Numb proteins interact with endocytic proteins via multiple domains. (A) Schematic diagrams of the mouse and Drosophila Numb proteins. Yellow boxed areas (including the green PTB domain) indicate high levels of conservation (63.3% amino acid identity). Red boxed areas and amino acid sequences indicate the locations of DPF and NPF motifs. The blue boxed area indicates putative SH3 binding sites on m-Numb. (B) Schematic diagrams of the Drosophila EH1 and Dap160 proteins. (C) Immunoblots showing copurification of m-Numb from brain extracts or d-Numb from embryonic extracts by GST fusions of EH1 or Dap160. + and −, presence and absence, respectively, of protein extracts. The regions of EH1 and Dap160 used to copurify Numb proteins correspond to the lines underneath the respective diagrams in panel B. (D) EH1 expression during Drosophila embryogenesis. A digoxigenin-labeled antisense RNA probe of EH1 was used. (a to c) Lateral views; (d) ventral view. The embryo in panel Db is representative of those derived from EH²/Cyo parents that show no expression of EH1.

Binding to endocytic proteins is not necessary for Numb function. (A to C and E to L) The notum of a wild-type (WT) adult fly and nota of those overexpressing WT and various mutant forms of Numb proteins. Overexpression was achieved by using ScaGal4 at either 18°C (A to C and E to I), 25°C (L), or 29°C (J and K). (D) Quantitative analysis of the effects of d-Numb and d-Numb^ΔC overexpression in independently generated transgenic lines. Each bar represents the average number of hairs from five nota, and overexpression was achieved by using ScaGal4^109-68 at 25°C. ScaGal4^109-68 causes proteins to be expressed at levels lower than those obtained by use of ScaGal4 and consequently causes less-severe defects. (M to O) Yeast two-hybrid assays showing the effects of various mutations on protein-protein interactions between Numb proteins and their binding partners. The control is either an empty vector or lamin. ND, not determined. EH1 and Nak are Drosophila proteins, Siah1 and LNX2 are mouse proteins, and α-Ada is from a human.

Numb proteins can specify cell fates independently of endocytosis and proteasomes. (A to J) Notum of a wild-type (WT) adult fly and nota of those overexpressing either DTS5 alone, d-Numb alone, or DTS5 together with d-Numb^ΔC or m-Numb D/NPF^AAA. Panels B, D, F, H, and J are higher-magnification images of the respective boxed areas in panels A, C, E, G, and I. Arrows and arrowheads indicate defects in the macrochaete and microchaete lineage, respectively. Closed arrowheads in panel D point to double-hair, double-socket or one-hair, three-socket phenotypes, whereas open arrowheads point to two- or four-socket phenotypes. All these are prototypic defects caused by increases in Notch signaling in the SOP lineage. (K to M) Elav antibody staining (in red) showing the number of neurons underneath the notum surface. Overexpression was achieved by using ScaGal4. Embryos and larvae were raised at 18°C, but within 10 h after pupa formation, the temperature was shifted to 29°C, the nonpermissive temperature for DTS5.

Drosophila EH1 and Dap160 negatively regulate Notch signaling. (A to D) Nota of adult flies carrying various combinations of wild-type (+) and mutant alleles of EH1, H, and Su(H). Each hair in the wild type represents an es organ. (E) Quantitative analysis of genetic interactions between EH1 and H mutant alleles. Blue bars represent numbers of hairs on the notum and scutellum, whereas yellow bars represent numbers of double sockets (without hair). Each bar represents the average from 10 nota, except for those for the wild-type (WT) and EH^1/2 alleles, which represent averages from 4 nota. (F to M) Nota of adult WT flies or those overexpressing EH1 and/or Dap160 from 1 or 2 copies of the UAS-EH1 and UAS-Dap160 transgenes. Images in panels K and M show the number of neurons (in red; by Elav antibody staining) underneath the notum surface (in blue). Arrowheads in panels G, I, and L point to clusters of hairs, whereas dashed circles indicate balding areas. Brackets in panel K show that a single neuron accompanies each hair in the WT. Images are shown at ×4, except for those in the insets and panels K and M, which are shown at ×20. EH1 and Dap160 were overexpressed by using ScaGal4 at 25°C (F, H to M) or 29°C (G). (N) Schematic drawings of the effects of changes in Notch signaling on the specification of SOP cells (light green) during lateral inhibition. (O) Schematic drawings of the SOP lineage that produces es organs on the notum and the effects of changes in Numb and Notch activities. In the WT, d-Numb (in red) localizes asymmetrically in dividing SOP cells and segregates to daughter cell IIB to promote its fate. Subsequently, cells IIA and IIIB asymmetrically segregate newly synthesized d-Numb to distinguish their daughter cells. S, socket cell; H, hair cell; Sh, sheath cell; N, neuron. The glial cell (G) is not part of the es organ, and IIB divisions appear to be insensitive to Numb activity.

Rescue of the Drosophila numb mutant phenotypes by Numb proteins lacking the endocytic protein-binding motifs. (A to C) Drosophila notum overexpressing d-numb dsRNA either alone (A and B) or together with the D/NPF^A/AAA mutant form of m-Numb (C). Images in panel B are the boxed areas of panel A at a higher magnification. Open and closed arrowheads point to defects in the microchaete and macrochaete lineage, respectively. Each arrowhead points to a mutant socket cell. Overexpression was achieved by using ScaGal4 at 18°C. (D to I) Neuronal rescue. Elav staining (in green) of wild-type (WT) (D and E), d-numb mutant (F and G), and m-Numb^ΔC-rescued (H and I) embryos. All are stage 15 embryos. Arrowheads point to the two neurons in the two dorsal-most es organs, and brackets show the lateral chordotonal organs. (J to L) Cell fate transformation in the two dorsal-most es organs. Elav staining (in green) shows the presence of two neurons (arrowheads) among the eight Cut-positive cells (in red) in WT (J) and m-Numb^ΔC-rescued (L) embryos but not in the d-numb mutant (K). Elav is present in both the nucleus and the cytoplasm. Thus, Elav-stained cells appear bigger than those stained with Cut, which is present only in the nucleus. (M and N) Sheath cell rescue. Prospero staining (in red) shows that sheath cells, which accompany neurons (in green) in the lateral chordotonal organs (brackets) in the WT (M), are also rescued by m-Numb^ΔC (N). Images in panels E, G, I, and J to N are shown at higher magnifications.