgf8 expression in E10.5 (AE), E11.5 (FJ) and E12.5 (KO) wild-type and Tbx22/2 hindlimbs. (F, G, H) Arrowheads show prolonged AER
expression of Fgf4/9/17 in the posterior limb bud at E11.5. (K, N) Arrowheads show maintenance of Fgf4 and Shh expression at E12.5 in Tbx22/2
hindlimbs. Fgf8 expression is unchanged in Tbx2-mutant limb buds.
Tbx2 directly represses Grem1 in the posterior
mesenchyme of hindlimb buds
Since Tbx2 encodes a nuclear transcriptional repressor , we
sought to identify targets that may help to explain the observed
molecular changes. We judged it unlikely, that Shh itself is a target
of Tbx2 repression since Tbx2 and Shh are broadly coexpressed in
the posterior limb bud mesenchyme. Furthermore, unchanged
expression of Shh in E11.5 mutant hindlimbs cannot explain
upregulation of Fgf4 in the AER at this stage. Because AER
expression of Fgf4 is mediated by inhibition of Bmp signaling by
the secreted Bmp antagonist Grem1 [12,13], we explored the
possibility of Grem1 regulation by Tbx2. In E12.0 and E11.5
wildtype embryos, the expression of Grem1 in two crescent-shaped
domains at the dorsal and ventral surface of the limb mesenchyme
was sharply excluded from the Tbx2-positive posterior hindlimb
mesenchyme (Figure 4A and 4B, 4D and 4E). In Tbx22/2
hindlimbs, the expression of Grem1 was unaffected at E10.5 and
E13.0 (data not shown), but appeared specifically up-regulated at
E12.0 and E11.5 in the posterior region (Figure 4C and 4F;
arrows). On adjacent sagittal sections of E11.5 Tbx2cre/+ hindlimbs,
cre and Grem1 were expressed in neighboring, non-overlapping
domains (Figure 4G4I). In Tbx2cre/cre- embryos, however, Grem1
expression was shifted posteriorly and overlapped with the domain of
cre expression that itself was unchanged. Consistent with local
expansion of the Bmp-antagonist Grem1 the Bmp target gene Dkk1
 was absent in the mesenchyme underlying the posterior AER in
Tbx22/2 hindlimbs at E11.5 (Figure S3A). Expression of Bmp targets
Msx1 and Msx2  was unaffected (Figure S3B and S3C), suggesting
that the level of Bmps was still sufficient to activate these genes.
To explore if Tbx2 mediates Grem1 repression directly, we
performed chromatin immunoprecipitation experiments (ChIP)
using anti-Tbx2 IgG and E11.5 wild-type hindlimb tissue
(Figure 4J and 4K). We found that in posterior limb-bud halves
Tbx2 protein specifically interacted with the known Grem1 limb
bud enhancer  but not with control genomic regions. No
binding was observed to chromatin prepared from anterior limb
bud halves that lack Tbx2 expression, demonstrating specificity of
this assay. Together our results strongly suggest that Tbx2
terminates Fgf4/Shh signaling in the posterior limb bud by direct
repression of Grem1.
Figure 3. Polydactyly in Tbx2-deficient limbs depends on the Fgf signaling level. (AF) Skeletal analysis of E18.5 hindlimbs. Normal digit
development in the wild-type (A) and after heterozygous loss of Fgfr1 (Tbx2cre/+;Fgfr1fl/+, B). Duplicated digit 4 in Tbx2-deficient limbs (C, black arrow).
Loss of one Fgfr1 allele on the Tbx2-mutant background (Tbx2cre/cre;Fgfr1fl/+) rescues polydactyly (D), or even causes oligodactyly (E), as observed in
homozygous Fgfr1-mutant embryos (Tbx2cre/+;Fgfr1fl/fl, F). Morphology of the proximal part of the most posterior metacarpal suggests normal
formation of digit 5 (white arrows). (G) Phenotype frequencies in Tbx2cre/Fgfr1fl compound mutant limbs (oligodactyly 4 digits; normal hindlimb 5
digits; digit 4 duplication 6 digits). A total of 135 embryos was analyzed. All phenotypes were found in a bilateral symmetric fashion. Numbers on
the right side indicate observed and expected counts (in brackets) of embryos for each genotype. Double mutant embryos (Tbx2cre/cre;Fgfr1fl/fl) were
not obtained at E18.5.
