Gata6-Dependent GLI3 Repressor Function is Essential in Anterior Limb Progenitor Cells for Proper Limb Development

Gli3 is a major regulator of Hedgehog signaling during limb development. In the anterior mesenchyme, GLI3 is proteolytically processed into GLI3R, a truncated repressor form that inhibits Hedgehog signaling. Although numerous studies have identified mechanisms that regulate Gli3 function in vitro, it is not completely understood how Gli3 function is regulated in vivo. In this study, we show a novel mechanism of regulation of GLI3R activities in limb buds by Gata6, a member of the GATA transcription factor family. We show that conditional inactivation of Gata6 prior to limb outgrowth by the Tcre deleter causes preaxial polydactyly, the formation of an anterior extra digit, in hindlimbs. A recent study suggested that Gata6 represses Shh transcription in hindlimb buds. However, we found that ectopic Hedgehog signaling precedes ectopic Shh expression. In conjunction, we observed Gata6 and Gli3 genetically interact, and compound heterozygous mutants develop preaxial polydactyly without ectopic Shh expression, indicating an additional prior mechanism to prevent polydactyly. These results support the idea that Gata6 possesses dual roles during limb development: enhancement of Gli3 repressor function to repress Hedgehog signaling in the anterior limb bud, and negative regulation of Shh expression. Our in vitro and in vivo studies identified that GATA6 physically interacts with GLI3R to facilitate nuclear localization of GLI3R and repressor activities of GLI3R. Both the genetic and biochemical data elucidates a novel mechanism by Gata6 to regulate GLI3R activities in the anterior limb progenitor cells to prevent polydactyly and attain proper development of the mammalian autopod.


Introduction
Understanding the developmental mechanisms that regulate progenitor cells to generate organs with specific morphology and function is a central topic in developmental biology. The vertebrate limb has been serving as an excellent system for such studies. In particular, mesenchymal progenitor cells in limb buds are specified, patterned and expanded to generate each skeletal element with a distinct morphology at each defined position to create the stereotypical limb skeletal system. The mammalian autopod possesses five digits, termed as d1-d5, in an anterior to posterior order. The number and identity of digits have been used as a readout of specification, patterning, and proliferative expansion of progenitor cells [1].
Sonic Hedgehog (Shh) is expressed in the zone of polarizing activity (ZPA), located at the posterior mesenchyme of the limb bud, and acts as a major regulatory molecule for limb development [1,2]. Anterior-posterior specification of digit progenitors is regulated by the concentration and duration of progenitor exposure to SHH [3][4][5][6]. SHH also regulates the proliferative expansion of mesenchymal progenitor cells to generate a sufficient number of cells to develop into cartilage condensations [7,8]. Accordingly, ectopic expression of Shh in the anterior portion is associated with preaxial polydactyly, which is characterized by the formation of ectopic digits in the anterior of the limb [9]. By contrast, the most anterior digit (d1) develops in a SHH-independent manner [10,11]. Recent studies have shown that anterior genetic programs, such as Irx3-Irx5 and Sall4, are required for development of d1, at least in part, by excluding SHH signaling from the anterior mesenchyme [12,13].
The glioma-associated oncogene family (GLI) proteins are zinc finger DNA binding proteins, which play diverse roles in animal development and diseases [14]. Among the three Gli genes, Gli3 encodes a bi-functional molecule, acting as both an activator (GLI3A) and a repressor (GLI3R), whose balance depends on Hedgehog signaling [14]. In the presence of Hedgehog ligands, its signal transduction at primary cilia causes inhibition of proteolytic processing of GLI3 [15]. This results in the accumulation of a full-length activator form of GLI3 (GLI3A) in the posterior mesenchyme. In contrast, in the absence of Hedgehog signaling, GLI3 is subjected to proteolysis, generating a truncated repressor form (GLI3R), which accumulates in the anterior mesenchyme. Because GLI1 lacks a repressor domain and GLI2 predominantly functions as an activator [16,17], GLI3R is the major GLI repressor in the limb [18].
