Figure 5

Naïve mESCs differentiate to rostral ventral forebrain/GE progenitors and mature GABAergic neurons. (A) Schematic of the neural differentiation protocol, detailing morphogen and small molecules employed, to generate medial ganglionic eminence progenitors and neurons from naïve PSCs. (B) Immunocytochemistry images highlighting the expression of key GE transcription factors Nkx2.1 and Olig2 within young post-mitotic cell at day 10 of differentiation. (C) mESCs differentiated to GE-specific neurons widely expressed TUJ1 and GABA, (D) with many GABA + neurons co-expressing Nkx2.1, indicative of MGE interneurons. (E) Numerous post-mitotic TUJ1+ neurons co-expressed the MGE markers Nkx2.1 and Olig2 by day 14. Note images (B–E) show cultures overviews, illustrating the largely homogeneous nature of the differentiations, while inserts (Bi’-Ev’) show immunocytochemical labeling at the resolution of individual cells. Numerous GE-related genes showed appropriate temporal expression, including the transient upregulation of (F) Nkx2.1 and (G) Gsx2 as well as maintained, elevated expression of (H) Lhx6, (I) Olig2 and (J) Dlx2. (K) Gad67 was significantly upregulated by GE-like progenitors at day 10, with expression peaking at day 14 in mature neurons. Dashed grey line in (F) represents Nkx2.1 expression in “LGE-like” cultures, induced by earlier ventralization. (L) Reflective of an ‘MGE-like’ fate, rostral VF cultures appropriately expressed GAD67, but not CTIP2 or DARPP32. (L) In contrast, earlier ventralization of these VF progenitors resulted in cultures showing high expression of DARPP32, CTIP2 and GAD67, suggestive of a possible ‘LGE-like fate. Scale bar = 100 um. Data (n = 3) represented as mean ± SD. One-way ANOVA with Tukey post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001. MGE: medial ganglionic eminience; LGE: laternal ganglionic eminence.