Extended Data Fig. 15: RA regulates CBLN2, MEIS2, and DLG4 (PSD95) expression and synaptogenesis in human fetal neocortical neurons.

a, Schematics of human cortical neuron differentiation and modulation of RA signalling. From PCW 8 neocortical tissue, cortical neural stem cells were isolated and expanded with FGF2 for 20 days. Neural differentiation was initiated by the mitogen withdrawal. After 14 days of differentiation, cells were treated with varying doses of RA or RA receptor inhibitor (RARi, AGN193109) for another 14 days. From PCW 20 or 23 neocortical tissue, cortical neural progenitors and neurons were isolated and cultured without FGF2. After 16 days of culture, cells were treated with RA or RARi for another 14 days. Gene expression of cell type markers (b), RA receptors (c) RA regulated genes CBLN2 and MEIS2 (d) in PCW 8 cortical cells after 28 days of differentiation, measured by ddPCR. One way ANOVA and multiple comparison, compared to vehicle (0.01% DMSO)-treated control. Errors bars: S.E.M. *P < 0.05 (RXRA expression, RA1.0, P = 0.0192, RARi5, P = 0.0277; CBLN2 expression, RARi10, P = 0.0135; MEIS2 expression, RA1.0, P = 0.0456), **P < 0.01 (RARA expression, RARi10, P = 0.0039; MEIS2 expression, RARi5, P = 0.0057, PARi10, P = 0.0028), ***P < 0.001 (MEIS2 expression, RA0.5, P = 0.0003), ****P<0.0001. N=3 experimental replicates per condition. e, TUBB3/TUJ1 and DLG4/PSD95 expression in RA or RARi-treated PCW 8 cortical cells. Scale bar: 50 μm. f, DLG4/PSD95 transcript in PCW 8 cortical cells was significantly increased in RA 1 μM condition quantified by ddPCR. Two-tailed t-test, compared to the vehicle treatment. Errors bars: S.E.M. **P = 0.0083. N= 8 fields per condition. g, The ratio of total DLG4/PSD95+ puncta volume to total TUJ1+ volume in PCW 8 cortical cells was increased in the RA condition and reduced in the RARi condition. Two-tailed t-test, compared to the vehicle treatment. Errors bars: S.E.M. *P = 0.0152, ***P = 0.0001. h, MAP2, synaptophysin (SYP) and DLG4/PSD95 expression in RA or RARi-treated PCW 20 and 23 cortical cells. Scale bar: 50 μm. i, The ratio of total DLG4/PSD95+ puncta volume to total MAP2+ volume in PCW 20 and 23 cortical cells was increased in RA conditions and reduced in RARi conditions. Two-tailed t-test, compared to the vehicle treatment. Errors bars: S.E.M. *P <0.05 (PCW 20-RA2, P = 0.0221; PCW 20 -RARi5, P = 0.0193; PCW 23 -RA0.5, P = 0.0192), **P <0.01 (PCW 20-RARi10, P = 0.0018; PCW 23-RA1, P = 0.0081), ***P = 0.0004. N = 8 fields per condition. j, The ratio of total number of SYP+ and DLG4/PSD95+ colocalized synaptic puncta to total MAP2+ volume in PCW 20 and 23 neocortical cells was increased in RA conditions and reduced in RARi conditions. Two-tailed t-test, compared to the vehicle treatment. Errors bars: S.E.M. *P (PCW 20 -RA0.5, P = 0.0271; PCW 20-RA2, P = 0.0159; PCW 23-RA1, P = 0.0289; PCW 23-RA2, P = 0.0447), ***P = 0.0004, ****P < 0.0001. N= 8 fields per condition.