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First published online 2 December 2004
doi: 10.1242/dev.01546

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Unidirectional Notch signaling depends on continuous cleavage of Delta

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel

View larger version (25K): [in a new window]   Fig. 1. Several Drosophila ADAM metalloproteases cleave Delta and Serrate in S2 cells. (A) Phylogenetic tree of ADAM metalloproteases. Drosophila in bold, Anopheles in gray, C. elegans in italics and human underlined. The different human ADAMs have distinct homologs in each of the species. Note that ADAM10 has two homologs in Drosophila (Kuz and Kul) and in Anopheles. Similarly, Meltrin- has two homologs in insects. (B) Kuz and Kul have all the signature domains of ADAM metalloproteases. In the protease and disintegrin domains, Kul shows a higher degree of similarity to the human ADAM10. (C) Dl or Ser were expressed in S2 cells, and their capacity to serve as substrates for co-expressed Drosophila ADAM metalloproteases monitored by the appearance of ligand in the medium, and the concomitant disappearance from the cells. Kul, Kuz and DTACE displayed potent cleavage of both ligands. DMeltrin induced only low levels of Ser cleavage. The activity of the Mmd Meltrin homolog was not examined, because its expression is restricted to the central nervous system (Chase et al., 1987).

View larger version (94K): [in a new window]   Fig. 7. Kul is required for unidirectional Notch signaling at the wing margin. (A,C) In wild-type wing discs, Cut and Wg are expressed by the two rows of wing margin cells. (B,D) Expression of ds-kul by sd-GAL4, which is broadly expressed in the wing pouch, gave rise to a reduced pouch and loss or severe reduction of Cut and Wg expression (arrow). The adult wing (J) was dramatically reduced in size and exhibited only a rudimentary wing margin in very restricted domains (arrowhead). Arrow-wing hinge. (E,F) ds-kul expression by sal-GAL4 gave rise to a reduction in Wg expression. (G) In wt wing discs Dl is not detected at the wing margin (arrowhead). (H,I) Expression of ds-kul in the sal domain altered the distribution of Dl, which accumulated also in the wing margin (arrowhead), and expanded beyond the normal dorsal and ventral borders of Dl expression at the juxta margin (arrow). This experiment demonstrates that continuous cleavage of Dl by Kul is necessary for complete removal of the protein in the margin. In the absence of Kul, residual levels of Dl transcription give rise to accumulation of Dl, and lead to disruption of unidirectional Notch signaling. (K) Scheme: in wild-type wing discs, the margin cells express Wg and Cut, and suppress the expression of Dl. Wg activates Dl and Ser expression in the juxta-margin cells. High levels of Dl and Ser in these cells have a dominant-negative effect on Notch activation, but activate Notch signaling in the margin cells. Thus, a stable signaling loop is maintained. Elimination of Kul leads to the appearance of Dl in the margin, and alleviation of Notch signaling due to the dominant-negative effect of Dl.

View larger version (53K): [in a new window]   Fig. 2. Reduction of Kul levels leads to loss of wing veins. (A) Wild-type wing. (B) Expression of ds-kul in the central part of the wing by sal-GAL4 led to loss of veins (arrow). This phenotype suggests that Kul may normally be required for proper signaling by the Notch pathway. (C) Overexpression of Kul by sal-GAL4 led to an expansion of veins (arrow). (D) kul RNA is broadly distributed in the wing imaginal disc. (E) Expression of ds-kul at the anterior-posterior boundary of the wing disc by ptc-GAL4 led to elimination of endogenous kul RNA within this domain (arrowhead). (F) Expression of kul by the ptc-GAL4 driver exhibited a marked elevation in kul RNA. (G) Overexpression of Kuz by sd-GAL4 led to an expansion of veins (arrow), similar to Kul. (H) Expression of dskuz by MS1096-GAL4 led to thickening of the veins, similar to a kuz loss-of function phenotype. (I) The specificity of ds-kul was monitored in S2 cells. HA-tagged Kul (Kul-HA) is detected as a high molecular precursor, and as a mature protein lacking the pro-domain. Co-expression of ds-kul eliminated expression of the protein. By contrast, ds-kuz had no effect on the expression of Kul-HA. However, ds-kul had no effect on the expression of Kuz-HA, while ds-kuz eliminated the expression of the Kuz-HA protein. We conclude that ds-kul is specific. Expression of UAS-GFP was used to demonstrate similar transfection levels.