Tbx2 is a target of Bmp signaling
Bmp ligands are expressed in the AER and at the margins of the
limb mesenchyme  and represent good candidates as
activators of Tbx2 expression, similar to other developmental
contexts [32,33,34,35]. To explore this possibility, we analyzed the
effect of Bmp on Tbx2 expression by bead implantation
experiments. We found that Bmp4-soaked beads implanted into
the central mesenchyme of E10.5 forelimb buds caused
upregulation of Tbx2 after 16 hours of culture (Figure 5A). This effect was
further increased after ectoderm removal (Figure 5B). We
established micromass cultures of E10.5 limb bud mesenchyme
and studied Tbx2 expression by quantitative RT-PCR 2 hours
after addition of Bmp4 to the medium. A dose-dependent increase
of Tbx2 mRNA was observed that closely resembled induction of
the known Bmp targets Msx1 and Msx2 (Figure 5C). To study Bmp
requirement of Tbx2 expression, we added Dorsomorphin, a
selective inhibitor of bone morphogenetic protein (BMP) type I
receptors , during the last two hours of culture. We observed a
twofold reduction of basal Tbx2 expression in the presence of
1 mM Dorsomorphin that again resembled the response of Msx1
and Msx2 (Figure 5D). Thus, Bmp signaling is necessary and
sufficient to induce Tbx2 expression in the limb. Tbx2 via
repression of Grem1 therefore operates in a positive feedback loop
with Bmp(s) at the posterior margin of the limb.
Ectopic Tbx2 abrogates Grem1 expression in the early
limb bud mesenchyme
Next we used a cre/loxP-based misexpression approach to
analyze if expression of Tbx2 in the entire limb bud would interfere
with e-m signaling and digit development. In HprtTBX2 mice
expression of the human TBX2 cDNA (introduced into the
Xchromosomal Hypoxanthine guanine phosphoribosyl transferase) locus, is
inducible by cre-mediated recombination . We employed the
Prrx1-cre mouse line to drive transgene expression in the entire
limb mesenchyme . X-chromosome inactivation in females
Figure 5. Bmp signaling induces Tbx2 expression in the limb bud mesenchyme. (A, B) Whole mount in situ hybridization analysis
demonstrates that Bmp4-soaked beads (100 mg/ml) but not BSA-soaked beads (control) induce Tbx2 expression in the central limb bud mesenchyme
after 18 h of culture (black arrows). White asterisks show an expansion of Tbx2 expression into the proximal-central region following ectoderm
removal. (C, D) Influence of Bmp signaling on Tbx2 expression in micromass cultures of dissociated E10.5 limb bud mesenchyme. Bmp4 (C) or the
Bmp-inhibitor Dorsomorphin (D) was added during the last 2 hours of in total 18 hours of culture. The relative expression levels of Tbx2 and of the
known Bmp targets Msx1 and Msx2 were analyzed by qPCR analysis. Means 6 SD of four independent cDNAs. Significant changes compared to
control are labeled with asterisks (*: p,0.05; **: p,0.01; ***: p,0.001, as determined by t-test).
causes mosaicism, we therefore only analyzed hemizygous
Prrx1cre/+;HprtTBX2/Y male embryos that express the transgene in a
uniform manner (abbreviated as Prrx1-TBX2). By Western blot
analysis and immunostainings we confirmed ubiquitous expression
of TBX2 in the E10.5 limb mesenchyme at levels comparable to
the endogenous Tbx2 protein (Figure S4AS4D). At E18.5,
transgenic embryos exhibited oligodactyly with a dramatic
reduction of the limb length (Figure 6A6D). Forelimbs (3 digits
in 9; 4 digits in 1 out of 10 embryos) were stronger affected than
hindlimbs (4 digits in 5; 5 digits in 4 and 6 digits in 1 out of 10
embryos), most likely reflecting the relatively delayed onset of
Prrx1-cre mediated recombination in hindlimbs . We
invariantly observed a single zeugopodial element and a reduction of
pectoral and pelvic girdles. Forelimb stylo- and zeugopod elements
were fused, in the hindlimb the femur was strongly reduced.