Consistent with the important function of Gli3 in limb development, its mutations cause developmental defects in mice and humans [19][20][21]. In particular, Gli3 -/mice develop polydactyly [21]. Genetic studies in mice demonstrated that a predominant function of Gli3 is to repress Hedgehog signaling target genes [22,23]. Furthermore, it has been shown that the balance of GLI3A and GLI3R regulates digit number and identity [24][25][26]. Numerous studies have shown that multiple mechanisms regulate GLI3 functions in vitro, such as posttranslational modifications, degradation, cytoplasmic retention, and primary cilium-mediated processing (reviewed in [14,27,28]). In vivo studies in mice demonstrated that Gli3 genetically interacts with Hox genes, Zic3 and Alx4 during limb development [29][30][31]. Despite these studies, the in vivo control of Gli3 function during proper limb development is still to be elucidated.
The Gata family of zinc finger transcription factors is an important regulator of tissue and organ development. The Gata family is subdivided into the Gata1/2/3 subfamily and the Gata4/5/6 subfamily, which show expression in hematopoietic cell lineages and meso-endoderm lineages, respectively [32,33]. In particular, Gata6 is essential for endoderm formation and is also involved in the development of various mesoderm-and endoderm-derived organs, such as the cardiovascular system and pancreas [34][35][36][37]. Moreover, a recent study suggested that Gata6 functions as a negative regulator of Shh expression in limb buds by binding to its limb bud-specific cis-regulatory element, ZRS [38].
In this study, we found that broad deletion of Gata6 in the limb mesenchymal progenitors caused hindlimb-specific preaxial polydactyly, which is associated with ectopic SHH signaling in the anterior hindlimb bud. We discovered that Gata6 and Gli3 genetically interact to regulate normal patterning of the hindlimb. Furthermore, we show that direct association of GATA6 with GLI3R promoted nuclear localization and transcriptional repressor activity of GLI3R. Our work identified that genetic and biochemical interactions between Gata6 and Gli3 act as essential mechanisms to regulate GLI3R activity for proper autopod patterning.

Results
Inactivation of Gata6 in early mesoderm caused hindlimb specific preaxial polydactyly Prior studies have identified expression of Gata6 in developing limb buds [38][39][40]. Gata6 null embryos die during gastrulation [34,35]; therefore, we inactivated Gata6 in the meso-endoderm by using the conditional allele of Gata6 (Gata6 fl ) [41] and the Tcre line, which recombines in the early meso-endoderm [42]. We found that Tcre; Gata6 fl/fl mutants (hereafter referred to as Gata6 cKO) die around E12.5-14.5 with broad hemorrhage (Fig 1A and 1E). This result is consistent with a former study, demonstrating a role of proper dosage of Gata4 and Gata6 for vessel integrity [43]. We found that Gata6 cKO embryos exhibited polydactyly in the hindlimb, while forelimbs seem to be unaffected (Fig 1A-1C, 1E and 1G, S1 Table). Alcian blue staining demonstrated that the mutant hindlimbs possess patterned digits, d1-d5, and an extra digit on the anterior edge, which morphologically resembles d1. Based on the position and morphology, tarsal and metatarsal elements were also patterned. Two ectopic tarsal elements, likely the navicular and medial cuneiform, were present proximally to the ectopic 1 st metatarsal (Fig 1D and 1H). These observations indicate that the autopod is patterned along the anterior-posterior axis, and the absence of Gata6 induces the formation of an extra anterior digit with the associated tarsal and metatarsal elements.
We also examined gene expression at a later stage. At E11.5, we detected ectopic Shh expression in the anterior border of Gata6 cKO hindlimbs (n = 4, S1 Fig). Consistent with evident ectopic Shh expression, expression of Hoxd13 (n = 3), Gli1 (n = 6), Ptch1 (n = 6) and Gremlin1 (n = 3) was also detected in the anterior margin. This data indicates that ectopic Hedgehog signaling became evident at E10.5 in Gata6 cKO hindlimb buds, although ectopic Shh expression was undetectable. At a later stage (E11.5), ectopic Shh expression became evident and all SHH targets, examined in this study, were detected in the anterior margin.
Reduction of Shh dosage rescued ectopic SHH signaling but not ectopic anterior digit formation If ectopic Shh expression accounts for the preaxial polydactyly in Gata6 cKO hindlimbs, we would expect that reducing Shh dosage might rescue the phenotype. Therefore, we genetically reduced Shh dosage from the Gata6 cKO background using the Shh null allele [2]. Gata6 cKO; Shh +/mutants did not survive beyond E12.5, thus, we examined expression of SHH target genes (Gli1 and Ptch1) and expression of Sox9, an early marker of chondrogenic condensation [53].
