Fig. 4. Phox2b represses, while Ngn2 upregulates negative regulators of neurogenesis. (A-F) Hes5 overexpression inhibits neuronal differentiation and counteracts the increase in Delta1 expression in response to Phox2b. In situ hybridization with Delta1 (A',C',E') and NeuroM (B',D') probes on transverse spinal cord sections. The extent of transfection is shown by GFP in situ hybridization as indicated. Hes5 overexpression downregulates Delta1 (C') and NeuroM (D') expression. The promotion of Delta1 expression by mPhox2b (A') is reduced by co-transfecting Hes5 (E'). (F) Quantitative analysis of Delta1 expression in response to mPhox2b, Hes5 or mPhox2b plus Hes5. Results are expressed as the difference in Delta1 signal intensity between the transfected and the non-transfected sides. Data points represent mean values±s.e.m. (arbitrary units) from more than 35 sections from at least four transfected embryos. ^*P<0.05, ^**P<0.001 with respect to transfection of mPhox2b alone. (G-J') Id2 overexpression inhibits neuronal differentiation and counteracts the increase in Delta1 and NeuroM expression in response to Phox2b. In situ hybridization with Delta1 (G',I') and NeuroM (H',J') probes on transverse spinal cord sections. The extent of transfection is shown by GFP in situ hybridization as indicated. Id2 overexpression downregulates NeuroM expression (H'); expression of Delta1 is only marginally affected (G'). The promotion of Delta1 and NeuroM expression by mPhox2b (A',B') is reduced by co-transfecting Id2 (compare A' and B' with I' and J'). (K-N'') Phox2b represses chick Hes5b and Id2, while Ngn2 upregulates their expression. In situ hybridization with chick Hes5b (K',L',N') and Id2 (K'',L'',N'') probes alone or combined with anti-BrdU immunohistochemistry (M) (brown nuclei) on spinal cord sections of embryos electroporated with either mPhox2b (K-M) or mouse Ngn2 (N-N''). In situ hybridization with a GFP probe (L,N) or GFP fluorescence (K) show the transfected area. Repression of chick Hes5b and Id2 is already observed at 6 h.a.e. of mPhox2b (K',K'') and has become pronounced at 24 h.a.e (L',L''). (M) After an 1 hour BrdU pulse, virtually all BrdU-positive cells on the untransfected (left) side are chick Hes5b- or Id2-positive, while in the transfected area (right) chick Hes5b or Id2 expression is absent in both BrdU-positive and -negative cells. The broken lines in M outline the lumen of the neural tube. In contrast to mPhox2b, mouse Ngn2 overexpression promotes chick Hes5b (N') and Id2 (N'') expression.