Boc is important, but not absolutely required, for Shh-mediated CGNP proliferation

To investigate the role of Boc in cerebellum development, we examined the gross morphology of Boc^−/− cerebella. While Boc^−/− mice are viable and cannot be distinguished from their littermates, their cerebellum is smaller than Boc^+/- or WT animals (Fig. 2A and data not shown). Boc^−/− cerebella were 14.3±0.05% (p<0.001) lighter than that of Boc^+/- cerebella (Fig. 2B). When the mass of the cerebellum was normalized to the body weight (p<0.001), the relative cerebellar mass was still reduced, indicating that this difference is not due to an overall decrease in total body weight (Fig. 2B). The cerebellum and IGL surface areas measured from sagittal sections of Boc^−/− adult mice were also reduced when compared to Boc^+/- animals (Fig. 2C,D; p<0.001 and 0.05, respectively). Although the IGL surface area is diminished in adult mice, migration of granule neurons and cerebellum foliation did not appear to be affected in Boc^−/− mice.

Open in a separate window

Figure 2

Boc^−/− mice have a smaller cerebellum than control mice

(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).

The decrease in cerebellum size in the absence of Boc could be due, at least in part, to reduced cell proliferation and/or enhanced cell death. TUNEL staining showed no significant difference in the number of apoptotic cells between Boc^−/− and Boc^+/- cerebella (Fig. 2E,F). In contrast, measurement of BrdU incorporation in the EGL of Boc^−/− and Boc^+/- mice showed that 40±1% of Boc^+/- CGNPs were actively dividing, compared to only 30±3% of Boc^−/− CGNPs (p<0.05) (Fig. 2G,H). Phospho-histone H3 (pH3) staining also showed a significant reduction in the number of mitotic pH3-labeled cells per mm² of EGL in Boc^−/− mice compared to Boc^+/- mice (p<0.05) (Fig. 2H). Together, these in vivo data indicate that Boc plays a role in CGNP proliferation.

Since Boc modulates Shh signaling (Okada et al., 2006; Tenzen et al., 2006; Zhang et al., 2006), we next tested whether Boc mediates Shh-induced CGNP proliferation. We cultured CGNPs purified from Boc^−/−, Boc^+/- and Boc^+/+ mice in the presence of varying concentrations of recombinant Shh (ShhN) (Fig. 2I). While Shh treatment induced the proliferation of WT CGNPs over 6 fold compared to unstimulated CGNPs, Shh stimulation increased Boc^−/− CGNP proliferation only about 3 fold. Significant differences in the proliferation of Boc^+/+, Boc^+/- and Boc^−/− CGNPs was observed at all concentrations of ShhN used (Fig. 2I), indicating that Boc promotes proliferation of CGNPs in a gene copy-number dependent manner. Together with our in vivo data, these results indicate that Boc^−/− mice have a smaller cerebellum due to a decrease in Shh-dependent CGNP proliferation and that Boc acts cell-autonomously in CGNPs to regulate their proliferation.

Gas1 is important, but not absolutely required, for Shh-mediated CGNP proliferation

Whilst inactivation of Boc in CGNPs, which do not express Cdon, lead to a partial decrease in their proliferation, it did not abolish their response to Shh. Moreover, CGNP proliferation is not further decreased when Cdon is inactivated in Boc^−/− mice (Fig. S1). These results are not consistent with a model where Boc and Cdon act like their Drosophila orthologues Ihog and Boi and are absolutely required for Hh signaling in vertebrates (Camp et al., 2010; Zheng et al., 2010). This raises the possibility that, unlike Drosophila, additional or different Shh binding molecules (other than Ptch1, Boc and Cdon) are required for vertebrate cells to respond to Shh.

