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First published online 20 October 2004
doi: 10.1242/dev.01448

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Drosophila Deltex mediates Suppressor of Hairless-independent and late-endosomal activation of Notch signaling

¹ Department of Biological Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan
² School of Biological Sciences, University of Manchester, Stopford Building, Oxford Road, Manchester M13 9PT, UK
³ Department of Nutrition, School of Medicine, University of Tokushima, 3-18-15 Kuramoto, Tokushima 770-8503, Japan
⁴ Department of Neuroscience and Immunology, Kumamoto University, Graduate School of Medical Sciences, Honjo 2-2-1, Kumamoto 860-0811, Japan
⁵ Department of Physiology, Keio University, School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
⁶ Genome and Drug Research Center, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan
⁷ PRESTO, JST, 4-1-8 Honcho, Kawaguchi, Saitama, Japan

View larger version (68K): [in a new window]   Fig. 1. dx is indispensable for the normal activation of N signaling at the DV boundary. (A) Wild-type adult wing. (B) Adult wing of dx24/Y, a hypomorphic loss-of-function mutant of dx, showing recessive distal wing-blade notching (arrowhead). (C,E) Wg expression in the wild-type third-instar wing disc, detected in a narrow stripe along the DV boundary by an anti-Wg antibody (green). E is a higher magnification of C. (D,F) Wg expression (green) in the dx24/Y third-instar wing disc, showing a reduction in the Wg protein at the intersection of the AP and DV boundaries that corresponds to the distal tip of the wing (black and white arrowhead in B,D,F). F is a higher magnification of D. (G,I) Cut expression in the wild-type third-instar wing disc, detected in a narrow stripe along the DV boundary by an anti-Cut antibody (green). I is a higher magnification of G. (H,J) Cut expression (green) in the dx24/Y third-instar wing disc, showing an interruption at the intersection of the AP and DV boundaries that corresponds to the distal tip of the wing (black and white arrowheads in B,H,J). J is a higher magnification of H. (K) Schematic diagram of the vgBE-lacZ transgene constructs. Wild-type recognition sequences for Su(H) and its mutant derivative are shown in the upper and lower lines, respectively. Mutated nucleotides are shown in red. (L-N) Activation of vgBE at the DV boundary of the third-instar wing disc detected by vgBE-lacZ (green). (L) A wing disc of vgBE-lacZ. (M,N) A wing disc of vgBE-lacZ overexpressing DxPro (purple) under the control of ptc-GAL4 at 18°C, showing suppression of vgBE activity in the region overexpressing DxPro (white arrowhead). M and N show a single green channel and a merged image, respectively.

View larger version (88K): [in a new window]   Fig. 2. Overexpression of Dx ectopically activates the target genes of N signaling. (A-C) Within the Dx-overexpressing clones (green), vgBE (purple) was activated in a cell autonomous manner. C is a merged image of A and B. (D-F) Within cells overexpressing Dx (green), the wg promoter was ectopically activated. The promoter activity of wg was detected by wg-lacZ (purple). F is a merged image of D and E. (G-I) Within cells overexpressing Dx (green), the Dl promoter was ectopically activated. The promoter activity of Dl was detected by Dl-lacZ (purple). I is a merged image of G and H. Weak cell non-autonomous induction of Dl-lacZ was also observed. (J,L) Expression of N in wild-type wing discs. N was detected by an anti-NICD antibody (green). (K,L) Activation of the N promoter was visualized by N-lacZ (purple). (M-O) Within the Dx-overexpressing clones (green), N-lacZ (purple) was activated in a cell autonomous manner. O is a merged image of M and N. (P-R) Within the NICD-overexpressing clones (green), N-lacZ (purple) was activated cell autonomously. R is a merged image of P and Q. All images were obtained from third-instar wing discs with respective genetic manipulation.

