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Hum Mol Genet. 2012 April 15; 21(8): 1888–1896.
Published online 2012 January 10. doi:  10.1093/hmg/dds002
PMCID: PMC3313802

Preaxial polydactyly caused by Gli3 haploinsufficiency is rescued by Zic3 loss of function in mice

Abstract

Limb anomalies are important birth defects that are incompletely understood genetically and mechanistically. GLI3, a mediator of hedgehog signaling, is a genetic cause of limb malformations including pre- and postaxial polydactyly, Pallister–Hall syndrome and Greig cephalopolysyndactyly. A closely related Gli (glioma-associated oncogene homolog)-superfamily member, ZIC3, causes X-linked heterotaxy syndrome in humans but has not been investigated in limb development. During limb development, post-translational processing of Gli3 from activator to repressor antagonizes and posteriorly restricts Sonic hedgehog (Shh). We demonstrate that Zic3 and Gli3 expression overlap in developing limbs and that Zic3 converts Gli3 from repressor to activator in vitro. In Gli3 mutant mice, Zic3 loss of function abrogates ectopic Shh expression in anterior limb buds, limits overexpression in the zone of polarizing activity and normalizes aberrant Gli3 repressor/Gli3 activator ratios observed in Gli3+/− embryos. Zic3 null;Gli3+/− neonates show rescue of the polydactylous phenotype seen in Gli3+/− animals. These studies identify a previously unrecognized role for Zic3 in regulating limb digit number via its modifying effect on Gli3 and Shh expression levels. Together, these results indicate that two Gli superfamily members that cause disparate human congenital malformation syndromes interact genetically and demonstrate the importance of Zic3 in regulating Shh pathway in developing limbs.

INTRODUCTION

Zic (zinc finger protein of the cerebellum) and Gli (glioma-associated oncogene homolog) transcription factors are members of the Gli superfamily of proteins which share a highly conserved zinc finger domain and have critical roles in multiple developmental processes. The murine Zic was first identified as a zinc finger protein expressed in granule cells throughout cerebellar development (1). The expression pattern of the mouse Zic1-5 genes suggests their essential roles in body pattern formation (2). In humans, mutations in ZIC1-4 and GLI3 genes result in important classes of developmental abnormalities: ZIC1 and ZIC4 mutations result in Dandy–Walker malformation; ZIC2 causes holoprosencephaly; ZIC3 mutation or deletion results in heterotaxy syndrome, a disorder characterized by disruption of left–right axis patterning; GLI3 mutations result in complex anomalies of the brain and digits (Greig cephalopolysyndactyly and Pallister–Hall syndromes) as well as isolated polydactyly (38).

Gli3 is a downstream mediator of Sonic hedgehog (Shh) signaling, a role which has been particularly well documented in limb development (914). Anteroposterior (A/P) patterning in the limbs of amniotes is controlled through secretion of Shh protein by posterior limb bud mesoderm (zone of polarizing activity—ZPA). Studies in vivo and in vitro suggest that Gli3 negatively regulates the expression of both Shh (by restricting it to the posterior mesoderm) and its target genes through a repressor form (Gli3R) (11,12,15,16). In the absence of Shh signal, Gli3 protein is phosphorylated and cleaved constitutively to produce the transcriptional repressor (Gli3R). Shh activity blocks Gli3 processing, yielding a full-length activator form (Gli3A). The Gli3R is present at high levels in the anterior developing limb bud and at low levels in the posterior (15). The intracellular gradient of Gli3R, which opposes the extracellular gradient of Shh, ultimately controls A/P limb patterning in a manner not yet fully unraveled (10,13,15,17). It has been proposed that the gradient of a Gli3R/Gli3A ratio determines digit number and identity (10).

Shh expression in the developing limb bud is regulated via a complicated network of transcription factors and growth factors acting in concert, in which Gli3 plays its part. Others include Hand2 (Hand—heart and neural crest derivatives expressed transcript) and Tbx3 (Tbx—T box) providing competence for Shh in posterior limb bud mesenchyme (13,18,19), Fgf4 and Fgf8 (Fgf, fibroblast growth factor) factors from the apical ectodermal ridge inducing and maintaining Shh expression (20,21) and Alx4 (homeobox protein aristaless-like 4) and other factors preventing Shh expression in anterior and distal mesenchyme (22). In this paper, we document for the first time a potential role of Zic3 in regulating Shh expression in the developing limb buds.