Given that Gas1 binds Shh and modulates Shh signaling (Allen et al., 2007; Martinelli and Fan, 2007a, b; Seppala et al., 2007), we hypothesized that Gas1 may be this additional receptor. We first characterized the expression pattern of Gas1 in the developing cerebellum. Immunofluorescence stainings showed that Gas1 is restricted to the presumptive EGL of the cerebellar primordium at E14.5 and continues to be expressed in the EGL at E18.5 (Fig. 3A). At P6, like Boc, Gas1 localizes to Lim1⁺ cells in the EGL (Fig. 3B). Gas1 staining is most intense in the outer proliferative layer of the EGL (Lim1⁺, Pax6⁺, TAG1⁻ cells) and was not detected in TAG1⁺ migratory granule neurons and in Calbindin⁺ PCs (Fig. 3C).

Open in a separate window

Figure 3

Gas1 expression in the developing cerebellum

(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.

To determine whether Boc and Gas1 are co-expressed in CGNPs, we performed immunostainings on consecutive sections of cerebellum from Math1-Cre; mTmG E18.5 mice, where the CGNPs express GFP following Cre-mediated recombination. We used this strategy instead of double immunostainings as both anti-Boc and anti-Gas1 antibodies are produced in the same species. We found that both Boc and Gas1 co-localize with GFP⁺ cells, indicating that Gas1 and Boc are co-expressed in the same CGNPs (Fig. 3D).

Although the gross morphology of Gas1^−/− cerebella appears normal, they are smaller in size compared to control cerebellum and have decreased proliferation in the outer EGL (Liu et al., 2001). While this phenotype is reminiscent of that of Boc^−/− cerebella, no direct link has been made between the phenotype and the ability of Gas1^−/− CGNPs to respond to Shh. To directly test this, we performed proliferation assays on purified CGNPs from Gas1^−/− mice and control littermates. Our results show that Gas1 is essential for normal CGNP proliferation in response to Shh (Fig. 4C). Interestingly, the mutation of Gas1, similarly to the mutation of Boc, is not sufficient to abrogate the response of CGNPs to Shh.

Open in a separate window

Figure 4

Shh-dependent proliferation is completely lost in Gas1^−/−;Boc^−/− CGNPs

(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.

Shh-dependent proliferation is completely lost in Gas1^−/−;Boc^−/− CGNPs

To determine whether Boc and Gas1 might have partially redundant functions in Shh-dependent CGNP proliferation, we examined the cerebellum of E18.5 Gas1^−/−;Boc^−/− embryos, since these animals die at birth. Hematoxylin-eosin staining of Gas1^−/−;Boc^−/− cerebella revealed a significant loss of the EGL compared to controls (Fig. 4A). Although Gas1^+/-;Boc^−/− and Gas1^−/−;Boc^−/− cerebella showed no significant difference in the cross-sectional area of the whole cerebellum, the overall area of Gas1^−/−;Boc^−/− EGL was reduced by about 30% compared to controls (p<0.001) (Fig. 4B). Quantitation of the EGL along the postero-anterior axis showed that the difference in EGL thickness is greatest towards the anterior pole of the cerebellum (Fig. 4B and S2D). Marker analysis showed that Lim1 and Pax6 were properly expressed in the EGL of Gas1^−/−;Boc^−/− embryos compared to controls (Fig. S2A,B), thus, CGNPs are specified and localize normally. Furthermore, Cdon expression was not changed in the absence of Gas1 and Boc (Fig. S2C). However, the proliferation of Gas1^−/−;Boc^−/− CGNPs was severely decreased compared to Gas1^+/-;Boc^−/− CGNPs (p<0.001) (Fig. 4B). Moreover, the number of pH3⁺ cells per µm² of EGL surface area was lower in Gas1^−/−;Boc^−/− than Gas1^+/-;Boc^−/− animals (p<0.05) (Fig. 4B), demonstrating that the decrease in pH3⁺ cells in the EGL is not simply due to a total decrease in EGL area. These results indicate that Gas1 and Boc account for a large part of CGNP proliferation at this stage in vivo.

