STEM CELLS AND REGENERATION RESEARCH ARTICLE 4335

Development 140, 4335-4346 (2013) doi:10.1242/dev.099754 © 2013. Published by The Company of Biologists Ltd The G -coupled receptor GPRC5B contributes to neurogenesis in the developing mouse neocortex Nobuhiro Kurabayashi1, Minh Dang Nguyen2 and Kamon Sanada1,*

SUMMARY Neural progenitor cells in the developing brain give rise to neurons and glia. Multiple extrinsic signalling molecules and their cognate membrane receptors have been identified to control neural progenitor fate. However, a role for G protein-coupled receptors in cell fate decisions in the brain remains largely putative. Here we show that GPRC5B, which encodes an orphan G protein-coupled receptor, is present in the ventricular surface of cortical progenitors in the mouse developing neocortex and is required for their neuronal differentiation. GPRC5B-depleted progenitors fail to adopt a neuronal fate and ultimately become astrocytes. Furthermore, GPRC5B- mediated signalling is associated with the proper regulation of β-catenin signalling, a pathway crucial for progenitor fate decision. Our study uncovers G protein-coupled receptor signalling in the neuronal fate determination of cortical progenitors.

KEY WORDS: G protein-coupled receptor, Developing neocortex, Neurogenesis

INTRODUCTION GPCR/G-protein-mediated signalling cascades in neural During development of the mammalian neocortex, cortical development are now being uncovered. For instance, perturbations progenitor cells located in the ventricular zone generate both of G and non-receptor type regulators for G proteins have neurons and glia. Prior to the neurogenic phase, most progenitor been shown to affect the biology of neural progenitors in the cells self-renew to expand the progenitor pool. As corticogenesis developing neocortex (Shinohara et al., 2004; Sanada and Tsai, proceeds, progenitor cells predominantly divide asymmetrically to 2005; Konno et al., 2008; Postiglione et al., 2011). Moreover, self-renew and to produce either a neuron or an intermediate mutations in GPR56, which encodes an orphan GPCR, are progenitor cell that undergoes additional symmetric divisions to associated with a human brain malformation called bilateral generate neurons (Chenn and McConnell, 1995; Miyata et al., 2001; frontoparietal polymicrogyria (Piao et al., 2004; Li et al., 2008). Noctor et al., 2001; Noctor et al., 2004; Miyata et al., 2004). The Also, exogenous (LPA) exposure in an ex vivo neurogenic phase is followed by the gliogenic phase during which cerebral cortical explant results in decreased apoptosis, an increased cortical progenitors give rise to glial progeny (Schmechel and number of mal-positioned M-phase cells, an increased number of Rakic, 1979; Voigt, 1989; Malatesta et al., 2000; Marshall and neurons and eventually enhanced growth of the explant through Goldman, 2002; Noctor et al., 2004). The precise control of LPA1 and LPA2 GPCRs (Kingsbury et al., 2003; Kingsbury et al., progenitor cell fate is key in yielding the correct number of neurons 2004). These observations implicate GPCR signalling in multiple and glia during corticogenesis. aspects of neural progenitor biology. Although many major Fate decision instructed in cortical progenitor cells involves the membrane receptor-mediated signalling pathways have been action of both environmental and intrinsic cues (Edlund and Jessell, implicated in progenitor cell fate decision, the participation of 1999; Qian et al., 2000). Recently, many environmental signalling GPCRs in this process remains unclear. molecules (e.g. Notch ligands, Wnts, fibroblast growth factor, In the present study, we found that GPRC5B, an orphan GPCR, platelet-derived growth factor and cytokines) and their cognate is predominantly expressed in neural progenitors in the developing receptors have been identified to affect cell fate during mouse brain and that GPRC5B-depleted progenitors fail to adopt a corticogenesis (reviewed by Guillemot, 2007). Importantly, these neuronal fate. Further, GPRC5B-mediated signalling is coupled to signalling pathways often cross-talk and their integration determines the heterotrimeric G proteins G12/13 and affects β-catenin proper cell fate. Thus, uncovering novel signalling pathways that signalling, which is important for the neuronal differentiation of affect progenitor cell fate constitutes an important step in our progenitors during the neurogenic phase. Together, the present study understanding of the complex mechanisms underlying identified a GPCR that is required for neurogenic potential in neural neurogenesis. progenitors and provided evidence that GPCR-mediated signalling, G protein-coupled receptors (GPCRs) constitute the largest through cross-talk with β-catenin signalling pathways, contributes family of membrane receptors. Most GPCRs transduce extracellular to the neuronal differentiation of neural progenitors. signals into cells via activation of cognate heterotrimeric G proteins. MATERIALS AND METHODS Plasmids and in situ hybridisation 1Molecular Genetics Research Laboratory, Graduate School of Science, The The plasmid expressing GFP under the control of the CAG promoter University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan. 2Hotchkiss (pCAGIG) was a kind gift from Takahiko Matsuda (Kyoto University, Brain Institute, University of Calgary, Departments of Clinical Neurosciences, Cell Kyoto, Japan). Plasmids encoding pertussis toxin (PTX), Gα12QL, Gα-CTs Biology and Anatomy, Biochemistry and Molecular Biology, HMRB 153, 3330 and TOPdGFP-CAGmCherry were generous gifts from Randall T. Moon Hospital Drive NW, Calgary, Alberta, Canada, T2N 4N1. (University of Washington, WA, USA), Hiroshi Itoh (Nara Institute of *Author for correspondence ([email protected]) Science and Technology, Nara, Japan), Yoh Takuwa (Kanazawa University, Ishikawa, Japan) and Anjen Chenn (University of Illinois, IL, USA),

