Fig. 1. Phox2b misexpression promotes Ngn2 but not Cash1 expression. In this and the following figures, light stippled lines demarcate the transfected area. A,A',A'' means that these are either consecutive sections from the same embryo (for in situ hybridization) or the same sections photographed through different filters (for GFP fluorescence and fluorescent antibody labeling). A broken line indicates the lumen of the neural tube. The time of incubation after electroporation (h.a.e.) is indicated above each panel. (A-D) Ngn2 and Mash1 have proneural activity in the chick neural tube. Overexpression of mouse Ngn2 or Mash1 in spinal regions of the chick neural tube upregulates Delta1 (A,A',C,C'). At 24 h.a.e. of mouse Ngn2, the transfected cells start to relocate to the mantle layer (ML) (A), and migration is complete at 48 h.a.e. (B). At 18 h.a.e. of Mash1, the transfected cells are still in the neuroepithelium (C), but they have relocated to the ML at 48 h.a.e. (D). In control transfections, most cells were still in the neuroepithelium at 48 h.a.e. (see Dubreuil et al., 2000). (E-H') In situ hybridization with Ngn2 and Cash1 probes on spinal cord sections at different times after electroporation of mPhox2b. GFP fluorescence shows the extent of electroporation. (F',I) Forced expression of mPhox2b expands the Ngn2 expression domain at 6 h.a.e.; the effect becomes less pronounced at 18 h.a.e. (H'). Double-labeling with a Ngn2 probe and anti-Phox2b antibodies (I) shows that most cells expressing Ngn2 ectopically also express mPhox2b (arrowheads in right panel). The dorsalmost region of the neural tube appears refractory to Ngn2 induction. Cash1 is not induced at 6 h.a.e. (E') or at 24 h.a.e. (G'). Note that at the earlier time point (HH 15/16), Cash1 is not yet expressed at spinal levels of the neural tube (E'). (J) Quantitative analysis of Ngn2 induction by mPhox2b at different times after electroporation. Results are expressed as the difference in Ngn2 signal intensity between the transfected and the non-transfected sides (arbitrary units). Data points represent mean values±s.e.m. from more than 35 sections from at least five transfected embryos.