In addition to Shh, Insulin Growth Factor (IGF) and Notch signaling also promote CGNP proliferation (Corcoran et al., 2008; Solecki et al., 2001). Residual CGNP proliferation is observed in other mutant cerebella that lack Shh signaling (Corrales et al., 2004), thus, the proliferation observed in the EGL of Gas1^−/−;Boc^−/− cerebellum is probably independent of Shh signaling. To test whether Gas1^−/−;Boc^−/− cells have completely lost Shh responsiveness, we cultured CGNPs purified from E18.5 Gas1^+/+;Boc^−/− and Gas1^−/−;Boc^−/− cerebella with various ShhN concentrations. We found that while Gas1^+/+;Boc^−/− CGNPs proliferate in vitro in response to Shh, Gas1^−/−;Boc^−/− CGNPs show no enhanced proliferation in response to Shh (Fig. 4D). Importantly, the proliferative response of Gas1^−/−;Boc^−/− CGNPs to IGF-I, another factor able to stimulate CGNP proliferation, remained similar to that of control cells (Fig. 4E). Furthermore, treatment with purmorphamine, a Smo agonist, induced the proliferation of Gas1^−/−;Boc^−/− CGNPs (p<0.01) (Fig. 4F), indicating that Boc and Gas1 function upstream of Smo. Together, our data indicates that the presence of either Gas1 or Boc is absolutely required for Shh to promote CGNP proliferation. Given that Shh signaling in the cerebellum begins only at E17.5 and that Shh signaling plays an even more important role in CGNP proliferation after birth than at E18.5 (Corrales et al., 2004; Flora et al., 2009; Lewis et al., 2004), we anticipate that the EGL of Gas1^−/−;Boc^−/− mice would be much more severely reduced post-natally. However, because Gas1^−/−;Boc^−/− mice die at birth, conditional alleles will be required to directly test this.

To test whether the lack of a proliferative response of Gas1^−/−;Boc^−/− CGNPs to Shh in vitro is consistent with loss of Shh signaling in vivo, we examined the expression of Gli1, a Shh transcriptional target (Corrales et al., 2004), by RNA in situ hybridization. While control cerebella had intense Gli1 signal in the EGL, Gli1 expression was not detected in Gas1^−/−;Boc^−/− cerebella (Fig. 4A), confirming the inactivation of Shh signaling in Gas1^−/−;Boc^−/− cerebella.

Boc and Gas1 interact with Ptch1 and form distinct receptor complexes

We next investigated the molecular mechanism by which Boc and Gas1 act and, more specifically, whether they associate with Ptch1 to constitute the Shh receptor complex. We found that Boc and Gas1 can each co-immunoprecipitate with Ptch1, indicating that Boc and Gas1 can physically interact with Ptch1 (Fig. 5A). Importantly, these interactions are specific to Ptch1, as both Dispatched-1 (Disp1) and Smo, two multi-span transmembrane proteins also involved in Shh signaling, failed to interact with either Boc or Gas1 (Fig. S3). Furthermore, the addition of Shh did not modify the ability of Ptch1 to interact with Boc, suggesting that their interaction is constitutive (Fig. 5B).

Open in a separate window

Figure 5

Gas1 and Boc interact with Ptch1

(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.

Mapping studies showed that the second large extracellular loop of Ptch1 (L2), which is necessary for binding to Shh (Marigo et al., 1996), was not required for the interaction with Boc. Ptch1ΔL2-HA, a Ptch1 construct where L2 is deleted, interacted with Boc to an extent similar to full length Ptch1-HA (Fig. 5C). This is consistent with the binding of Shh to Ptch1 not being necessary for Ptch1 to interact with Boc. We next mapped the domain(s) of Boc mediating its interaction with Ptch1. BocΔCyto-GFP, a mutant lacking the cytoplasmic domain of Boc, interacted with Ptch1 as strongly as full-length Boc-GFP (Fig. 5D), indicating that the cytoplasmic domain is not required for its association with Ptch1.

