Fig 2: Ptch1/2 and co-receptors are differentially expressed in PGCs and somatic cells.a Expression of Ptch1, Ptch2, Gas1, Boc and Cdon demonstrated by immunofluorescence analyses of primary cultures of dissected E10.5 mouse GR, with co-staining of SSEA1. Representative images from three independent experiments are shown. Scale bar, 50 µm. b Single-cell suspensions from freshly-dissected GR of E10.5 GFPStella embryos were sorted by FACS (Supplementary Fig. 4) and the expression of Ptch1, Ptch2, GFP and Stella in GFP-positive (PGCs) and GFP-negative (somatic cells) populations analysed by quantitative RT-PCR. Each data point represents one experiment. Values normalised to 18s rRNA were plotted with error bars representing SD (n = 3) after unpaired t-test (*P < 0.05, **P < 0.005, ***P < 0.001). c Immunofluorescence analyses of Gas1, Ptch2, p-Src, Boc and SSEA1 on the GR tissue sections of WT and Gas1 KO embryos at E10.5. The merged images are shown without DAPI signal for improved clarity. Representative images from three independent experiments are shown. Scale bar, 10 µm. d Dot plots showing the relative fluorescence intensity values of Gas1, Ptch2, p-Src, Boc signal observed in PGCs which were normalised with the fluorescence intensity values of SSEA in each cell. Gas1 KO embryos showed a significant decrease in signal intensity in Gas1 (WT (n = 12), KO (n = 5), ****P < 0.0001) and p-Src (WT (n = 7), KO (n = 12), **P = 0.0054) but no significant changes in Ptch2 (WT (n = 6), KO (n = 6), P = 0.0564) and Boc (WT (n = 6), KO (n = 9), P = 0.2583). Error bars represent SD after unpaired t-test with Welch’s correction. Source data are provided as a Source Data file.

Fig 3: Ptch2/Gas1 and Ptch1/Boc induce differential Hh downstream signalling responses.a NIH3T3/Cas9 cells with Ptch1 KO or Gas1 KO were transfected with empty vector, Ptch2 or Ptch1 expression construct and stimulated with Shh for 40 min and 24 h. Profile of Gli1 and Gli3 transcripts at the respective time points was analysed by quantitative RT-PCR normalised to Gapdh. Data represent the mean fold induction compared with the untreated control. Error bars represent SD of three independent experiments. Statistical analysis by unpaired t test (*P < 0.05, **P < 0.01, ***P < 0.001). Dot plots showing individual data points are included in the Source Data file. b NIH3T3/Cas9 cells with Ptch1 KO or Gas1 KO were transfected with empty vector, Ptch2 or Ptch1 expression construct and stimulated with Shh as indicated. The signalling responses at the respective time points were analysed by western blotting of total cell lysates using specific antibodies as shown, with b-Actin loading control. Representative blots from three independent experiments are shown. Source data are provided as a Source Data file.

Fig 4: Defective noncanonical HH signalling in Wdr11–/– PGCs. (A) Representative images of immunofluorescence of PTCH2, GAS1 and p-Src on the WT and Wdr11–/– GR sections. The merged images are shown without DAPI signal to improve the clarity. Scale bar, 10 µm. (B) The relative fluorescence intensity values were normalised with those of SSEA1 in each cell as in (A), since the expression of SSEA1 was not itself impacted by the Wdr11 KO. Data for PTCH2 (n = 8 WT; n = 10 KO), GAS1 (n = 10 WT; n = 12 KO) and p-Src (n = 11 WT; n = 12 KO) represented as means ± SD after unpaired t test with Welch’s correction (****P < 0.0001). (C) PGCs (labelled by SSEA1, red) in the GR primary cultures of WT and Wdr11–/– were analysed by the respective antibodies as indicated after the addition of solvent dimethylformamide (DMF) or recombinant SHH protein (Shh-N) for 10 min. Representative images with merged signals (yellow) are shown with DAPI (blue). Scale bar, 10 µm. (D) The normalised relative intensity values of respective proteins in each cell as in (C) were compared for WT (DMF n = 8; Shh-N n = 9) and KO (DMF n = 9; Shh-N n = 9). Values are means ± SD after unpaired t test with Welch’s correction (****P < 0.0001).

Fig 5: PTCH2/SHH signalling induces GAS1 expression. (A) NIH3T3/Cas9 cells expressing PTCH1 or PTCH2 individually were treated with ( +) or without (–) Shh-N and analysed by Western blotting for the endogenous proteins as indicated. B-actin as a loading control. (B) The mRNA levels of Boc, Cdo and Gas1 in WT NIH3T3 cells cultured in either 10% newborn calf serum-containing medium (NCS) or 0.5% serum-containing medium (SFM) for 24 h were analysed by RT-PCR and agarose gel electrophoresis. H2O was used as the template in the negative control (Left panel). Gas1 mRNA levels in WT NIH3T3 cultured in NCS or SFM were quantitatively assessed by real-time qRT-PCR (n = 3; unpaired student’s t test, P = 0.000066) (Right panel). (C) Western blot analyses of the cell lysate and the conditioned medium (CM) collected from WT NIH3T3 cells which were transfected with either an empty vector or SHH expression construct and cultured in SFM for 48 h. The full-length (37 kd) and the cleaved form (34 kd) of GAS1 protein are detected in CM. (D) Western blot analyses of the CM or cell lysates of NIH3T3 cells treated with varying concentrations of TMI005 (ADAM17 specific inhibitor) as indicated, for 48 h in SFM. The pro-form (135kd) and the mature form (100kd) of ADAM17 are detected in the lysate.