Accepted 19 August 2013 respectively. pBS/U6 plasmid and mouse Gprc5b subcloned into the Development 4336 RESEARCH ARTICLE Development 140 (21) pCDNA3 plasmid were generously provided by Yang Shi (Harvard Medical Cell culture, transfection and cell-based analyses School, MA, USA) and Yuko Harada (Kobe University, Kobe, Japan), HEK293T cells on sterile coverslips in the wells of 24-well plates were respectively. transiently transfected using Lipofectamine 2000 (Invitrogen). The full-length open reading frame of mouse Gprc5b was subcloned into For analysis of the subcellular distribution of β-catenin in Vero cells, cells a pCAGEN plasmid (a kind gift from Takahiko Matsuda) that directs on sterile coverslips in the wells of 24-well plates were transiently subcloned expression from the CAG promoter. A plasmid encoding a transfected with plasmids encoding GFP (pEGFP-C1, Clontech; 0.1 silent mutant of GPRC5B was generated by the QuikChange mutagenesis μg/well), GPRC5B/pCDNA3 (0.3 μg/well) and Gα12QL (0.025 μg/well) technique using primers 5Ј-GGATGGGTCTTTGTGATTTTCCATGCCA- using FuGENE 6 (Roche). Cultures were fixed 48 hours later and subjected TTCC-3Ј (forward) and 5Ј-GGAATGGCATGGAAAATCACAAAG - to immunostaining. For analysis of the subcellular distribution of β-catenin in cortical progenitor cells, E14 embryos were electroporated with plasmids ACCCATCC-3Ј (reverse). Gprc5b subcloned into the pBluescript II KS(–) encoding GFP (pCAGIG; 5 μg/μl), GPRC5B (5 μg/μl), and Gα12-CT (10 vector was used for generating RNA probes, and in situ hybridisation was μg/μl). Following electroporation, neocortical cells were prepared (Sanada carried out as described (Asada et al., 2007). Plasmids encoding GPRC5B and Tsai, 2005), plated at a density of ~2.0×106 cells on sterile coverslips shRNA were generated by inserting the annealed oligonucleotides into a precoated with poly-ornithine and fibronectin in wells of 24-well plates and pBS/U6 plasmid (Sui et al., 2002). Oligonucleotides were as follows: maintained in DMEM/F12 supplemented with N2, B21 and bFGF (FGF2) Ј GPRC5B shRNA, 5 -GGTCTTTGTCATCTTCCATGAAGCTTCATGGA- (20 ng/ml). Neocortical cells were fixed at 2 days in vitro (DIV2) with 2% Ј Ј AGATGACAAAGACCCTTTTTG-3 and 5 -AATTCAAAAAGGGT - paraformaldehyde in PBS for 20 minutes at 37°C after 4 hours of treatment CTTTGTCATCTTCCATGAAGCTTCATGGAAGATGACAAAGACC-3Ј; with MG132 (25 μM), permeabilised with 0.5% Triton X-100 in PBS for GPRC5B shRNA#2, 5Ј-GGCTTTTCCAATGGAAGCTTAAGCTTAA - 15 minutes, and blocked with 3% BSA/0.1% Triton X-100 in PBS, followed GCTTCCATTGGAAAAGCCCTTTTTG-3Ј and 5Ј-AATTCAAAAA - by immunostaining. GGGCTTTTCCAATGGAAGCTTAAGCTTAAGCTTCCATTGGAAA- For analysis of the identity of GPRC5B-depleted cells in vitro (Fig. 4), AGCC-3Ј. neocortical cells were prepared by incubating neocortices with papain in Hank’s balanced salt solution, plated at a density of 0.8×106 cells on sterile In utero electroporation coverslips precoated with poly-D-lysine in wells of 24-well plates, and In utero electroporation was performed as described previously (Sanada maintained in DMEM/F12 supplemented with N2, B27 and 10% FBS. At and Tsai, 2005). Concentrations of plasmids were: GFP-expressing plasmid DIV6, cultures were fixed and subjected to immunostaining. (pCAGIG: 5 μg/μl for embryonic day (E) 11/E13 and 2.5 μg/μl for E14), GPRC5B shRNA plasmid (5 μg/μl), plasmid expressing silent mutant RT-PCR GPRC5B (1.5 μg/μl), control shRNA plasmid (pBS/U6: 5 μg/μl) and Total RNA (1 μg) was reverse-transcribed with SuperScript II (Invitrogen) TOPdGFP-CAGmCherry (5 μg/μl). Animal experiments were conducted using oligo(dT)16 primer. cDNAs were then subjected to PCR analysis (35 in accordance with guidelines set by The University of Tokyo. cycles) using the primers listed in supplementary material Table S1. β-catenin activity assay GPRC5B antibody production and immunostaining E14 embryos were electroporated with a dual reporter plasmid, TOPdGFP- Antibody against GPRC5B was generated by Sigma Genosis as described CAGmCherry, together with various additional plasmids. Embryos were below. Synthetic peptides corresponding to amino acids Gly395-Trp410 of harvested 24 hours later. Brain cryosections (20 μm) were immunostained GPRC5B were conjugated to keyhole limpet haemocyanin and used to with antibodies against PAX6 and GFP. Images of GFP and mCherry in immunize rabbits. GPRC5B antibody was affinity-purified from the serum individual sections were obtained under identical parameters with a 40× by a column coupled with synthetic peptides Gly395-Trp410 (for western objective (EC Plan-Neofluar, Zeiss) on a Zeiss LSM5 confocal microscope. blotting and immunostaining of cultured cells) or Pro401-Trp410 (for Mean fluorescence intensity of mCherry and GFP in the soma of individual immunohistochemistry of brain sections). cells was measured using Zeiss Zen2010 software. In addition, the average Brains were fixed with 4% paraformaldehyde in PBS for 30 minutes (for background fluorescence intensity was measured in individual sections E14-18 brains) or 2 hours (for postnatal brains) at room temperature. Thick using areas with no electroporated-cells present and was subtracted from cryosections (20 μm for E15-18 brains; 30 μm for postnatal brains) were the mCherry and GFP intensity measurements. Cells with saturated signal prepared. For immunostaining of postnatal day (P) 14 brain sections with were not used for the analysis. The ratio of fluorescence intensity of GFP anti-GPRC5B, brain sections (40 μm) were prepared with a vibratome. For to that of mCherry in individual cells was defined as the β-catenin activity NeuN (RBFOX3 – Mouse Genome Informatics) staining, brain sections index. were pretreated with a Tris/EDTA solution (10 mM Tris, 1 mM EDTA, pH 9.0) at 70°C for 15 minutes. For staining for CUX1, GFAP and S100, brain Statistical analysis sections were pretreated with HistoVT One solution (Nakalai Tesque) at All data are shown as mean ± s.e.m. Statistical analyses were carried out 70°C for 15 minutes. Immunohistochemistry/immunostaining were with Excel (Microsoft). Comparison of two groups was analysed using a performed as described (Sanada and Tsai, 2005). Primary antibodies were two-tailed t-test. P<0.05 was considered statistically significant. rabbit anti-GPRC5B (1:100-1:1000), mouse anti-NeuN (1:1000; Chemicon), rabbit anti-GFAP (1:2000; Sigma-Aldrich), rabbit anti-GFP RESULTS (1:1000; Invitrogen), rat anti-GFP (1:2000; Nakalai Tesque), rabbit anti- Expression of GPRC5B in cortical progenitors of MAP2 (1:100; Millipore), rabbit anti-PAX6 (1:5000; Covance), rabbit anti- the developing neocortex CUX1 (1:200; Santa Cruz Biotechnology), rat anti-PDGFRα (1:800; BD Pharmingen), rabbit anti-S100 (1:200; DAKO), rabbit anti-cleaved caspase GPRC5 receptors, such as GPRC5A, GPRC5B and GPRC5C, are 3 (1:100; Cell Signaling), rabbit anti-TBR1 (1:1000; Abcam), goat anti- orphan GPCRs. GPRC5A was initially identified by differential SOX2 (1:200; Santa Cruz Biotechnology), mouse anti-S100β (1:1000; display analysis of a small lung carcinoma cell line and is expressed Sigma-Aldrich), mouse anti-pan cadherin (1:200; Sigma-Aldrich), mouse almost exclusively in the human lung (Cheng and Lotan, 1998). anti-myc (9E10, 1:200; Santa Cruz Biotechnology), mouse anti-TUJ1 GPRC5B and GPRC5C are mainly expressed in the central nervous (TUBB3 – Mouse Genome Informatics) (1:5000; Covance), rabbit anti- system and peripheral tissues, respectively (Robbins et al., 2000). To TBR2 (1:500; Chemicon) and mouse anti-β-catenin (1:100; BD examine the expression of these receptors in neural progenitor cells Transduction Laboratories). The sections/cells were mounted in Prolong in the developing mouse brain, PCR analysis of cDNAs derived Gold Mounting Solution (Invitrogen). Images were obtained with a Zeiss from cultured progenitor cells, E16 cortices, adult brains and lungs

