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First published online 10 August 2005
doi: 10.1242/dev.01977

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Mutual repression between msh and Iro-C is an essential component of the boundary between body wall and wing in Drosophila

Centro de Biología Molecular Severo Ochoa, CSIC and UAM, Cantoblanco, 28049 Madrid, Spain

View larger version (73K): [in a new window]   Fig. 1. Expression of msh and ara/caup in the wing disc. Images show accumulation of Msh (green), Ara/Caup (red) and Wg (blue). (A) Late second instar disc. The border between the Msh and Ara/Caup domains is relatively sharp (arrowhead). (B) Mid-third instar discs. There is low level Msh accumulation in the dorsal wing pouch (arrow) and in the posterior notum (arrowhead). (C) Late third instar disc images. Channels showing Msh and Ara/Caup expressions are separately illustrated at bottom of the panel. Yellow lines indicate the positions of z-axis optical sections. These are shown at left (top) and right (middle) of the panel, with separate and merged red and green channels. White curved lines highlight the profiles of the disc epithelium sections. vh, ventral hinge, pl, pleura; dh, dorsal hinge; n, notum; ln, lateral notum; mn, medial notum; wp, wing pouch; a, anterior; p. posterior; white asterisks, tegula; blue asterisks, dorsal radius. msh expression is maximal in the dorsal and ventral prospective hinges (white arrowheads) and lower in the posterior notum (white arrow), while that of ara/caup is maximal at the prospective lateral notum (yellow arrowhead). Blue arrow indicates the region of decreased Iro-C expression in the lateral notum of late third instar discs. Blue horizontal line indicates the fold that approximately coincides with the notum/hinge boundary. In the notum/dorsal hinge z-section, there is strong notal expression of ara/caup that does not overlap with the strong expression of msh. This expression is also contiguous but does not overlap at the z-section through the ventral hinge and pleura (top, left). Wg marks the wing pouch epithelium (blue arrowhead) and this is contiguous with the domain of msh expression. Therefore, this should correspond to the ventral hinge and ara/caup expression should correspond to the pleura.

View larger version (73K): [in a new window]   Fig. 2. msh68 M+ clones interfere with the development of the notum and wing hinge. Clones were visualized by the f marker. (A) General view of the proximal wing, wing hinge (within the square) and left heminotum of a wild-type fly. (B) Wing with an abnormally wide hinge attached to the scutum and, ectopically, to the scutellum (arrow) of a fly carrying msh68 clones. Arrowheads indicate mutant scutellar bristles. (C) A posteriorly displaced wing (compare with A) attached to a thorax with a reduced scutellum (white arrowhead). Positional reference: dorsocentral bristles (red arrowhead). (D,E) High magnification views of a wild-type hinge and the mutant hinge shown in C (area within a rectangle) after mounting of the cuticle. The tegula is marked (t) as a positional reference. Rectangle in A indicates approximate area of the image in D of an unrelated fly. Broken lines indicate approximate borders of clones. Sclerites 1 (arrow) and 2 (arrowhead) are clearly visible in D, but these are absent in E. Red asterisk indicates the position where they would be expected to occur. Red arrowheads indicate ectopic chaetae. Green arrowhead indicates a probable scutellar macrochaeta. dr, wild-type dorsal radius and its clusters of sensilla campaniformia; only a few dorsal radius-like sensilla are seen in E (black arrowhead). (F,G) Lateral and dorsal views, respectively, of ectopic notum structures (arrows), which developed adjacent and just above the wing hinge (red arrowhead). The presence of f macro- and microchaetae indicate that the tissue is mutant for msh and suggests its notal identity. Tegulae (t), scutellum (asterisks) and costa (arrowhead) are marked for orientation. (H,I) Wild-type notum and notum with msh68 M+ clone(s) that removed macro and microchaetae. White arrowheads indicate positions of missing supraalar macrochaetae in I. Red arrowhead indicates an extra macrochaeta. Asterisks indicate extant dorsocentral macrochaetae. (J) Clone(s) associated with extra scutellar bristles (white arrowhead), an ectopic wild-type bristle (red arrowhead) and disruption of the scutum-scutellar suture (arrow). (K) Frequencies of anomalies (excepting modifications of the bristle pattern) associated with msh68 M+ clones induced at the indicated developmental times after egg laying. In parenthesis, number of heminota examined with one or more anomalies. Essentially all clones that comprised relatively large regions of the notum displayed one or more of the listed anomalies.

View larger version (94K): [in a new window]   Fig. 3. msh is required for the proper growth and patterning of the dorsal hinge. Third instar wing discs were stained (red channel) for the indicated proteins. In A,B,D,F, msh68 M+ clones are marked by the absence of green. (A) Pattern of Wg accumulation. The expression seems only slightly decreased within the msh clone (white arrowheads). (B) Tsh accumulation is not appreciably modified by the msh clones. In A and B, the fold of the epithelium that separates the notum and hinge territories (blue arrowhead) is absent, but that which separates the hinge and the wing pouch (yellow arrowheads) is present. (C,D) Proneural clusters of Sc accumulation in a wild-type disc (C) and a disc with large msh68 M+ territories (D). d, distal tegula; p, proximal tegula; r dorsal radius; a, anterior postalar cluster. Arrowheads indicate lateroanterior proneural clusters. (E) zfh-2 expression in the dorsal wild-type wing hinge (arrowheads). (F) zfh-2 expression is not modified in msh68 M+ clones (arrowheads). (G) Overexpression of UAS-msh (green channel; ap-Gal4 driver, at 17°C) induces ectopic expression of zfh-2 in the wing pouch (arrowhead) and weakly in the notum territory (arrow).

