The fertilized HMM mutant quail eggs were provided by Avian Bioscience Research center (ABRC) at Nagoya University. Embryos were staged according to Ainsworth et al. (2010). The HMM mutant shows autosomal recessive inheritance, although the causal gene, hmm, is still unknown. Heterozygous embryos showed no phenotype and were indistinguishable from wild-type embryos. Therefore we used a mixture of wild-type and heterozygous embryos as a control. Experimental procedures for isolating embryos were performed in accordance with guidelines set forth by the Regulations on Animal Experiments at Nagoya University. The embryo research was approved by Nagoya University Animal Experiment Committee (approval number 17).

Limb buds were fixed with 4% PFA overnight and then embedded in 1% low-melting agarose (Lonza). Excess agarose around the limb was removed with a razor blade. After that, agarose containing the limb buds was attached to the swivel base with Loctite for optical projection tomography (OPT) scanning (Henkel). Limb buds were treated with 100% MeOH for 3 h, and samples cleared in a 1:2 solution of benzyl alcohol (Wako): benzyl benzoate (Wako) overnight. A 3D image was taken with the OPT scanner 3001 (Bioptonics) and visualized by Avizo software (Maxnet).

Victoria blue staining was performed as described previously (Suzuki et al., 2008). Embryos were dissected in PBS and fixed in 10% Formalin overnight at room temperature. Embryos were stained overnight with 1% Victoria blue (Sigma) solution containing 1% HCl, and 70% EtOH. Embryos were washed overnight with 1% HCl in 70% EtOH solution following overnight treatment with 100% methylsalicylate to render them transparent.

In situ hybridization was performed as described previously (Suzuki et al., 2008). The following probes were used for in situ hybridizations: Hoxa13 (Yokouchi et al., 1991), Hoxd13 (Nelson et al., 1996), Fgf8, Gli3, Alx4, Lhx9, Ptch2, and Bmp2 (kindly gifted by Dr. John. F. Fallon, University of Wisconsin-Madison), Hand2 (kindly gifted by Dr. Kazuko Koshiba-Takeuchi, University of Tokyo), Shh, Gli1, and Ptch1 (kindly gifted by Dr. Yuki Sato, Kyushu university), Pax6 (kindly gifted by Dr. Yoshio Wakamatsu, Tohoku university), Pax3, Pax7, and Dbx2 (kindly gifted by Dr. Harukazu Nakamura, Tohoku university), Islet1 (589–1551 bp, GenBank NM_205414), and MyoD (155–1051 bp, GenBank NM_204214).

The ZPA was isolated from the posterior side of the donor limb bud with a sharpened tungsten needle. Isolated ZPA was placed in ice-cold Tyrode solution (137 mM NaCl, 2.7 mM KCl, 1 mM MgCl₂, 1.8 mM CaCl₂, 0.2 mM Na₂HPO₄, 12 mM NaHCO₃, 5.5 mM d-glucose) and divided into several pieces. A piece of the ZPA was stained by squirting it with DiI solution (1% DiI dissolved with 70% EtOH) in Tyrode solution, and it was implanted at the anterior side of the host limb bud with a tungsten needle.

Primary embryonic fibroblasts were isolated from the back region between the forelimb and the hindlimb at St. 35. Back tissues were dissected in PBS. After tissues were minced with a razor blade, they were incubated in 0.25% trypsin-EDTA solution (Wako) for 15 min at 37°C. An equal volume of 100% fetal calf serum was added. The supernatant of the cell suspension was plated on 3.5-cm glass-base tissue culture dish (IWAKI). The next day, cells were fixed with 4% PFA for 10 min at room temperature.

Limb buds were dissected in ice-cold PBS and fixed with 4% PFA for 15 min on ice. The PFA solution was immediately removed and fresh ice-cold PBS was added. The limb buds were treated with 30% sucrose in PBS overnight at 4°C and then embedded in compound for frozen sections (Leica). Samples were then sectioned by cryostat for immunohistochemistry.

Cells in frozen sections or fixed primary fibroblasts were permeabilized by treating with 0.2% TritonX-100 (Wako) for 20 min at room temperature, and then blocked with 3% BSA in PBS for 30 min. Anti-SHH antibody 5E1 (1:100) (DSHB), Anti-GLI3 antibody (1:100) (Santa Cruz sc-20688), and anti-acetylated Tubulin clone 6-11B-1 (1:1000) (Sigma) were diluted in 3% BSA/PBS solution. Samples were incubated overnight at 4°C with the primary antibody. The next day, samples were incubated with the secondary antibody (Alexa Fluor 488) (Thermo Fisher Scientific) diluted at 1:500 in 3% BSA/PBS solution for 2 h at room temperature. After 1 μg/ml of DAPI in PBS was added to the samples for 15 min, the samples were mounted with fluorescein mounting medium (Dako) and fluorescent images were taken with an Olympus FV1000 confocal microscope.