To further characterize the region of Boc that interacts with Ptch1, we performed binding assays with various derivatives of Boc-Fc fusion proteins encompassing the Boc extracellular domain and cells expressing Ptch1-GFP. Deletion analysis of the Boc extracellular domain revealed that removal of the FNIIIc domain (mutant Boc FNIII(ab)), shown to be required and sufficient for Shh binding (Okada et al., 2006), only marginally affected Ptch1 binding, while truncation of both the FNIIIa and FNIIIb domains (mutant FNIII(c)) abolished it almost entirely (Fig. 5G). Boc-Fc constructs containing either the FNIIIa or FNIIIb domains alone bound to Ptch1 at levels that were about 60% of that of Boc ecto-Fc. Together our data indicate that the Boc FNIIIa and FNIIIb domains are required and sufficient to mediate its interaction with Ptch1. In addition, the Boc FNIIIc domain, which is necessary for Shh binding, is not required for the Boc-Ptch1 interaction, further supporting a Shh-independent interaction between Boc and Ptch1.

We next tested whether Boc interacts with Gas1 and did not detect an interaction between Boc and Gas1 either in the absence or presence of Ptch1 (Fig. 5E, top panel, lanes 5–6), despite detecting a strong interaction between Boc and Ptch1 (Fig. 5E, middle panel, lane 6). These experiments suggest that Boc/Ptch1 complexes do not contain detectable amounts of Gas1 and that Boc, Ptch1 and Gas1 are unlikely to form a tripartite complex.

To further confirm these results, we performed the complementary experiment and looked for the presence of Boc in Gas1/Ptch1 complexes. Lysates of cells transfected with Ptch1-GFP, Boc-Flag and Gas1 were first immunoprecipitated with anti-Gas1 antibodies and, despite detecting a strong interaction between Gas1 and Ptch1 (Fig. 5F, IP#1 middle panel, lane 6), we did not detect an interaction between Boc and Gas1 in the absence nor presence of Ptch1 (Fig. 5F, top panel lanes 5–6). To confirm that Boc is indeed able to interact with Ptch1 in these lysates and test whether both Boc/Ptch1 and Gas1/Ptch1 complexes are present in the same cell lysates, we recovered the supernatants from the anti-Gas1 immunoprecipitation (IP#1) and subjected them to a second immunoprecipitation, this time with anti-Flag antibodies to immunnoprecipitate Boc (Fig. 5F; see Fig. S4 for a schematic). We found that the Ptch1-GFP remaining in the supernatant efficiently co-immunoprecipitated with Boc (Fig. 5F, IP#2 middle panels, lane 6). Together, our data indicates that while Boc and Gas1 can both interact with Ptch1, it is unlikely that Boc, Gas1 and Ptch1 form a tripartite complex. Moreover, these results suggest that the Boc-Ptch1 and the Gas1-Ptch1 complexes are distinct molecular entities.

Gas1 is an essential vertebrate Shh binding protein

Previous studies have shown that forced expression of Boc, Cdon, or Gas1 can potentiate Shh signaling and that inactivation of Cdon or Gas1 can decrease Shh signaling in the neural tube (Allen et al., 2007; Martinelli and Fan, 2007a; Tenzen et al., 2006). Although these studies suggested a modulatory role for Boc, Cdon, and Gas1 in Shh signaling, whether Ptch1 alone is sufficient in the absence of any of these receptors remained an open question. Our study, together with a companion study by Allen et al. showing that embryos mutant for Boc, Cdon, and Gas1 completely lack Shh signaling in the neural tube, demonstrate that in vertebrates these receptors do not function solely as auxiliary Shh receptors but are absolutely required for Shh signaling in vivo.

