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: Boc mediates Hh ligand reception by Ptch1.a Representative images of at least two independent immunoblotting experiments in which NIH3T3/Cas9 cells with Ptch1 KO were transfected with mouse Ptch1 expression construct and stimulated with Shh for 40 min and 24 h. Interactions of Ptch1 with endogenous Smo, Boc and Gas1 were analysed by IP and WB. IP was performed using specific antibodies as indicated, with WB using respective antibodies as shown. b A schematic diagram illustrating the protein interactions before and after Shh treatment. Source data are provided as a Source Data file.

Fig 4: Hh pathway genes and co-receptors are expressed in the PGC migratory niche in the mouse.a RT-PCR analyses of Hh pathway genes in E9.5 hindgut (HG), E10.5 urogenital ridge (UG) and E11.5 genital ridge (GR). b RT-PCR analysis of Ptch co-receptors, Boc, Cdon and Gas1 in E10.5 UG. c Immunofluorescence staining of Shh, Gas1, Boc and Cdon on E9.5 and E10.5 embryo sections. Scale bar, 50 µm. d Co-staining of germ cell marker SSEA1 and Gas1, Boc or Cdon on the genital ridge area of E10.5 embryos. Scale bar, 50 µm. e In situ hybridisation of Shh on the transverse section of E10.5 embryo counterstained with haematoxylin. The PGC migratory route from HG heading towards UG is illustrated by circled dots. n neural tube, nt notochord, a aorta, gr genital ridge, hg hindgut. Scale bar 100 µm.

Fig 5: Gas1 mediates Hh ligand reception by Ptch2.a Representative images of at least two independent immunoblotting experiments in which NIH3T3/Cas9 cells with Ptch1 KO were transfected with Myc-tagged Ptch2 expression construct and stimulated with Shh protein in serum-free medium for 40 min. Interactions of Ptch2 with endogenous Smo, Gas1 and Boc were analysed by immunoprecipitation (IP) and Western blotting (WB). IP was performed using specific antibodies as indicated and analysed by WB with respective antibodies as shown. The images in the bottom panel are from Ptch1 KO cells transfected with empty vector, which did not show any co-precipitation of endogenous Gas1 and Ptch2 regardless of Shh treatment, confirming the specificity of these antibodies (bottom panel). b A schematic diagram illustrating the protein interactions before and after Shh treatment. c NIH3T3/Cas9 cells with Gas1 KO were transfected with Myc-tagged Ptch2 and stimulated with Shh protein in serum-free medium for 40 min. IP was performed with anti-Myc antibody. Representative blots from two independent experiments are shown. d A schematic diagram illustrating the protein interactions in Gas1 KO cells. Source data are provided as a Source Data file.