Mutations in Gli3 and/or Shh are known to affect digit number and identity. The semidominant mouse mutation Extra toes-J (Xt-J) generates a Gli3 null allele resulting in preaxial digit 1 iterations in heterozygotes (23). The limbs of Gli3Xt-J/Gli3Xt-J (hereafter designated Gli3−/−) exhibit severe polydactyly and loss of digit identities (23). The complete loss of Shh function in the Shh−/− mutant mouse results in severe skeletal deficiencies distal to the stylopod–zeugopod junction (elbow/knee joints); all zeugopod and autopod elements are either missing, fused or lack normal identity, except for a single digit 1 in the hindlimb (10,24).

Gli3−/− embryos express Shh ectopically at the anterior mesoderm of developing limb buds (11), without effect on skeletal patterning in the absence of Gli3, since Shh−/−;Gli3−/− and Gli3−/− limbs are virtually indistinguishable (10). The current paradigm argues that Gli3R exerts a potent negative effect on the number of digits and the loss of Gli3R activity is the direct cause of polydactyly in both Gli3−/− or Shh−/−;Gli3−/− mutants.

The functional relationship of the Zic and Gli zinc finger proteins, particularly during development, is not understood. Gli proteins bind a consensus nonamer target DNA sequence (GLI-BS—Gli-binding site) (25) to which Zic proteins can also bind (1), although with lower affinity. Zic proteins significantly enhance gene expression, most efficiently in the presence of GLI-BS, but also from promoters without GLI-BS (26), suggesting they may function as transcriptional coactivators. Co-expression of Zic and Gli in vitro leads to synergistic enhancement or mutual suppression of GLI-BS-mediated transcription depending on the cell type (26). Zic and Gli proteins also physically interact through their zinc finger domains and regulate each other's subcellular localization and transcriptional activity (27).

In this paper, we attempted to shed a new light on Zic3 function by studying its modifying effect on Gli3 expression and activity, as well as Shh expression in the developing limb buds—a role of Zic3 not investigated previously. To study the role of Zic3 during development, we previously generated Zic3 null mice (Zic3−/− and Zic3-/Y) carrying a targeted deletion of the entire Zic3 gene (28). The Zic3 null phenotype closely resembles defects seen in patients with X-linked heterotaxy, which include complex congenital heart disease, disturbances of laterality, neural tube abnormalities and vertebral defects. The null phenotype indicates that Zic3 plays an important role in axial midline development and left–right patterning. To study Zic3 expression pattern, we generated a novel Zic3 reporter transgenic mouse line, Zic3-LacZ-BAC (LacZ—β-galactosidase; BAC—bacterial artificial chromosome), expressing β-galactosidase under the control of Zic3 regulatory sequences and we demonstrate that Zic3 is expressed in the distal limb bud mesenchyme in a pattern spatially and temporally overlapping with Gli3.

To gain a better understanding of Zic3 and Gli3 interactions in vivo, we investigated whether mice carrying mutations of both Zic3 and Gli3 would present with features distinct from the single-gene mutants. We demonstrate that Zic3 loss of function rescues polydactyly in Gli3+/− neonates and abrogates ectopic Shh expression in anterior limb buds of Gli3−/− embryos. At the molecular level, loss of function of Zic3 leads to normalization of Shh expression and Gli3R/Gli3A ratio in Gli3+/− limb buds. Finally, we show increases in Gli3 transcripts in limb buds of Zic3 null embryos. These results uncover an important role for Zic3 in modulating the balance between Gli3A and Gli3R in developing limb buds and in regulating Shh expression level.

RESULTS

Zic3 and Gli3 expression overlap during limb patterning

Zic3 and Gli3 expression in developing embryos were analyzed using whole-mount in situ hybridization (WISH) at embryonic day 10.5 (E10.5) and E11.5 (Fig. 1), the critical window for Shh-mediated A/P patterning. In the whole embryo, expression of Zic3 and Gli3 overlap and are present in somites, forebrain, midbrain, hindbrain, spinal cord, limb buds and the developing eye (Fig. 1). Gli3 is broadly expressed in the developing limb bud mesenchyme (Fig. 1B and D) at these stages. Zic3 expression in limb bud distal mesenchyme (Fig. 1F and H) overlaps with that of Gli3.