Fertilized eggs were incubated for 3.5 days. The limb buds of St. 23 embryos were isolated in ice-cold PBS with a tungsten needle and then collected in 1.5 mL collection tubes. After solubilizing the cells with 200 μl of lysis buffer (50 mM HEPES (pH 7.4), 150 mM NaCl, 10 mM EDTA, 1% Triton X-100, 200 mM sodium fluoride, 10% glycerol, 20 mM sodium pyrophosphate, 2 mM phenylmethane sulfonyl fluoride, 4 mM Na₃VO₄, 0.1 mg/ml leupeptin, and 15 mM benzamidine), the cell lysate was centrifuged at 14,000 g for 10 min. The supernatant was assayed for protein content using a Bio-Rad protein assay kit. The proteins were then resolved with SDS-polyacrylamide gel electrophoresis and electrotransferred to polyvinylidene difluoride membranes. After blocking with 5% (w/v) non-fat dry milk in Tris-buffered saline-Tween buffer (20 mM Tris (pH 7.6), 0.14 M NaCl, and 0.1% (w/v) Tween 20), the membranes were treated with primary antibodies, Anti-GLI3 antibody (1:1000) (Santa Cruz sc-20688), and Anti-αTubulin clone B-5-1-2 (1:2500) (Sigma). The proteins were visualized using an ECL Western blotting detection system.

We previously reported that the hmm^−/− embryo shows syndactylous polydactyly (Tsudzuki et al., 1998). It is still unclear how these phenotypes are induced in hmm^−/− limb bud. In both talpid² and talpid³ mutants, the limb bud is wider in size along the AP axis before cartilage condensation starts (Francis-West et al., 1995; Caruccio et al., 1999). This wider limb bud leads to expansion of the autopod, resulting in polydactyly (Litingtung et al., 2002). Therefore, we first examined the shape of the limb bud in the hmm^−/− embryo. To compare the shape of the entire limb bud between the mutant and wild-type, we took OPT images (Sharpe et al., 2002) of the hind limb at St. 23, after the limb bud emerged from the body wall (Figures 1A,B). The hmm^−/− limb bud was slightly wider than the hmm^+/+;+/− (simplified hereafter as hmm^+/−) limb bud along the AP axis (Figure 1A). The hmm^−/− limb bud was also expanded along the dorso-ventral (DV) axis compared to the hmm^+/− limb bud (Figure 1B). To elucidate the mechanisms underlying the expansion of the limb bud along the AP axis, we compared the limb field's AP width between hmm^+/− and hmm^−/− embryos. We found that the anterior boundary of the hmm^−/− forelimb bud (FLB) was expanded cranially compared to that of the hmm^+/− FLB (Figures 1C,E). The AP width of the hindlimb bud (HLB) appeared to be the same in both hmm^+/− and hmm^−/− embryos (Figures 1D,F), implying that a different mechanism is involved in the expansion of the hindlimb along the AP axis. In the talpid² mutant, the expansion of Fgf8 expression at the apical ectodermal ridge (AER) along the AP axis is observed along with formation of a wider limb bud (Caruccio et al., 1999). Therefore, we checked the expression of Fgf8 at St. 23 and St. 26 in the hmm^−/− limb bud, and found that it was expanded into both the anterior and posterior ends close to the body wall (Figures 1G–J). Expansion of Fgf8 expression was continued into St. 26, along with wider autopod formation in the hmm^−/− embryo (Figures 1K–N). Taken together, these results suggest that mesenchymal cells of the hmm^−/− limb bud propagate more than in the wild-type and give rise to a wider limb bud with an extended AER along the AP axis. In addition, the anterior boundary of the forelimb field is expanded in the hmm^−/− embryo when the FLB is initiated.

In the autopod of the hmm^−/− embryo many of the digits are shortened during development (Tsudzuki et al., 1998). We therefore analyzed detailed patterns of polydactylous digits along the AP axis. In the hmm^+/− HLB, digital rays 1, 2, 3, and 4 are formed correctly to their unique full lengths along the AP axis (Figure 2A). In contrast, we observed indistinguishable shortened digital rays in the hmm^−/− HLB. We also found webbing along the AP axis of the digital ray sequence in the hmm^−/− HLB. These results imply that anterior-posterior polarity is disrupted in the hmm^−/− limb bud.