LSM5 confocal microscope. was performed. In agreement with previous reports (Cheng and Development Role of GPRC5B in neurogenesis RESEARCH ARTICLE 4337

Fig. 1. GPRC5B expression in the developing mouse brain. (A) Expression of Gprc5a, Gprc5b and Gprc5c was analysed by RT-PCR using cDNAs derived from mouse cortical progenitor cells and the tissues indicated. (B) Expression of Gprc5b and Gprc5b_v2 in the neocortex at different developmental stages. (C,D) In situ hybridisation analyses with Gprc5b probes. (C) Coronal section of E10 embryo (left) and magnified view of the head region (right). (D) E14 brain sections probed with sense (left) and antisense (right) RNA. (E) E14 neocortical sections immunostained with anti- GPRC5B (green) and anti-pan cadherin (red) antibodies. Images of the entire immunostained cerebral wall are shown. (F) High-magnification images of apical regions of GFP plasmid-electroporated (left four panels) and GFP plasmid + GPRC5B shRNA-electroporated (right four panels) progenitors in E14 neocortical sections immunostained with antibodies against GPRC5B and cadherin. GFP-labelled descending processes are outlined with dashed lines. Insets are magnified views of the apical region of the GFP-labelled processes. FB, forebrain; V, ventricle; LV, lateral ventricle; SC, spinal cord. Scale bars: 100 μm in E; 20 μm in F.

Lotan, 1998), Gprc5a was detected in the lung, but not in the brain respectively (supplementary material Fig. S1; see also Fig. 6A). (Fig. 1A). By contrast, Gprc5b and Gprc5c were detected in the When E14 neocortical sections were immunostained, GPRC5B adult brain. Importantly, Gprc5b, but not Gprc5a and Gprc5c, was immunofluorescent signals were mainly detected at the luminal expressed in E16 cortex and by cultured cortical progenitors. surface of the ventricular zone (VZ) (Fig. 1E). Almost no GPRC5B GPRC5B possesses a brain-specific C-terminal splice variant immunoreactivity was observed in sections immunostained with (GPRC5B_v2) that differs in C-terminal 14 amino acids (Cool et antibody preincubated with its immunogen (supplementary material al., 2010). PCR analysis showed that Gprc5b_v2 was hardly Fig. S2). Detailed analyses of GFP-labelled progenitors revealed detectable in the neocortex during embryonic and perinatal periods that GPRC5B immunofluorescent signals were punctate and present but was expressed in the adult neocortex. These results are near the apical surface of the descending processes (Fig. 1F). In consistent with previous reports showing that the mRNA and protein addition, these signals were detected in close proximity to cadherin, levels of GPRC5B_v2 in the brain are detected at ~P14-15 and peak which is highly concentrated at adherens junctions formed by at adulthood (Cool et al., 2010; Sano et al., 2011). Thus, GPRC5B, descending processes of neighbouring progenitors (Chenn et al., but not GPRC5B_v2, is the predominant form expressed in the 1998). The specificity of the antibody was also confirmed in developing neocortex. neocortical sections that were electroporated with the GPRC5B In situ hybridisation analysis using an antisense probe revealed shRNA-expressing plasmid (see below). In GPRC5B shRNA cells, strong expression of Gprc5b mRNA at proliferative regions cadherin was normally detected near the luminal surface of the VZ, surrounding the ventricles of E10 and E14 mouse forebrain whereas GPRC5B immunofluorescent signals were strikingly (Fig. 1B-D). To localise GPRC5B protein in the developing diminished (Fig. 1F). Together, these results indicate that GPRC5B neocortex, an affinity-purified rabbit anti-GPRC5B antibody was is preferentially expressed in cortical progenitors of the developing generated using its C-terminal region as the immunogen (see brain, particularly at the luminal surface of progenitors. Materials and methods). The antibody specifically detected We also examined the localisation of GPRC5B at P14. Intense GPRC5B transiently expressed in HEK293T and COS7 cells, as GPRC5B immunoreactivity was detected at the apical surface of