The delayed onset of recombination in the hindlimbs led us to
study the early consequences of TBX2 misexpression in forelimbs
that demonstrated a complete lack of autopod outgrowth at E12.5.
In sharp contrast to the Tbx2 loss-of-function situation,
Lysotracker Red staining showed apoptotic mesenchymal cells
underneath the entire AER at this stage in Prrx1-TBX2 embryos
(Figure 6E and 6F). In E10.5 wild-type forelimbs, apoptosis was
confined to the proximal mesenchyme as previously reported 
(Figure 6G). Age matched Prrx1-TBX2 embryos exhibited strongly
reduced forelimb buds and widespread apoptosis throughout the
mesenchyme as observed by LysoTracker Red and by TUNEL
staining (Figure 6H and Figure S4F).
A highly similar phenotypic spectrum of limb defects including
reduced limb length and autopod outgrowth, oligodactyly, fusion
of zeugopodial elements, as well as early and widespread
mesenchymal apoptosis was reported in Grem12/2 mutants
[12,13], suggesting that loss of Grem1 expression may account for
the observed effects in Prrx1-TBX2 embryos. Indeed, we found
strong reduction of Grem1 expression in E10.5 transgenic forelimb
buds (Figure 6K and 6L) and residual expression at E12.5 at the
base of the autopod (Figure 6I and 6J). Thus, Tbx2 represses
Grem1 expression distally, whereas the more proximal expression
domain at E12.5 might be controlled by an independent
mechanism. Next, we analyzed the effects of TBX2 misexpression
on known downstream effectors of Grem1. At E10.5, expression of
both Fgf4 and Shh was strongly reduced (Figure 6M6P), again
closely resembling the situation in Grem12/2 limbs. Reduction of
the target genes Spry4 and Ptch1 [39,40] confirmed a decrease in
Fgf4 and Shh signaling (Figure S5). Fgf9 and Fgf17 expression was
reduced but expression of Fgf8 and of the more broadly expressed
Fgf target Etv4 (Pea3)  was unaffected, demonstrating that
Tbx2 acts specifically on posterior e-m signaling (Figure S5C and
S5D). To study if reduction of Grem1 is associated with increased
Bmp signaling we analyzed Msx1 and Msx2 expression, and found
that both genes were upregulated in the distal limb bud
(Figure 5Q5T), i.e. in regions normally devoid of Msx1/2
expression due to Grem1-mediated Bmp-antagonism (compare
Figure 5K) [12,13].
Notably, we did neither observe transformations of digit identity
nor changes in limb positioning (data not shown), as reported for
Tbx2 and Tbx3 misexpression in the chick model [19,20]. Hence,
control of digit formation by local repression of Grem1 is the
primary function of Tbx2 in the mouse.
Precise termination of the e-m signaling loop involving Shh,
Grem1 and Fgfs is crucial to restrict limb bud size and to assure
a normal digit number. Studies in chick and mouse have
indicated that downregulation of Grem1 drives termination of
this loop but suggested two different molecular mechanisms:
The one is based on the observation that Shh expressing cells as
well as their descendants are unable to express Grem1 [15,42].
Proliferative expansion of ZPA-derived cells  would thereby
displace the source of Grem1 secretion from the AER to a point
where the distal range of Grem1 diffusion is eventually
exceeded. As a consequence, Bmp signaling increases and
suppresses AER-Fgfs, followed by termination of Shh expression
and proliferative expansion. Although this model is supported
by the sequence of signal terminations in the chick, the factor
responsible for the cell-autonomous repression of Grem1 in the
Shh lineage cells has remained enigmatic (Scherz et al., 2004).