In order to identify the specific region where anterior-posterior polarity is disrupted, we performed victoria blue staining to visualize condensing cartilage at St. 35 (Figure 2B). In the forelimb, the hmm^−/− embryo has four to eight digits with syn-, poly-, and brachdactyly, whereas the hmm^+/− embryo has three digits (Table 1). The forelimb of hmm^−/− has one shortened, thick humerus in the stylopod and an unidentifiable ulna/radius in the zeugopod. The hindlimb of the hmm^−/− embryo also has one shortened, thick femur in the stylopod and an unidentifiable fibula/tibia in the zeugopod. The autopod of the hmm^−/− hindlimb has seven to eleven digits (Table 1). Based on the morphological criteria of digit identity (number, size, and shape of the phalanges Suzuki, 2013), we assumed that the hmm^−/− autopod has lost digit identity. We also found that the metacarpal/metatarsal bones were fused and the metacarpal/metatarsal-phalangeal joints were missing in the hmm^−/− autopod (Figure 2B arrowhead). We observed the phalangeal joint in both the forelimb and the hindlimb, but its formation was incomplete. These results indicate that anterior-posterior polarity of both the forelimb and the hindlimb is disrupted in the hmm^−/− embryo.

To understand the molecular mechanisms of anterior-posterior pattering deficiency in hmm^−/− limbs, we next examined gene expression patterns of marker genes specifically expressed at the anterior/posterior sides. Genetic antagonism between Hand2 and Gli3 is necessary to establish AP polarity at the early limb bud stage (te Welscher et al., 2002a). We found that expression of Hand2 was restricted at the posterior side in the hmm^+/− limb bud. In contrast, in the hmm^−/− limb bud we observed strong expression of Hand2 at the posterior side and weak expression at the anterior side (Figures 3A–D). In the hmm^+/− limb bud, Gli3 was not expressed at the posterior side where Hand2 was expressed (Figures 3E,F). In contrast, Gli3 was expressed throughout the limb bud at the early stages in the hmm^−/− limb bud (Figures 3G,H). Alx4 was expressed at the anterior mesoderm at St. 23 in the hmm^+/− limb bud (Figures 3I,J), but its expression was downregulated though still detectable in the hmm^−/− limb bud (Figures 3K,L). At St. 25, Alx4 was continuously expressed at the anterior side of the stylopod and zeugopod in the hmm^+/− limb bud (Figures 3I′,J′). In the hmm^−/− limb bud, its expression was still observed in the FLB (Figure 3K′) but not detectable in the HLB (Figure 3L′). Lhx9 was expressed at the anterior side at St. 22 (Figures 3M,N) and at the anterior autopod at St. 25 (Figures 3M′,N′). In contrast to the expression of Alx4, expression of Lhx9 was expanded to the posterior side in the hmm^−/− limb bud (Figures 3O–P′). In particular, Lhx9 expression was observed at the posterior end of the hmm^−/− hindlimb autopod (Figure 3P′). Expression of Hoxd13 was restricted to the posterior side in the hmm^+/− limb bud at St. 22 (Figures 3Q,R), and at St. 25 its expression was observed at the posterior mesoderm in the autopod (Figures 3Q′,R′). Expression of Hoxd13 was similarly restricted to the posterior side in the hmm^−/− limb bud (Figures 3S–T′). We checked the autopod area for Hoxa13 expression in the contra-lateral side (Figures 3U–X), and we saw that the autopod expanded along the AP axis in the hmm^−/− limb bud at St. 25 (Figures 3W,X). However, expression of Hoxd13 was still restricted to the posterior side (Figures 3S′,T′), the same as in the hmm^+/− autopod (Figures 3Q′,R′). At St. 29 Hoxd13 was expressed throughout the hmm^+/− autopod except on the anterior side of digit 1 (Figures 3Y,Z). However, it was expressed all the way to the anterior end of the hmm^−/− autopod (Figures 3a,b) along with Hoxa13 (Figures 3c–f). From these results, we conclude that the hmm^−/− limb bud partially maintains AP polarity at the limb bud stage (St. 20–25), but loses AP polarity at the late autopod stage (St. 29).

As described above, AP polarity in the limb bud is disrupted in the hmm^−/− embryo. It is known that expression of hand2, Gli3, and Alx4 is altered by SHH signaling, which establishes anterior-posterior polarity in the limb (Takahashi et al., 1998; te Welscher et al., 2002a). We therefore examined the expression patterns of target genes downstream of SHH in the limb bud. Expression of Shh was restricted to the posterior edge of the hmm^+/− limb bud (Figures 4A,B). In the hmm^−/− limb bud, Shh was expressed at the posterior mesoderm; however, expression was restricted to a more proximal region of the FLB than in the hmm^+/− limb bud (Figure 4C). On the other hand, the expression domain of Shh was expanded proximally in the HLB (Figure 4D).

