Nedd8 affects transduction of the Hh pathway in Drosophila eye development. (A–D) The Ci^FL protein, but not ci expression, is upregulated in the Nedd8 mutant cells (revealed by the lack of GFP expression, in green). (A,B) A late third-instar eye disc containing Nedd8 mutant clones and stained by anti-Ci^FL antibody (2A1) in red. The open arrowheads mark the position of the MF in this and following figures. In the mutant cells anterior to the MF, Ci^FL accumulation (arrows) reaches a level identical to that observed in the MF where Hh signaling is fully induced. In the posterior mutant cells, Ci^FL accumulation is persistent at a lower level abutting the MF (arrowheads) and not detected in more posterior cells (asterisks). Note that the anterior clones are obviously larger than the posterior clones, suggesting a survival defect in the Nedd8 mutant cells. (C,D) A late third-instar eye disc containing Nedd8 mutant clones and double stained for ci-LacZ expression in red (C) and Ci^FL in blue (D). (C) The ci-LacZ expression level is ubiquitous throughout the whole eye disc, and remains unchanged in the Nedd8 mutant clones (C, arrow), in which the Ci^FL protein accumulates (D, arrow). (E–H) Accumulated Ci^FL responds to Hh signaling to induce MF fate in Nedd8 mutant clones. (E–G) A late third-instar eye disc containing Nedd8 mutant clones was double stained for F-actin in blue (F) and dpp-LacZ in red (G). (E) The Nedd8 mutant clones are marked by the lack of GFP expression and outlined by a white line in E–G. (F) F-actin staining (blue) by phalloidin shows that the Nedd8 mutant cells constrict precociously. (G) Expression of dpp-lacZ (red) is induced in the mutant cells abutting the MF anteriorly. (H) In the Nedd8 mutant clones, as shown by lack of arm-lacZ expression (green), expression of the proneural protein Atonal (red) is induced (arrows).

Nedd8 modifies Cul1 and controls its stability. (A) Western blot by αCul1 antibodies. Cell extracts were prepared from first-instar larvae of wild-type (left lane) and Cul1^EX mutant (right lane). The levels of Cul1 proteins of 90 kD are greatly reduced in Cul1^EX cell extract. (B) Conjugation of Nedd8 to Cul1. Western blot of cell extract prepared from the eye discs and brain lobes of third-instar larvae by αCul1 antibodies (left lane). Immunocomplex of Cul1 from precipitation by αCul1 antibodies is blotted by αNedd8 antibodies (middle lane). An arrow indicates the putative Nedd8-modified Cul1 form. In the control experiment without the addition of αCul1 antibodies in precipitation, no Nedd8 positive signal can be detected (right lane). (C) Immunoblotting of cell extract prepared from first-instar larvae of wild-type (left lane) and Nedd8^AN015 (right lane) by αCul1 antibodies. The Nedd8-modified form of Cul1 is missing in Nedd8 larvae (right lane, arrow). (D) In the absence of Nedd8 modification, the Cul1 level is increased. Western blot analysis of cell extract from third-instar eye discs and brain lobes by αCul1 antibodies indicates that the Cul1 protein is accumulated in Nedd8^AN015/Nedd8^EP(2)2063 (right lane). (E,F) A late third-instar eye disc containing Nedd8 mutant clones and stained with αCul1 antibodies (red). Cul1 accumulates in the Nedd8 mutant clones (red in E) that are marked by the lack of GFP expression (green in F).

SCF^Slimb complex and a Cul3-based degradation machinery to control the Ci^FL stability in the anterior and posterior cells of the eye discs, respectively. (A–D) The Cul1-based, SCF^Slimb complex functions in the anterior cells to promote Ci^FL proteolytic processing. In the Cul1^EX (A) and slimb¹ (C) mutant clones located anterior to the MF, Ci^FL accumulation is detected (red), whereas in the posterior clones, no accumulation can be observed. The mutant clones of Cul1^EX (B) and slimb¹ (D) are marked by the lack of GFP expression. (E,F) In the Cul3^gft2 mutant clones, marked by the lack of GFP expression (green), only posterior clones accumulate Ci^FL (red). (G,H) The ubiquitous expression patterns of Cul1 (G) and Cul3 (H) in the eye discs as revealed by in situ hybridization with antisense probes of Cul1 and Cul3, respectively. In the control experiments, the sense probes gave no signals (data not shown). (I) The phylogenetic tree of the Drosophila Cullin family (denoted by Dm) to their human counterparts (Hs).

A proposed model for Ci degradation in Drosophila eye development (see Discussion for details).