assessed by western blotting and immunocytochemistry, cells lining the lateral ventricle, which were positive for S100β, an Development 4338 RESEARCH ARTICLE Development 140 (21) antigen that is highly expressed in ependymal cells (supplementary confirming the efficacy of the shRNA in vivo. In addition, the radial material Fig. S3). GPRC5B was not detected in SOX2-positive cells polarity of progenitor cells appears to be preserved following in the subependymal zone and in cells positive for GFAP, a marker GPRC5B depletion, as the normal apical localisation of cadherin for astrocytes and subventricular zone (SVZ) progenitors (Mirzadeh was retained (Fig. 1F). et al., 2008). These data indicate the expression of GPRC5B in In another set of experiments, we introduced GPRC5B shRNA or ependymal cells in the postnatal brain. control shRNA construct, together with the GFP-expressing plasmid, into the VZ of E13 mouse brains by in utero GPRC5B knockdown inhibits the generation of electroporation. At E15 neocortical sections were prepared and neurons immunostained with an antibody against PAX6, a marker for To investigate a putative role for GPRC5B in cortical progenitors in neocortical progenitors. In GPRC5B shRNA-electroporated the developing neocortex, we examined the effects of GPRC5B neocortices, a smaller fraction of GFP-labelled cells remained as knockdown on cell fate. For this purpose, DNA-based RNAi undifferentiated PAX6-positive progenitors when compared with plasmids that express shRNA against Gprc5b were generated. the fraction found in control shRNA-expressing neocortices GPRC5B transiently expressed in HEK293T cells was efficiently (14.2±1.0% versus 23.1±0.7%, respectively; n=4 embryos, silenced by the GPRC5B shRNA construct. By contrast, the P<0.001) (Fig. 2A). These results suggest that GPRC5B expression of mutant GPRC5B, with two silent mutations within knockdown slightly promotes the differentiation of cortical the target sequence of the GPRC5B shRNA, was not affected by progenitors. Of note, following GPRC5B shRNA electroporation the shRNA (supplementary material Fig. S1). When GPRC5B we did not observe an increase in the number of cleaved caspase 3- shRNA was electroporated into the VZ of E13 embryos, levels of positive cells (data not shown). This result indicates that apoptosis GPRC5B were very low or barely detectable near the luminal did not account for the reduction in progenitors caused by GPRC5B surface of the shRNA-transfected progenitors (Fig. 1F), thereby depletion.

Fig. 2. GPRC5B knockdown promotes progenitor differentiation. Plasmid expressing either GPRC5B shRNA (right) or control shRNA (left) was electroporated, together with the GFP-expressing plasmid, into E13 embryos. E15 brain sections were immunostained with antibodies against (A) PAX6, (B) TBR2 or (C) CUX1. Images of the entire cerebral wall are shown, with the boxed regions magnified to the right. Images are projections of four serial z-sections (1 μm intervals) for PAX6/TBR2 and two z-sections (1 μm interval) for CUX1. The graphs on the right show the fraction of cells positive for PAX6 (A), TBR2 (B) or CUX1 (C) among GFP-positive cells. Mean ± s.e.m. (n=3-5). *P<0.05, ***P<0.001. Con, control shRNA; RNAi, GPRC5B shRNA. Scale bars: 20 μm for entire cerebral wall; 10 μm in magnified views. CP, cortical plate; IZ, intermediate zone; VZ, ventricular zone. Development Role of GPRC5B in neurogenesis RESEARCH ARTICLE 4339

To determine whether the neuronal differentiation of progenitors shRNA cells were positive for MAP2 (<5%) and CUX1 is promoted upon GPRC5B knockdown, we assessed the production (26.8±2.0%; n=3 embryos, P<0.001) (Fig. 3A,B). In addition, GFP- of intermediate/basal progenitors by immunohistochemistry using labelled cells in the IZ were negative for PAX6 (Fig. 3C). These an antibody to TBR2 (EOMES – Mouse Genome Informatics), a phenotypes (i.e. decreased CUX1-positive cells, mislocalisation of specific marker for intermediate progenitors that are destined to GFP-labelled cells in the IZ) were recapitulated with a second become neurons (Englund et al., 2005). Remarkably, TBR2-positive shRNA construct (GPRC5B shRNA#2) (supplementary material populations of GFP-labelled cells in GPRC5B shRNA brains were Figs S1, S4), confirming that the observed phenotypes are specific decreased as compared with control shRNA brains (Fig. 2B). In to GPRC5B. Taken together, these results indicate that GPRC5B- addition, whereas the majority of GFP-labelled cells in control depleted progenitors, rather than remaining undifferentiated, shRNA brains were positive for CUX1 (Fig. 2C), which is a marker differentiate into non-neuronal cells. for both progenitors and neurons destined for layers 2-4 (Nieto et al., To confirm that impaired neurogenesis results specifically from 2004; Shen et al., 2006), a significantly smaller fraction of GPRC5B GPRC5B knockdown, we performed a rescue experiment in which shRNA cells showed detectable levels of CUX1 (Fig. 2C). Thus, GPRC5B shRNA was co-expressed with a mutant GPRC5B that has introduction of GPRC5B shRNA into progenitors impairs two silent mutations within the GPRC5B shRNA target sequence neurogenesis in the developing neocortex. (supplementary material Fig. S1). Co-expression of shRNA- To assess the long-term effects of GPRC5B knockdown on insensitive GPRC5B almost completely reversed the decrease in neurogenesis in vivo, E18 cortices electroporated at E14 were CUX1-positive cells caused by GPRC5B shRNA (supplementary immunostained with antibodies against neuronal markers. Almost material Fig. S4C,D). Thus, impaired neurogenesis in GPRC5B all GPRC5B shRNA cells were abnormally located in the shRNA progenitors is caused by loss of GPRC5B function. intermediate zone (IZ), instead of being correctly positioned in the To further support the notion that GPRC5B is crucial for neuronal cortical plate (CP) (Fig. 3). Whereas the vast majority of control differentiation, cells electroporated with GPRC5B shRNA/control shRNA cells were immunostained with MAP2 (>98%) and CUX1 shRNA at E14 were isolated from E16 neocortices, cultured until (76.2±2.1%; n=3 embryos), only a small fraction of GPRC5B DIV6 and immunostained with an antibody against the neuronal

