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First published online 13 April 2005
doi: 10.1242/dev.01825

Development 132, 2319-2332 (2005)
Published by The Company of Biologists 2005
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The ubiquitin ligase Drosophila Mind bomb promotes Notch signaling by regulating the localization and activity of Serrate and Delta

Eric C. Lai1,*, Fabrice Roegiers2,3, Xiaoli Qin1, Yuh Nung Jan2 and Gerald M. Rubin1

1 Department of Molecular and Cell Biology, Howard Hughes Medical Institute, 545 Life Sciences Addition, University of California at Berkeley, Berkeley, CA 94720-3200, USA
2 Department of Physiology and Biochemistry, Howard Hughes Medical Institute, 1550 4th Street, Room GD481, University of California, San Francisco, Box 0725, San Francisco, CA 94143-0725, USA
3 Fox Chase Cancer Center, Cellular and Developmental Biology Program, 333 Cottman Avenue, Philadelphia, PA 19111, USA



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  		Fig. 1. Summary of D-mib structure-function studies. All Mib proteins display the same domain structure seen in D-mib. The different domains are color-coded as follows: Mib/Herc2 domains (MH), green; zz zinc finger (ZF), orange; Mib domain, light grey; ankyrin repeats, blue; RING fingers, red. We assayed the activities of the depicted portions of D-mib in vivo and in vitro, and the results are summarized on the right; selected data are shown in Figs 2, 3, 4. Dl, Delta; Ser, Serrate.

 


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  		Fig. 2. D-mib is required for multiple Neur-independent, Notch-dependent developmental processes. (A,C,E,G,I,K) Wild-type; (B,D,F,H,J,L) D-mib1 homozygotes. D-mib1 pharate adults are largely eyeless (B) and wingless (D). D-mib mutants (F) also display defective leg development and lack joints (arrows compare with E). Leg structures are abbreviated as follows: Ti, tibia; t1-t5, the five tarsal segments; cl, claw. (G-L) Wing imaginal discs stained for Nubbin (G,H), Cut (I,J) and Senseless (K,L). Note that wing margin (WM) expression of Cut and Senseless is absent in D-mib1 discs, but that sensory organ precursors (arrows) are singularized normally in this mutant.

 


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  		Fig. 3. Effects of ectopic D-mib and D-mib{Delta}RF on Notch-regulated developmental patterning. (A) Wild-type adult head. (B) sca-Gal4>UAS-D-mib head is missing several macrochaetae. (C) sca-Gal4>UAS-D-mib{Delta}RF exhibits macrochaetae tufting. (D) Wild-type wing. WM, wing margin; asterisk marks a wing vein. (E) bx-Gal4/Y; UAS-D-mib wing displays longitudinal vein breaks (asterisk) and lacks crossveins. (F) bx-Gal4/Y; UAS-D-mib{Delta}RF is vestigial and completely lacks a wing margin; the remaining wing tissue present is composed mostly of severely thickened wing veins (asterisk). (G) Close-up of the L3 vein in a dpp-Gal4/+ wing; arrowheads point to two campaniform sensilla. The normal thickness of vein is denoted with a bracket. (H) dpp-Gal4, UAS-D-mib wing lacks campaniform sensilla. (I) dpp-Gal4, UAS-D-mib{Delta}RF wing displays an extremely thickened L3 vein and a vast surplus of campaniform sensilla; both features are indicative of failed Notch signaling. (J-L) Third instar wing imaginal discs stained for Sens; only the wing pouch region is shown. (J) In wild type, sensory organ precursors for L3 sensilla are indicated (arrow). (K) sca-Gal4>UAS-D-mib lacks some L3 sensory precursors. (L) sca-Gal4>UAS-D-mib{Delta}RF shows ectopic L3 sensory precursors. As the sensory multiplication defect is more prominent at later times, the disc in panel L is slightly older than those of panels J and K. (M) Cut expression at the prospective wing margin (WM) in wild type. (N) dpp-Gal4, UAS-D-mib shows normal wing margin development. (O) dpp-Gal4, UAS-D-mib{Delta}RF disc shows a gap in the wing margin in D-mib{Delta}RF-expressing cells (asterisk).

