(A) Diagram of the developing cerebellum at E14.5 and E18.5. Immunostaining of Boc and Cdon in sagittal sections of the developing mouse cerebellum shows that Boc is expressed in the EGL, ChP and RL at E14.5 and expression is maintained in the EGL and ChP at E18.5. Cdon is expressed in the RL and ChP at E14.5 and E18.5. (B) At P6, Xgal staining (left, counterstained with nuclear fast red) and immunofluorescence on sagittal cerebellum sections from Boc^+/- and Cdon^+/- βgeo (β-galactosidase-neomycin) mice reveal Boc and Cdon expression. Boc is expressed in Lim1⁺ cells (B,C) and Cdon in the ChP (B). (C) P3 WT mouse cerebellum sections co-immunolabeled with Boc (red) and various cerebellar cell markers (green) showing high Boc expression in proliferating CGNPs (Lim1⁺, Pax6⁺) and lower expression in PC (CaBP⁺, Lim1⁺) and differentiated granule cells (Pax6⁺ in the IGL). Scale bars: (A) 100 µm (B) 250 µm, (C) 50 µm. EGL, external germinal layer; IGL, internal granular layer; ChP, choroid plexus; RL, rhombic lip; PC, Purkinje cells; CGNP, cerebellar granule neurons; CaBP, Calbindin; VZ, ventricular zone.

(A) Immunostaining of sagittal sections showing Gas1 expression in the RL and EGL of the developing cerebellum at E14.5 and E18.5. (B,C) P4-6 WT cerebellum sections immunostained for Gas1 (red) and various cerebellar cell markers (green). Gas1 is highly expressed by proliferating CGNPs (Lim1⁺ cells; Pax6⁺ in EGL). (D) Immunostaining of E18.5 cerebellum sections from Math1-Cre;mTmG mice show that Boc and Gas1 are expressed in Math1⁺,GFP⁺ CGNPs in the EGL. EGL, external germinal layer; IGL, internal granular layer; PC, Purkinje cells; RL, rhombic lip; VZ, ventricular zone; ChP, choroid plexus. Scale bars: (A, C and D) 100 µm, (B) 500 µm.

(A) Boc and Gas1 interact with Ptch1. COS7 cells were transfected with the indicated constructs and lysates were immunoprecipitated (IP) with an anti-GFP antibody and immunoblotted (IB) with anti-Gas1, anti-Flag or anti-GFP antibodies. (B) Boc interacts with Ptch1 in a constitutive manner. COS7 cells expressing Ptch1-HA and Boc-Flag were treated with ShhN and subjected to anti-HA IP and either anti-Flag or anti-HA IB. (C) The Ptch1 L2 region is not required for the Boc-Ptch1 interaction. Anti-HA IP was perfomed on COS7 lysates expressing Boc-Flag and Ptch1-HA or Ptch1ΔL2-followed by anti-Flag or anti-HA IB. (D) The Boc cytoplasmic tail is not required for the Boc-Ptch1 interaction. COS7 cells expressing Ptch1-HA and Boc-GFP or BocΔcyto-GFP were subjected to anti-GFP IP and either anti-HA or anti-GFP IB. (E, F) Ptch1 forms receptor complexes with either Boc or Gas1 but not both. Boc-Flag was co-transfected with Gas1 with or without Ptch1-GFP in COS7 cells. (E) Lysates were IP with anti-Flag antibodies, followed by anti-Gas1 or anti-GFP IB. (F) Lysates were first immunoprecipated (IP #1) with anti-Gas1 antibodies. Supernatants from IP#1 were subjected to a second immunoprecipitation (IP #2) with anti-Flag antibodies. Both anti-Gas1 (IP #1) and anti-Flag (IP #2) immunoprecipitates were IB with anti-Gas1, anti-Flag and anti-GFP antibodies. See Fig. S4 for a schematic of this experiment. (A–F) Protein expression inputs were verified by IB with the indicated antibodies. ns, non-specific. (G, left) Diagram of WT Boc, Boc-Fc and Boc-Fc mutant proteins. (G, right) The Boc-Ptch1 interaction is mediated by the Boc FNIII(ab) domains. COS7 cells expressing Ptch1-GFP were incubated with conditioned mediated containing Boc-Fc proteins. Bound proteins were labeled with HRP-conjugated anti-Fc antibody and peroxidase activity measured.

(Left) In the absence of Shh, Boc, Cdon and Gas1 interact with Ptch1 to form Boc/Ptch1, Cdon/Ptch1 or Gas1/Ptch1 complexes. The Boc/Ptch1 interaction is mediated by the FNIIIa and FNIIIb domains of Boc. (Right) Shh binding to Boc/Ptch1, Cdon/Ptch1, and Gas1/Ptch1 receptor complexes leads to the de-repression of Smo, which in turn activates signal transduction events that result in Gli-mediated transcription. In the case of Boc and Cdon, the interaction with Shh is direct and is mediated by the FNIIIc domain of Boc and Cdon. Unlike Ihog and Boi which promote the relocalization of Ptc to the surface of Drosophila cells, Boc and Gas1 do not seem to relocalize Ptch1 to the surface of mammalian cells (Fig. S5A-C).

