Fig. 3. Helt selects the GABAergic versus glutamatergic phenotype in the mesencephalon. (A) Gene targeting strategy. All exons encoding the Helt ORF were replaced with a GFP cDNA, and recombination was confirmed by Southern blotting (data not shown). The null phenotype in homozygous mutant mouse embryos was confirmed by complete loss of Helt expression (right-hand panels). (B) Helt is required for induction of GABAergic neurons and suppression of glutamatergic differentiation. In the Helt^-/- mutant mesencephalon, Gad1⁺ neurons are only detected in the ventral-most m5 region of the presumptive GABAergic domains, whereas Vglut2⁺ neurons are generated in essentially all domains of the mesencephalon at E11.5. At E12.5, although Gad1⁺ neurons start to emerge in a broad area of the ventral GABAergic domains (m3 to m5), the frequency is still lower than that in wild-type control embryos and ectopic Vglut2⁺ neurogenesis continues. Dorsal Gad1⁺ neurons are completely absent in the mutants. (C) Helt suppresses glutamatergic neurogenesis and induces GABAergic neurons. In the transgenic embryos expressing Helt under the control of the nestin enhancer, the number of Vglut2⁺ cells is decreased and the number of Gad1⁺ cells is increased instead. Note that GABAergic neurons are efficiently generated and glutamatergic neurons are suppressed in the ML just outside of the VZ positive for exogenous Helt, suggesting a cell-autonomous effect of Helt.