Removing one allele of Shh from the Gata6 cKO background resulted in posteriorly restricted expression of Gli1 and Ptch1, and the ectopic anterior expression became undetectable (n = 4, Fig 2A-2C and 2E-2G). However, ectopic chondrogenic condensation in the anterior portion was still detected by Sox9 expression at E12.5 (n = 3, Fig 2I-2K). Removing both alleles of Shh from the Gata6 cKO background resulted in the loss of Gli1 and Ptch1 expression and single digit condensation, the same phenotype as Shh -/limbs (n = 3, Fig 2D, 2H and 2L) [10,11]. These results indicate that Shh functions downstream of Gata6 during preaxial polydactyly development. However, ectopic chondrogenic condensation in the anterior portion of Gata6 cKO; Shh +/hindlimbs suggests that additional mechanisms could be involved in the preaxial polydactyly in Gata6 cKO hindlimbs.
Gli3 genetically interacts with Gata6 in forelimbs and hindlimbs GLI3 is a major regulator of Hedgehog signaling, and thus, Gli3 might be involved in preaxial polydactyly in Gata6 cKO hindlimbs. To test this hypothesis, we genetically removed Gli3 from the Gata6 cKO background. Gli3 +/hindlimbs developed a small spike in the anterior region [21,54], while most of the Tcre; Gata6 +/fl hindlimbs were indistinguishable from the wild-type hindlimbs at E14.5-15.5 (Fig 3F-3H, Table 1). Tcre; Gata6 +/fl ; Gli3 +/compound heterozygous hindlimbs developed an extra digit in the anterior region ( Fig 3I). Unexpectedly, we also found that this interaction operates in forelimbs. Gli3 +/forelimbs developed d1, which  was associated with small ectopic cartilage condensation at the distal tip. Contrary to this, Tcre; Gata6 +/fl ; Gli3 +/compound heterozygous forelimbs developed an evident extra digit with incomplete penetrance (Fig 3A-3D) or an extra digit that partially fused with endogenous d1 with incomplete penetrance (S2 Table). These results demonstrate a genetic interaction between Gli3 and Gata6 in fore-and hind-limbs.

GATA6 and GLI3 functionally and physically interact in vitro
Ectopic Shh expression in the Gata6 cKO background could affect gene expression patterns in hindlimb buds. Therefore, we set up in vitro experiments to further investigate how Gata6 regulates Gli3 function. We first set up luciferase reporter assays using 12xGLI-binding site luciferase [31]. Transfecting a C-terminally truncated form of human GLI3 that could function as GLI3R caused significant reduction of the reporter activities, while transfecting human GATA6 did not affect the reporter activities. Co-transfecting GLI3R and GATA6 caused further reduction of the reporter activities (Fig 5A). These results are consistent with the in vivo data and support the idea that GATA6 functionally interacts with and contributes to GLI3R activities. Next, we tested whether GATA6 and GLI3R physically interact by co-immunoprecipitation assays. HEK293T cells were transfected with Flag-tagged GATA6, Myc-tagged GLI3R or GFP. Flag-GATA6 and Myc-GLI3R were co-immunoprecipitated, demonstrating that GATA6 and GLI3R can interact (Fig 5B and 5C). We also confirmed that the interaction occurs in vivo. GLI3R was detected in immunoprecipitated complex from E10.25-10.5 wild-type hindlimb buds using ant-GATA6 (Fig 5D). To further characterize their interaction, we mapped the GLI3R interaction domain in GATA6. For this purpose, we generated serial deletion mutants (Fig 5E), and performed co-immunoprecipitation assays with Flag-GLI3R. The ΔN1 and ΔN2 mutants showed a strong interaction with Flag-GLI3R. The ΔN3 and ΔC1 mutants exhibited weak interaction, and we did not detect any interactions of Flag-GLI3R with ΔN4 and ΔC2 (Fig 5F).
We also generated intra-molecular deletion mutants. These mutants lack the GLI3R-binding domain (GBD) 1, which includes the second putative transactivation domain (ΔGBD1), or both GBD1 and GBD2 (ΔGBD1/2). We did not detect any interaction of ΔGBD1/2 with GLI3R, although ΔGBD1 exhibited a weak interaction with GLI3R ( Fig 5G). These results suggest that the zinc finger domain 1 (ZFD1) is critical to interact with GLI3R. The weak interaction of ΔN3, ΔC1 and ΔGBD1, which possess the ZFD1, also suggests that both the N-and Cterminal regions around the ZFD1 contribute to the interaction with GLI3R, in collaboration with the ZFD1.

