Endocytosis of DeltaD deletion mutants.

(A,E,I,M,Q,U) Distribution of zdd2 (green) in COS7 cells transfected with DeltaD (A) or DeltaD ΔA, ΔB, ΔC, ΔD or ΔA-D deletion mutants (E, I, M, Q, U). Surface DeltaD was first labelled by incubation with zdd2 at 4°C for 30′ then, following washout of unbound zdd2, internalization was allowed for 30′ at 37°C. Nuclei were labelled with DAPI (blue). (B-D, F-H, J-L, N-P, R-T, V-X) Distribution of zdd2 (green) in COS7 cells co-transfected with DeltaD constructs and Mib1 (red) following internalization as described above. Each set of 3 panels, respectively, shows distribution of the DeltaD construct (green), Myc-Mib1 (red)/nuclei (blue), and the merged image. See materials and methods for details.

Endocytosis of DeltaD point mutants.

Distribution of zdd2 (green) in COS7 cells transfected with DeltaD-NN (A), DeltaD-KK (E) and DeltaD-NN/KK following the internalization protocol described in Fig 3. Nuclei were labelled with DAPI (blue). Distribution of zdd2 (green) in COS7 cells co-transfected with DeltaD-NN (B-D), DeltaD-KK (F-H), DeltaD-NN/KK (J-L) and Mib1 following internalization as described above. Each set of 3 panels, respectively, shows distribution of the DeltaD construct (green), Myc-Mib1 (red)/nuclei (blue), and the merged image. (M) Summary of expression classes found in DeltaD full length, deletion and point mutants assayed following the internalization protocol described above. P-values for pairwise comparison based on Fisher’s Exact test of independence. P >. 05 does not meet the criteria for the Null hypothesis that pairs contain an equivalent distribution of expression classes.

Ectopic expression of deltaD deletion and point mutant recapitulates neurogenic phenotype of DeltaD (ΔA-D).

(A) The prospective distribution of neurons revealed by the distribution of huC as revealed by in situ hybridization probe (purple) in control embryos injected with only β-galactosidase mRNA. (B-F) huC in embryos co-injected with β-galactosidase and deltaD (B), deltaD-C (C), deltaD B (D), deltaD NN/KK (E) or DeltaD ΔA-D (F) mRNA. Distribution of ectopic mRNA injected in one cell at the two-cell stage revealed by X-Gal distribution (blue). Dorsal view, rostral to the left. Embryos are at approximately the 3 somite stage. (G) Quantification of the effect of ectopic expression of mRNA encoding various forms of DeltaD on the distribution of early neurons. Red indicates fraction with a neurogenic phenotype (increased density of neurons), Green—fraction with no obvious effect on neuron density, Blue- fraction with suppression of neurogenesis (reduced neuron density). P-values for pairwise comparison based on Fisher’s Exact test of independence. P >. 05 does not meet the criteria for the Null hypothesis that pairs contain an equivalent distribution of phenotype classes.