As an alternative mechanism, recent loss-of-function
experiments in the mouse have supported the existence of an
inhibitory Fgf/Grem1 signaling loop that becomes progressively
activated once the positive Shh/Grem1/Fgf loop has induced
sufficiently high levels of Fgfs . This model can elegantly
rationalize the regulation of limb bud size via interconnected,
self-terminating signaling loops. However, it fails to explain the
selective absence of Grem1 from the posterior limb bud
The parallel upregulation and prolonged expression of Grem1,
Fgf4/9/17 and Shh in Tbx2-deficient hindlimbs and their
coordinated downregulation upon limb-specific TBX2
overexpression identifies Tbx2 as an essential factor for the termination of the
e-m signaling loop (summarized in Figure 7). Given the virtual
overlap of Tbx2 and Shh cell lineages (see Figure S2AS2D and
), we propose that Tbx2 renders Shh-descendant cells unable to
induce Grem1. Consistently we found that the transcriptional
repressor Tbx2 binds to the Grem1 limb enhancer in vivo. This
437 bp element has been identified previously by genome-wide,
limb-specific ChIP analysis of Gli3, the transcription factor that
mediates Shh-dependent gene transcription in the limb bud .
In transgenic mice the element largely recapitulates the complex
Grem1 limb expression pattern, which argues for an integration of
both activating (Gli3) as well as repressive modules (Tbx2).
Absence of Tbx2 expression from the anterior limb margin can
explain earlier observations that Shh loaded beads are sufficient to
induce Grem1 in this region but not in the posterior limb
mesenchyme . Importantly, we noted that in Tbx2-deficient
hindlimbs the posterior mesenchyme directly underneath the AER
remained Grem1 negative (Figure 4F). This suggests that the
negative Fgf-Grem1 signaling loop  stays active in Tbx22/2
embryos and argues that both termination mechanisms (see above)
operate in parallel in adjacent domains of the limb mesenchyme to
achieve spatio-temporal control of Grem1 expression.
The hindlimb-specific requirement for Tbx2 cannot easily be
explained at this point. It may result from differences in the
proliferative expansion of fore- and hindlimbs, differential Shh/
Fgf signaling activities, or the existence of additional repressors
that might operate redundantly with Tbx2 in forelimbs. Tbx3 is
unlikely to compensate for the loss of Tbx2 given the absence of
Tbx3 expression at the distal margin of forelimb buds (Figure S1).
However, we can clearly exclude a function of mouse Tbx2 in
specification of digit identity, as previously suggested from
experiments in chicken embryos . Although some of the
observed differences may relate to species-specific variations in the
underlying molecular programs, experimental caveats due to
unphysiological levels of protein obtained after retroviral mis- and
overexpression cannot be excluded at this point.
We have shown that Bmp signaling activates Tbx2 expression in
the limb mesenchyme. This notion is supported by reduction of
Tbx2 expression in embryos with reduced levels of Bmp signaling
in the limb . Since Tbx2 expression is normal in
Prrx1cre;Bmp2fl/fl;Bmp4fl/fl or Prrx1-cre;Bmp2fl/fl;Bmp72/2 mice ,
several Bmp ligands are likely to operate redundantly. Recent
transgenic analysis of the Tbx2 promoter led to the identification of
Smad binding sites [34,35] that mediate Tbx2 expression in the
limb (supporting material in ) further stressing the relevance of
Bmp signaling for Tbx2 expression in this organ. In chick embryos
the non-AER marginal ectoderm activates Tbx2 expression,
whereas transplantation of the AER or Fgf-soaked beads repress
Tbx2 expression . In contrast to chick embryos, however,
murine Tbx2 expression is not excluded from mesenchymal
regions underling the AER and extends more distally,
demonstrating species-specific differences in Tbx2 regulation. Abrogation
of Shh signaling by cyclopamine treatment has shown that Shh is
dispensable for posterior Tbx2 expression . The fact that
implantation of Shh-soaked beads can induce Tbx2 expression in
the anterior mesenchyme  may therefore be secondary to
induction of Bmp expression in these regions . Thus, Bmp
signaling constitutes a major positive input for Tbx2 limb
expression but additional factors may feed-in to fine-tune its
expression. However, the mutual relationship: Bmp-induction of
Tbx2 and Tbx2 repression of Grem1-mediated Bmp-antagonism,
constitute a positive feedback loop that acts locally to diminish and
terminate Shh/Fgf4 signaling (Figure 7). Postaxial polydactyly,
expansion of Sox9 expression and reduced apoptosis in Bmp
pathway mutants [44,47] represent phenotypic similarities that
support engagement in a common pathway.