Figure 1.
Zic3 and Gli3 expression overlap during embryonic stages crucial for A/P limb patterning and digit specification (E10.5–E11.5), as shown by WISH. (AD) Gli3 expression in whole embryos and limb buds. (EH) Zic3 expression at stages ...

Zic3 expression in limb buds was further validated using a novel Zic3 reporter transgenic mouse line, Zic3-LacZ-BAC (See Materials and Methods and Supplementary Material, Fig. S1). Eight independent Zic3-LacZ-BAC lines were analyzed and the limbs of three representative lines are shown in Supplementary Material, Figure S2. These analyses confirm that Zic3 is indeed expressed in limb bud distal mesenchyme.

Zic3 synergizes with Gli3 to activate transcription

We investigated the ability of Zic3 to modulate Gli transcriptional activity, using in vitro reporter assays. Zic3 and Gli expression constructs were used in transactivation assays with the 12Gli-RE-TKO-luciferase (12Gli-luc) reporter, containing multimerized GLI-BSs (Fig. 2). Zic3 activates the reporter 7-fold, consistent with its known ability to bind the canonical Gli-binding sequence (26). Transfection with Gli1 results in ~40-fold activation of transcription. Co-transfection of Zic3 and Gli1 results in no significant increase in transcriptional activation (P = 0.374) compared with Gli1 alone. In contrast, Gli3 acts as a weak repressor of the reporter. Co-transfection of Zic3 and Gli3 results in nearly 30-fold activation of transcription (Fig. 2A). This effect is significantly greater than with Zic3 alone (P < 0.0001), indicating a synergistic effect of the two proteins.

Figure 2.
Zic3 synergizes with Gli3 to activate transcription in vitro. HeLa cells were co-transfected with Zic3, Gli1 or Gli3 expression constructs and Gli12luc (A) or Ptch-luc (B). Histograms show fold activation compared with the reporter alone. Results represent ...

In vivo, one downstream target of Gli-mediated Shh signal transduction is the receptor Patched. The Patched promoter contains a single GLI-BS. We utilized a Patched promoter-luciferase construct to test the ability of Gli3 and Zic3 to synergize in transcriptional activation (Fig. 2B). Qualitatively similar results were obtained. Transfected independently, Zic3 results in a 2-fold activation of the reporter, whereas Gli3 is responsible for a 5-fold repression of transcription. Co-transfection results in a 4-fold increase in activation (P < 0.0001). These results suggest that co-transfection with Zic3 transforms Gli3 from a functional repressor to an activator in vitro.

Zic3 loss of function rescues Gli3+/− polydactyly

To determine Zic3 and Gli3 interactions in vivo, we asked whether mice carrying mutations of both Zic3 and Gli3 present with a phenotype distinct from single-gene mutants. The polydactylous limb phenotype of Gli3 mutants both in mice and in humans has been well documented (5,23,29). Mice heterozygous and homozygous for the XtJ deletion in Gli3 exhibit varying numbers of preaxial extra digits. We analyzed the fore- and hindlimb phenotypes of WT, Zic3 null, Gli3+/−, Gli3−/−, Zic3 null;Gli3+/− and Zic3 null;Gli3−/− neonates or E19 embryos (Table 1, Fig. 3 and Supplementary Material, Fig. S4). Limb abnormalities were not observed in Zic3 null mice (Fig. 3D–F). Gli3+/− limbs show duplication of digit 1 (arrows in Fig. 3G–I). This phenotype is rescued by Zic3 loss of function (Fig. 3J–L) (P = 0.025 for number of digits in Zic3 null; Gli3+/− versus Gli3+/− animals). In some cases, a subtle phenotype of widened first digit was identified in Zic3 null;Gli3 +/− limbs, but no polydactyly was seen (Table 1 and Supplementary Material, Fig. S4). Gli3−/− limbs exhibit severe polydactyly combined with the loss of normal A/P digit identity (Fig. 3M–O). Zic3 loss of function does not rescue the Gli3−/− phenotype (Fig. 3P–R) (P = 0.102 for number of digits in Zic3 null;Gli3−/− versus Gli3−/− animals), although a trend toward subtle partial rescue cannot be definitively excluded.