 


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  		Fig. 4. The N terminus of D-mib mediates physical association with both Delta and Serrate. Co-immunoprecipitation was performed on lysates from 293T cells transfected with polyoma-tagged DSL ligands and Myc-tagged D-mib proteins. Structures of D-mib variants are depicted in Fig. 1. In transfected cells, D-mib proteins appear as single bands (A, lanes 6-10), Delta is present in full-length form and as a cleavage product corresponding to its intracellular domain (B, lanes 16-20), and Serrate appears as a series of relatively closely migrating bands (C, lanes 26-30). (A) Delta efficiently co-immunoprecipitates D-mib-N, D-mib{Delta}3RF and D-mib{Delta}RF (lanes 2-4). (B) Delta is efficiently co-immunoprecipitated by D-mib-N, D-mib{Delta}3RF and D-mib{Delta}RF (lanes 12-14); D-mib-N also associates with the cleaved intracellular domain of Delta (lane 12). Full-length D-mib interacts weakly with Delta (lane 11), but levels of Delta are also decreased in the presence of D-mib (lane 16). (C) Serrate is co-immunoprecipitated by D-mib-N, D-mib{Delta}3RF and D-mib{Delta}RF (lanes 22-24), and D-mib reduces overall levels of Serrate (lane 26). In all cases, the interaction between DSL ligands and D-mib-N is strongest.

 


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  		Fig. 5. D-mib promotes the signaling activity of DSL ligands. Shown are wing imaginal discs doubly stained for Cut (A-F) and DNA (G-L); only the wing pouch region is shown in (A-F). (A,G) Wild type. Expression of Cut at the wing margin is denoted with two arrowheads, and the ventral (V) and dorsal (D) regions of the wing pouch are marked. (B,H) dpp-Gal4>UAS-Delta disc is strongly overgrown (compare disc sizes of G and H). A strong ectopic margin is seen in the dorsal wing pouch (D) posterior to the dpp-Gal4 domain. (C,I) dpp-Gal4>UAS-Delta, UAS-D-mib disc is similarly overgrown; however, strong ectopic margins are now induced in the ventral compartment (V) both anterior and posterior to the dpp-Gal4 domain. (D,J) dpp-Gal4>UAS-Delta, UAS-D-mib{Delta}RF shows complete suppression of Delta-induced disc overgrowth (compare J with H), and complete inhibition of Delta-induced margin development. In addition, there is a large gap in the endogenous wing margin in the dpp-Gal4 domain (D, asterisk), as seen with UAS-dpp-Gal4>UAS-D-mib{Delta}RF discs (see Fig. 6O). (E,K) dpp-Gal4>UAS-Serrate disc is strongly overgrown and shows ectopic ventral margins (V). (F,L) dpp-Gal4>UAS-Serrate, UAS-D-mib{Delta}RF shows complete suppression of Serrate-induced disc overgrowth and margin induction, and a gap in the endogenous wing margin can be seen (F, asterisk).

 


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  		Fig. 6. In vivo regulation of Serrate and Delta localization and stability by D-mib. In all discs excepting E and F, Delta is in red and Serrate in green. (A-D) Tests of endogenous D-mib function. Wing imaginal discs from wild-type (A,C) and D-mib1 (B,D). Apart from the defective wing pouch development, Dl expression is fairly normal in D-mib discs (B), but elevated levels of Serrate are present and localized primarily to the plasma membrane (D). Imaging of Delta and Serrate in A-D was performed identically, so that relative protein levels between discs is comparable. (E-P) Tests of ectopic D-mib function. In these experiments, D-mib isoforms are expressed in a stripe at the anteroposterior compartment boundary using dpp-Gal4. (E,F) dpp-Gal4, UAS-nGFP double stained for GFP (E) and GFP + Delta (F). The L3 wing vein expression of Delta is contained within the domain of dpp-Gal4 activity. (G-L) Wing discs double stained for Serrate and Delta. In all cases, the L3 wing vein domain is marked with an asterisk, and insets depict magnified apical views from this region. (G,H) Wild type. (I,J) dpp-Gal4, UAS-D-mib discs show a reduction of Serrate from the apical membrane and a strong decrease in both the apical and total level of Delta. (K,L) dpp-Gal4, UAS-D-mib{Delta}RF disc displays strongly increased levels of both Serrate and Delta. Endogenous margin-specific expression of Serrate and Delta is also interrupted (arrowhead). Note the large apical intracellular aggregates of Serrate (K, inset). (M-P) Effect of D-mib proteins on exogenous Delta. In these panels, one copy of hs-Delta is present, and animals were heat-shocked at 38°C for 40 minutes, then allowed to rest for the indicated period of time prior to dissection and fixation. The regions shown in (N-P) correspond to the boxed region in F. (M) hs-Delta/+, 40 minute rest. Delta is present at the plasma membrane of all cells. (N) hs-Delta, dpp-Gal4, UAS-D-mib, 40 minute rest. All Delta within the D-mib-expressing domain is vesicular. (O) hs-Delta, dpp-Gal4, UAS-D-mib, 90 minute rest. All Delta within the D-mib-expressing domain has been degraded. (P) hs-Delta, dpp-Gal4, UAS-D-mib{Delta}RF, 120 minute rest. D-mib{Delta}RF fails to efficiently induce either the internalization or degradation of Delta.

