Fig 2: Shh signaling is necessary and sufficient for cortical folding in the ferret cerebral cortex.(A–C) pCAG-EGFP plus either pCAG-Shh-N or pCAG control vector was electroporated at E33, and the brains were dissected at P16. (A) Coronal sections of the electroporated brains were stained with anti-GFP antibody and Hoechst 33342 (magenta). Boxed areas in the upper panels are magnified in the lower panels. Asterisks indicate the positions of sulci. The number of sulci was increased in the Shh-N-transfected GFP-positive area (Shh, EP). EP, electroporated side; non-EP, non-electroporated side. Scale bars = 5 mm (upper) and 2 mm (lower). (B) Quantification of the number of cortical folds. The local GN ratio was significantly increased by Shh-N. n = 3 animals for control, n = 4 animals for Shh. Bars present mean ± SEM. **p<0.01, Student's t-test. (C) Coronal sections of the electroporated brains were stained with anti-Brn2 (cyan) and anti-Ctip2 (red) antibodies and Hoechst 33342 (blue). Cortical layer structures were preserved in the Shh-N-electroporated area of the cerebral cortex. Asterisks indicate the positions of sulci. Scale bar = 1 mm. (D, E) pCAG-EGFP plus either pCAG-HhipΔC22 or pCAG control vector was electroporated at E33, and the brains were dissected at P16. (D) Coronal sections of the electroporated brains were stained with anti-GFP antibody and Hoechst 33342 (magenta). Cortical folding was markedly suppressed in the HhipΔC22-transfected GFP-positive area (HhipΔC22, EP, arrow). Arrowheads indicate cortical folds in the control cortex and in the non-electroporated side of the cortex. EP, electroporated side; non-EP, non-electroporated side. Scale bars = 5 mm (upper) and 2 mm (lower). (E) Quantification of cortical folding. The local SD ratio and the local GS ratio were significantly smaller in HhipΔC22-transfected samples. n = 3 animals for each condition. Bars present mean ± SEM. ns, not significant. *p<0.05,**p<0.01, Student's t-test. (F, G) pCAG-EGFP plus either pCAG-Shh-N or pCAG control vector was electroporated at E33, and the brains were dissected at P16. Coronal sections were subjected to Hoechst 33342 staining plus immunohistochemistry for Ctip2 and Brn2. (F) Quantification of the thicknesses of layer 2/3, layer 4, layer 5 and layer 6. The ratios of the thicknesses of the electroporated side relative to those of the non-electroporated side are shown. Note that the thickness of layer 2/3 was markedly increased by Shh-N. (G) Ratio of upper layer thickness (layer 2/3) to lower layer thickness (layer 4–6). n = 3 animals for each condition. Bars present mean ± SD. *p<0.05, **p<0.01; ns, not significant; Student's t-test. (H, I) pCAG-EGFP plus either pCAG-HhipΔC22 or pCAG control vector was electroporated at E33, and the brains were dissected at P16. Coronal sections were subjected to Hoechst 33342 staining plus immunohistochemistry for Ctip2 and Brn2. (H) Quantification of the thicknesses of layer 2/3, layer 4, layer 5 and layer 6. The ratios of the thicknesses of the electroporated side relative to those of the non-electroporated side are shown. Note that the thickness of layer 2/3 was selectively reduced by HhipΔC22. (I) The ratio of upper layer thickness (layer 2/3) to lower layer thickness (layer 4–6). n = 3 animals for each condition. Bars present mean ± SD. *p<0.05; ns, not significant; Student's t-test.

Fig 3: Shh pathway regulates cell proliferation and cell survival in lung fibroblasts.Proliferation of CCL206 lung fibroblasts was assessed by cell counting (A, D) and cell survival by MTT assay (B, E) after treatment with recombinant Shh (500 ng/ml) or with 100 nM, 1 µM or 10µMcyclopamine (cyclop) for 5 days. Results are presented in percentage as relative proliferation and relative survival compared to non-treated cells (A, B) or to vehicle (D, E) *p<0,1; **p<0,05. Representative phase-contrast microscope pictures upon Shh treatment (C) and upon 10 µM cyclopamine treatment (F) are shown.

Fig 4: The supernatant of H520 cells activates Hedgehog pathway in lung fibroblasts. Lung fibroblasts were serum-starved for 24 hours and then treated or not with the supernatant of H520 cells for 24, 48 or 72 hours. (A) RT-qPCR was performed to analyze Gli1, Gli2, Gli3 and Ptch1 mRNA levels in CCL206 treated with H520 supernatant. Results are presented as fold of RNA levels in treated cells compared with non-treated cells for each time point. **p<0,05; ***p<0,01. (B) Primary human fibroblasts from lung squamous carcinoma were serum-starved for 24 hours and treated or not with H520 supernatant for the indicated times. RT-qPCR was performed to assess Gli1, Gli2, Gli3 and Ptch1 mRNA levels. Results are presented as fold of mRNA levels in treated cells compared with non-treated cells for each time point. *p<0,1, **p<0,05. (C) The knockdown of Shh was performed in H520 cells with siRNA. The efficiency of Shh knockdown in H520 cells was confirmed by western blot realized with the supernatant of H520 cells transfected with negative control siRNA (NC) or with the siRNA of Shh. (D) CCL206 fibroblasts were treated for 24, 48 and 72 hours with the supernatant of either H520 cells transfected with a negative control siRNA or with the supernatant of H520 cells transfected with the siRNA of Shh. RT-qPCR was performed to evaluate changes in Gli1, Gli2, Gli3 and Ptch1 mRNA levels. Results are presented as fold of mRNA levels (2∧∧Ct) in CCL206 cells treated with the supernatant of H520 transfected with the siRNA of Shh compared with fibroblasts treated with the supernatant of H520 transfected with the negative control siRNA. *p<0,1; **p<0,05; ***p<0,01.

Fig 5: Lung fibroblasts respond to Shh treatment.Mouse lung fibroblasts CCL206 were treated or not with 500 ng/ml of recombinant mouse Shh or 500 ng/ml of human Shh for 24, 48 and 72 hours. (A) mRNA levels of Gli1, Gli2, Gli3 and Ptch1 upon treatment were assessed by RT-qPCR. Results are presented as fold of differences in mRNA levels (2∧∧Ct) of treated cells compared with non-treated cells for each time point. *p<0,1; ***p<0,01. (B) Western blot was performed to evaluate changes in Gli1 and Ptch1 protein levels in CCL206 fibroblasts treated or not with mouse Shh (500 ng/ml). ß-actin was used as a loading control. (C) Primary human fibroblasts were treated or not with recombinant human Shh (500 ng/ml) for 24, 48 and 72 hours. RT-qPCR was performed to assess the mRNA levels of Gli1, Gli2, Gli3 and Ptch1. Results are presented as fold of mRNA differences (2∧∧Ct) in treated cells compared with non-treated cells for each time point. *p<0,1; **p<0,05; ***p<0,01.