Interaction between GATA6 and GLI3R regulates subcellular localization of GLI3R
Our analyses indicated the presence of genetic and physical interactions between Gata6 and Gli3. Given that both GATA6 and GLI3R act as transcription factors, we next examined subcellular localization of these proteins after co-transfecting HEK293T cells with Flag-GLI3R and either full length or mutant forms of Myc-GATA6.
We observed three patterns of localization (Fig 6A and 6B, S3 Fig). First, co-transfection of either full length GATA6, ΔN1-GATA6 or ΔN2-GATA6, which can interact with GLI3R and possess the nuclear localization signal (NLS), resulted in predominant nuclear localization of both GLI3R and GATA6. Second, we co-transfected ΔN3-GATA6 or ΔN4-GATA6, which possess the NLS, but have either very weak or undetectable interactions with GLI3R. In these transfection assays, GLI3R localization became either predominantly cytoplasmic or localized similarly in both the cytoplasm and nucleus, although GATA6 was predominantly detected in the nucleus. Third, we co-transfected ΔC1-GATA6 or ΔC2-GATA6, which lack the NLS and have very weak or undetectable interactions with GLI3R. We detected GATA6 predominantly in the cytoplasm, consistent with the lack of NLS. GLI3R was also predominantly located in the cytoplasm or located similarly in the nucleus and cytoplasm.
These results indicate a correlation between GLI3R nuclear localization and nuclear GATA6 that possesses a GLI3R-interaction ability. This correlation suggests that physical association between GATA6 and GLI3R contributes to nuclear localization and the repressor activities of GLI3R. We next tested this idea in vivo by examining GLI3R nuclear localization. The earliest molecular alteration in Gata6 cKO hindlimb buds in our study is ectopic Gli1 and Ptch1 expression at E10.5 (Fig 1). Therefore, we re-examined Gata6/GATA6 expression, although their mRNA expression patterns were examined in previous studies [38][39][40]. Gata6 mRNA was detected in the anterior-proximal part of hindlimb buds at E10.25 (34 somite stage) (S4A Fig), but the strong signals in endoderm -derived tissues seem to mask the limb bud signals. Therefore, we also performed immunofluorescence of GATA6 in combination with limb bud mesenchyme markers, such as Fibroblast growth factor10 (FGF10) [57] or Dual specificity phosphatase6 (DUSP6) [58][59][60]. Co-staining with these markers on transverse sections indicates that GATA6 is present in the ventral side of the proximal region in anterior hindlimb buds at E10.25 (S4B and S4C Fig). The GATA6 signal was undetectable in limb buds in the middle-posterior region.
In the anterior proximal region of limb buds at E10.25, we detected GLI3R predominantly in the nucleus or similarly in the nucleus and cytoplasm (Fig 6C, 6E, 6G and 6I). By contrast, Gata6 cKO hindlimb buds showed a reduced percentage of cells with predominant nuclear GLI3R signals. Accordingly, we detected an increased percentage of cells with nuclear/cytoplasmic GLI3R (Fig 6D, 6H and 6I). Western blot analysis of nuclear extracts from the anterior part of hindlimb buds showed reduced GLI3R levels in Gata6 cKO, compared to wild-type embryos (Fig 6J). Although the presence of nuclear GLI3R in Gata6 cKO hindlimb buds indicates Gata6-independent GLI3R nuclear localization mechanisms in the anterior mesenchyme, reduced GLI3R levels provide evidence that Gata6 contributes to GLI3R nuclear localization. These results are consistent with the in vitro data, and further support the idea that Gata6 regulation of GLI3R nuclear localization contributes to GLI3R activities during normal limb development.