While loss of Grem1 (following TBX2 misexpression) causes a
general increase in Bmp signaling, as indicated by induction of
distal Msx1/2 expression, both genes were unaffected in Tbx22/2
limbs. Here, absence of the Bmp target Dkk1 was observed in the
sub-AER mesenchyme, indicating that Dkk1 expression requires
higher levels of Bmp signaling. In fact, the loss of Dkk1 expression
might explain reduced apoptosis in the Tbx22/2 sub-AER
mesenchyme as ectopic Dkk1 induces programmed cell death
. Moreover, the postaxial polydactyly in Dkk1 mutants  is
compatible with a role downstream of Tbx2.
Mesoaxial polydactyly has been suggested as a characteristic
feature of Oral-Facial-Digital syndrome (OFDS) type IV (OMIM
258860) . Interestingly, a variant case of OFDS, that partially
resembles OFDS type IV and type II (also known as Mohr
Syndrome, OMIM 252100) shows a malformation spectrum
including endocardial cushion defects, cleft palate and central
polydactyly with bifurcated Y-shaped metacarpals of the forth digit
 phenocopying Tbx2 loss-of-function in the mouse [17,21,51].
Together, our data allow the integration and refinement of
existing models for termination of distal limb outgrowth, and
emphasizes how local differences of signaling activities are
translated into the architecture of the adult skeleton, i.e. the
number or digits. They show that central polydactyly like preaxial
and postaxial variants arise from perturbation in components of
the signaling loops including Shh, Grem, Bmp and Fgf signaling.
Materials and Methods
Mice and genonotyping
Mice carrying a null allele of Tbx2 (Tbx2tm1.1(cre)Vmc, synonyms:
Tbx22, Tbx2cre) , a floxed allele of Fgfr1 , the transgenic
lines Tg(Prrx1-cre)1Cjt/J) (synonym: Prx1-Cre)  and the reporter
lines R26lacZ (synonym: R26R)  and Tg(CAG-Bgeo/GFP)21Lbe)
(synonym: Z/EG)  and mice with integration of the human
TBX2 gene in the Hprt locus (Hprttm2(CAG-TBX2,-EGFP)Akis, synonym:
HprtTBX2)  were maintained on an outbred (NMRI)
background. For timed pregnancies, vaginal plugs were checked in the
morning after mating; noon was taken as embryonic day (E) 0.5.
Pregnant females were sacrificed by cervical dislocation; embryos
were harvested in phosphate-buffered saline, decapitated, fixed in
4% paraformaldehyde overnight, and stored in 100% methanol at
220uC before further use. Genomic DNA prepared from yolk sacs
or tail biopsies was used for genotyping by polymerase chain
Limb culture experiments
Limb buds from E10.5 wild-type NMRI embryos were dissected
in PBS and placed on Nucleopore filters (Whatman, pore size
1.0 mm) on top of a stainless steel mesh at the air-liquid interface in
3.5 cm cell culture dishes. The surgical removal of the ectoderm
was performed with forceps in DMEM/10% FCS, after
incubation of limb buds in 2% Trypsin/PBS (w/v) for 20 min at 4uC.