Conservation of Nedd8 in evolution and Drosophila Nedd8 mutant phenotypes. (A) Sequence comparison of Nedd8. Drosophila Nedd8 shares 88%–98% identity to other Nedd8 from yeast to mammals. Also indicated are the point mutations in Nedd8^AN024 and Nedd8^AN015 alleles. Conceptually, Nedd8^AN015 encodes a C-terminus-truncated protein by a nonsense mutation at aa 49. Thus, Gly76, the essential residue required for conjugation to the substrate protein (Wada et al. 1998), was missing in this mutant. In Nedd8^AN024, the translation start codon was replaced by a missense mutation. (B) The 4-d-old larvae of wild-type (left) and Nedd8^AN024 (right). Note growth of the Nedd8^AN024 larva was arrested in the first instar stage. Bar, 0.5mm. (C–F) The growth and survival defect of Nedd8 mutant cells. (C,D) The adult nota of w f hs-FLP; + FRT^40A/M2(Z)f^+30b FRT^40A (C) and w f hs-FLP; Nedd8^AN015 FRT^40A/M2(Z)f^+30b FRT^40A (D). (C) Almost all cells in the adult notum are marked with forked (note curly forked bristles) compared to D, in which no Nedd8^AN015 mutant clones can be observed. (E,F) The adult eyes of w ey-FLP; + FRT^40A/cl2L3 p[w^+30C] FRT^40A (E) and w ey-FLP; Nedd8^AN015 FRT^40A/cl2L3 p[w^+30C] FRT^40A (F). The wild-type clones containing white ommatidia occupy almost the whole eye in E, compared to F in which Nedd8^AN015 clones (marked by white ommatidia) are absent, leaving a defective eye that contains only Nedd8^AN015/+ cells in red ommatidia. (G–Q) Accumulation of Ci^FL, Arm, and CycE, and disruption of twist mRNA expression in Nedd8 mutant cells. The wing (G–L) or eye discs (M–O) carrying Nedd8 mutant clones marked by the lack of GFP (H,K) or β-galactosidase expression (N). Accumulation of Ci^FL (red in G,I) appears only in mutant clones located in the anterior compartment of the wing disc (for imaginal discs in this and following figures, anterior is to the left). Accumulation of Arm (red in J,L) appears in the cytosol of Nedd8 mutant cells (arrows), in contrast to the cell surface-associated Arm in wild-type cells (arrowheads). (M, O) Accumulation of CycE is also detected in the Nedd8 mutant clones in the eye disc. (P,Q) In situ hybridization of twist mRNA in wild-type embryos (P) and Nedd8 mutant embryos laid by Nedd8^AN015/Nedd8²⁰³ females (Q).

The Nedd8 pathway on Ci^FL proteolytic processing is downstream of the Hh receptor Smo and PKA activity. (A–D) Ci^FL accumulation in Nedd8 mutant cells is independent of smo activity. (A,B) A late third-instar eye disc carrying smo and Nedd8 double mutant clones and stained for Ci^FL in red. Ci^FL accumulates in all double mutant clones (marked by the lack of GFP staining in B) regardless of their locations in the eye disc. (C,D) A late third-instar eye disc carrying smo clones and stained for Ci^FL in red. In the smo mutant cells marked by the lack of GFP expression (green in D), Ci^FL expression is missing in clones anterior to and in the MF (arrows). However, Ci^FL accumulation is detected in mutant cells in the posterior clones (arrowheads) except near the posterior margin (asterisks). (E–H) Nedd8 is required for PKA activity in promoting Ci^FL proteolytic processing. (E,F) A late third-instar eye disc expressing PKA catalytic domain (UAS-mC*) driven by eq-GAL4 in the equator region, as visualized by the coexpressed GFP (green in E). Constitutive activation of PKA suppresses Ci^FL accumulation in MF (red in F). (G,H) A late third-instar eye disc expressing UAS-mC* under the control of eq-GAL4 and containing Nedd8 mutant clones. Ci^FL accumulation (red) persists in the Nedd8 mutant clones even when PKA is constitutively activated in the mutant cells (arrows). The accumulated Ci^FL level is similar to that in the mutant cells without mC* expression (arrowheads). The Nedd8 mutant clones are marked by the lack of arm-lacZ expression (green in H), and the effect of misexpressed PKA catalytic subunit can be observed by the downregulated Ci^FL level in MF cells near the Nedd8 clone.

Accumulation of Ci^FL, Arm, and CycE in Cul1 mutant cells. Cul1^EX mutant clones are marked by the lack of GFP expression in wing discs (green in B,E) and eye disc (green in H). (A–C) Ci^FL (red in A,C) accumulates in the Cul1^EX mutant clones (indicated by arrows) in the anterior compartment of the wing discs. (D–F) In Cul1^EX mutant cells, Arm is upregulated (red in D,F) in cytosol (arrows) of wing disc cells. (G–I) Enhancement of CycE protein level (red in G,I) is observed in Cul1^EX mutant clones (arrows) in the eye disc.

Regulation of Ci^FL degradation in posterior cells of the eye disc. (A,B) PKA activity is not required for Ci^FL degradation in the posterior cells. To inhibit PKA activity, the dominant negative form of PKA regulatory domain is expressed in random clones marked by GFP expression (see Materials and Methods). The protein level of Ci^FL (red) is upregulated in anterior (arrow) but not posterior clones (arrowhead). (C–F) Ci^FL degradation is independent of PKA phosphorylation. The PKA phosphorylation site-mutated form of Ci, Ci^−3P, is expressed by eq-GAL4 in the equator, as shown by the coexpressed GFP (C). (D) Misexpressed Ci^−3P is refractory to PKA-mediated proteolytic processing in the anterior cells, as shown by antibody staining for Ci^FL. However, no Ci^−3P expression can be detected in the posterior cells. (E) The Ci^−3P is fused with an HA tag in the N terminus, and no signal can be detected with an antibody against HA in the posterior cells, suggesting that both the full-length and the short form of Ci are degraded completely. (F) The merged image of C,D,E.