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First published online June 1, 2005
doi: 10.1242/10.1242/dev.01860

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Distinct roles for Mind bomb, Neuralized and Epsin in mediating DSL endocytosis and signaling in Drosophila

Howard Hughes Medical Institute, Department of Genetics and Development, Columbia University College of Physicians and Surgeons, 701 West 168th Street, New York, NY 10032, USA

View larger version (98K): [in a new window]   Fig. 1. dmib is required for normal DSL-Notch signaling during wing development. (A) Predicted transcript and protein product of the Drosophila mind bomb (dmib) locus. The dmib coding sequence (thick black line) is present on four exons interrupted by three introns (thin lines). The Dmib protein contains conserved Ankyrin repeats (dark green) and RING finger domains (light green). dmibEY09780 is an EP transgene that is inserted 96 bp upstream of the inferred translation start site; dmibL70 and dmibL53 are EMS-induced stop codons at Q954 and Q1163 deleting all three, and/or just the third, RING finger domain(s), respectively. All three dmib alleles behave as amorphs (Materials and methods), and all three gave indistiguishable results in each of the various experiments shown in this and subsequent Figures. (B) Adult mesonotum with several clones of dmibEY09780 cells. The mutant cells are marked by expression of a UAS-y+ transgene, which darkens cuticle, especially bristles; the mutant territories are outlined in white. dmib mutant cells form normal patterns of macro- and micro-chaetes, except that the density of microchaetes is somewhat higher than normal and some macrochaetes (particularly scutellar bristles) are occasionally duplicated (not shown); however, each bristle organ itself appears morphologically normal. (C) Wild-type wing. (D) Wing with clones of dmibL53 cells (marked by UAS-y+ expression) associated with wing notching (asterisk) and vein thickening (arrow), both phenotypes indicating a loss of DSL-Notch signaling activity. We note here that entirely mutant, dmib- animals can survive to the pupal stage (Materials and methods), and that some of these differentiate as pharate adults; the same is also true of dmib- animals obtained from dmib- female germ cells. As expected from the phenotype of mutant clones, these show severely truncated wings, consisting only of small stumps of wing hinge tissue, as well as an abnormally high density of microchaetes and occasional duplication of macrochaetes on the mesonotum. In addition, such mutant animals develop only rudimentary eyes containing around 25-50 ommatidia and form legs with truncated and fused leg segments. All of these phenotypes indicate deficiencies in well-characterized DSL-Notch signaling events during wing, eye and leg development. (E) Wing derived from a nub-Gal4/UAS-dmibRNAi wing imaginal disc, in which the UAS-dmibRNAi transgene was expressed uniformly throughout the prospective wing, causing severe wing notching and vein thickening. (F) Wing derived from a nub-Gal4/UAS-dmib wing imaginal disc. Over-expression of dmib throughout the prospective wing partially suppresses vein formation, the reciprocal phenotype to that caused by expression of dmibRNAi, indicative of an abnormal gain in DSL-Notch signaling.

View larger version (113K): [in a new window]   Fig. 2. Requirements for dmib activity in DSL endocytosis and signaling across the dorsoventral compartment boundary. (A-C) Wing disc containing a clone of dmibEY09780 cells [here, and in all subsequent figures, clones are marked by nuclear GFP expression (green) unless otherwise stated, the discs are oriented dorsal side up and anterior to the left]. (A,C) Ser (red) is normally expressed at moderate levels throughout the dorsal compartment (upper half) and is upregulated in cells neighboring the dorsoventral (D-V) compartment boundary (horizontal stripe) and in pro-vein cells (vertical stripes). (B,C) Cut (blue) is expressed in dorsal and ventral cells flanking the D-V compartment boundary in response to Ser signaling from dorsal cells and Dl signaling from ventral cells. DSL-Notch signaling across the D-V compartment boundary also upregulates Dl and Ser expression on each side, creating a positive feedback loop necessary for for peak activation of Notch and Cut expression on both sides. The dmibEY09780 clone abuts the D-V boundary and blocks Cut expression on both sides (B), indicating a failure in DSL-Notch signaling. (A',A'') Apical and sub-apical planes of section of the region boxed in white in A are shown at higher magnification; Ser accumulates at abnormally high levels in association with the apical cell surface in dmibEY09780 cells and at the expense of Ser-positive cytosolic puncta within the cells (the clone border is outlined in white). (D) Wing disc containing multiple clones of dmibEY09780 cells (green). As in A-C, dmibEY09780 clones that abut the D-V boundary are associated with a loss in Cut expression (red); however, as shown at higher magnification in the boxed inset, dmibEY09780 cells next to wild-type cells along the clone border express Cut (appears as yellow) indicating that they received DSL signals sent from neighboring wild-type cells. (E-G) Wing disc containing clones of dmibEY09780 cells (green). (E,G) Dl (red) is normally expressed at moderate levels in all cells in both compartments, and is upregulated along the D-V boundary and in pro-veins. (F,G) Wingless (Wg) expression (blue), like Cut, is induced in response to DSL-Notch signaling across the D-V boundary; a dmibEY09780 clone that crosses the D-V boundary blocks Wg expression on both sides. (E',E'') Apical and sub-apical planes of section of the region boxed in white in E are shown at higher magnification; Dl accumulation on the apical cell surface and in cytosolic puncta appears unchanged in dmibEY09780 cells relative to neighboring wild-type cells (the clone border is outlined in white, as in A',A''). (H) Wing disc in which UAS-dmibRNAi and UAS-lacZ (green) are co-expressed along the antero-posterior compartment border under the control of ptcG4; Cut expression (red) is abolished in dmibRNAi lacZ expressing cells flanking the D-V boundary.

