Fig. 5. Cells expressing GABAergic markers are very reduced in differentiated Sox2 mutant cultures. Double-immunofluorescence with general neuronal markers (β-tubulin, rows 1, 2; MAP2, rows 3, 6; red), GABA (rows 1-4) and calretinin (5-6), in normal and mutant day 9-differentiated cultures. Histograms: percentage of positive cells, with wild-type average of 100%. Most β-tubulin-positive cells in normal (top) are GABA positive. In mutant (second row), two immature-looking β-tubulin-positive cells are very weakly GABA positive (or negative) (arrows), in contrast to the adjacent well-arborized GABA-positive cell. In normal cultures, most GABA- and virtually all calretinin-positive cells (rows 3, 5) express the mature neuronal marker MAP2; these cells are extremely reduced in mutant cultures (rows 4, 6 and histogram). Results from n=4 normal and n=4 mutant mice (see Table S1 in the supplementary material).