(A) Comparison of whole cerebella and (C) sagittal sections from adult Boc^+/- and Boc^−/− mice. (B) Cerebellum weight and normalized cerebellum weight relative to body weight. n=11 cerebella/group. (D) Cerebellum surface area (left) and IGL surface area (right) measured from three medio-lateral matching levels. n=4 cerebella/group. (E) Apoptotic CGNPs from P3 Boc^+/- and Boc^−/− mice visualized by TUNEL staining. (F) Number of apoptotic cells/mm² in the EGL from TUNEL-stained sections from 5 Boc^+/- and 4 Boc^−/− mice. (G) Proliferating CGNPs in the EGL from P3 Boc^+/- and Boc^−/− mice visualized by anti-BrdU staining. (H) (left) Percentage of BrdU⁺ cells in the EGL. n=4 animals/group. (right) Number of pH3⁺ cells/mm² in the EGL of 5 Boc^+/- and 4 Boc^−/− P3 mice. (I) CGNPs purified from Boc^+/+, Boc^+/- and Boc^−/− mice cerebella at P4 were cultured in the presence of 0–90 nM ShhN. Proliferating cells were measured by ³H-thymidine incorporation. Data is represented as fold induction in CGNP proliferation compared to untreated cells. Scale bars: (A) 2mm, (C) 1mm, (E,G) 250 µm. IGL, internal granular layer; EGL, external germinal layer. p valules measured from Student’s t-test (B,D,F,H) and two-way ANOVA (I).

(A) Haematoxylin-eosin staining on sagittal sections of E18.5 cerebellum revealing a thinner EGL in Gas1^−/−;Boc^−/− cerebella than control. Anti-pH3 immunostaining of sagittal sections of Gas1^+/-;Boc^−/− and Gas1^−/−;Boc^−/− cerebella counterstained with DAPI. RNA in situ hybridization showing the loss of expression of the Shh transcriptional target Gli1 in Gas1^−/−;Boc^−/− cerebella at E18.5. (B) Quantification of: cerebellum surface area, EGL surface area, pH3⁺ cells in EGL, pH3⁺ cells per µm², and EGL thickness along the postero-anterior axis, n=4 animals/group. (C) CGNPs purified from Gas1^+/+;Boc^+/+ (n=3), Gas1^+/-;Boc^+/+ (n=4) and Gas1^−/−;Boc^+/+ (n=4) mice cerebella at E18.5 were cultured with 0, 3, 10, 30 nM ShhN. Proliferating cells were visualized by immunostaining with an anti-Ki67 antibody. Data is represented as fold CGNP proliferation over untreated control (C, D and E) or DMSO control (F). (D) Similar to (C) but CGNPs were purified from Gas1^+/+;Boc^−/− (n=3), Gas1^+/-;Boc^−/− (n=3) and Gas1^−/−;Boc^−/− (n=3) mice cerebella at E18.5. (E) CGNPs were purified from control (Ctl; Gas1^+/+;Boc^−/− and Gas1^+/-;Boc^−/−) (n=3) and Gas1^−/−;Boc^−/− (n=3) mice cerebella at E18.5 and treated with 0, 20, 50 or 100 ng/ml of IGF-I. (F) Similar to (E) but CGNPs were treated with either DMSO, 0.150 µM purmorphamine, or 30 nM ShhN. p values measured from Student’s t-test (B), two-way ANOVA (C, E), and ANOVA (D, F). EGL, external germinal layer. Scale bars: top two rows=500 µm, bottom two rows=100 µm.

(A, B, C) Structural representation of ShhN. Residues colored in light, medium and dark green represent amino acids contacting Boc/Cdon FNIII(c) domain. E90 is represented in light green (A). R154 is represented in dark green (B). (C) Amino acids important for Ptch1 binding are colored in red while residues dispensible for the Ptch1-Shh interaction are illustrated in blue. (D) Anti-ShhN immunoblots showing the expression of purified ShhN WT, E90A, and R154E in comparison to ShhN C24II (top panel). Anti-ShhN immunoblot showing conditioned medium (CM) containing ShhN-AP WT, E90A, and R154E in comparison to ShhN C24II (bottom panel). (E) Stimulation of Gli-luciferase reporter activity in C3H 10T1/2 cells treated with 17, 50 and 150 nM ShhN WT, E90A or R154E mutants. (F) Differentiation of C3H 10T1/2 cells treated with 12.5, 25 and 50 nM ShhN WT, E90A or R154E mutants. (E–F) Each condition was performed in duplicate, n=3. (G) Induction in rat P4 CGNP proliferation upon treatment with 6.25, 12.5, 25 and 50 nM of ShhN WT, E90A or R154E mutants. Proliferation was measured by ³H-thymidine incorporation. Each condition was performed in triplicate, n=3. (E–G) Two-way ANOVA comparing the activity of ShhN E90A to ShhN WT was performed. (H) Table summarizing binding of ShhN mutants to Ptch1, Boc, Cdon and Gas1 and their functional activity.