View larger version (60K): [in a new window]   Fig. 4. msh downregulates ara/caup in the hinge territory. Red: accumulation of Ara/Caup. (A) Wild-type disc. The notum Ara/Caup domain (arrow) is widely separated (white line) from the Nub (blue) wing pouch domain. (B) Disc harboring large msh68 M+ clones (absence of green). The notal Ara/Caup domain reaches almost to the wing pouch (arrowhead). Asterisks indicate the area incapable of expressing ara/caup. (C) Small msh68 clones (absence of green), induced 48/72 hours AEL, autonomously derepress ara/caup at the presumptive hinge, except when located (arrowhead) in the area shown in B (asterisk). (D) Disc overexpressing UAS-mshi driven by ap-Gal4. Msh almost completely disappears from the dorsal hinge (d) and Ara/Caup accumulates there. A slightly convex line joining the arrowheads would approximately demarcate the notum/dorsal-hinge border. Arrow indicates presumptive ventral hinge and pleura with unmodified msh and ara/caup expressions. (E) Clone overexpressing UAS-msh (green, GFP marker, induced at 12-36 hours AEL) removed or strongly inhibited notal Ara/Caup accumulation (arrowhead). (F) Drawing of a series of non-Minute msh68 clones reveals the areas (purple) competent to express ara/caup. (G) Early overexpression of UAS-msh, ap-Gal4 driver at 25°C (green, UAS-GFP marker) interfered with the growth of the notum territory (arrowhead; compare with wild-type disc in A, arrow). ara/caup expression always persisted in the proximal-most part of the notum territory, a result similarly observed with clones expressing UAS-msh in this location (text). This suggests that Iro-C is differentially controlled in different regions of the notum.

View larger version (106K): [in a new window]   Fig. 5. Regulation of msh by ara/caup and ap. (A) iroDFM3 clone (absence of red; induced at 36-60 hours AEL). msh is autonomously upregulated (arrowhead). (B) Small, late-induced (60-84 hours AEL) iroDFM3 clones (absence of red) near the hinge border (arrowhead) similarly derepress msh. (C) Clones overexpressing UAS-ara (induced at 36-60 hours AEL) autonomously repress msh in the hinge territory (arrowheads). (D) iroDFM3 clones (absence of green) do not survive in the notum region of discs deficient for Msh (UAS-mshi was driven with ap-Gal4). Clones are found only in the dorsal hinge and in the wing pouch regions (arrows). The wild-type twin spots (bright green, arrowhead) indicate that mitotic recombination events took place in the notum territory. (E) Late third instar wing disc from a homozygous apUGO35 larva. msh expression (red) is absent in essentially all the dorsal compartment of the disc (arrowhead), but it is present in the ventral hinge (arrow). ara/caup expression is shown in green. n, notum territory; vh, ventral hinge. (F) Chipe55 clones (absence of green; induced 24-48 hours AEL) failed to activate msh at the dorsal hinge (arrowhead) and dorsal wing pouch (blue arrowhead), but were without effect at the ventral hinge (arrow), consistent with the independence of msh expression in this territory from ap.

View larger version (43K): [in a new window]   Fig. 6. Known genetic interactions that define/maintain the notum/dorsal hinge subdivision of the wing disc. (A) In the second instar, EGFR signaling activates the `pronotum' genes Iro-C (Wang et al., 2000; Zecca and Struhl, 2002a; Zecca and Struhl, 2002b), and Dpp signaling, which is active only in the distal part of the disc, confines the expression of Iro-C to the proximal part of the disc (Cavodeassi et al., 2002), thus defining the notum territory. Also in the second instar, EGFR signaling, by means of ap, activates msh in the dorsal hinge. The proximal border of the msh domain abuts the Iro-C territory and the mutual repression between these genes contributes to maintain and stabilize the border between the Iro-C and the Msh territories. As discussed in the text, this border should define and/or maintain the notum/dorsal hinge subdivision of the disc (red line). (B,C) In support of this model, B and C show the expressions (red) in first/second instar discs of dpp-lacZ (B), which occurs mostly in the distal part of the disc, and of ap-lacZ (C), which takes place most strongly in a more central region. Within this region, msh will later be activated at high levels (Fig. 1A, and not shown). The contour of the discs has been marked with broken lines. (D) Adult structures that correspond to the domains of Iro-C (notum, orange) and msh (dorsal hinge, blue). The dorsal hinge has been equated to the msh domain. Its distal limit with the proximal wing (light blue), approximately corresponds with the inner circle of Wg expression (Fig. 1C) (del Álamo Rodríguez et al., 2002).