In Drosophila, Ihog and Boi, the orthologs of Boc and Cdon, are absolutely required for Hh signaling (Camp et al., 2010; Zheng et al., 2010). If Hh receptor requirements were entirely conserved, it would have been expected that vertebrate cells lacking Boc and Cdon would not be able to respond to Shh. However, the inactivation of Boc in CGNPs (which do not express Cdon) leads to a partial decrease in their proliferative response to Shh. Moreover, inactivation of Cdon in Boc^−/− cerebella did not further decrease CGNP proliferation compared to inactivation of Boc alone (Fig. S1). Hence, unlike Drosophila which absolutely require either Ihog or Boi for Hh signaling, vertebrate cells still respond to Shh in absence of Boc and Cdon. These results highlight a fundamental difference between Drosophila and vertebrate Hh signaling and raised two possible mechanisms for Hh reception in vertebrates: Either Ptch1 is sufficient for Shh signaling or, unlike Drosophila, additional Shh binding molecules enable vertebrate cells to respond to Shh. Our data, together with results from Allen et al. (Allen et al., submitted), support a model where Gas1 acts with Boc and Cdon as essential Shh receptors. We showed that the combined inactivation of Boc, together with Gas1, completely abolishes the ability of CGNPs to respond to Shh, and that a mutant Shh ligand which binds Ptch1 (but not Boc, Cdon nor Gas1) is insufficient to activate Shh signaling. Thus, Gas1 – which is not present in the Drosophila genome – is an essential vertebrate Shh binding protein.

Other differences also exist between Hh signaling in invertebrates and vertebrates. The crystal structure of Hh in complex with Ihog, and Shh in complex with Cdon show that while Shh interacts with the third FNIII domain of Cdon and Boc (McLellan et al., 2008; Okada et al., 2006; Tenzen et al., 2006), Hh interacts with the non-orthologous first FNIII domain of Ihog and Boi (McLellan et al., 2006; Yao et al., 2006). In addition, the mode of Hh binding by the FNIII domains is not conserved between Drosophila and vertebrates (McLellan et al., 2008).

Previous studies in Drosophila S2 cells have shown that expression of Ihog results in a dramatic relocalization of Ptc to the cell suface (Zheng et al., 2010). Surprisingly, neither Boc nor Gas1 expression resulted in an increased relocalization of Ptch1 to the surface in at least two mammalian cell types (Fig. S5A,B). Surface biotinylation experiments also led to the same conclusion (Fig. S5C). While it is possible that tagging Ptch1 with GFP may interfere with its relocalization, we think that this is unlikely to occur, as Ptch1-GFP appears properly targeted to the cell surface given that we were able to detect an interaction between Boc and Ptch1-GFP in our cell surface binding assays (Fig. 5G). Thus, in contrast to what was observed for Ihog and Ptc, Boc and Gas1 do not appear to play a major role in relocalizing Ptch1 to the cell surface. Taken together, these results highlight important differences between Drosophila and vertebrate Hh signal reception.

Another fundamental difference is the requirement for the primary cilium for vertebrate Hh signaling (Eggenschwiler and Anderson, 2007). While Boc and Gas1 do not relocalize Ptch1 to the cell surface, it will be interesting to determine whether they contribute to its localization at the primary cilium (Corbit et al., 2005; Rohatgi et al., 2007).

Another interesting subject for future investigation is whether posttranslational modifications and multimerization of Shh (Dessaud et al., 2008) affect its interaction with the receptor complexes described in this study.

Are there specialized functions for Boc, Cdon, and Gas1?

Although Boc and Gas1 seemed to act redundantly in our CGNP proliferation experiments, it is possible that, in other cellular contexts, Boc, Gas1, or Cdon might impart additional non-redundant functions to the Shh signaling pathway. For example, Boc is required for Shh-mediated axon guidance (Fabre et al., 2010; Okada et al., 2006). Shh guides axons through a Src-family kinase (SFKs) dependent and Gli-independent pathway, where SFKs couple Shh signaling to cytoskeletal changes that elicit axon turning (Charron et al., 2003; Yam et al., 2009). It is possible that axon guidance by Shh is a specific function of Boc, since inactivation of Cdon did not disrupt axon guidance (Fabre et al., 2010; Okada et al., 2006). Thus, it is likely that different Shh receptor complexes, besides eliciting a canonical Shh signal transduction cascade, also impart additional functions. We speculate that the acquisition of novel functions for the vertebrate Shh signaling pathway during evolution might have paralleled the appearance of additional Shh binding proteins, such as Gas1.

