Journal: Brain

Article Title: POU3F2 regulates canonical Wnt signalling via SOX13 and ADNP to expand the neural progenitor population

doi: 10.1093/brain/awaf221

Figure Lengend Snippet: POU3F2 activates expression of SOX13 and ADNP to regulate canonical Wnt signalling in NPCs. ( A ) Potential direct effectors of POU3F2 were prioritized if they belonged to the TCF-dependent signalling in response to Wnt Reactome pathway (R-HSA-201681). ( B ) Quantification of ADNP protein expression measured via western blotting in POU3F2 WT and POU3F2 MUT NPCs, mean ± SEM ( n = 4 differentiations). Paired t -test, * P < 0.05. ( C ) Quantification of SOX13 protein expression measured via western blotting in POU3F2 WT and POU3F2 MUT NPCs, mean ± SEM ( n = 5 differentiations). Paired t -test, ** P < 0.01. ( D ) Schematic of experimental design for overexpression of a gene of interest followed by the SUPERTOPFLASH canonical Wnt reporter assay. ( E ) Baseline canonical Wnt signalling, as measured by a SUPERTOPFLASH assay, in POU3F2 WT and POU3F2 MUT NPCs expressing GFP (negative control) or ADNP, mean ± SEM ( n = 3 differentiations). Mixed-effects model with post hoc Sidak's test, ns = not significant. ( F ) Activated canonical Wnt signalling, as measured by a SUPERTOPFLASH assay, in POU3F2 WT and POU3F2 MUT NPCs expressing GFP (negative control) or ADNP, mean ± SEM ( n = 3 differentiations). Mixed-effects model with post hoc Sidak's test, * P < 0.05, ** P < 0.01. ( G ) Baseline canonical Wnt signalling, as measured by a SUPERTOPFLASH assay, in POU3F2 WT and POU3F2 MUT NPCs expressing GFP (negative control) or SOX13, mean ± SEM ( n = 3 differentiations). GFP control data represented here is included in . Mixed-effects model with post hoc Sidak's test, * P < 0.05, ** P < 0.01. ( H ) Activated canonical Wnt signalling, as measured by a SUPERTOPFLASH assay, in POU3F2 WT and POU3F2 MUT NPCs expressing GFP (negative control) or SOX13, mean ± SEM ( n = 3 differentiations). GFP control data represented here is included in . Mixed-effects model with post hoc Sidak's test, * P < 0.05, *** P < 0.001. ( I ) Genomic structure of SOX13 . Box above gene indicates relative position of gRNA target sequence. Western blot validation of SOX13 knockdown (GAPDH = loading control). ( J ) Gene ontology pathway enrichment results of differential gene expression in SOX13 MUT NPCs, separated by directionality of log 2 fold change. ( K ) GSEA of Wnt-activated targets and G2/M progenitor markers in SOX13 MUT versus SOX13 WT differential expression. ( L ) Model of POU3F2-mediated regulation of canonical Wnt signalling and progenitor expansion via transcription of ADNP and SOX13 . Schematics created in BioRender. Benoit, C. (2025) https://BioRender.com/35q22ly , https://BioRender.com/baxexcu . gRNA = guide RNA; GSEA = gene set enrichment analysis; MUT = mutant; NPC = neural progenitor cell; SEM = standard error of the mean; WT = wild-type.

Article Snippet: Primary antibodies diluted in blocking buffer were applied overnight at 4°C: POU3F2 (CST, Cat. No. 12137S, 1:1000), GAPDH (Proteintech, Cat. No. 60004-1-Ig, 1:10 000), β-catenin (CST, Cat. No. 8480S, 1:1000), Nestin (Novus Biologicals, Cat. No. MAB1259, 1:1000), TUJ1 (Novus Biologicals, Cat. No. NB100-1612, 1:1000), POU3F2 (Santa Cruz, Cat. No. sc-393324, 1:1000), HA-Tag (CST, Cat. No. C29F4, 1:500), ADNP (Abcam, Cat. No. ab300114, 1:500), SOX13 (Proteintech, Cat. No. 18902-1, 1:1000).

Techniques: Expressing, Western Blot, Over Expression, Reporter Assay, Negative Control, Control, Sequencing, Biomarker Discovery, Knockdown, Gene Expression, Quantitative Proteomics, Mutagenesis