Table 1.
Polydactyly phenotype information
Figure 3.
Loss of function of Zic3 rescues the Gli3 haploinsufficient limb phenotype. Dorsal (A, D, G, J, M and P) and ventral (B, E, H, K, N and Q) views and skeletal preparations (C, F, I, L, O and R) of E18-P1 mouse forelimbs of indicated ...

Zic3 loss of function abrogates ectopic Shh expression in limb buds

Proper location and level of Shh expression are requirements for normal limb patterning. We used WISH to interrogate the location of expression, and real-time PCR to investigate the expression level. With regard to location of expression, Gli3−/− embryos are known to express Shh ectopically in the anterior mesoderm of developing limb buds (11). Shh expression pattern is normal in Zic3 null limb buds (Fig. 4C and D). In Gli3−/− embryos, ectopic Shh expression is present in anterior forelimbs and hindlimbs (Fig. 4E and F, arrows). In Zic3 null;Gli3−/− embryos, this ectopic Shh expression is abrogated (Fig. 4G and H).

Figure 4.
Zic3 loss of function abrogates ectopic Shh expression in developing Gli3−/− limb buds. Shh expression in forelimbs and hindlimbs of E10.5 WT (A and B), Zic3 null (C and D), Gli3−/− (E and F) and Zic3 null;Gli3−/− ...

Zic3 loss of function rescues Shh overexpression in posterior limb buds of Gli3+/− embryos

Shh is the major regulator of Gli3 processing. We next asked whether the level of Shh expression is affected by Gli3 and Zic3 loss of function. We examined the level of Shh transcript in posterior hindlimbs of E11.5 WT (n = 5), Zic3 null (n = 3), Gli3+/− (n = 4), Gli3−/− (n = 3), Zic3 null;Gli3+/− (n = 3) and Zic3 null;Gli3−/− (n = 3) embryos by quantitative real-time PCR. We observed upregulation of Shh expression in posterior limb buds of Gli3+/− and Gli3−/− E11.5 embryos (11-fold and 6-fold, respectively), as well as 2.7-fold upregulation in Zic3 null posterior limb buds (Fig. 5). Our finding that Shh expression level in Gli3+/− limb buds was higher than in Gli3−/− was unexpected, since Gli3 is thought to downregulate Shh expression (16). In Gli3+/− and Gli3−/− embryos lacking Zic3 function, Shh expression in posterior limb buds is rescued to the level comparable with WT (Fig. 5). Therefore, anatomic analysis shows rescue or partial rescue of polydactyly (Fig. 3), and investigation of Shh location (Fig. 4) and expression levels (Fig. 5) demonstrate relative rescue of molecular abnormalities.

Figure 5.
Shh overexpression in E11.5 posterior limb buds of Gli3 mutant embryos is rescued in the absence of Zic3, as determined by real-time PCR. Expression of Shh is significantly increased in Gli3+/− and Gli3−/− posterior limb buds compared ...

Zic3 loss of function increases Gli3 expression level in the limb buds

We examined Gli3 expression level in the developing limb buds of WT (n = 4) and Zic3 null (n = 8) embryos by real-time PCR. In Zic3 null limb buds, Gli3 expression is significantly increased compared with WT (1.33-fold to 1.74-fold, mean 1.45-fold, P = 0.0014) (Supplementary Material, Fig. S3).

Zic3 loss of function restores normal Gli3A and Gli3R levels in Gli3+/− embryos

To determine the effect of Zic3 loss of function on Gli3 protein processing in Gli3+/− limb buds, western blot analysis was performed using a Gli3-specific antibody (gift from B. Wang) on protein extracts from dissected limb buds of E11.5 WT (n = 3), Gli3+/− (n = 4) and Zic3 null;Gli3+/− (n = 4) embryos. Anti-Gli3 antibody recognizes full-length Gli3 (Gli3A; 190 kDa), Gli3 repressor (Gli3R; 83 kDa) and truncated mutant Gli3 protein (Xt-J; 66 kDa) (Fig. 6A). Densitometric analysis of Gli3 bands normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) shows the expected graded distribution of Gli3R along the A/P axis in limb buds of all genotypes, with a higher level of Gli3R in the anterior domains (Fig. 6B). Gli3R/Gli3A ratios are increased in both anterior and posterior halves of Gli3+/− limb buds compared with WT, with the P-value showing a trend toward significance in the posterior half (P = 0.067) (Fig. 6C). Importantly, a normal Gli3R/Gli3A ratio is restored in Zic3 null;Gli3+/− limb buds, and Gli3R/Gli3A ratios in WT compared with Zic3 null;Gli3+/− are not statistically different (P = 0.146 for posterior limb buds).