Discussion
In this study, we found hindlimb-specific preaxial polydactyly in Gata6 mutants. The skeletal phenotype of Gata6 mutants was restricted to hindlimbs, and the forelimbs developed normally. Several possibilities would account for such limb type-specific phenotypes. For instance, a recent study showed that Gata4 is differentially expressed in forelimb buds (high) and hindlimb buds (low) [38]. Gata4 and Gata6 are functionally redundant during heart development and for vascular integrity [36,43]; therefore, Gata4 might compensate for loss of Gata6 in forelimb buds [38]. Another possibility is that differences in the sensitivity to Hedgehog signaling contribute to different phenotypes in fore-and hind-limbs. It is suggested that levels of Hedgehog signaling are higher in hindlimb mesenchyme than forelimb mesenchyme [12], and that hindlimbs are more sensitive to changes in the levels of Hedgehog signaling. Higher Hedgehog signaling, in combination with reduced GLI3R, might have contributed to hindlimb-specific polydactyly in Gata6 cKO. This idea is consistent with ectopic digit formation in Tcre; Gata6 +/fl ; Gli3 +/forelimbs, in which GLI3R activities would be lower than and SHH signaling levels would be higher than Gli3 +/forelimbs. These two scenarios are not mutually exclusive, and they might cooperate together to ensure proper Hedgehog signaling and pentadactyly in mammalian limbs.
Our study proposes two mechanisms by which Gata6 regulates proper autopod patterning. One mechanism is by enhancing GLI3R activities to repress Hedgehog signaling in the anterior mesenchyme, and the other is by negative regulation of Shh expression in the anterior mesenchyme.
Genetic studies have shown that preaxial polydactyly is associated with ectopic expression of Shh in the anterior mesenchyme [9]. Expression of Shh is positively and negatively regulated in the posterior and anterior mesenchyme, respectively. Twist1, Alx4, Gli3, Tulp3 and Etv4-Etv5 act as negative regulators, for their loss of function caused ectopic Shh expression [23,47,49,51,61]. Genetic and biochemical studies have shown that Hand2 and Hoxd13 positively regulate Shh expression through the limb bud-specific cis-regulatory element, ZRS [44,62]. Anterior Shh expression could be induced by loss of negative regulators or ectopic expression of positive regulators [63]. Given that these regulators did not exhibit significant alteration in Gata6 cKO hindlimb buds, the preaxial polydactyly phenotype in Gata6 cKO limbs is unlikely to be induced through these genes. A recent study suggested that Gata6 represses Shh in the limb through binding to ZRS [38]. Our data is consistent with this report, and demonstrated that Shh and its targets are ectopically expressed in Gata6 cKO hindlimb buds at E11.5. Restoration of normal expression pattern of Gli1 and Ptch1 in Gata6 cKO; Shh +/hindlimbs also supports the idea that Gata6 is upstream of Shh.
The second role is repressing ectopic Hedgehog signaling by enhancing repressor function of Gli3. Ectopic Shh expression in the Gata6 cKO background affects data interpretations; however, compound heterozygous mutant analyses could enable separate analysis of the two mechanisms and support the second mechanism. Previous studies have shown Gli3 to genetically interact with other genes during limb development. Studies on Hox genes suggested that the Gli3 -/polydactyly phenotype is mediated by Hoxd9 and Hoxd10 [29,64]. In addition, it has been shown that polydactyly of Gli3 -/limbs becomes milder on the Alx4 -/or Zic3 -/background [30,31], which suggested that the Gli3 -/polydactyly phenotype requires Alx4 or Zic3.
In contrast to these reports, loss of one allele of Gata6 enhanced the polydactyly phenotype of Gli3 +/hindlimbs. Therefore, unlike previous genetic studies, our study identified Gata6 as a negative factor for polydactyly development. Given that GLI3R prevents extra-digit formation in the anterior mesenchyme [55], our results suggest that Gata6 cooperates with GLI3R activities.
It is believed that d1 develops in a Shh-independent manner, while development of d2-d5 requires Shh [5,6,10,11]. Genetic manipulation of Gli3 in mice provided evidence that high levels of GLI3R in the anterior of limb buds is necessary for proper d1 development and ensuring pentadactyly [24,55,65]. Expression pattern of Pax9, which requires high levels of GLI3R [56], indicates that Gata6 contributes to GLI3R activities in the anterior of hindlimb buds. In particular, Pax9 was undetectable in Tcre; Gata6 +/fl ; Gli3 +/hindlimb buds, similar to Gata6 cKO and Gli3 -/hindlimb buds. These altered expression pattern of Pax9 correlates with ectopic digit condensation and preaxial polydactyly, and further supports the idea that Gata6 cooperate with Gli3 for proper GLI3R activities in the anterior of hindlimb buds.
