msh specifies dorsal cell fate in the Drosophila wing
Marco Milán1,*,
Ulrich Weihe1,*,
Stanley Tiong2,
,
Welcome Bender2 and
Stephen M. Cohen1,
1 European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany
2 Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA
Present address: Exilixis Inc., 170 Harbor Way, PO Box 511, South San Francisco, CA 94083-0511, USA
* These authors contributed equally to the work
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Fig. 1. Effects of removing msh: symmetric ventral-ventral wings. (A) Cuticle preparation of a wild-type wing. AWM, anterior wing margin; L2-L5, longitudinal veins 2-5. (B,C) Detailed views of dorsal structures of the wing in (B) wild type and (C) msh mutant clones. (Upper panels) Anterior wing margin (AWM); (center panels) alulas; (lower panels) veins. (B) In the wild type the AWM differentiates three rows of bristles. Two are dorsal (d); a row of thick mechanosensory bristles adjacent to the compartment boundary and a row of thin curved chemosensory bristles. The ventral row (v) is composed of thin bristles interspersed with chemosensory bristles in every fifth position. A schematic representation of the AWM is shown below. Red circles denote dorsal bristles, big circles indicate mechanosensory bristles and small circles indicate chemosensory bristles. Filled circles denote the chemosensory bristles located in the ventral surface, and open circles the mechanosensory bristles. (Center panel) The alula has a single row of bristles on the ventral surface and no dorsal bristles (not shown). (Bottom panel) Magnification of the dorsal side of vein L3. Corrugation of the L3 vein is asymmetric on dorsal (d) and ventral (v) surfaces of the wild-type wing. The corrugated surface (indicated in red in the diagrams at bottom) consists of 2-3 rows of more darkly pigmented cells. The opposite surface consists of one row of cells. In wild-type wings, veins L3, L5 and the distal part of L4 are corrugated dorsally and veins L2 and proximal L4 are corrugated ventrally. (C) Mutant clones were generated in f36 hs-FLP (I); FRT 82 msh 68/FRT 82 P(f+) larvae. msh mutant cells were marked with forked. In the AWM small arrows indicate the clone. The blue arrows indicate chemosensory bristles and large arrowheads indicate dorsal bristles outside the clone. A schematic representation of the AWM mutant for msh is shown below. Both surfaces differentiate ventral bristles (v). (Center panel) Magnification of an alula covered with clones mutant for msh shows that both dorsal and ventral surfaces differentiate bristles. (Lower panel) Magnification of the dorsal side of vein L3 shows part of the clone mutant for msh (red arrows); wild-type cells in the vein are indicated by black arrows. Note the transition from dorsal to ventral corrugation as shown in the diagram at the bottom.
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Fig. 2. Apterous regulates msh expression. (A-D) msh expression in third instar wing discs using an msh antisense RNA probe. d, dorsal; v, ventral. (A) Wild-type. Levels of msh expression are low in the dorsal compartment of the wing pouch, except along the dorsal anterior wing margin where expression is higher. In addition, a small patch of msh expressing cells is observed in the ventral compartment (in the anterior mesopleura, arrow) and in the dorsal notum. (B) dpp-Gal4 UAS-apterous disc. dpp-Gal4 is expressed adjacent to the AP boundary in the anterior compartment. Ectopic expression of msh in the ventral compartment is indicated by a red star. The level is comparable to the low dorsal level. Note the difference between the ectopic msh expressing tissue and the normal ventral tissue adjacent to the anterior mesopleura (arrow). (C) apterous mutant disc. apugo is a null allele. Expression of msh is lost in the dorsal compartment, but not in the anterior mesopleura (arrow) and part of the notum. (D) Dlw1/+ disc. msh expression is lower in the dorsal wing pouch. In situ hybridizations to msh were done in parallel in A and D. (E) msh-lacZ expression in a wild-type late third instar wing disc visualized by anti-ß-gal (red). (F) msh-lacZ expression in a wing disc expressing dLMO under patchedGal4 control. patchedGal4 is expressed in anterior cells adjacent to the AP boundary and directs high levels of dLMO expression (green). Endogenous dLMO is expressed at moderate levels in dorsal cells and at low levels in ventral cells. (Right) Repression of msh-lacZ in the patchedGal4 domain is indicated by an arrow. A, anterior; P, posterior.