Affi-Gel blue beads (100200 mm diameter, Bio-Rad) were rinsed
in PBS and incubated at room temperature for 1 h in either
recombinant human BMP4 (100 mg/ml, AbD Serotech) or in
1 mg/ml BSA (control). Beads were rinsed in PBS before
implantation into the limb mesenchyme. The culture was
performed at 37uC and 5% CO2 in organ culture medium
(DMEM/10% FCS, 16 solutions of Penicillin/Streptomycin,
Glutamax, sodium pyruvate, and non-essential amino acids
Limb micromass cultures
Micromass cultures were established by dissociation of E10.5
fore- and hindlimb buds in DMEM/10% FCS, after incubation in
2% Trypsin/PBS (w/v) for 5 min at 37uC. A single cell suspension
was obtained by gentle pipetting; clumps of ectoderm were
removed after sedimentation. Cells were adjusted to 1.56107
cells/ml in organ culture medium (as above), before 10 ml spots
were placed on 24 well plates. Cells were incubated for 1 hour at
37uC to allow adherence, before the wells were filled with
medium. Recombinant BMP4 (as above) or Dorsomorphin
(Sigma) were added to the medium after 16 h of culture, 2 hours
before RNA isolation. Total RNA was extracted from single
micromass cultures with PeqGOLD reagent (Peqlab). RNA
(500 ng) was reverse transcribed using oligo dT primer and
RevertAid M-MuLV Reverse Transcriptase (Fermentas) following
the manufacturers recommendations. Relative gene expression
was measured using iQ2SYBR Green reagent (Biorad) and
calculated using the DDCT method by normalization to Hprt
expression. The error bars show standard deviation from 4
In situ hybridization, skeletal Preparations,
bgalactosidase and apoptosis assays, and
Skeletal preparations with Alcian blue and Alizarin red,
bgalactosidase stainings, detection of apoptosis by the TUNEL
assay, and in situ hybridization analyses on whole embryos and on
10 mm paraffin sections were performed as previously described
[56,57,58]. All experiments were performed on at least three
independent embryos. For experiments that showed variable
results, numbers of used specimens are mentioned in the text. For
detection of apoptotic cells, embryos were collected in PBS and
incubated for 30 min at 37uC in prewarmed PBS containing
2.5 mM LysoTracker Red (Sigma), followed by several washes in
PBS. Tbx2 antibody (#07-318, Millipore) was used 1:100 for
immunostainings on 5 mm paraffin sections. Signals were
amplified by Tyramide Signal Amplification (PerkinElmer).
Distal pieces of E11.5 wild-type hindlimb buds were separated
in anterior and posterior halves (as indicated in Figure 4J) and
collected in 3 separate pools of 8 embryos each and treated with
4% paraformaldehyde overnight. ChIP experiments using an
antiTbx2 antibody were performed essentially as previously described
. Primer sequences for the Grem1 enhancer were
GGCCAAATAACCACACAGGAAAC, corresponding to a 447 bp fragment
previously tested in transgenic animals . Primer pairs of control
regions were TGAAAACCCCAAGGAGTCTG,
CATGGGCAGGATACTACGCT (193 bp product, 25.5 kbp distal) and
GGCACTGGATAAAACTCCCA (273 bp product, 24.3 kbp distal from the Grem1 limb
Whole-mount specimens were photographed on Leica M420
with Fujix digital camera HC-300Z. Whole-mount
GFP-epifluorescence was documented on a Leica MZFLIII macroscope
equipped with a Leica DFC300 camera. Sections of in situ
hybridizations were photographed using a Leica DM5000
microscope with a Leica DFC300FX camera. All images were
processed in Adobe Photoshop CS.
Figure S1 Tbx2 and Tbx3 exhibit dynamic expression in the
developing mouse limb bud. Comparative expression analysis of
Tbx2 and Tbx3 in whole forelimb (AF) and hindlimb buds (GL)
of wild-type mouse embryos by in situ hybridization. Probes used
and embryonic stages are indicated in the figure. At the posterior
limb bud margin the distal limit of expression is highlighted by
blue arrowheads, demonstrating that Tbx2 expression extends
further distally compared to Tbx3. The expression of Tbx3 in the
AER is indicated by black arrowheads. At E12.5, Tbx2 shows
strong expression in the IDM4 (asterisks). Note that the murine
expression patterns of Tbx2 and Tbx3 expression were inverted in a
previous publication .
