Fig. 7: Hippo signaling effector YAP1 is downstream of cholinergic signaling.

a Scheme showing the ACh, NIC exposure and nAChR knockdown in primary dissociated neural cultures, derived from whole cortices. Cells were treated from Day 0 to Day 5 and protein lysates were prepared RIPA buffer. b Western blotting for Hippo co-activator YAP1 showed an upregulation with NIC treatment and a downregulation with KD of CHRNA7 and CHRFAM7A, compared to respective control samples. B-ACTIN was used as a loading control. c In-silico analysis of published single-cell sequencing data shows the RNA expression of several known Hippo effectors in relevant neuronal cell types. YAP1, FAT3, CRB2 and DCHS1 are highly expressed in RGs and oRGs⁹⁶. d Representative images showing protein expression of YAP1, DCHS1, SOX2 and EOMES in primary cortical tissue at GW23, n = 3. e Scheme showing the rescue experiment, where cortical organotypic slices were either treated with broad (ACh, NIC) agonists or a YAP1 inhibitor (TAT- PDHPS1) or a combination of both, for 5 consecutive days. Samples were fixed with 4% PFA and processed for immunofluorescence. f Immunostaining shows upregulation of YAP1 protein expression and increased nuclear translocation (arrows) post agonist treatment in oSVZ, compared to untreated controls. TAT, Acetylcholine+TAT and Nicotine+TAT show relatively similar YAP1 expression and localization as the untreated control, n = 3. g Quantifications post ACh, NIC, TAT, ACh+TAT and NIC + TAT samples compared to untreated controls show full rescue of HOPX+ cells over total DAPI in ACh+TAT-treated, and partial rescue in NIC + TAT-treated samples (Source Data), two-sided t-test, *p = 0.05, **p = 0.01, ns not significant, n = 3. h Representative immunostaining for RFP, YAP1 and DAPI show downregulation of YAP1 protein in RFP+ cells in shCHRNA7 and shCHRFAM7A samples (arrows), compared to RFP+ cells in shCONTROL. (All scale bars are mentioned on the image panels).