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. 2017 Mar 24;18(5):712–725. doi: 10.15252/embr.201643585

Figure 5. GPR37 modulates neural fate in neural progenitor cells.

Figure 5

  • A, B
    qPCR expression analysis of Gpr37 in the indicated mouse tissues or isolated NPCs. Gpr37 expression was normalized against Gapdh (mean values ± SD, n = 3; one‐way ANOVA followed by Holm–Sidak test).
  • C
    Immunofluorescence microscopy of endogenous GPR37 (red) with LRP6, calreticulin (ER), TGN38 (Golgi), EEA1 (early endosome), clathrin (endocytic vesicles), or pan‐cadherin (cell junctions), shown in green, in NPCs from disassociated neurospheres. Yellow signal shows co‐localization (arrows). Scale bar: 10 μm.
  • D
    Immunofluorescence microscopy of endogenous LRP6 (green) and GPR37 (red) in neurospheres upon siGpr37 treatment. DNA (Hoechst) is shown in blue. Scale bar: 100 μm.
  • E
    Immunoblot analysis of LRP6 protein levels in NPC cells upon siRNA knockdown of GPR37 or LRP5/6. Note that siGpr37 reduces LRP6 protein levels, without affecting its mRNA expression (see Fig EV5D). Shown are representative experiments that were carried out three times.
  • F, G
    qPCR expression analysis of the Wnt target gene Sp5 (F) and the indicated neural fate markers (G) in NPCs upon siRNA knockdown of GPR37 or LRP5/6. Neurospheres were treated for 5 h with control or Wnt3a‐conditioned media. Expression was normalized against Hprt (mean values ± SE, n = 5; *P < 0.05, ***P < 0.001, one‐way ANOVA followed by Holm–Sidak test).
  • H
    Immunofluorescence microscopy of MAP2 (green) and NG2 (red) in differentiating NPCs. Neurospheres were treated with the indicated siRNA and cultured in neuronal differentiation medium (see Materials and Methods). Bottom right, percentage of MAP2‐positive cells per colony (mean values ± SE, n = 5; **P < 0.01, one‐way ANOVA followed by Holm–Sidak test). Scale bar: 100 μm.