Boc, but not Cdon, is expressed in proliferating CGNPs of the cerebellum
To investigate the receptor requirements for the proliferative effect of Shh in CGNPs, we first analyzed Boc and Cdon expression in the developing cerebellum. CGNPs arise from the rhombic lip (RL) between embryonic day (E) 13.5–14.5 and migrate anteriorly over the cerebellar anlage, forming the highly proliferative external germinal layer (EGL) (Roussel and Hatten, 2011
). Starting at E17.5 and continuing during early postnatal development, Purkinje cells (PCs) lining the EGL stimulate CGNP proliferation by secreting Shh (Dahmane and Ruiz i Altaba, 1999
; Kenney and Rowitch, 2000
; Wallace, 1999
; Wechsler-Reya and Scott, 1999
). Following a proliferative burst, CGNPs stop dividing, differentiate into granule neurons, migrate inwards past the PC layer and populate the internal granular layer (IGL).
We first examined Boc and Cdon expression in the cerebellar anlage of E14.5 mouse embryos. Immunostainings of sagittal sections showed that while Boc was expressed in the presumptive EGL, RL and the ventricular zone of the roof of the 4th ventricle, Cdon expression was restricted to the RL (). At E18.5, a stage at which CGNPs proliferate in response to Shh, we detected Boc expression in the EGL and, albeit at lower level, in the PC layer of the developing cerebellum. In contrast, Cdon expression was limited to the tip of the RL.
Boc and Cdon expression in the developing cerebellum
Analysis of post-natal day (P) 6 Boc+/-
gene-targeted mice encoding a β-galactosidase (β-Gal)-neomycin reporter gene fusion (β-geo) (Okada et al., 2006
) revealed strong β-Gal activity in Boc+/-
cerebellum, but was limited to the choroid plexus of Cdon+/-
cerebellum. Immunostainings confirmed this expression pattern and revealed that Boc localized to cells expressing Lim1, a marker for CGNPs and PCs (). Interestingly, while highest levels of Boc were detected in the outer proliferative region of the EGL (Lim1+
cells), lower levels were observed in differentiated migratory granule cells (TAG1+
cells) and in PCs (Calbindin+
cells). These results show that Boc, but not Cdon, is highly expressed in proliferating CGNPs of the cerebellum.
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 ( and data not shown). Boc−/− cerebella were 14.3±0.05% (p<0.001) lighter than that of Boc+/- cerebella (). 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 (). The cerebellum and IGL surface areas measured from sagittal sections of Boc−/− adult mice were also reduced when compared to Boc+/- animals (; 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.
Boc−/− mice have a smaller cerebellum than control mice
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 (). 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) (). 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) (). 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−/−
mice in the presence of varying concentrations of recombinant Shh (ShhN) (). 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+/+
CGNPs was observed at all concentrations of ShhN used (), 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
; 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 (). At P6, like Boc, Gas1 localizes to Lim1+
cells in the EGL (). Gas1 staining is most intense in the outer proliferative layer of the EGL (Lim1+
cells) and was not detected in TAG1+
migratory granule neurons and in Calbindin+
Gas1 expression in the developing cerebellum
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 ().
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 (). Interestingly, the mutation of Gas1
, similarly to the mutation of Boc
, is not sufficient to abrogate the response of CGNPs to Shh.
Shh-dependent proliferation is completely lost in Gas1−/−;Boc−/− CGNPs
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 (). Although 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) (). 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 ( 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) (). Moreover, the number of pH3+
cells per µm2
of EGL surface area was lower in Gas1−/−;Boc−/−
animals (p<0.05) (), 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−/−
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 (). 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 (). Furthermore, treatment with purmorphamine, a Smo agonist, induced the proliferation of Gas1−/−;Boc−/−
CGNPs (p<0.01) (), 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 (), confirming the inactivation of Shh signaling in Gas1−/−;Boc−/−
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 (). 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 ().
Gas1 and Boc interact with Ptch1
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 (). 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 (), 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 (). 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 (, top panel, lanes 5–6), despite detecting a strong interaction between Boc and Ptch1 (, 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 (, IP#1 middle panel, lane 6), we did not detect an interaction between Boc and Gas1 in the absence nor presence of Ptch1 (, 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 (; see Fig. S4
for a schematic). We found that the Ptch1-GFP remaining in the supernatant efficiently co-immunoprecipitated with Boc (, 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.