Figure S2 (AD) Tbx2 expressing cells contribute to posterior
digits 3, 4 and 5. (A, B) X-Gal stainings to detect -galactosidase
activity in E13.0 Tbx2cre/+;R26lacZ/+ fore- and hindlimbs. Cells
previously expressing Tbx2 as detected by b-galactosidase activity
are present in the anterior and posterior flank mesenchyme and in
the posterior half of the autopod encompassing the anlagen of digit
4 and 5, and partially of digit 3. (C, D) GFP-epifluorescence
analysis in E18.5 Tbx2cre/+;ZEGGFP/+ embryos detects the final
contribution of the Tbx2-cre+ cell lineage to fore- and hindlimbs.
Digit 3 is partially, digits 4 and 5 are completely derived from Tbx2
expressing cells. Note that the Tbx2-positive domains in the
anterior and posterior flank mesenchyme do not substantially
contribute to the E18.5 limb since they are most likely removed by
apoptosis during development. (E, F) In situ hybridization analysis
of Eomes expression as marker for digit 4 identity. At E12.5
wildtype hindlimb buds show a proximal domain of Eomes expression
(arrow) that indicates formation of digit 4. Absence of signal in
Tbx2cre/cre;Fgfr1fl/fl hindlimbs argued for a specific loss of digit 4 in
Figure S3 (AC) Expression analysis of the Bmp target genes
Dkk1, Msx1 and Msx2 by whole mount in situ hybridization in
E11.5 wild-type and Tbx22/2 hindlimbs. Magnified regions (1 and
2) in (A) show loss of mesenchymal Dkk1 expression in the posterior
limb bud region (arrow) of Tbx22/2 mutants but maintained
expression in the adjacent AER (asterisks). (BC) Unaffected
expression of Msx1 and Msx2.
Figure S4 TBX2 protein levels and apoptosis in misexpression
embryos. (A, B) Analysis of endogenous Tbx2 and transgenic
TBX2 protein expression. Anterior and posterior halves of E11.5
forelimbs were collected (as shown in the scheme in A), and lysates
were analyzed by Western blot. TBX2 misexpression in
Prrx1TBX2 (Prrx1-cre/+;HprtTBX2/Y) embryos was found at physiological
levels. b-actin Western blot is shown as a loading control. (C, D)
Immunostaining of endogenous and ectopic Tbx2 expression (red
signal) in control and Prrx1-TBX2 embryos. Sagittal E10.5
forelimb sections are shown. Posterior restriction of Tbx2 in the
wild-type (C) and ubiquitous mesenchymal expression of
transgenic TBX2 protein (D). Same Tbx2 antiserum used as in (B). (E,
Figure S5 Selective disruption of posterior e-m signaling
following TBX2 misexpression in the limb. (AF) In situ
hybridization analysis of Spry4, Ptch1, Fgf9, Fgf17, Fgf8 and Etv4
expression in E10.5 wild-type and Prrx1-TBX2 (Prrx1-cre/
+;HprtTBX2/Y) forelimbs. Spry4, Ptch1, Fgf9 and Fgf17 are strongly
reduced (red arrowheads) following TBX2 misexpression, whereas
the levels of Fgf8 and Etv4 are not strongly affected. Note that in
wild-type limb buds the expression of Spry4 is more restricted to
the posterior-distal mesenchyme as compared to Etv4 that is
expressed beneath the entire AER.
We thank Vincent Wakker for his contribution to the data, Aravind Shekar
and Achim Gossler for technical help, and Mark-Oliver Trowe for critical
comments on the manuscript.
Conceived and designed the experiments: HFF AK. Performed the
experiments: HFF TH-WL MKS SP KS-G MP. Analyzed the data: HFF
TH-WL MKS SP AK. Contributed reagents/materials/analysis tools:
VMC. Wrote the paper: HFF AK.
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