View larger version (55K): [in a new window]   Fig. 3. Structure-function analysis of Kul. The activity of Kul was monitored in S2 cells, by its capacity to release Dl to the medium and reduce the levels of the protein in the cells. Following expression of Dl alone, some cleavage can be detected, presumably by endogenous ADAM proteins. The levels of cleaved Dl are markedly elevated following co-expression of full-length Kul. A mutation leading to the elimination of the catalytic activity (Kul E-A), removal of the cytoplasmic tail (Kul Ex), or inactivating the cleavage site of the pro-domain (Pro-Kul), abolished the capacity of Kul to cleave Dl. Furthermore, in all three cases the activity of the endogenous ADAMs was also compromised.

View larger version (91K): [in a new window]   Fig. 4. Kul is required for unidirectional Notch signaling in the pupal wing. Expression of Dl in the wing veins was followed by anti-Dl (red), while the activation of Notch signaling was monitored by anti-ß-Gal staining of the Su(H)m8-lacZ reporter (blue). The L4 vein is indicated by an arrowhead. (A,C) In wild-type wings, Dl is expressed in 2-3 cell rows, marking the future veins. Su(H)m8 is excluded from the veins and is induced in up to five cell rows adjacent to the vein on each side. (B,D) In wings expressing ds-kul in the sal domain, dramatic alterations in both patterns were observed. Dl is expanded beyond the normal vein region (asterisk). Consequently, the cells expressing ectopic Dl trigger activation of Notch signaling within the vein (arrow). In some regions, expression of Dl is eliminated in the vein (arrowhead), possibly as a result of ectopic Notch signaling. (E) Scheme: in wild-type wings, Dl is expressed by the vein cells, activating Notch in the adjacent cells to induce an inter-vein fate within the pro-vein territory. Following Kul overexpression, the levels of Dl are reduced and Notch signaling is compromised, leading to an expansion of vein cell fate within the pro-vein territory (Fig. 2C). By contrast, when ds-kul is expressed, the levels of Dl rise in the cells adjacent to the veins, leading to Notch activation within the vein, and to the ectopic induction of an inter-vein fate (Fig. 2B and Fig. 4B,D).

View larger version (69K): [in a new window]   Fig. 5. Kul cleaves Delta but not Serrate in the wing disc. (A) Dl expression (red) in a wild-type wing disc. (B) Overexpression of Kul in the wing pouch eliminated the normal Dl protein in the wing margin and future veins. Note that in the notum, where MS1096-GAL4 is not expressed, normal levels of Dl were retained (arrow). (C,D) Overexpression of Kul by sal-GAL4 eliminated Dl in the sal domain. (E-G) Expression of Dl (red) in flipout clones (marked by GFP expression). (H-J) Co-expression of Kul (blue) and Dl in flipout clones eliminated the Dl protein. Thus, Kul mediates directly cleavage of Dl, rather than affecting the expression of Dl. (K-M) Clones of cells expressing Kul (green) showed elimination of Dl but not Ser. The loss of Dl was confined to the clone, demonstrating the cell-autonomous activity of Kul. (N-P) Overexpression of Kuz eliminated both Dl and Ser within the clones.

View larger version (74K): [in a new window]   Fig. 6. Effects of Kul overexpression in the wing imaginal disc. (A) Ser expression (green) in a wild-type wing disc. (B) Overexpression of Kul by MS1096 resulted in a uniform distribution of Ser, which is punctate. The effect is more pronounced in the dorsal part of the pouch, where expression of the driver is higher. (C-G) Kul was overexpressed in the central part of the wing disc by sal-GAL4. (C) Ser expression is expanded within the sal domain. Instead of the typical membrane-associated distribution of Ser (arrow), it is detected in a punctate intracellular pattern (arrowhead). (D) Merged image showing Ser (green) and Cut (red). (E) Cut expression is expanded throughout the domain of Kul overexpression (arrow). (F,G) Expression of Wg (red) is expanded within the same domain (arrow).