Fig. 3. GPRC5B knockdown impairs neurogenesis. Plasmid expressing either GPRC5B shRNA or control shRNA was electroporated, together with the GFP-expressing plasmid, into E14 embryos. E18 brain sections were immunostained with antibodies against (A) MAP2, (B) CUX1 or (C) PAX6. Images of the entire cerebral wall are shown. Magnified views of the CP or IZ regions (boxed areas) are shown to the right. Con, control shRNA; RNAi, GPRC5B shRNA. Scale bars: 20 μm. Development 4340 RESEARCH ARTICLE Development 140 (21)

Fig. 4. GPRC5B-depleted cells fail to adopt a neuronal fate. (A) Plasmid expressing control shRNA, GPRC5B shRNA, GPRC5B shRNA#2 and both GPRC5B shRNA and GPRC5Bres (GPRC5B that contains two silent mutations within the GPRC5B shRNA-targeted sequence) was electroporated, together with the GFP-expressing plasmid, into E14 embryos. Neocortical cell cultures were prepared from E16 embryos and immunostained at DIV6 with antibodies against TUJ1 and GFAP. (B) Magnified views of the cells indicated by arrowheads a-h in A. (C) The fraction of GFP-positive cells that were also positive for GFAP (left) or TUJ1 (right). **P<0.01, ***P<0.001. Mean ± s.e.m. Scale bars: 50 μm in A; 20 μm in B.

marker TUJ1. More than 90% of control cells were TUJ1 positive, GPRC5B knockdown cells ultimately become whereas a smaller population of GPRC5B shRNA cells were TUJ1 astrocytes positive (Fig. 4). Surprisingly, a significantly larger population of We then examined the fate of GPRC5B shRNA-electroporated cells GPRC5B-depleted cells was positive for GFAP (GPRC5B shRNA, in the postnatal period. The control or GPRC5B shRNA construct 51±0.7%; GPRC5B shRNA#2, 32±4%; control shRNA, 5.9±1.4%; was electroporated into E14 embryos and the pups were harvested n=3 embryos). In addition, almost all of the cells were negative for at P14 or P27. In P14 control brains, almost all GFP-labelled cells PDGFRα, an oligodendrocyte lineage marker (data not shown). were located in layers 2-4 and were positive for the mature neuronal There was no significant difference in the percentage of apoptotic marker NeuN (Fig. 5A,C). By contrast, at P14 ~50% of GPRC5B cells between control and GPRC5B knockdown GFP-labelled cells knockdown cells were located within or near the SVZ, with the (<2.6%). Importantly, the increase in the GFAP-positive population remaining cells found in the lower part of the CP (Fig. 5A, among GFP-labelled cells upon GPRC5B depletion was almost arrowheads). Importantly, 66.0±3.3% (n=3 embryos) of the cells in completely reversed by co-expression of the GPRC5B mutant the CP displayed the bushy morphology that is reminiscent of insensitive to GPRC5B shRNA (Fig. 4). These results indicate that mature astrocytes (Fig. 5B,C). Immunohistochemical analysis GPRC5B is required for the proper neuronal differentiation of confirmed that almost all bushy cells were positive for the mature progenitors and that GPRC5B depletion generates cells with the astrocyte markers GFAP and S100 (Fig. 5D,E), but negative for

potential to differentiate into astrocytes in vitro. NeuN (Fig. 5C) and PDGFRα (supplementary material Fig. S5). Of Development Role of GPRC5B in neurogenesis RESEARCH ARTICLE 4341

Fig. 5. GPRC5B knockdown generates astrocytes. Plasmid expressing either GPRC5B shRNA or control shRNA was electroporated, together with the GFP-expressing plasmid, into E14 embryos, and embryos were harvested at P14. (A) Representative images of GFP-labelled cells throughout the entire cerebral wall in brains electroporated with control shRNA (left) and GPRC5B shRNA (middle and right). Arrowheads indicate GFP-labelled bushy cells. (B) Typical morphology of GFP-labelled cells in the CP and SVZ regions in GPRC5B shRNA neocortices. Shown are cells with bushy morphology in the CP (top), cells that do not have bushy morphology in the CP (middle), and cells in the SVZ region (bottom). (C) GFP-labelled cells in electroporated neocortices immunostained with anti-NeuN antibody. Shown are high-magnification images of GFP-labelled cells in control shRNA brain (row 1) and GPRC5B shRNA brains (row 2, cells with bushy morphology; row 3, cells without bushy morphology; row 4, cells in the SVZ region). (D,E) GFP-labelled cells in GPRC5B shRNA neocortices immunostained with antibodies against GFAP (D) and S100 (E). High-magnification images of GFP-labelled cells with bushy morphology are shown. WM, white matter; SVZ, subventricular zone. Scale bars: 100 μm in A; 20 μm in C-E. note, the vast majority of GFP-labelled cells without bushy shRNA (GFAP, 7.5±2.4%; NeuN, 85±6.1%; n=6 embryos; morphology in the SVZ and CP were negative for GFAP, NeuN and supplementary material Fig. S7). Together with the fact that almost PDGFRα (Fig. 5C; supplementary material Fig. S5; data not all GFP-labelled cells in control shRNA cortices were positive for shown). Collectively, among the GPRC5B-depleted cell NeuN (>96%) and negative for GFAP (>99%), we conclude that a populations, we found a very small fraction positive for NeuN significantly large population of cells derived from GPRC5B- (6.2±3.1%, n=3 embryos) and a much larger fraction positive for depleted progenitors fail to adopt a neuronal fate and instead GFAP (29.4±12.8%, n=3 embryos) and S100 (29.2±12.9%, n=3 ultimately become astrocytes. embryos). Moreover, in P27 brains, ~50% of GPRC5B-depleted To further support this conclusion, we performed a clonal analysis cells were positive for GFAP (49.9±0.3%, n=3 embryos), 22% were of GPRC5B-depleted progenitor cells (supplementary material Fig. positive for NeuN (22.5±4.0%, n=4 embryos) and none were S8). GFP-labelled progenitor cells electroporated with either control positive for PDGFRα (supplementary material Fig. S6). Finally, the shRNA or GPRC5B shRNA at E14 were prepared at E15 and increase in the GFAP-positive cell population upon GPRC5B cultured at clonal density. The cellular composition of the GFP- depletion observed at P27 was consistently reversed by co- labelled clones was analysed at DIV5. GFP-labelled control cells

