Extended Data Fig. 12: Hs-HARE5-dependent control of WNT signaling during neurogenesis.

a, Experimental paradigm for testing canonical WNT signaling using TCF/LEF-H2B-EGFP reporter, following inhibition by IWR1 in HEK cells. b, Quantification of WNT activated cells treated with DMSO, 3 µM IWR1 and 30 µM IWR1. (n = 3) independent transfections. Each dot represents the average value of multiple imaging fields from each transfection. *P = 0.0378. c, Experimental paradigm for testing canonical WNT signaling in E10.5 mouse primary NPCs in vitro. d, Representative image of E11.5 control and HARE5^Hs/Mm NPCs labeled with WNT reporter (green), mCherry (red), SOX2 (grey) and DAPI (blue). Inset, high magnification view of canonical WNT activated cells. e, Quantification of EGFP+ RGCs in E11.5 control (n = 5) and combined HARE5^Hs/Mm and HARE5^Hs/Hs (n = 8) cortices from 3 litters. Each litter is normalized with control littermates. **P = 0.0029. f, Experimental paradigm for testing canonical WNT signal activity of E14.5 by IUE in vivo. g, Representative sections of E15.5 control and HARE5^Hs/Mm brains electroporated with pCAG-mCherry (red), WNT reporter (green), SOX2 (grey) and DAPI (blue). h, Quantification of EGFP + SOX2+ RGCs in E15.5 control (n = 5) and combined HARE5^Hs/Mm and HARE5^Hs/Hs (n = 7) cortices from 3 litters. i, Volcano plots showing the differential RNA expression levels of various WNT signaling ligands across E12.5 to E14.5 of control (left) and HARE5^Hs/Hs (right). Results derived from RNAseq experiment in Extended Data Fig. 9 with n = 3 embryos each genotype and developmental stage, from 5 litters. j-k, RT-qPCR of mFzd8 (j) and canonical WNT signaling targets (k) after 2 days of transfection in E10.5 mouse NPCs with control and mFzd8 for (n = 3) individual transfections. *P = 0.0152 (j); *P = 0.0484 (Tcf1); *P = 0.025 (Dkk1); *P = 0.0157 (cMyc); *P = 0.0221 (Sox2). i, Experimental paradigm for measuring canonical WNT signaling by WNT reporter using TCF/LEF-H2B-EGFP reporter, following activation by CHIR99021 in human NPCs. m, Quantification of WNT activated cell proportion in D12 NPCs treated with DMSO (n = 3) and 5 µM CHIR99021 (n = 3) from 2 differentiations of 2 control lines. Each dot represents the average value of imaging fields from each cell line. **P = 0.0033. Scale bars: 100 µm (a,d), 50 µm (g-inset), 20 µm (d-inset), 10 µm (g). All values represent the mean ± s.d., Statistics on independent NPC experiments; colors, littermates (e), cell lines (m). P values were calculated based on the one-way ANOVA with Dunnett’s multiple comparisons test (b). Student’s unpaired, two-tailed t-test (e, h, j, k,m). Panel c adapted with permission from ref. ⁶³, The Company of Biologists.

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