Figure 3. RA induces GABAergic differentiation in neurosphere-derived cells from the LGE.

(A–B) Neurosphere-expanded wild-type (WT) cells derived from the E14.5 LGE give rise to Gad67-positive and Tuj1-positive cells after 1 wk of differentiation; under the same conditions no Gad67-positive cells were detected in differentiating cultures derived from Raldh3−/− (KO) LGEs. (C–D) Gad67 was not detected in differentiating cultures of E14.5 neurospheres from either wild-type or Raldh3−/− cortex. (E–H) Cells derived from E14.5 Raldh3−/− LGE and cortex neurospheres both express nestin and GFAP at indistinguishable levels from cells derived from wild-type neurospheres. (I–J) RA induces GABAergic differentiation of LGE-derived neurospheres; addition of 100 nM RA for 1 wk in the differentiation medium of expanded LGE cells resulted in an increase of Gad67+/Tuj1+ neurons in both wild-type and Raldh3−/− cultures. (K–L) Cortex-derived neurospheres treated with 100 nM RA exhibited no Gad67 detection. (M) Quantification of the percentage of LGE neurosphere-derived Tuj1-positive neurons also positive for Gad67 showed that in differentiating medium containing only fetal calf serum (FCS) with no added RA, the percentage of Gad67+/Tuj1+ cells derived from Raldh3−/− LGE neurospheres was significantly lower than that of wild-type LGE neurospheres. Following addition of 100 nM RA to the differentiation medium for 1 wk, the majority of Tuj1-positive neurons derived from both wild-type and Raldh3−/− LGE neurospheres were also Gad67-positive. The percentage was calculated by dividing the immunopositive cell number with the total number of DAPI-stained nuclei. Values are listed as mean ± SEM; * p<0.05; ** p<0.01; *** p<0.001.