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First published online 8 April 2004
doi: 10.1242/dev.01100

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brinker and optomotor-blind act coordinately to initiate development of the L5 wing vein primordium in Drosophila

¹ Section of Cell and Developmental Biology, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0349, USA
² University of California, San Francisco, Third and Parnassus, San Francisco, CA 94143-0448, USA

View larger version (64K): [in a new window]   Fig. 1. ab is the L5 organizing gene. (A) A wild-type (wt) wing. The L2-L5 longitudinal veins are indicated. (B) An adult ab1/ab1 mutant wing. (C) ab expression in a wild-type third instar wing imaginal disc, visualized with an antisense ab probe (arrowhead indicates the ab stripe). (D) ab expression is greatly reduced in an ab1/ab1 mutant wing disc. (E) Double labeling of ab RNA (blue) and Dl protein (brown), shows that these genes are co-expressed in cells corresponding to the L5 primordium. Inset shows a higher magnification of the L5 primordium. The L1, and L3-L5, vein primordia are indicated. (F) rho is expressed in all longitudinal vein primordia in a wild-type third instar larval wing disc, detected with an antisense rho probe. Arrows indicate the approximate location of the L5 primordium in this and subsequent panels. (G) rho expression is lost in the L5 primordium of ab1/ab1 mutant wing discs. Wing discs are oriented with anterior at the top and dorsal to the left in this and subsequent panels. (H) Ubiquitous expression of ab with MS1096-GAL4 eliminates rho expression in all vein primordia. (I) Dl is expressed in the L1 and L3-L5 wing vein primordia. (J) Dl expression is lost in the L5 primordium of ab1/ab1 mutant wing discs. (K) Dl expression in all vein primordia is greatly reduced in discs ubiquitously expressing ab in MS1096-GAL4; UAS-ab wing discs. Weak Dl expression is visible in the ventral compartment of the disc, consistent with the lower levels of MS1096-GAL4 expression in ventral versus dorsal cells. (L) Blistered (Bs) protein is expressed at high levels in intervein cells, but is strongly downregulated in the L2-L5 vein primordia. (M) Bs downregulation in the L5 primordium is lost in ab1/ab1 mutant wing discs. (N) Bs expression is greatly reduced in all cells of MS1096-GAL4; UAS-ab wing discs. (O) A third instar wing imaginal disc misexpressing ab in flip-out clones (arrows) stained for ß-Gal (green) and Dl (blue). Dl is expressed in a cell-autonomous fashion within a subset of ß-Gal-expressing cells. Additional double-label experiments reveal that all cells in flip-out clones expressing ß-Gal also express Ab at high levels (O.C., unpublished). (P) Dl channel only for the disc shown in O. (Q) A third instar wing imaginal disc misexpressing ab in a flip-out clone (arrows) stained for ß-Gal (green) and the intervein marker Bs (red). Bs is downregulated in a cell-autonomous fashion within all cells of the clone. (R) Bs channel only for the disc shown in Q. (S) Ectopic veins form in a cell-autonomous fashion within small ab-expressing flip-out clones marked by being f36a (outlined).

View larger version (91K): [in a new window]   Fig. 2. The L5 primordium forms within the omb domain adjacent to brk-expressing cells. (A) A third instar wing imaginal disc triple labeled for Kni protein (red), Dl protein (blue) and sal-lacZ expression (green, ß-Gal). (B) sal-lacZ (ß-Gal) channel alone for the disc shown in A. (C) Kni and Dl channels for the disc shown in A. Kni is expressed anterior to cells expressing high levels of sal, but there is a significant gap between the posterior edge of the sal expression domain and the L5 primordium. (D) A third instar wing imaginal disc triple labeled for Kni protein (red), Dl protein (blue) and omb-lacZ expression (green, ß-Gal). (E) omb-lacZ (ß-Gal) channel alone for the disc shown in D. (F) Kni and Dl channels for disc shown in D. omb expression extends well past the Kni L2 stripe anteriorly, but just includes the L5 primordium posteriorly. (G) A third instar wing imaginal disc triple labeled for Kni protein (red), Dl protein (blue) and brk-lacZ expression (green, ß-Gal). (H) brk-lacZ (ß-Gal) channel alone for the disc shown in G. (I) Kni and Dl channels for disc shown in G. The L5 primordium lies along the outside border of the high level brk expression domain. (J) A third instar wing imaginal disc of omb-lacZ flies, double labeled for ß-Gal protein (brown) and ab RNA (blue, arrowhead). The ab stripe runs just within the domain of strong omb expression. (K) A third instar wing imaginal disc of brk-lacZ flies, double labeled for ß-Gal protein (brown) and ab RNA (blue, arrowhead). The ab stripe runs just adjacent to the domain of strong brk expression.

