Fig. 5. In vitro-prepared CP-like neurons changed subtype marker expression upon exposure to microglia.

a Immunostaining for d4Venus (Gadd45g; green), Tbr2 (red), MAP2 (cyan), and DAPI (blue) in E14 Gadd45g-d4Venus Tg mouse cerebral walls. The broken lines indicate apical and basal surfaces. Scale bar, 50 µm. b The protocol for the in vitro preparation of CP-like neurons, which were obtained from Gadd45g-d4Venus⁺ cells isolated from the E14 Gadd45g-d4Venus cortex by FACS, after culture for 2 days. c A time-lapse series showing the reduction in Gadd45g-dVenus expression during culture. Scale bar, 20 µm. d CX3CR1-GFP⁺ microglia harvested from E15 CX3CR1-GFP pallial walls by FACS were added to the CP-like neuronal cultures. e Representative immunostaining (shown in pseudo color) for βIII-tubulin (cyan), Tbr1/Satb2/Cux1 (red), and Ctip2 (green). The arrowhead indicates microglia. Scale bar, 30 µm. f The proportion of the βIII-tubulin⁺ cells that expressed Tbr1, Ctip2, Satb2, or Cux1 in cocultures with or without microglia (two-sided Mann–Whitney U test; n = 10 independent experiments; P = 0.0021 for Tbr1, P = 7.6 × 10⁻⁵ for Ctip2, P = 7.6 × 10⁻⁵ for Satb2, P = 1.5 × 10⁻⁴ for Cux1). g An experimental schematic of the coculture of youngest (0 day, Gadd45g-d4Venus⁺) neurons and CX3CR1-GFP⁺ microglia. h Representative immunostaining (shown in pseudo color) for βIII-tubulin (cyan), Tbr1/Satb2/Cux1 (red), and Ctip2 (green). The arrowhead indicates microglia. Scale bar, 30 µm. i The proportion of βIII-tubulin⁺ cells that expressed Tbr1, Ctip2, Satb2, or Cux1 in cocultures with or without microglia (two-sided Mann–Whitney U test; n = 10 independent cultures; P = 0.529 for Tbr1, P = 0.289 for Ctip2, P = 0.631 for Satb2, P = 0.912 for Cux1). Data are presented as the mean values ± S.D. Source data are provided as a Source Data File.