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.

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.

GPRC5B-depleted cells fail to adopt a neuronal fate. (A) Plasmid expressing control shRNA, GPRC5B shRNA, GPRC5B shRNA#2 and both GPRC5B shRNA and GPRC5B^res (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.

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.

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.

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.