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First published online February 2, 2004
doi: 10.1242/10.1242/dev.00979

Development 131, 767-774 (2004)
Published by The Company of Biologists 2004
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Proximodistal subdivision of Drosophila legs and wings: the elbow-no ocelli gene complex

Ulrich Weihe1, Ruslan Dorfman2, Mathias F. Wernet3, Stephen M. Cohen1,{dagger} and Marco Milán1,*,{dagger}

1 European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany
2 Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel
3 Department of Biology, New York University, New York, USA



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  		Fig. 1. el and noc expression in presumptive appendages. (A) Genomic organization of the elbow and no ocelli loci. The el transcript consists of three exons spanning 3.3 kb [EST clone RE67722 (Dorfman et al., 2002Go)]. noc is located 83 kb downstream of el and consists of two exons. The el1 deletion removed ~25 kb of DNA between the two coding regions and specifically affects expression of both genes in the wing imaginal disc, suggesting that it removes a common regulatory element shared by the two loci. el and noc were coexpressed at all stages examined. (B) Diagram of the proximal-distal subdivision of both wing and leg primordia by signaling molecules. In the leg imaginal disc, the combined activity of Dpp and Wg induces the expression of Dll in the presumptive appendage primordium (blue) and restricts the expression of Hth to the periphery of the disc, which will give rise to the adult body wall (red). In the wing imaginal disc, Wg activity induces the expression of Nub in the wing primordium (blue) and Vein (Vn) activity induces the expression of Hth in the periphery of the disc, which will give rise to the adult body wall (red). (C) Second instar leg disc labeled with antibodies to visualize Dll protein (blue), El protein (green) and Hth protein (red). Noc expression was identical to El expression (not shown). (D) Second instar wing disc labeled to visualize nocGal4 using UAS-GFP (green), Nub protein (blue) and Hth protein (red). Note that the expression domain of Noc is slightly broader than the domain of Nub and overlaps Hth expression. Hth repression lags behind Tsh repression and thus underestimates the size of the wing field at early stages (Wu and Cohen, 2002Go). (E) Mature third instar leg imaginal disc labeled as in C. Note the change in the relative expression pattern of El and Dll with respect to the earlier stage in C. (F) Mature third instar wing disc labeled as in (D). El and Noc are expressed in a ring corresponding to the wing hinge and in wedge shaped domains centered on the dorsal-ventral boundary.

 


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  		Fig. 2. Regulation of El and Noc at early stages. (A) dppGal4-driven expression of the Wg-pathway inhibitor GSK3/Shaggy (Sgg) in an early third instar wing disc. Anti-El staining (purple) is reduced in the dpp expression domain (GFP, green). (B) dppGal4-driven expression of Wg in a late second instar wing disc. El staining is ectopically induced in body wall regions in a non-autonomous manner close to the dpp expression domain. (C) dppGal4-driven expression of the Wg-pathway inhibitor GSK3/Shaggy in an early third instar leg disc. El is reduced in the dpp expression domain (arrow). (D) dppGal4-driven expression of the Dpp-pathway inhibitor Brinker (Brk) in an early third instar leg disc. El is reduced in the dpp expression domain (arrow).

 


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  		Fig. 3. Early el noc double mutant clones in the wing. (A,C) Wing discs with clones of cells lacking elbow and no ocelli (el3.3.1 noc{Delta}64) at an early stage. Nub protein is shown in green; Tsh protein in red. Clones are marked by the absence of ß-gal (blue). (A) Mutant clones (asterisks) sorted out from the Nub-expressing wing pouch. Twin clones (arrowheads) remain in the Nub-expressing domain. (B) Summary of the relative positions of clones of cells lacking el and noc (white circles) and their twins (black circles) when born in the wing primordium (green) or in the body wall (red). (C) Wing disc with a large el3.3.1 noc{Delta}64 double mutant clone in the wing pouch. Tsh (red) was ectopically expressed and Nub (green) was lost in the clone. Comparable clones also showed ectopic Hth expression (not shown). (D) Wing disc with large Minute+ el3.3.1 noc{Delta}64 double mutant clones. Asterisks mark clones abutting the wing pouch and remaining in the body wall region. Hth protein is shown in red. (E) Summary of the location of Minute+ el3.3.1 noc{Delta}64 double mutant clones in the body wall region of wing imaginal discs.

