Figure 3.

eFGF2 or MEK inhibition alters cortical layer phenotype

(A) Under Basal conditions few phosphorylated ERK⁺ (pERK⁺) progenitors are observed, while the majority of cells are phosphorylated S6⁺ (pS6⁺). FGF2 treatment increased pERK expression, while MEK inhibition (MEKi) blocked pERK expression.

(B–D) Western blot showing upregulated pERK and pS6 after 24 h of FGF2 treatment (B). MEKi abolished pERK expression but had no effect on pS6. Western blot quantification of pERK (C) and pS6 (D) (n = 3 independent experiments).

(E) Differentiation conditions of cortical progenitors showing Basal, eFGF2, and MEKi conditions (±DAPT).

(F–K) Sorted PAX6⁺ progenitors under Basal conditions (±DAPT) (F and G), or treated with eFGF2 (H and I) or PD0325901 (J and K) were assessed for laminar fate at D55. (G) Quantification showed that progenitors under Basal conditions generated DL and UL neurons, with DAPT at D35 biasing TBR1⁺ neurons. (I) eFGF2 treatment generated TBR1⁺ neurons almost exclusively ± DAPT. (K) MEKi-treated progenitors predominantly generated CTIP2⁺ neurons ± DAPT, while in the absence of DAPT UL BRN2⁺ cells were observed (n = 3 independent experiments).

(L) qRT-PCR analysis of cortical genes confirmed cell quantifications (n = 3 independent experiments). Relative to Basal, eFGF2 (−DAPT) increased BRN2 and decreased SATB2 expression, suggestive of an IPC population rather than UL fate. MEKi (+DAPT) decreased the alternative deep (TBR1) and superficial (BRN2) laminar fates. In contrast, MEKi (−DAPT) increased BRN2 and SATB2 expression, reflective of UL fates.

(M) Increased NESTIN and decreased MAP2 gene expression confirmed cell-cycle exit and maturation in DAPT cultures (n = 3 independent experiments).

Data are mean ± SEM, one-way ANOVA with Dunnett's correction, n = 3; ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001. Scale bars, 100 μm (A) and 50 μm (F–J).