Figure 6.
Increased Gli3R/A ratio in Gli3+/− limb buds is rescued in the absence of Zic3. (A) Representative western blot of protein extracts from anterior (A) and posterior (P) limb bud halves of E11.5 embryos. (B) Levels of Gli3A and Gli3R proteins in ...

DISCUSSION

The ZIC transcription factor family plays important roles in human development. Mutations in ZIC3 cause X-linked heterotaxy (HTX1 MIM 306955) and isolated congenital heart defects as a result of its role in left–right patterning and interaction with the Nodal signal transduction pathway (28,30,31). Previous analyses of Zic and Gli function have suggested that Zic proteins may act as transcriptional co-activators in conjunction with Gli proteins (26). Together with the expression patterns of Zic1-3 genes (2) overlapping with regions of Shh pathway activity, these findings suggest a role for Zic transcription factors in the regulation of, or response to, Shh signaling. In this study, we present evidence that Zic3 alters Gli3 function in vitro and modulates its phenotypic effects on limb development in vivo, thereby identifying a novel genetic regulatory interaction.

Zic3 is involved in regulating Gli3 expression (Supplementary Material, Fig. S3) and thus the rescue of phenotypic and molecular abnormalities in Zic3 null;Gli3+/− embryos may stem from compensation for Gli3 haploinsufficiency by Zic3-mediated upregulation of Gli3 transcript levels. Gli3 western results are also consistent with this model, showing normalization of Gli3A and Gli3R levels in Zic3 null;Gli3+/− limb buds. We find that Zic3 loss of function normalizes many changes seen in the developing Gli3+/− limbs (Figs 3, ,55 and and6),6), as well as in Gli3−/− limb buds (Fig. 4). Figure 7 summarizes the expression analyses relevant for Zic3-mediated rescue of preaxial polydactyly. In Gli3−/− embryos, the polydactylous phenotype persists despite the fact that Zic3 loss of function rescues ectopic expression of Shh in anterior limb buds and Shh overexpression in the ZPA. These findings imply that the presence of functional Gli3 protein is necessary for phenotypic rescue and are in agreement with Litingtung et al. (10), who argue that Shh has no effect on skeletal patterning in the absence of Gli3.

Figure 7.
Model of preaxial polydactyly rescue by Zic3 loss of function. Levels of Shh, Zic3 and Gli3R are represented by dots. In Gli3+/− limb buds, Shh expression is upregulated, resulting in lower than WT levels of Gli3R in anterior limb bud and polydactyly ...

The increase of Gli3R/Gli3A ratio observed in Gli3+/− neonates with polydactyly was unexpected since this phenotype has previously been associated with a Gli3R/Gli3A ratio shifted in favor of Gli3A (32). Our results suggest that the decrease in the absolute Gli3R level is more important in the development of preaxial polydactyly than any change in the Gli3R/Gli3A ratio.