How does Gata6 cooperate with Gli3? Our data support the idea that GATA6 physically interacts with GLI3R, facilitates the nuclear localization of GLI3R, and enhances the repressor activities of GLI3R. Reduced nuclear GLI3R localization in Gata6 cKO hindlimb supports the idea that this interaction-mediated nuclear GLI3R localization would also occur in vivo. A recent study showed that Gata4, 5, and 6 can repress Gli-dependent reporter activation in vitro [66]. This study suggested that GATA inhibits SHH-dependent GLI activator function by protein interaction in the chick presomitic mesoderm. Based on this report and our study, GATA might modulate both GLI3R (this study) and SHH-dependent GLI activator [66] in a contextdependent manner. Since expression of Gata genes is reported in other Gli3-positive developing tissues, such as the branchial arch, somite and central nervous system [16,67,68], Gata regulation of GLI3R might be a shared mechanism during the development of other organs.

Ethics statement
Animal breeding was performed according to the approval by the Institutional Animal Care and Use Committee of the University of Minnesota. Compressed CO 2 gas from a cylinder followed by cervical dislocation was the methods of euthanasia for mice. All efforts were made to minimize suffering.

Mouse lines and embryo
The mouse lines for Gata6 fl [41], Gli3 - [69] and Tcre [42] were maintained on a mixed genetic background. Skeletal preparation was done as previously published [70]. Whole mount in situ hybridization was done as previously published [13].

Expression constructs
The full-length human GATA6 construct and the human GLI3 construct were published [31,71]. The GLI3R construct was generated by deleting the 3' part of full-length cDNA, and cloned into 3xFlag CMV7. GATA6 deletion constructs were generated by PCR-based cloning and cloned in pcDNA3.1 or pCS2.
For subcellular localization analysis in vitro, images were acquired form six arbitrary areas from two plates. Nuclear/cytoplasmic localization of GLI3R and GATA6 was blindly evaluated in cells that were doubly transfected with GLI3R and GATA6 (or its mutants) except for samples that are transfected with GLI3R alone. For in vivo samples, nuclear/cytoplasmic localization of GLI3R was evaluated similarly in the anterior-proximal domain where GATA6 signals in wild-type hindlimb buds were detected. In Gata6 cKO embryos, the anterior-proximal domain, similar to wild-type embryos, was selected for GLI3R subcellular localization. The quantification was performed similar to in vitro samples.
Luciferase reporter assay NIH3T3 cells in 48-well plates were transfected with the 12xGLI-binding site-TK minimum promoter-luciferase [31] with pRL-TK, GATA6 and/or GLI3R expression constructs by using Fugene6 (Promega). Forty hours after transfection, cells were subjected to analysis using the Dual-Luciferase Reporter Assay System (Promega). Experiments were performed in triplicate, and statistical significance was analyzed by One-way ANOVA followed by the Tukey's comparison.
Co-immunoprecipitation assay and nuclear GLI3R detection HEK293T cells were transfected with expression constructs by using the standard calcium phosphate method. Cell lysates, prepared after two days, were passed through 25 gauge syringes to ensure protein extraction from the nucleus, and co-immunoprecipitation assays were performed by using Dynabeads protein G (Invitrogen) and anti-Flag (Sigma, M2, F3165, 2μg) or anti-Myc tag (Abcam, ab9106, 1 μg) antibodies. Proteins were resolved by SDS-PAGE, transferred to PVDF membranes (Millipore, MA, USA), reacted with anti-Myc tag or anti-Flag antibodies, followed by HRP goat anti-mouse or rabbit IgG, and a chemiluminescence detection.
For co-immunoprecipitation assays with in vivo samples, hindlimb buds were collected from wild-type embryos at E10. 25-10.5. After pooling, the samples were lysed and subjected to co-immunoprecipitation procedures [73] using anti-GATA6 (Cell Signaling, #5851) and Dynabeads protein G. The protein complex was eluted, and detected by Western using anti-GLI3 (R&D Systems, AF3690, dilution 1:100) and the PicoLUCENT PLUS HRP detection kit (G-Bioscience) according to the manufacturer's instructions.
For nuclear GLI3R detection by Western, anterior one third of hindlimb buds at E10.25-10.5 were collected, and the nuclear fraction was prepared after dissociating cells by using the NE-PER kit (Thermo Fischer) according to the manufacturer's instructions. The nuclear extracts were analyzed by Western using anti-GLI3 (R&D Systems, AF3690) and anti-Histone H3 (Abcam, ab-1791).  Table. Number of forelimbs with indicated phenotypes at E14. 5-16.5. Embryos at E14.5-16.5 were collected and scored. (DOCX)