expression of the GPRC5B mutant insensitive to the GPRC5B gave rise to 56±4.0% neuronal clones, 11±1.0% astroglial clones Development 4342 RESEARCH ARTICLE Development 140 (21) and 22±6.0% mixed clones (containing neurons and astrocytes). (Gilchrist et al., 2002). Based on this mechanism, we examined Depletion of GPRC5B by shRNA dramatically reduced the which G proteins mediate GPRC5B signalling by using C-terminal percentage of neuronal clones (41±3.9%) and increased the peptides of α-subunits (Gαs-CT, Gαq-CT, Gα12-CT and Gα13-CT) percentage of astroglial clones (30.0±2.7%). Expression of the (Sugimoto et al., 2003) as well as pertussis toxin, which inactivates GPRC5B insensitive to GPRC5B shRNA almost completely Gαi/o via the specific catalysis of ADP-ribosylation of the α- abolished the increase in astroglial clones. Importantly, the average subunit. When either Gαs-CT or Gαq-CT was co-transfected with size of total clones and astroglial clones was not significantly GPRC5B, no effect on GPRC5B-induced cell rounding was different between GPRC5B shRNA and control cultures (GPRC5B observed (Fig. 6A,B). Likewise, co-transfection of the plasmid shRNA: total 4.0±0.8 cells/clone, astroglial 3.3±0.2 cells/clone; encoding pertussis toxin did not affect cell morphology. By contrast, control: total 4.1±0.2 cells/clone, astroglial 4.0±0.3 cells/clone). GPRC5B-induced cell rounding was largely prevented by co- These results indicate that the increased number of astrocytes upon expression of Gα12-CT or Gα13-CT (Fig. 6A). When plasmid GPRC5B depletion in vivo (Fig. 5) is unlikely to be due to enhanced encoding pertussis toxin or Gα-CTs was solely transfected into cell proliferation. Of note, the percentage of apoptotic cells was not COS7 cells, the morphology of the transfected cells was normal significantly changed upon GPRC5B depletion (1.3±0.2% versus (data not shown). These results indicate that GPRC5B couples with 0.8±0.4% in control and GPRC5B shRNA, respectively). Taken the G12/13 class of heterotrimeric G proteins. together, these results strengthen the idea that GPRC5B depletion Wnt/β-catenin signalling has been shown to promote the neuronal generates cells with reduced neurogenic potential that are prone to differentiation of progenitors at later stages of corticogenesis differentiate into astrocytes. (Hirabayashi et al., 2004; Israsena et al., 2004). Interestingly, Gα12/13 signalling is linked to β-catenin signalling. Indeed, activated Gα12/13 β-catenin signalling is potentiated by GPRC5B is known to bind to the cytoplasmic tails of several members of the signalling cadherin family and to induce the dissociation of cadherin-bound β- Most GPCRs transmit signals through the activation of catenin, which in turn causes an increase in β-catenin-mediated heterotrimeric G proteins. G protein α-subunits are generally transcriptional activation (Meigs et al., 2001). These observations divided into four families (Gi/o, Gs, Gq/11 and G12/13) based on raise the hypothesis that β-catenin signalling might be one of the their sequence similarity. Overexpression of many of the GPCRs in downstream pathways elicited by GPRC5B-G12/13 in neural cells results in the activation of their cognate G proteins in a ligand- progenitors. In support of this, GPRC5B alters the subcellular independent manner (Milligan, 2003). We found that distribution of β-catenin in Vero cells (Fig. 7A,B). In mock- overexpression of GPRC5B induces significant rounding of COS7 transfected cells stained with β-catenin antibody, β-catenin signals cells (~80%) (Fig. 6A,B), a phenotype reminiscent of cells were predominantly localised at the cell cortex. Expression of expressing mutationally activated Gα12/13 (Kranenburg et al., GPRC5B induced a marked shift in β-catenin immunoreactivity: β- 1999; Meigs et al., 2005; Stemmle et al., 2006). Peptides catenin signals, which are normally found at the cell periphery, corresponding to C-terminal regions of Gα (Gα-CTs) can serve as adopted a more diffuse pattern throughout the cell (Fig. 7A,B). competitive blockers for GPCR-G protein signalling, as they Consistent with a previous report (Meigs et al., 2001), a similar selectively bind to the G protein-binding site on cognate GPCRs redistribution of β-catenin was induced upon expression of and suppress signal transduction through a given G protein mutationally activated Gα12 [Gα12(Q229L)] (Fig. 7A,B).