Shh mRNA expression is also affected in limb buds of Zic3, Gli3 and Zic3/Gli3 mutants. Previous work proposes an autoregulation of Shh expression in the ZPA, via modulation of ZPA domain expansion, cell fate and cell death (33). Furthermore, only a fraction of ZPA cells capable of expressing Shh actively produce Shh pre-mRNA at any given time, implying that Shh expression is a dynamic process in which the transcript level may fluctuate within each ZPA cell (34). Thus, the upregulation of Shh expression seen in Gli3 mutant embryos could result from increased Shh transcript levels within cells or from an expanded ZPA domain. Normalization by Zic3 loss of function reflects an important role in modulating Shh expression level. We expected that complete loss of Gli3R in Gli3−/− embryos would further upregulate Shh expression compared with Gli3+/− embryos, but we observed the opposite (Fig. 5). These results suggest that qualitative upregulation is more important than the specific level, which may depend on transient fluctuations of Shh expression. In addition to transcript level and cell number, the length of Shh exposure is a third important variable. Specification of the anterior digits depends upon differential concentrations of Shh, whereas the length of time of exposure to Shh is critical for differential patterning of the most posterior digits (35). Mutations of Gli family members could therefore influence digit patterning by altering the number of Shh-expressing cells in the ZPA, changing the level of Shh expression within cells, or affecting the duration of Shh expression. Finally, it should be noted that the alterations identified are specific to mRNA and protein levels require further assessment. Gli proteins function within a regulatory network to coordinate limb patterning. Although the mechanistic basis of Gli3 function in the limb is not completely understood, important Gli3 interactions with other transcription factors in the limb mesenchyme with effects on morphogenesis and pattern, such as Plzf (promyeloytic leukemia zinc finger) (36) and Hoxd12 (Hoxd—homeobox D) (37), have been identified. With this new identification of a Gli3–Zic3 interaction within the limb, the relationship of Zic3 proteins to this molecular circuitry requires investigation. For example, both Zic3 and Hoxd12 physically interact with Gli3 via its N-terminal zinc finger domains, suggesting a potential for competitive interactions that will require future study. There are a number of similarities between Hoxd12–Gli3 and Zic3–Gli3 interactions. Hoxd12 interacts physically and genetically with Gli3 during limb development, and can convert the Gli3 repressor into an activator of Shh target genes. Gli3 and Hoxd12 expression overlap in the developing limb bud, as do Gli3 and Zic3. Both Hoxd12 and Zic3 affect the phenotype of Gli3+/− mutants but not Gli3−/− mutants. The presence of functional Gli3 protein is required for Hoxd12 to exert effects on digit morphology and on Shh expression. Likewise, Zic3 loss of function rescues the Gli3 mutant phenotype only in the presence of Gli3 protein (in Gli3+/−). Finally, genetic redundancy within the limb buds may be important for both Hoxd genes (Hoxd10-13) and Zic genes. Each of the five murine Zic genes contains five C2H2 zinc fingers. Based on homology within the zinc finger domain, Zic1, 2 and 3 are more similar to each other and appear to form a subfamily (38). The expression studies document that Zic2 and Zic3 overlap in the developing limb buds at E10.5–E11.5, whereas Zic1 is not expressed in limb buds at that time (2). The expression overlap and the structural similarities of Zic2 and Zic3 indicate the potential for functional compensation in the developing limb buds, a hypothesis which requires additional investigation.

In conclusion, this study identifies Zic3 as an important potential genetic modifier of the polydactyly phenotype and shows that Zic3 deficiency rescues preaxial polydactyly by increasing Gli3R (Fig. 7). In addition, these results identify a potential function for Zic3 in regulating Shh pathway in vivo. We propose that Zic3 plays a significant role in the network of transcription factors regulating limb development by interacting with Gli3 to regulate transcription, to fine-tune Gli3 and Shh expression levels and to maintain the proper balance between Gli3A and Gli3R in developing limb buds.

MATERIAL AND METHODS

Animals

Mouse embryos of E10.5, E11.5, E19 and P1 (postnatal day 1) were collected for gene expression or phenotype analyses. The Zic3 null mice have been described previously (28). These mice are maintained on a mixed 129;C57BL/6 background due to lethality on an inbred background. The Zic3-LacZ-BAC transgenic mice were generated using a pBACe3.6 vector carrying a modified 200 kb insert of mouse genomic DNA with Zic3 gene located at the center of the transgene (Supplementary Material, Fig. S1). The original BAC clone was purchased from BAC PAC resources. Exon 1 of Zic3 was replaced with β-galactosidase coding sequence at the translational start site, using an Escherichia coli recombination system (39). Transgenic founders were generated by the University of Michigan Transgenic Animal Model Core and eight independent transgenic lines were established on an FVB/N background.