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Fig. 3. Ectopic msh produces symmetrical dorsal-dorsal wings. c765-Gal4/+; UAS-msh/+ wing. All features are of dorsal identity. In the anterior wing margin each surface differentiates two rows of bristles: a row of densely packed mechanosensory bristles and a row of chemosensory bristles. The pattern of vein L3 corrugation, shown in the diagram, is completely dorsal (d) on both surfaces. The alula differentiates almost no bristles in the ventral surface.
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Fig. 4. Dorsal wing (Dlw) alleles. (A) Genomic organization of the msh gene. The msh transcript consists of 2 exons spanning approx. 10 kb. Arrows indicate breakpoints associated with Dlw alleles. Other predicted genes in the region are indicated. msh expression in the wing discs was monitored using mshlacZ89, an imprecise excision line of P{lacZ}rH96 that keeps the lacZ reporter gene. (B) Wild-type wing. AWM, anterior wing margin. PWM, posterior wing margin. The AP compartment boundary is shown by a line. (C,D) patchedGal4/uas-dLMO; Dlw1/+ wing. dLMO is overexpressed in the anterior compartment. dLMO inhibits Apterous activity. PWM', ectopic posterior wing margin induced in the dorsal surface along the AP compartment boundary. The wing is overgrown owing to ectopic expression of Wg along the patchedGal4 stripe. Dorsal and ventral surfaces do not contact normally. (D) Magnification of the dorsal side of vein L3 of the wing shown in C. In a wild-type wing, vein L3 is corrugated and has three campaniform sensillae on the dorsal surface (see Fig. 1). In the Dlw1/+ wing, campaniform sensillae (arrows in C and D) and corrugation are characteristic of the dorsal surface of vein L3. Their appearance in the Dlw1/+ wing indicates that Msh activity is present, despite the loss of Apterous activity.
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Fig. 5. msh restores dorsal identity in the absence of Apterous. (A) apGal4/apUGO35 wing. (B) apGal4/apUGO35 uas-msh wing. The arrow indicates wing margin bristles of dorsal identity shown at higher magnification on the right. (C) apGal4/apUGO35 EP-fng wing. The anterior wing margin (AWM) differentiated ventral-type bristles in both dorsal and ventral surfaces (middle, illustrated below). (D) apGal4/apUGO35 EP-fng/uas-msh wing. Bristles in the dorsal AWM had dorsal identity. AWM bristles were more densely packed than in wild type. Note also the reduced size of the dorsal compartment and the ectopic bristles in the wing blade. (E) apGal4/+; uas-msh wing. The reduced size of the dorsal compartment, the ectopic bristles in the wing blade and increased bristle density in the AWM were similar to those in D. This is presumably caused by strong overexpression of msh under apGal4 control. Weaker overexpression using c765-gal4 did not produce these defects (see Fig. 3). Ectopic bristles in the A compartment are mechanosensory bristles; those located in the P compartment are thin bristles. The DV identity of the posterior bristles could not be determined.
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FIG. 6. CONCLUSIONS. IN VERTEBRATE LIMBS TWO RELATED APTEROUS GENES, LMX1 AND LHX2, HAVE BEEN SHOWN TO ACT SEPARATELY TO DEFINE DORSAL IDENTITY AND LIMB OUTGROWTH. IN THE DROSOPHILA WING, APTEROUS INDUCES LIMB OUTGROWTH BY CONTROLLING DV SIGNALING AND SPECIFIES DORSAL IDENTITY. DORSAL IDENTITY IS DEFINED BY THE APTEROUS TARGET GENE MSH/DLW.
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© The Company of Biologists Ltd 2001
