Fig 2: Deletion of Cic in the neurogenic period biases NSCs to glial lineage selection. a Localized deletion of Cic in neural stem cells via in utero electroporation into VZ cell of Cic-floxed embryos; targeted cells carry a fluorescent marker and express either cre recombinase (Cre), dominant negative ETV5 (DNETV5), or not (Empty control), depending on the electroporated plasmid. b–l Immunofluorescence staining and quantitation of cells in the electroporated patch. Sox2 + stem cells b, c and Sox9 + glioblasts d, e in n = 4 per group 2 days after E13 electroporation, analyzed at E15. Late-born Tbr1 + neurons f, g, Aldh1 + astrocytes h, i, Olig2 + oligodendrogial lineage cells j, k, and Pdgfra + oligodendrocyte precursors cells l detected 5 days after E13 electroporation, analysis at E18. Data from n = 5 for control and n = 6 mice for Cre for Tbr2 staining; n = 6 for control and n = 5 for cre for Aldh1 staining; and n = 4 per group for Olig2 and Pdgfra staining. Statistical analyses between control and experimental groups performed by ANOVA with Tukey’s post hoc test. Scale bars: 50 µm. Source data are provided as a Source Data file. Data shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ns–not significant. Labels: Ctx–cortex, LV–lateral ventricle, VZ–ventricular zone

Fig 3: MAPK activation increases ERK negative feedback and activates STAT3(A) A simplified schematic showing the MAPK pathway and its negative feedback regulators DUSP6, SPRY2, and ETV5. Created with BioRender.com.(B) Western blot analysis on SCLC cell lines as indicated upon MEKDDS217D/S221D expression (0, 1, 2, 3 days). MEKDD, total and phosphorylated ERK1/2, DUSP6, SPRY2, ETV5 are shown with ß-actin used as loading control. MEKDD, total and phosphorylated ERK1/2 were taken from Figure 1D for presentation purposes.(C) Heatmap of SCLC cell lines showing log2 fold change for relative change in gene expression after MEKDDS217D/S221D expression (24 h) for the indicated genes. Unpaired t test was performed between ASCL1- and non-ASCL1-SCLC cell lines with p value as indicated.(D) Western blot analysis on ASCL1-driven SCLC cell line H69 cultured as adherent, suspension, and mixed upon MEKDDS217D/S221D expression. Total and phosphorylated ERK1/2, DUSP6, and SPRY2 are shown with ß-actin used as loading control. Total and phosphorylated ERK1/2 were taken from Figure 2C for presentation purposes.(E) Phosphokinase array in H720 and H82 upon MEKDDS217D/S221D expression after 24 h. Phosphorylated ERK1/2 (T202/Y204) and STAT3 (S727) are indicated.(F) Mean pixel density derived from (D) for phosphorylated ERK1/2 (T202/Y204) and STAT3 (S727) after 24 h of MEKDDS217D/S221D expression in the indicated cell lines (±SEM). Unpaired t test was performed so that ***p < 0.001, ****p < 0.0001.(G) Western blot analysis on SCLC cell lines as indicated upon MEKDDS217D/S221D expression (0, 1, 2, 3 days). Total and phosphorylated ERK1/2, total and phosphorylated STAT3, and total and phosphorylated AKT are shown with ß-actin used as loading control. Total and phosphorylated ERK1/2 were taken from Figure 2B for presentation purposes.

Fig 4: Etv5 is necessary and sufficient for proliferation and cell fate bias downstream of Cic loss. a–f Cic-null NSCs (CicnullEmpty), Cic-null NSCs with dominant negative Etv5 (CicnullDN-Etv5), and Cic-wildtype NSCs overexpressing Etv5 (Etv5 overpression) were grown in lineage-directed culture conditions and assessed for their ability to differentiate to neurons, astrocytes, and oligodendrocytes as determined by immunostaining for bIII-Tubulin (Tuj1), Gfap, and Olig2, Pdgfra, and Mbp. Scale bar: 10 µm. Analysis of Tuj1 + cells from NSCs in neuronal differentiating condition, NDC a, d; analysis of Gfap + cells from NSCs in astrocytic differentiating condition, ADC b, e; and analyses of Olig2 + , Pdgfra + , and Mbp + cells from NSCs in oligodendrocyte differentiating condition, ODC c, f from n = 3 biological replicates, with three technical replicates each, for cell culture studies. g, h Representative images and quantitation of EdU incorporation 2 days post electroporation of wildtype ETV5 or empty control plasmid, both carrying mCherry as a marker, into E13 CICFl/Fl VZ. Note: mCherry fluorescence and EdU staining were false-colorized to green and red after grayscale imaging. Scale bar: 50 µm. Data from n = 4 mice per each group. Scale bar: 50 µm. i, j Representative images and quantitation of EdU incorporation 2 days post-electroporation of Cre only or of Cre co-electroporated with DNETV5 into E13 CICFl/Fl VZ. Data from n = 4 mice per each group. Scale bar: 50 µm k EdU incorporation assay in cultured Cic-wildtype NSCs without or with ETV5 overexpression from n = 3 biological replicates. l EdU incorporation in Cic-floxed NSCs with Cre, and without or with DNETV5 expression from n = 3 biological replicates. Data shown as mean ± SD. Statistical analyses performed either t test in h, j, k, l; or with ANOVA with Tukey’s post hoc test in d, e, f. ns–not significant, *p < 0.05, **p < 0.01, ***p < 0.0001. Source data are provided as a Source Data file. ADC–astrocytic differentiation condition, NDC–neuronal differentiation condition, ODC–oligodendrocytic differentiation condition. VZ–ventricular zone, LV–lateral ventricle

Fig 5: Etv5 is a direct target of Cic transcriptional repression. a, b Etv1, Etv4, and Etv5 mRNA expression levels a and protein expression levels b in cultured CIC-null and -control cells after 48-hour exposure to oligodendrocytic differentiating conditions; transcript levels normalized to average of three (Actin, GAPDH, Tubulin beta chain) housekeeping genes; data from n = 3 biologic replicates. Statistical analyses performed by unpaired t test. Data shown as mean ± SD. *p < 0.05. c ChIP-PCR for Cic at the Etv5 promoter. d Electroporation of Cic shRNA or non-targeting (NT) shRNA at E13 followed by in situ hybridization for Etv5 and GFP at E15; Etv5 transcripts are upregulated in areas of Cic knockdown indicated by the GFP expression; data from n = 3 mice per group. e Immunofluorescence staining for Etv5 and GFP protein expression 2 days after Cre electroporation into E13 VZ of Cic-floxed embryos. GFP + cells show increased Etv5 protein. Scale bar: 50 µm. f Immunofluorescence staining for Etv5 expression in Sox2 + NSCs at mid-neurogenesis. g–i Immunofluorescence staining for Etv5 expression Gfap + astrocytes g, Pdgfra + OPCs h, and CNPase + oligodendrocytes i in P56 adult cortex. Scale bar: 50 µm. j Quantitation of Etv5 nuclear staining for n = 20 cells for each cell type marker shown; each point represents one cell quantitated. Data from n = 5 mice per group. Data shown as mean ± SD. Source data are provided as a Source Data file. Statistical analyses performed with ANOVA with Tukey’s post hoc test. ns–not significant, ****p < 0.0001. SVZ–subventricular zone, LV–lateral ventricle, Ctx–cortex. k Schematic depiction of relationship of Cic and Etv5 expression as neural stem cells differentiate to mature myelinating oligodendrocytes