Fig. 6. GPRC5B couples with the G12/13 class of heterotrimeric G proteins. (A) COS7 cells were transiently transfected with plasmids encoding GFP (pCAGIG, 0.5 μg/well), GPRC5B (0.5 μg/well), pertussis toxin (PTX or PT; 0.5 μg/well) and myc epitope-tagged Gα-CTs (Gs, Gq, G12, and G13; 1.5 μg/well). Cells were fixed 48 hours later and then immunostained with antibodies against GFP (green), GPRC5B (blue) and myc epitope (red). Expression of GPRC5B induces cell rounding, whereas co-expression of either Gα12-CT or Gα13-CT prevents GPRC5B-induced rounding of the cells. (B) The fraction of GFP-labelled cells showing a rounded morphology was measured from at least four separate experiments (a total of 66-210 cells were counted in each

condition). Mean ± s.e.m. ***P<0.001 versus GPRC5B cells. Scale bar: 100 μm. Development Role of GPRC5B in neurogenesis RESEARCH ARTICLE 4343

Fig. 7. Effects of GPRC5B on the distribution of β-catenin. (A) Effects of GPRC5B on the distribution of β-catenin in Vero cells. Vero cells transiently transfected with plasmids expressing the indicated proteins were immunostained with anti-β-catenin antibody (red). Arrowheads indicate GFP-labelled cells that show increased cytoplasmic signals for β-catenin. (B) The β-catenin fluorescence intensity of the intracellular area (except for the plasma membrane area) of GFP- positive cells and that of neighbouring GFP- negative cells were measured, and the ratio of these intensity values is plotted (red circles). Yellow circles with error bars indicate mean ± s.e.m. ***P<0.001 versus control by two-tailed Welch’s t- test. (C) Effects of GPRC5B on the distribution of β- catenin in cortical progenitor cells. Plasmids expressing the indicated proteins were electroporated, together with the GFP-expressing plasmid, into E14 embryos. Neocortical cell cultures were prepared from the brains immediately after electroporation. Neocortical cells were then fixed at DIV2 and immunostained with antibodies against GFP (green), β-catenin (red) and SOX2 (blue). Representative images are shown. Green and white arrowheads indicate GFP-positive and GFP-negative dividing progenitors, respectively. Magnified views of the cells indicated by arrowheads a-f are shown to the right. (D) The β- catenin fluorescence intensity of the intracellular area (except for the plasma membrane area) of GFP-positive dividing cells and that of nearby GFP- negative dividing cells were measured, and the ratio of these intensity values is plotted as in B. Scale bars: 20 μm in A; 10 μm in C.

It is known that the expression of activated Gα12 causes a with observations made on Rat-1 fibroblast cells (Meigs et al., substantial increase in β-catenin-mediated transcription in SW480 2001), a slight but significant increase in β-catenin-mediated human colon carcinoma cells that lack functional APC, as well as a transcription was detected in Vero cells upon expression of slight increase in Rat-1 fibroblast cells (Meigs et al., 2001). We then GPRC5B as well as upon expression of activated Gα12 sought to examine whether the expression of GPRC5B in Vero cells (supplementary material Fig. S9). We also examined the distribution causes an increase in β-catenin-mediated transcription. We utilised pattern of β-catenin in cultured progenitor cells. In dividing a dual reporter construct (TOPdGFP-CAGmCherry) that drives the progenitor cells, intense β-catenin immunoreactivity was detected at expression of destabilised GFP (dGFP) under the control of a β- the cell cortex. Introduction of GPRC5B increased β-catenin catenin-responsive promoter (TOP, a TCF/LEF consensus site immunoreactivity in the cytosol of neural progenitors (Fig. 7C,D). upstream of a minimal c-Fos promoter) and directs mCherry This phenotype was prevented by co-expression of Gα12-CT expression through a constitutive CAG promoter (Mutch et al., (Fig. 7C,D), thereby confirming a potential role for GPRC5B- 2009). In this system, mCherry serves as a transfection control, and G12/13 signalling in the regulation of β-catenin. the ratio of dGFP to mCherry fluorescence intensity was used to To evaluate the role for GPRC5B in β-catenin signalling in vivo, quantify the β-catenin signalling levels in individual cells (Mutch et we electroporated the TOPdGFP-CAGmCherry reporter plasmid

al., 2009; Yokota et al., 2009; Woodhead et al., 2006). Consistent into E14 embryos and harvested the embryos 24 hours later. In Development 4344 RESEARCH ARTICLE Development 140 (21)

Fig. 8. Effects of GPRC5B knockdown on β-catenin signalling in neural progenitor cells. (A) E14 embryos were electroporated with plasmids expressing control shRNA (row 1), GPRC5B shRNA (row 2), GPRC5B shRNA#2 (row 3), and both GPRC5B shRNA and GPRC5Bres (GPRC5B that contains two silent mutations within the GPRC5B shRNA-targeted sequence) (row 4), along with the β-catenin activity reporter construct TOPdGFP-CAGmCherry. Embryos were harvested 24 hours later. Brain sections were stained with antibodies against GFP (green) and PAX6 (blue). Images of the VZ are shown. Two examples of mCherry+/PAX6+ cortical progenitors are indicated by white arrowheads. An example of mCherry+/PAX6– differentiated cells is indicated by the open arrowhead. Scale bar: 20 μm. (B) Magnified images of the cells indicated by arrowheads in A. mCherry+/PAX6+ cells are in the left six columns, and mCherry+/PAX6– cells are in the right three columns. The expression of dGFP in PAX6-positive control cells is substantially downregulated by GPRC5B depletion (compare row 1 with rows 2 and 3), and this is largely reversed by the introduction of GPRC5Bres (row 4). Less dGFP expression is observed in PAX6-negative cells. (C) The ratio of the fluorescence intensity of dGFP to that of mCherry in the soma of individual cells expressing mCherry was defined as the β-catenin activity index and is shown. Mean ± s.e.m. (n=3 embryos). *P<0.05, ***P<0.001. cortices electroporated with both TOPdGFP-CAGmCherry and converge with β-catenin signalling to promote the neuronal control shRNA plasmids, PAX6-positive cortical progenitors in the differentiation of progenitors. VZ showed significantly higher dGFP expression than PAX6- negative differentiated cells (Fig. 8). This is consistent with a DISCUSSION previous study showing that β-catenin signalling levels are The present study demonstrates that GPRC5B is selectively downregulated in differentiated cells (Woodhead et al., 2006). expressed in neural progenitor cells of the developing neocortex. Importantly, a marked suppression of dGFP expression was detected GPRC5B-depleted cortical progenitors give rise to cells that fail to in GPRC5B-depleted PAX6-positive progenitor cells (Fig. 8). In adopt a neuronal fate. Instead, they are prone to differentiate into addition, downregulation of β-catenin signalling in GPRC5B astrocytes as revealed by the analysis of cell type identity at E18, knockdown progenitors was significantly reversed by co-expression P14 and P27. In brief, our findings implicate GPCR-mediated of the mutant GPRC5B insensitive to GPRC5B shRNA (Fig. 8). signalling in the control of the neurogenic potential and cell fate of These results underscore a role for GPRC5B in regulating the β- cortical progenitors. catenin-mediated transcription that is important for the neuronal In the developing neocortex, β-catenin signalling plays multiple