Mice

Boc and Cdon (Okada et al., 2006; Zhang et al., 2010) and Gas1 (Martinelli and Fan, 2007a) mice were described previously.

Plasmids and reagents

Recombinant ShhN C24II and IGF-I were from R&D Systems. pEGFP-mCdon, pEGFP-mBoc and pCA-gap-EGFP were previously described (Okada et al., 2006). pEGFP-mPtch1, mGli1 and pcDNA3-mGas1 were kindly provided by C.C. Hui and V. Wallace. pEGFP-Smo was kindly provided by P. Beachy.(Okada et al., 2006)

Histology and immunohistochemistry

β-gal activity detection and immunochemistry on sections were performed according to protocols described previously (Charron et al., 2003; Fabre et al., 2010; Okada et al., 2006). Antibody dilutions: rabbit anti-mouse Lim1 (1:500, T. Jessell lab), rabbit anti-mouse Pax6 (1:100, Chemicon), rabbit anti-mouse Calbindin (1:200, Chemicon), goat anti-mouse TAG1 (1:400), rabbit anti-GFP (1:1000, Invitrogen), goat anti-mouse Boc (1:100, R&D), goat anti-mouse Cdon (1:250, R&D), goat anti-mouse Gas1 (1:200, R&D). Prior to performing immunostainings with anti-Boc antibody, sections were subjected to antigen retrieval for 1hr at 98°C in a sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0), cooled at RT for 20 minutes and washed extensively with PBS.

Isolation of CGNPs and in vitro proliferation assays

CGNPs were isolated from either E18.5 or P4 mouse or rat cerebella using a modified protocol previously described (Wechsler-Reya and Scott, 1999). Briefly, isolated cerebella were cut in small pieces and treated with 0.25% trypsin and DNaseI. Following trituration, single cell suspensions were centrifuged through a 30% to 65% Percoll step gradient. Cells harvested at the 30% interphase were resuspended in Neurobasal supplemented with B27, 0.5 mM L-Glutamine and Pen/Strep and plated in 96-well plates pre-coated with 100 µg/ml poly-D-Lysine. For CGNP proliferation assays with IGF-I, cells were resuspended in Neurobasal supplemented with 0.06% D-glucose, 100 µg/ml apo-transferrin, 16 µg/ml putrescine, 30 nM sodium selenite, 20 ng/ml progesterone and 1 mg/ml BSA. For ³H-thymidine incorporation assays, cells were seeded at 4×10⁵ cells/well in a 96-well plate in triplicate and treated with ShhN C24II for 48h. CGNPs were pulsed with 1 µCi/ml ³H-thymidine (PerkinElmer) for the last 12h. Incorporation was measured using the Filtermate harvester (PerkinElmer) and TopCount NXT beta counter (Packard). Alternatively, CGNPs were seeded at 2.5×10⁴ cells/well of a 96 well/plate and cultured as described above. Following 48h in culture, cells were fixed with 4% PFA, blocked with 10% PHT and immunostained with mouse anti-Ki67 antibody (1:100, Becton-Dickinson).

We are grateful to M. Cayouette, C. Jolicoeur, A. Kania, P. Fabre, E. Diaz, K. Tsanov and J.J. Guo for providing advice and/or reagents, S. McConnell and A. Okada for Boc and Cdon mice, and D. Leahy for providing the Shh-Cdon crystallographic data prior to publication. We thank J. Barthe, J. Cardin and F. Bourque for expert technical assistance. We thank D. van Meyel, P.T. Yam, M. Cayouette and A. Kania for critical comments on the manuscript. This work was supported by a grant from the Canadian Cancer Society Research Institute (CCSRI). BCW is supported by NIH grant DA-004443 to P. Schiller. APM is supported by NIH grant NS-033642. LI and ML are supported by Fonds de Recherche en Santé du Québec (FRSQ) post-doctoral training awards. FM is a Research Fellow of The Terry Fox Foundation. FC was a Canadian Institutes of Health Research (CIHR) New Investigator and is now an FRSQ Research Scientist.