 


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  		Fig. 7. Mutual phenotypic suppression of wild-type Neur and D-mib proteins with their RING-deleted counterparts. Shown are adult wings of the depicted genotypes, with a focus on wing vein determination and wing margin development. (A) dpp-Gal4/+ wing. Arrowhead denotes the anterior crossvein and asterisk marks the distal wing margin. (B) dpp-Gal4, UAS-neur wing is mostly wild type. (C) dpp-Gal4, UAS-D-mib wing shows mild wing vein loss (arrowhead). (D) dpp-Gal4, UAS-neur{Delta}RF wing exhibits a distal wing notch (asterisk) and a very mildly thickened L3 vein. Wing notching induced by neur{Delta}RF is completely suppressed by the co-expression of UAS-neur (E, asterisk), as well as by UAS-D-mib (F, asterisk); wing vein loss is often observed in the latter (F, arrowhead). (G) dpp-Gal4, UAS-D-mib{Delta}RF wing displays an enormous wing notch (asterisk) and a severely thickened L3 wing vein remnant (arrow). Both phenotypes are partially suppressed by the co-expression of one copy of UAS-neur (H, arrow and asterisk) and are almost completely suppressed by the co-expression of two copies of UAS-neur (I, arrow and asterisk). Note that some wing vein loss is even evident in the latter genotype (I, arrowhead).

 


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  		Fig. 8. Functional replacement of Neur by D-mib. Shown are dorsal thoraces from adult flies (A-D) or 36-hour APF pupae (E-P) containing ubx-FLP-generated MARCM clones of cells that are simultaneously mutant for neur and express sca-Gal4 and relevant UAS-transgenes (please refer to the Materials and methods for details of the genetics). (A) Clones of the weak allele neurA101 exhibit a bristle tufting phenotype (asterisk), wherein multiple sensory organs are present at individual positions. (B) The tufting phenotype of neurA101 clones is completely rescued by expression of UAS-neur. (C) Clones of the stronger allele neurIF65 are bald (asterisk), due to conversion of outer sensory cell fates into neurons. (D) The balding phenotype of neurIF65 clones is rescued back to a mild-tufting phenotype (asterisk) by expression of UAS-D-mib. (E-G) neurIF65 sensory clusters marked by GFP expression (due to MARCM activation of sca-Gal4>UAS-pon-GFP: E, asterisk) fail to express Su(H), a marker of socket cell fate (red, F,G); arrow in F indicates a Su(H)+ nucleus. (H-J) neurIF65 sensory clusters with sensory specific expression of D-mib show rescue of Su(H) expression. Note that small clusters of Su(H)+ cells are usually seen, indicating a partial rescue of the strong neurIF65 lateral inhibition defect. (K-P) X-Z confocal sections through individual neur mutant sensory clusters expressing GFP and stained for ELAV, a neuronal marker. (K,N) A single neuron and the two large cell bodies of the socket and shaft cells are present in wild type. (L,O) A large mass of neurons is found in a neurIF65 sensory cluster, and large cell bodies indicative of outer cell fates are absent. (M,P) Rescue of the neurIF65 neurogenic defect and socket/shaft differentiation by ectopic D-mib.

 

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© The Company of Biologists Ltd 2005