View larger version (115K): [in a new window]   Fig. 3. omb interacts genetically with ab. (A) A homozygous ab1/ab1 mutant wing lacks the distal section of L5. (B) Heterozygous ab1/+ flies have a fully penetrant wild-type wing phenotype. (C) Heterozygous omb1/+ flies also have a fully penetrant wild-type wing phenotype. (D) An omb1/omb1 mutant female wing has a notched wing margin, but no L5 truncation. (E) A brkm68/+; ab1/ab1 wing has a phenotype similar to that of ab1/ab1 homozygotes. (F) A trans-heterozygous omb1/+; ab1/+ female wing with a distal L5 truncation. This is a reproducible phenotype that occurs with low penetrance (3%). (G) An omb1/+; ab1/ab1 female wing has an enhanced L5 vein truncation relative to that observed in ab1/ab1 controls (compare with A). (H) An omb1/Y; ab1/ab1 double mutant male wing has a greatly enhanced L5 truncation phenotype relative to that observed in ab1/ab1 flies (compare with A).

View larger version (44K): [in a new window]   Fig. 4. Misexpression of omb or brk preferentially eliminates or displaces the L2 and L5 veins. (A) A wing from an MS1096-GAL4; UAS-omb fly is severely reduced in size and lacks posterior veins. (B) A wing from a C765-GAL4; UAS-brk fly displays a central shift of the L2 and L5 veins resulting in L2 approaching or fusing with L3 (i.e. 0-4 cells apart compared with 10-12 cells apart in wild type), and L5 approaching or fusing with L4 (i.e. 0-9 cells apart compared with 18-20 cells apart in wild type). By contrast, the space between the central L3 and L4 veins remained relatively unaltered (14-16 cells apart compared with 16-17 cells in wild type). All distance measurements between veins were made in the central region of the wing. (C) A third instar larval wing imaginal disc (anterior at the top) from a brk-lacZ; VgB-GAL4; UAS-GFP individual, grown at 22°C, stained for ß-Gal expression (red) and examined for GFP fluorescence (green). Note the posterior domain in which green VgB>GFP expression abuts the posterior edge of the brk expression domain (arrow), and note that expression of GFP continues along the margin. Stronger and wider GFP expression was observed in discs of the same genotype raised at 25°C (data not shown). (D) A wing from a VgB-GAL4; UAS-brk fly grown at 25°C displays a central shift of the L2 and L5 veins resulting in fusion of L2 with L3, and of L4 with L5. (E) A wing from a VgB-GAL4; UAS-omb fly grown at 22°C, which has a notched margin, truncated L5 vein (arrow) and a long ectopic vein posterior to L5 (arrowhead). Scale bars: 0.5 mm.

