Fig. 3. Effects of Phox2b overexpression in the hindbrain. (A-C'') Phox2b promotes migration to the ML and induces endogenous Islet1/2 and Phox2b expression. In situ hybridization on transverse sections of the caudal hindbrain with GFP (A), mPhox2b (B) and chicken Phox2b (B') probes and double-labeling with anti-Islet1/2 (C) and anti-mPhox2b (C') antibodies. At 48 h.a.e. of mPhox2b into the hindbrain, the transfected cells have relocated to the ML (B), an effect not seen in control transfections with GFP alone (A). The mPhox2b-expressing cells also express the endogenous Phox2b gene (B'). The ventral expression domain of endogenous Phox2b corresponds to the vm precursors. Islet1/2 is expressed in ventral motoneurons and by the cells that ectopically express mPhox2b (C,C'). (D-G') Phox2b upregulates Ngn2 and Cash1 in combination with Nkx2.2. In situ hybridization on transverse sections of the caudal hindbrain using mPhox2b (D-F), mouse Nkx2.2 (G), Ngn2 (D') and Cash1 (E',F',G') probes. mPhox2b alone upregulates Ngn2 (D'), but not Cash1 (E'). Nkx2.2 (0.5 mg/ml) alone does not induce Cash1 (G'), but does so when co-electroporated with Phox2b (F').