View larger version (103K): [in a new window]   Fig. 3. Dmib is required for sending, but not receiving, DSL signals that induce Cut expression in the wing disc. (A) Clones of cells ectopically expressing Dl (green) induce Cut expression (red) in adjacent wild-type cells in the dorsal compartment (upper half). The abnormally high levels of Dl expression generated under these conditions autonomously inhibits Notch transduction and Cut expression by cells within the clones. (B'-B''') Clones of dmibL53 cells that ectopically express Dl fail to induce ectopic Cut expression and interrupt normal Cut expression when they abut the D-V boundary. Ectopic Dl expressing dmib- cells, unlike simple dmib- cells, do not express Cut even when adjacent to wild-type, Cut-expressing border cells along the D-V boundary, presumably because Dl over-expression in these cells autonomously inhibits Notch transduction, as it does in otherwise wild-type cells. (C) Clones of cells ectopically expressing Ser induce Cut expression in wild-type cells in the ventral compartment (lower half); Cut expression within the clone is inhibited, as in A. (D'-D''') Clones of dmibL53 cells that ectopically express Ser fail to induce ectopic Cut expression and interrupt normal Cut expression when they abut the D-V boundary. Cut expression is autonomously inhibited within the clones, as in B. (E) Clones of cells expressing NECN activate Cut expression autonomously (appears yellow). (F'-F''') Clones of dmibL53 cells that over-express NECN activate Cut expression autonomously, as in E. (G) Clones of cells expressing DlR+ activate ectopic Cut expression in adjacent wild-type cells in the dorsal compartment; Cut expression within such clones is inhibited, as in A,C. (H'-H''') Clones of dmibL53 cells that express DlR+ induce ectopic Cut expression ectopically in adjacent wild-type cells within the dorsal compartment, as in G, indicating that they bypass the requirement for Dmib.

View larger version (91K): [in a new window]   Fig. 4. Ectopic Neur can substitute for Dmib during wing disc development. (A-D) A clone of dmibL53 cells that ectopically expresses Neur (green) does not abolish Cut expression (blue) where it crosses the D-V compartment boundary. Ser expression (red) also appears normal in these cells. (E-H) Clones of dmibL53 cells that ectopically express Neur (green) do not block Cut expression (blue) when they abut or cross the D-V boundary. Normal Dl expression (red) is observed within the clones. (I-L) Clones of dmibL53 cells (green) that ectopically co-express Neur and Dl (red) induce ectopic Cut expression (blue) in adjacent wild-type cells in both dorsal and ventral compartments.

View larger version (134K): [in a new window]   Fig. 5. Dmib is required for normal bulk endocytosis of Ser but not Dl. (A) Clones of dmibEY09780 cells (marked black by the absence of ßGal, green) in a wing disc in which Dl (red) is over-expressed in the wing primordium under nub-Gal4 control. (B,C) Higher magnification of the region boxed in white in A; apical and sub-apical planes of focus are shown in B and C, respectively. Accumulation of Dl on both the apical cell surface, as well as in cytosolic puncta, appears unaffected by the absence of Dmib. (D) Clones of dmibEY09780 cells (marked as in A) in a wing disc in which Ser (red) is over-expressed in the wing primordium under nub-Gal4 control. Surface accumulation of Ser is dramatically enhanced by the absence of Dmib. (E-E'') Higher magnification of the region boxed in white in D, showing enhanced surface accumulation of Ser on the apical surface of dmibEY09780 cells. (F-F'') Little, or no, change is apparent in the accumulation of Ser in cytosolic puncta in dmibEY09780 cells.