View larger version (79K): [in a new window]   Fig. 3. Dx activates N signaling in a Su(H)-independent manner. (A) A mid-third-instar wing disc of vgBE-lacZ. ß-GAL protein is shown in green. (B,C) Overexpression of Dx (purple) along the AP boundary in a mid-third-instar wing disc ectopically activated the vgBE (green) in both dorsal and ventral compartments of the wing pouch. B and C show a single green channel and a merged image, respectively. (D) In the wing disc dissected from a vgBE Su(H)m-lacZ transgenic line, ß-GAL (green) was not detected along the DV boundary. (E,F) Overexpression of Dx (purple) along the AP boundary did not activate vgBE Su(H)m-lacZ (green). E and F show a single green channel and a merged image, respectively. (G-J) Within Su(H)47/Su(H)47 mutant clones (marked by an absence of green fluorescence) generated in the late-third-instar wing disc, an overexpression of NICD (blue) failed to activate the vgBE along the AP boundary (purple). J is a merged image of G, H and I. (K-N) Within Su(H)47/Su(H)47 mutant clones (marked by an absence of green fluorescence) generated in the late-third-instar wing disc, an overexpression of Dx (blue) still activated the vgBE (purple). N is a merged image of K, L and M. (O) Overexpression of Dx (purple) along the AP boundary induced ectopic expression of Wg mostly, but not exclusively, in the ventral region of the wing pouch (green). (P) Misexpression of double-strand RNA of N along the AP boundary resulted in the suppression of Wg expression (green) in the late-third-instar wing disc. (Q,R) Overexpression of Dx (purple) with double-strand RNA of N failed to induce the ectopic expression of Wg (green) in the late-third-instar wing disc. Q and R show a single green channel and a merged image, respectively. (S-V) Expression of the wg gene (blue) detected by in situ hybridization in the late-third-instar disc. (S) wg expression in the wild-type wing disc. (T) Overexpression of Dx along the AP boundary induced a weak ectopic wg expression mostly in the ventral region of the wing pouch. (U) Overexpressed NFL induced a weak ectopic wg expression. (V) Co-expression of Dx and NFL resulted in a synergistic enhancement of N signaling revealed by strong wg expression along the AP boundary. (W-Z) Within DlREV10 and SerRX106 double mutant clones (marked by an absence of green fluorescence) generated in the late-third-instar wing disc, an overexpression of Dx (blue) still activated the vgBE (purple). Z is a merged image of W, X and Y. UAS-dx (B,C,E,F,O-R,T-V,W-Z) and UAS-NIR (P-R) were driven by ptc-GAL4. UAS-dx (K-N) and UAS-NICD (G-J) were driven by dpp-GAL4.

View larger version (101K): [in a new window]   Fig. 4. Dx modulates the intracellular distribution of N. (A-L) Confocal microscopic images of third-instar wing discs overexpressing Dx. The boundaries of the region overexpressing Dx are shown by white lines. (A,C,E,G) Overexpression of Dx (blue in E and G) resulted in the depletion of N (green in A and G), but not Dl (purple in C and G), from the apical cell surface. G is a merged image of A, C and E. (B,D,F,H) In the basal plane, overexpression of Dx (blue in F and H) increased N (green in B and H) in intracellular vesicles. In the cells overexpressing Dx, Dx, but not Dl, was frequently co-localized with N in these vesicles (the left part of B-H). In contrast, N co-localized with Dl in the wild-type cells (the right part of B-H). Similar results were obtained when N was stained either with antibodies against the intracellular domain or against the extracellular domain of N. H is a merged image of B, D and F. (I-L) An optical cross-section of a wing disc overexpressing Dx, stained as in A-H. N (green) and Dl (purple) staining are shown in I and J, respectively. K is a merged image of I and J. L shows Dx (blue) staining merged with K. (M-P) A third-instar wing disc overexpressing Dx (blue) was incubated with fluorescein Dextran (green) to label the endocytic compartments. Fluorescein Dextran (green) and endogenous N (purple) were co-stained. Some of the vesicles containing Fluorescein Dextran and N, although not all, were also marked with Dx (shown by white arrowheads). P is a merged image of M, N and O. UAS-dx was driven by ptc-GAL4.