View larger version (84K): [in a new window]   Fig. 5. brk induces L5 formation in adjacent omb-expressing cells. Clonal analysis of omb and brk function in L5 development. Adult wings and wing imaginal discs are oriented with the anterior at the top. In adult wings, ventral clones are outlined in blue and dorsal clones are outlined in red. Clones in wing discs are marked by the absence of GFP expression. (A) Wing with an anterior dorsal omb- clone. The L2 vein extends uninterrupted through the clone. (B) kni expression (red labeling) is normal inside an anterior omb- mutant clone (arrowhead) in a third instar larval wing imaginal disc. (C) A wing with posterior dorsal omb- clones. The clone anterior to L5 (arrowhead) does not cause any vein phenotype, whereas the clone that crosses L5 results in vein truncation (arrow). (D) Dl expression (blue labeling) is lost within a posterior omb- mutant clone (arrowhead) in a third instar larval wing imaginal disc. (E) A wing with a posterior ventral brk- clone. An ectopic vein runs strictly inside and along the clone border. (F) Dl is ectopically expressed (blue labeling) within and encircling a posterior brk- clone (arrowhead) in a third instar larval wing imaginal disc. (G) A wing with an anterior ventral brk- clone. An ectopic vein lies along the border, within the clone (red arrowhead), except near the branch-point with L2 (black arrowhead) where it runs just outside of the clone. (H) kni is ectopically expressed (red labeling) within an anterior brk- clone (arrowhead) in a third instar larval wing imaginal disc. (I) A wing with overlapping dorsal and ventral posterior omb- brk- double mutant clones. Ectopic disorganized veins form within the clone interior. (J) Dl (blue labeling) is expressed within a posterior omb- brk- double mutant clone (arrowhead) in a third instar larval wing imaginal disc. Dl is expressed in unorganized pattern, within the interior of the clone. (K) A wing with anterior ventral and dorsal omb- brk- double mutant clones. A short segment of vein runs within the interior of the dorsal clone. (L) kni is not misexpressed (red labeling) within anterior omb- brk- double mutant clones (arrowheads) in a third instar larval wing imaginal disc. (M) Posterior ventral and dorsal brk- clones in ab1/ab1 mutant flies. The vein running along the posterior clone border is truncated (red arrowhead) in a similar location as the endogenous L5 vein (black arrowhead). In addition, a segment of vein forms within the center of the ventral clone. The phenotypes for the brk- clones shown in this panel and in E are representative clones scored posterior to or overlapping the L5 vein. Among a total of 44 such brk- clones generated in a wild-type background, 31 (70%) had veins extending for more than half of the proximal-distal length of the clone. These veins all formed inside and along the clone borders. The remaining 13 brk- clones had shorter segments of vein, which also ran along and within the clone borders. Among 15 comparably situated brk- clones generated in an ab- background, 3 (20%) had veins that extended along more than half the length of the clone border. The remaining clones had only short segments of vein and only 3 (20%) had disorganized vein material forming within the interior of the clone. No such internal disorganized veins were observed in any of the brk- clones generated in a wild-type background. (N) Ab expression (blue) along the border of a brk- clone located posterior to L5. No such ectopic Ab expression was observed in brk- omb- double mutant clones (O.C., unpublished).

View larger version (55K): [in a new window]   Fig. 6. A model for induction of the L5 primordium along the omb/brk border. The expression domain of the two Dpp signaling target genes omb (blue) and sal (green) is defined by their sensitivity to activation by Dpp and repression by Brk (red domains), in extreme anterior (A) and posterior (P) regions of the wing disc. L5 forms along the posterior border between omb and brk domains (solid blue line) following activation of the L5 organizer gene ab. brk-expressing cells on one side of the border produce a short range signal (Y) to which they cannot respond. This signal diffuses to the neighboring cells and, in combination with Omb, activates expression of ab along the border (yellow stripe). The green dotted line, which marks the posterior extent of strong sal expression, corresponds to a cryptic paravein border located midway between L4 and L5 (Sturtevant et al., 1997). This model is similar to that suggested for L2 formation along the border of the high level sal expression domain (Lunde et al., 1998). In the case of L2, sal-expressing cells produce a short range signal (X), to which they cannot respond due to repression by Sal, and this signal induces the expression of the L2 organizer gene kni along the border of cells just anterior to the sal domain (yellow stripe). The blue dotted line, which is indicated within the domain of strong omb expression along its anterior border and adjacent to cells expressing high levels of brk, may correspond to a cryptic paravein border located between L2 and the wing margin.