View larger version (124K): [in a new window]   Fig. 6. Epsin is required for normal bulk endocytosis of Ser. (A-D) Wing disc stained for endogenous Ser expression (red) containing clones of lqf- cells (green). lqf- clones that abut or cross the D-V boundary impair Cut expression (blue). (A',A'') Apical and sub-apical planes of section of the region boxed in white in A are shown at higher magnification, and the borders of two clones of lqf- cells are outlined in white; Ser shows enhanced accumulation in lqf- cells relative to neighboring wild-type cells at the apical cell surface. (E,E') Clones of lqf- cells that ectopically express Ser (green) fail to induce ectopic Cut expression (blue) and interrupt normal Cut expression when they abut the D-V boundary. (F) Clones of lqf- cells (marked black, by the absence of ßGal, green) in a wing disc in which Ser (red) is over-expressed in the wing primordium under nub-Gal4 control. Surface accumulation of Ser is dramatically enhanced by the absence of Epsin. (G,H) Apical and sub-apical planes of section of the region boxed in white in F are shown at higher magnification in G and H, respectively. Ser accumulates dramatically at the apical cell surface of lqf- cells, but no change is apparent in the number or intensity of staining of Ser-positive puncta within the cells (the small island of strong Ser staining in H is due to surface staining in a local fold in the disc).

View larger version (156K): [in a new window]   Fig. 7. Over-expression of Dmib enhances endocytosis of DSL ligands. (A-A''') Wing disc in which Dl (red) is over-expressed in anterior cells along the A-P compartment boundary under the control of ptc-Gal4, inducing ectopic expression of Cut (green) in neighboring posterior cells in the dorsal compartment. A'' and A''' show the region boxed in A' at higher magnification at apical and sub-apical planes of focus: Dl accumulates predominantly at the cell surface. (B-B''') As in A, except that Dmib is co-over-expressed with Dl; under these conditions, Dl accumulation at the cell surface is depleted (compare A'' and B''), allowing Dl-positive puncta to be readily detected just below the apical cell surface in B''. (C-C''') As in A, except that Ser (red) rather than Dl is over-expressed under ptc-Gal4 control, and ectopic Cut expression (green) is induced in the ventral compartment. Note the cell surface accumulation of Ser in C''. (D-D''') As in C, except that Dmib is co-over-expressed with Ser; under these conditions, Ser accumulation at cell surface is depleted (compare C' and D''), and cytosolic puncta visible just beneath the apical surface in C''.

View larger version (32K): [in a new window]   Fig. 8. Roles of Dmib, Neur, Epsin and endocytosis in DSL signalling. (A) Different utilization of distinct endocytic pathways by Ser and Dl. Both ligands can enter the cell by at least three internalization pathways (arrows). Dmib/Neur ubiquitinates Lysine residues (K) in the cytosolic domains of both ligands, targeting them for endocytosis via the action of Epsin (red arrows) or by other factors, defining two distinct routes. Both ligands also contain additional internalization signals that target them for endocytosis in a ubiquitin- and Epsin-independent manner, defining a third route. As indicated by the thickness of the arrows, Ser normally enters the cell predominately via the two ubiquitin-dependent pathways, and mostly via the Epsin-dependent pathway. By contrast, Dl does so predominately via the remaining, ubiquitin-independent pathway. Nevertheless, only those molecules of Ser and Dl that enter via the Epsin pathway can signal. (B) Models for Epsin-mediated activation of Notch by DSL ligands. Two general classes of models are shown, distinguished by whether the Epsin-mediated endocytic event required for signaling occurs before or after the ligands are internalized. In the first class (left), Epsin-mediated clustering of DSL ligands into coated pits or other specializations, or Epsin-mediated invagination of these structures into the cell, might provide a particular micro-environment (red shading) or mechanical stress (red arrow) that is essential for inducing cleavage or shedding of the ectodomain of Notch. In the second class (right), Epsin might direct, or accompany, DSL proteins into a particular recycling pathway (red arrow) that is essential to convert or repackage them into ligands that can activate Notch upon return to the cell surface.

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