View larger version (107K): [in a new window]   Fig. 5. Overexpressed Dx increases the number of endocytic vesicles containing N and stabilizes the N in these vesicles. (A-L) The expression of N+-GV3 protein (green) in eye discs was detected by anti-GAL4. Dx was driven by the GMR promoter (purple in I-L). (A,E,I) Wild-type (A) and GMR-dx (E,I) eye discs before heat shock. No expression of N+-GV3 (green) was detected. I is a merged image. (B,C) In wild-type eye discs, N+-GV3 expression (green) was observed throughout the eye disc, except for the morphogenetic furrow, 30 minutes after heat shock. The N+-GV3 expression was primarily localized to the plasma membrane and within a small number of intracellular vesicles. C is a higher magnification of B. (D) In the wild-type eye disc, no N+-GV3 expression (green) was observed 12 hours after heat shock. (F,G,J,K) In GMR-dx eye discs, N+-GV3 expression (green) was observed throughout the eye disc, except for the morphogenetic furrow, 30 minutes after heat shock. In the region expressing Dx (purple), N+-GV3 expression was primarily localized to the intracellular vesicles. G and K are a higher magnification of F and J, respectively. J and K are merged images. (H,L) In the region expressing Dx (purple), N+-GV3 (green) was still detected. L is a merged image. (M-Q) In situ hybridization of wild-type (M,N,Q) and GMR-dx (O,P) eye discs using antisense (M-P) and sense (Q) GAL4 probes. N+-GV3 mRNA was detected using the antisense GAL4 probe. (M,O) In wild-type and GMR-dx eye discs, N+-GV3 mRNA was observed throughout the eye disc 30 minutes after heat shock. (N,P) In wild-type and GMR-dx eye discs, no N+-GV3 mRNA was detected 12 hours after heat shock. (Q) In situ hybridization using a sense-strand probe for GAL4. (R,S) Within the dx–/dx– clones (patches without green fluorescence), the number of vesicles containing N+-GV3 (purple) was reduced, compared with wild-type and dx–/+ cells. (T) The number of vesicles containing N+-GV3 per cell was compared in seven independent measurements. The relative number of vesicles in the dx–/dx– cells are shown as percentages.

View larger version (139K): [in a new window]   Fig. 6. Dx co-localizes with N in the late-endosomes. (A-L) Confocal microscopic images of third-instar wing discs overexpressing Dx. (A-D) Some of the Dx (blue in C and D) immunoreactivity co-localized with Clc-GFP (green in A and D) and N (purple in B and D). D is a merged image of A, B and C. (E-H) Drosophila Hk exists exclusively in the early-endosomes. Neither N (green in E and H) nor Dx (blue in G and H) co-localized with Hk (purple in F and H). H is a merged image of E, F and G. (I-L) Drosophila Rab7-GFP accumulates in the late-endosomal compartment. Dx (blue in K and L) and endogenous N (purple in J and L) co-localize in the late-endosome (green in I and L). L is a merged image of I, J and K. (M-P) A fusion protein of Dx and YFP (green in M, O and P) was expressed in the Drosophila S2 cell line. The lysosomes were visualized by LysoTracker (purple in N, O and P). O is a merged image of M and N. P is a merged picture of the optical microscopic image of a cell with O. (A-L) UAS-dx was driven by ptc-GAL4. (M-P) UAS-YFP-dx was driven by pWA-GAL4.

View larger version (138K): [in a new window]   Fig. 7. Blockage of N delivery to the late-endosome inhibits the activation of Dx-mediated N signaling. (A-C) Overexpression of Rab5S43N, a dominant-negative form of Rab5, along the AP boundary of the third instar wing disc did not show a detectable effect on endogenous vgBE activation (green in A and C). Endogenous expression of Dx was also detected (purple in B and C). (D-F) Co-expression of Dx (purple in E and F) and Rab5S43N along the AP boundary resulted in inhibition of the ectopic activation of vgBE (green in D and F) associated with Dx overexpression. (G-J) In the apical region of the epithelial cells overexpressing Dx (blue in I and J) with Rab5S43N, N (green in G and J) accumulated in unusually large Hk-positive vesicles (purple in H and J), where Dx was co-localized with N (shown by white arrowheads). (K-N) In the basal region of the cells overexpressing Dx (blue in M and N) with Rab5S43N, the accumulation of N (green in K and N) in the late-endosomes was not detected (compare to Fig. 4B). UAS-dx and UAS-Rab5S43N were driven by dpp-GAL4 (A-F) or ptc-GAL4 (G-N).