Binding of Shh to Ptch1 is not sufficient to activate Shh signaling
Our results indicate that Boc and Gas1 are required for Shh-mediated CGNP proliferation and that they form independent complexes with Ptch1. While Boc and Gas1 are essential components of these receptor complexes, they could function as partners of Ptch1, but not necessarily as receptors that bind to Shh. To determine whether the binding of Shh to Gas1 and/or Boc (and Cdon) is required for a Shh response, we generated a mutant Shh protein unable to bind Boc/Cdon/Gas1 but retaining the ability to bind Ptch1. If this mutant Shh molecule with altered specificity no longer activates signaling, it would suggest that Shh binding to Boc/Cdon/Gas1 is required for pathway activation. Conversely, if this mutant form of Shh activates the pathway, it would support a model where binding to Ptch1 alone is sufficient for Shh signaling.
The amino acids responsible for mediating the interaction between Shh and Boc/Cdon have been identified from co-crystal structures of Shh and the third FNIII domain of Cdon and Boc () (Kavran et al., 2010
; McLellan et al., 2008
). Although similar structural data is unavailable for Shh in complex with Ptch1, mutagenesis of Shh surface amino acids has identified residues required and residues dispensable for the binding of Shh to Ptch1 () (Bosanac et al., 2009
). Since Shh E90 is a contact amino acid between Shh and Cdon/Boc (McLellan et al., 2008
) that is not required for binding to Ptch1 () (Bosanac et al., 2009
), we predicted that a mutation at this site might affect binding to Boc and Cdon, but not to Ptch1. In contrast, Shh R154 is a contact amino acid between Shh and Boc/Cdon (McLellan et al., 2008
) that is also required for Ptch1 binding () (Bosanac et al., 2009
); thus, a R154 mutation is expected to affect binding of Shh to Boc, Cdon and Ptch1.
A Shh mutant which binds Ptch1 but which fails to bind Boc, Cdon and Gas1 does not induce Shh signaling
We introduced mutations of these residues into alkaline-phosphatase (AP)–tagged ShhN (ShhN-AP) and tested their binding to Boc, Cdon, Gas1, and Ptch1 ( and Table S1
). Consistent with our structural predictions, ShhN-AP R154E was unable to bind to Boc, Cdon and Ptch1. Also in agreement with our predictions, ShhN-AP E90A did not bind to Boc and Cdon, but retained the ability to bind Ptch1, with a dissociation constant not signficantly different (p>0.05) from that of WT ShhN ( and Table S1
). We also assessed the binding of our Shh mutants to Gas1 and found that they behaved similarly towards Gas1 as they did with Boc and Cdon: ShhN-AP E90A and R154E were both unable to bind Gas1. Thus, according to the binding characteristics of our Shh mutants, some common amino acids may mediate the interaction of Shh with Gas1, Boc, and Cdon, a finding consistent with previous reports (Kavran et al., 2010
; McLellan et al., 2008
We next examined the effect of the E90A and R154E mutations on Shh signal transduction. We introduced these mutations into untagged ShhN and recombinant proteins were purified (). To measure the signaling activity of the ShhN E90A and R154E mutants, we performed transcription reporter assays using cells stably transfected with a Gli-luciferase reporter plasmid. While WT ShhN activated Shh-mediated transcription in a concentration-dependent manner, ShhN E90A and R154E mutants were unable to do so (). We next tested the ability of our Shh mutants to promote the osteoblastic differentiation of C3H 10T½ cells and neither ShhN E90A nor R154E were able to induce alkaline phosphatase expression, a marker of differentiation (). Finally, we assayed the ability of these altered-specificity Shh ligands to induce CGNP proliferation. We found that while WT ShhN activated Shh-mediated proliferation in a dose-dependent manner, both ShhN E90A and R154E mutants were unable to induce proliferation (). Together, these data show that ShhN E90A, which interacts with Ptch1 but not with Boc, Cdon, and Gas1, fails to induce Shh signaling and Shh-dependent cellular responses. This indicates that binding of Shh to Ptch1 alone is not sufficient to activate Shh signaling, suggesting that binding to Boc, Cdon, or Gas1 cell surface proteins is absolutely required for Shh-dependent signal transduction to occur.