Mice heterozygous for the Extra toes-J mutation (Gli3Xt-J) were purchased from The Jackson Laboratory. Zic3 null and Gli3Xt-J heterozygous mice were intercrossed for more than six generations to obtain double-heterozygous animals on a mixed background (Zic3+/−;Gli3+/−), and further intercrosses were performed to obtain Zic3 null;Gli3−/− and Zic3 null;Gli3+/− embryos and neonates. All experiments used wild-type littermate controls.

All mice were housed in the Association for Assessment and Accreditation of Laboratory Animal Care (AALAC)-accredited Cincinnati Children's Hospital Research Foundation Animal Facility and experiments were approved by the Institutional Animal Care and Use Committee.

WISH, β-galactosidase staining and skeleton preparations

WISH was performed as described previously (28). Probes were labeled using a DIG RNA Labeling Kit (Roche Applied Science). A Zic3 probe spanning 471 bp in the 3′ region of the gene was generated by PCR using forward primer 5′-TCTAGATTCCTTACAATGTCAGT-3′ and reverse primer 5′-AGAAGCACTTTAACCATGAG-3′. The Shh probe was described elsewhere (40), as was the Gli3 probe (41). Staining for β-galactosidase was performed on Zic3-LacZ-BAC embryos using standard protocols (28). Skeletons of E19 embryos and neonates were prepared according to published protocols (42).

Luciferase assays

HeLa cells were transfected using Lipofectamine Plus (Invitrogen) according to the manufacturer's protocol. Co-transfection experiments were performed with 200 ng each of the reporter and 100 ng each of the expression constructs and with 20 ng of pRL-TK Renilla luciferase (Promega) as an internal standard. Total DNA per transfection was kept constant by adding empty vectors. Reporters included 12Gli-luc, a construct generated by ligating 12 multimerized oligonucleotides corresponding to Gli consensus-binding sequence (43), and Patched-luciferase reporter with one Gli3-binding sequence in the promoter region (44). The Zic3, Gli1 and Gli3 expression constructs have been described previously (8,26). Luciferase activities were measured using the Dual Luciferase Reporter Assay System (Promega) 48 h after transfection. The relative fold activation is presented as the ratio of the normalized value of reporter and expression construct to reporter alone. All results represent three independent transfection experiments, compared for statistical significance by Student's t-test.

Quantitative real-time PCR

Limb buds of E11.5 embryos were collected in RNAlater® (Ambion). We harvested posterior autopod halves for Shh expression analysis and whole-limb buds for Gli3 expression analysis. RNA extractions were performed using Totally RNA™ Kit (Ambion). cDNA was generated with High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Real-time PCR was performed using ABI PRISM® 7000 Sequence Detection System (Applied Biosystems) and Power SYBR® Green PCR Master Mix (Applied Biosystems), with intron-spanning primer pairs (see Supplementary Material for sequences). Six independent reactions were run for each biological sample. Shh and Gli3 gene expression results were normalized to Gapdh. Histograms represent relative expression ± standard error.

Quantification of Gli3 protein by western blot

Anterior and posterior E11.5 autopod halves (Fig. 6A) were lysed in radioimmunoprecipitation assay buffer containing complete protease inhibitor cocktail (Roche). Samples in 1× Laemmli buffer with 5% β-ME were boiled for 5 min and subjected to electrophoresis through 8% SDS–PAGE under reducing conditions and transferred onto a polyvinylidene fluoride membrane (Millipore). Gli3 protein was detected using a rabbit anti-Gli3 antibody (1:250; gift from B. Wang) (15) and goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:5000; Santa Cruz Biotechnology) followed by the ECL Plus detection system (Amersham). Anti-GAPDH antibody (1:2500; Abcam) was used as a loading control. Bands were quantitated from exposed films. Images were digitized using CCD technology-based Molecular Imager GelDoc XR+ system (BioRad). Densitometry was performed using the ImageQuant software (Molecular Dynamics). Histogram peak background correction was applied. Levels of Gli3A and Gli3R were normalized to GAPDH. Results were compared for statistical significance by Student's t-test.

FUNDING

This work was funded by RO1 HL088639 from the National Institutes of Health (S.M.W.).

Supplementary Material

Supplementary Data:

ACKNOWLEDGEMENTS

We thank Jennifer Purnell for expert technical assistance. We thank Dr Baolin Wang for anti-Gli3 antibody.

Conflict of Interest statement. The authors have no conflicts to disclose.

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