Fig. 3.
Activation of Notch signaling at the ventral roof plate border. a–l Transverse sections through the dorsal neural tube at the level of the forelimb in wildtype (WT) and Wnt1-Cre; Mib1fl/fl mouse embryos, immunostained for N1ICD (white or red) and DAPI (blue). N1ICD staining is shown in combination with DAPI (a, c, e, g, i, k) or as a single color image for clarity (b, d, f, h, j, l). Low levels of N1ICD are evident in the neural tube at E9.75 (a–d); however, at E10.5 (e–h) and E11.5 (i–l), N1ICD is broadly expressed throughout the dorsal neural tube in wildtype embryos, whereas this is diminished in Wnt1-Cre; Mib1fl/fl embryos. m–x Transverse sections through the dorsal neural tube at the level of the forelimb in wildtype (WT) and Wnt1-Cre; Mib1fl/fl mouse embryos, immunostained for DLL1 (red or white) and DAPI (blue). DLL1 staining is shown in combination with DAPI (m, o, q, s, u, w) or as a single color image for clarity (n, p, r, t, v, x). At all ages examined, in wildtypes, DLL1 is expressed throughout the neural tube except for its dorsal-most region corresponding to the RP (boundary demonstrated by dashed lines in n, r, v). In Wnt1-Cre; Mib1fl/fl embryos, DLL1 protein accumulates at the cell membrane due to lack of Mib1-induced endocytosis, and by E11.5 there are no DLL1-negative cells present in the dorsal-most neural tube. y, z Quantification of DLL1-negative cells in the dorsal-most neural tube at E10.5 and E11.5. At E10.5, there is no difference between genotypes, whereas at E11.5 there is a significant reduction in the number of DLL1-negative cells, and hence of the presumptive RP, in Wnt1-Cre; Mib1fl/fl embryos. N = 6; ns, not significant; ****p < 0.0001. aa Double immunostaining for Dll1 (green) and N1ICD (red) at E11.5; note that the dorsal limit of expression of both markers coincides (dotted lines). bb, cc, dd Gdf7 ISH (blue or green) and N1ICD immunostaining (red) of adjacent sections showing a side-by-side expression of both markers with no apparent overlap (dotted lines). Bar = 50 μm