 


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  		Fig. 4. Truncated appendages. (A) Cuticle preparation of a wild-type leg. (B) Cuticle preparation of a wild-type adult wing. (C) Cuticle preparation of a leg with Minute+ el3.3.1 noc{Delta}64 double mutant clones marked with forked. induced early in development (60 hours AEL). Distal elements are deleted (co, coxa; tr, trochanter; fe, femur; ti, tibia; ta, tarsal segments). (D) Cuticle preparation of a wing carrying large Minute+ el3.3.1 noc{Delta}64 double mutant clones marked with forked, induced early in development (60 hours AEL). The remaining wing tissue is heterozygous. The notum was not affected.

 


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  		Fig. 5. Early el noc double mutant clones in the leg. (A) Schematic representation of a leg showing the distribution of Minute[+] el3.3.1 noc{Delta}64 double mutant clones induced early in development (60 h AEL). Co, Coxa; Tr, Trochanter; Fe, Femur; Ti, Tibia; Ta, tarsal segments. (B) Summary of the relative position of clones of cells lacking el and no ocelli (grey circles) and their twins (black circles) when born in the proximal or distal leg. ta, tarsal segments. (C) Leg disc with Minute[+] el3.3.1 noc{Delta}64 double mutant clones. Mutant cells were found in femur and tibia, but not in the tarsal segments (ta). Hth protein (red). Clones are marked by the absence of ßGal (blue). (D) Summary of the locations of Minute+ el3.3.1 noc{Delta}64 double mutant clones in the proximal region of leg imaginal discs. The position of each clone is depicted as one semi-transparent layer of grey. The darker an area is, the more clones were found in that region. (E) Leg disc with an el3.3.1 noc{Delta}64 double mutant clone induced early in development (60 hours AEL). Hth (red) was ectopically expressed in some clones (arrow). Note that the twin spot cannot be seen in this picture because the el3.3.1 noc{Delta}64 double mutant clones sort out from the epithelium and are therefore found in a different focal plane. Clones induced earlier were not recovered.

 


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  		Fig. 6. Phenotypes of later el noc double mutant clones. (A) Cuticle preparation of a wild-type wing (left) and magnification of the wing hinge (right). PCo, MCo and DCo indicate proximal, medial and distal costa; Teg, tegula; veins are numbered vI-V. (B,C) Cuticle preparations of wings with large Minute+ el3.3.1 noc{Delta}64 double mutant clones induced at 84 hours (early third instar time in the Minute genotype). Clone marked by forked (shaded pink). Note the reduced size of the wings compared to the wild-type wing in A (same magnification). Right: detail of the wing hinge regions. Note deletion of hinge structures. (D) Cuticle preparation of an el1 mutant wing (left) and amplification of the wing hinge (right). Note deletion of hinge structures and reduced wing size compared to wild-type wings. (E) Cuticle preparation of a wild type adult leg. (F) Cuticle preparation of a leg carrying a double mutant clone extending through coxa, femur and tibia, but not tarsal segments. The trochanter is missing. Mutant tissue is shaded pink. Arrowheads indicate joints (not visible at this magnification; Co, Coxa; Tr, Trochanter; Fe, Femur; Ti, Tibia; 1st-5th, tarsal segments).

 


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  		Fig. 7. elbow1 is a regulatory double mutant of both el and noc. (A) Immunostaining of wild-type third instar wing imaginal discs with anti-El (red) and anti-Noc (green) antibodies. (B) Immunostaining of el1 homozygous mutant third instar wing imaginal discs with anti-El (red) and anti-Noc (green) antibodies. Note that the mutant does not show the wedge-like staining along the dorsal-ventral boundary that is characteristic of wild-type third instar wing discs. Since both, El and Noc stainings are affected, we conclude that el1 is a regulatory double mutant affecting both genes.

 

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© The Company of Biologists Ltd 2004