differentiation of progenitors. Thus, GPRC5B signalling may roles in such as the neuronal differentiation and proliferation of Development Role of GPRC5B in neurogenesis RESEARCH ARTICLE 4345 cortical progenitors. At early stages of cortical development, β- its function in neural progenitor cells. It has been revealed that mice catenin signalling is essential for progenitor proliferation (Chenn with a conditional knockout for Gα12 and Gα13 in the nervous and Walsh, 2002; Zechner et al., 2003; Mutch et al., 2009; Mutch et system display neuronal ectopia in the developing neocortex (Moers al., 2010). At later stages of cortical development, β-catenin et al., 2008). This phenotype is mainly caused by the overmigration signalling contributes to neuronal differentiation (Hirabayashi et al., of neurons and is accompanied by a disrupted pial basement 2004; Israsena et al., 2004). It is well known that β-catenin is an membrane, the abnormal protrusion of radial processes of neural essential component of two distinct cellular systems: the Wnt progenitors into the ectopia and the displacement of Cajal-Retzius signalling pathway and the cadherin-based adherens junctions. The cells in the subarachnoid space (Moers et al., 2008). As the effects cadherin-bound pool of β-catenin is thought to serve as a reservoir of Gα12 and Gα13 knockout on progenitor fate were not studied by for signalling-competent β-catenin and to be functionally connected Moers and collaborators, our study reveals for the first time a to the Wnt/β-catenin signalling pathway (Barth et al., 1997; Nelson potential contribution of Gα12 and Gα13 in the neuronal and Nusse, 2004). It is also noteworthy that activated Gα12/13 can differentiation of progenitors. induce the dissociation of cadherin-bound β-catenin, which in turn In conclusion, our study sheds light on the roles of GPCR causes an increase in β-catenin-mediated transcriptional activation signalling in neurogenesis and fate determination. Our findings of (Meigs et al., 2001). Our study shows that GPRC5B is coupled with a novel GPRC5B-G12/13-β-catenin signalling pathway in Gα12/13 and that its signalling induces a redistribution of β-catenin progenitor cell fate regulation could provide unique insights into in cortical progenitor cells. Furthermore, GPRC5B depletion the complex mechanisms underlying neurogenesis. The requirement attenuates β-catenin-mediated transcriptional activation in the of GPCR-mediated signalling, which is a main target for drugs, for cortical progenitors of E14 neocortices in vivo. Thus, one of the the neurogenic potential of neural progenitors also has implications downstream actions of GPRC5B in progenitors is to reinforce β- in the context of novel avenues to manipulate stem cells with a view catenin signalling, probably by releasing cadherin-bound β-catenin. to therapeutic value. In support of this model, GPRC5B localises in close proximity to cadherin at adherens junctions of progenitors. Potential cross-talk Acknowledgements We thank So-ichi Tamai and Naoyuki Asada for critical reading of the between GPRC5B and Wnt/β-catenin signalling is also supported manuscript; and Drs Anjen Chenn, Takahiko Matsuda, Randall T. Moon, Hiroshi by the fact that GPRC5B physically associates with the Wnt Itoh, Yuko Harada, Yang Shi and Yoh Takuwa for plasmids. receptor in HeLa cells (Harada et al., 2007). In E12 brains electroporated at E11, the fate of GPRC5B-depleted Funding cells was not altered in terms of PAX6-positive neural progenitor This work was supported in part by Grant-in-Aid for Young Scientists (B) (to N.K.), Scientific Research (B) (to K.S.) and Scientific Research on Innovative populations (42.1±1.4% versus 38.9±4.4% in control versus GPRC5B Areas ‘Neural Diversity and Neocortical Organization’ (to K.S.) from the shRNA, respectively; n=3-4 embryos, P=0.46), TBR2-positive Ministry of Education, Culture, Sports, Science and Technology of Japan; by intermediate progenitor populations (34.5±2.5% versus 42.5±5.3% Precursory Research for Embryonic Science and Technology from Japan Science in control and GPRC5B shRNA, respectively; n=3-4 embryos, and Technology Agency; and by Takeda Science Foundation. M.D.N. holds a P=0.19) and TBR1-positive early neuronal populations (10.1±0.8% Canadian Institutes of Health Research Operating Grant and a Scholarship from the Alberta Innovates Health Solutions. versus 13.8±4.2% in control and GPRC5B shRNA, respectively; n=3- 5 embryos, P=0.30). These results suggest that the GPRC5B Competing interests statement signalling cascade might not be activated at early stages of The authors declare no competing financial interests. corticogenesis, but rather underscore the integration of the GPRC5B Author contributions and β-catenin signalling pathways at later stages of corticogenesis to N.K., M.D.N. and K.S. designed experiments and wrote the manuscript; N.K. regulate the neurogenic potential of cortical progenitors. and K.S. performed experiments. Noticeably, GPRC5B-depleted progenitors, instead of remaining undifferentiated, gave rise to cells that were prone to differentiate Supplementary material into GFAP-positive astrocytes in vitro and in vivo. 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Hence, acute reduction of GPRC5B has enabled us to dissect out PLoS ONE 5, e10351. Development 4346 RESEARCH ARTICLE Development 140 (21)

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