Gli1, Ptch1, Ptch2, and Bmp2 are known to be downstream target genes of SHH signaling that are expressed in response to SHH signaling (Chiang et al., 2001). In the hmm^+/− limb bud, the expression domain of Gli1 was expanded to the middle part of the limb bud along the anterior-posterior axis (Figures 4E,F). In contrast, Gli1 was expressed uniformly at very low levels throughout the hmm^−/− limb bud (Figures 4G,H). Similarly, Ptch1 was expressed at the posterior half of the hmm^+/− limb bud (Figures 4I, J), but in the hmm^−/− limb bud was expressed uniformly at very low levels along the anterior-posterior axis (Figures 4K,L). In the hmm^+/− limb bud Ptch2 was expressed at the posterior mesoderm similarly to Ptch1 (Figures 4M,N), but expression was not detected in the hmm^−/− limb bud (Figures 4O,P). In the hmm^−/− limb bud, expression of Bmp2 was observed at the posterior edge in the FLB only, but in hmm^+/− limb buds expression was observed at the posterior mesoderm in both the FLB and the HLB (Figures 4Q–T). The expression of Gli1, Ptch1, Ptch2, and Bmp2 is reduced or lost in the limb bud of the Shh^−/− mouse embryo (Litingtung et al., 2002), therefore SHH signaling must be substantially reduced despite the detection of Shh expression in the hmm^−/− limb bud. Reduction of SHH signaling was also observed in the neural tube of the hmm^−/− embryo (Figure S1A).

We next examined limb bud expression of Gli3, a transcriptional factor that mediates SHH signaling. In the hmm^+/− limb bud Gli3 was expressed in the mesenchyme in a complementary pattern to Shh expression (Figures 4A,B,U,V). In contrast, in the hmm^−/− limb bud Gli3 was strongly expressed throughout the limb bud, including in the Shh-expressing region (Figures 4C,D,W,X). We therefore concluded that the syndactylous polydactyly phenotype in the hmm^−/− limb bud does not result simply from a loss of SHH signaling due to a defect in Gli3 expression.

In hmm^−/− embryos, cells in the limb bud showed a reduction in SHH signaling despite expression of Shh (Figure 4). We therefore hypothesized that cells in the hmm^−/− embryo might have lost SHH protein activity or the ability to respond to SHH protein. To test these hypotheses, we analyzed the polarizing activity of the hmm^−/− limb bud. When we grafted ZPA from the hmm^+/− FLB to the anterior side of the hmm^+/− FLB at St. 20, strong ectopic Ptch2 expression was observed (83%, n = 6) (Figure 5A). In contrast, implantation of ZPA from the hmm^+/− FLB to the hmm^−/− FLB did not induce ectopic Ptch2 expression (0%, n = 4) (Figures 5B,C). We next grafted ZPA from the hmm^−/− FLB to the hmm^+/− FLB. Unexpectedly, ectopic Ptch2 expression was not induced (0%, n = 8) (Figures 5D,E). These results show that mesenchymal cells in the hmm^−/− limb bud do not respond to SHH protein. Furthermore, despite the presence of Shh expression (Figures 4C,D), ZPA derived from the hmm^−/− limb bud cannot induce expression of SHH signaling downstream targets.

We next examined the presence of SHH protein using immunohistochemistry. We found that the protein was detectable by anti-SHH antibody in the limb bud and the notochord in both hmm^+/− (Figures 6A,B, Figure S1B) and hmm^−/− (Figures 6C,D, Figure S1B) embryos. These observations raise the following possibilities in the hmm^−/− embryo: first, the secretion pathway of the SHH protein is defective; second, the SHH protein is dysfunctional; and third, the SHH signaling pathway in the target tissues is disrupted.

It was recently reported that loss of primary cilia induces constitutive activation of SHH signaling through high expression of GLI3A in both talpid² and talpid³ mutants (Davey et al., 2006; Chang et al., 2014). Therefore, we visualized the primary cilia using immunohistochemistry targeting acetylated tubulin. However, we found that primary cilia were still present in both the hmm^+/− and hmm^−/− limb buds (Figure 7), suggesting that loss of primary cilia is not a cause of the hmm^−/− phenotype. We next performed western blotting for the GLI3 protein (Figure 8A), and found that the active form of GLI3, GLI3A, was highly expressed in both the anterior and posterior halves of the hmm^−/− limb bud compared to the hmm^+/− limb bud. In particular, high expression of GLI3A was observed on the anterior side of the hmm^−/− limb bud where Shh is not expressed. Expression of GLI3R was not detected in our experiments. This result raises the possibility that GLI3A protein does not function normally in the hmm^−/− limb bud.

Finally, we examined whether transport of the GLI3 protein from the cytoplasm to the nucleus is disrupted in the hmm^−/− embryo using immunohistochemistry. We observed that GLI3 protein is localized to the nucleus in both hmm^+/− and hmm^−/− primary fibroblast cells (Figure 8B). Taken together, our results imply that despite the presence of GLI3 protein in the nucleus, SHH signaling is abolished in the hmm^−/− embryo due to a loss of function of the GLI3 protein.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.