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The Drosophila proboscis is specified by two Hox genes, proboscipedia and Sex combs reduced, via repression of leg and antennal appendage genes

Arhat Abzhanov^*, Stacy Holtzman^* and Thomas C. Kaufman

Howard Hughes Medical Institute, Department of Biology, Indiana University, Bloomington, IN 47405, USA
^* These authors contributed equally to this work

View larger version (43K): [in a new window]   Fig. 1. (A-C) Drosophila labial discs, their proximodistal orientation, the resulting adult appendage, and wild-type expression patterns of Pb and Scr. (A) The labial discs are attached to the mouthparts of larvae. (B) Their point of attachment is at the proximal end of the disc, the peripodial stalk. (C) The labial discs fuse to form the proboscis, the appendage used for feeding. (D-F) Pb (green) and Scr (red) are co-expressed throughout wild-type labial discs except for the proximalmost cells (A. M. Pattatucci, PhD thesis, Indiana University, 1991; Cribbs et al., 1992; Johnston and Schubiger, 1996). L, labial disc; T, thoracic leg disc. White dots indicate stalks of labial discs only. The distal ends of most of the labial discs point towards the right.

View larger version (30K): [in a new window]   Fig. 2. Expression of Dac, Dll and Exd in wild-type and pb5 labial discs. (A) Dac (red) is not expressed in wild-type labial discs. (B) In wild-type labial discs, Dll is expressed in a compact crescent-shaped domain – the distalmost region of the disc. (C) Exd (blue) is present at very low levels in wild-type labial discs. (D,H) The overall pattern of Exd (blue), Dac (red) and Dll (green) in a pb5 labial disc (D) and wild-type (H) T2 leg disc. (E-G) Same labial disc showing Dac, Dll and Exd separately. (E) The Dac (red) domain in pb5 labial discs is relatively narrower that the leg disc (compare with the wild-type T2 leg disc in H). (F) In pb5 labial discs the Dll-expressing domain is larger than wild type and the protein accumulates at levels comparable with those seen in leg and antennal discs.(G) Exd is easily detectable in the proximal portion of the pb5 labial disc. (I-K) Same T2 disc as in H showing Dac, Dll and Exd separately for comparison.

View larger version (31K): [in a new window]   Fig. 3. The effect of ectopic accumulation of Pb on the expression of dac, Dll and exd. (A) There is a circular Dac (red) domain in wild-type leg discs (T2 is shown). (B) The pattern of accumulation of ß-galactosidase in the T3 leg disc of a dpp=>lacZ animal. Note the yellow nuclei expressing both ß-gal (green) and Dac (red). (C,D) dpp=>pb T2 and T1 leg discs showing a broken Dac (red) ring. The ring is interrupted by a stripe of Pb-expressing cells (green). (E) dpp=>lacZ is capable of driving lacZ (green) in Exd-expressing cells (red) of the T2 leg disc. (F) Little or no co-expression of Pb (green) and Exd (red) is seen in a T1 leg disc of dpp=>pb flies.(G) ß-gal (green) expression overlaps with that of Dll (red) in a dpp-lacZ T3 leg disc. Note multiple yellow nuclei expressing both Dll and ß-gal. (H) dpp=>pb showing Pb (green) in a stripe across a T3 leg disc stained with anti-Dll antibody (red). Although the normal expression pattern of Dll is altered, there is co-expression in many cells.

View larger version (61K): [in a new window]   Fig. 4. Regulation of Scr by Pb. (A-C) Expression of Dll (green), Dac (red) and Scr (blue) in a pb5 labial disc. (B) The Dac (red) expression domain completely overlaps the expression domain of Dll (green). (C) Scr (blue) and Dac (red) expression in a pb5 labial disc. Note the gap in Scr accumulation that coincides with the band of Dac-positive cells and that there is no detectable Scr accumulation in the distal end of the disc. For comparison with wild-type Scr expression in the labial disc, refer to Fig. 5A. (D-F) Same disc showing individual channels of Dll, Dac and Scr. (G) Wild-type T3 leg disc stained with anti-Scr (green) and anti-Dac (red) antibodies. There is no overlap (yellow) of accumulation. (H) The Dac (red) ring is interrupted by the Scr (green) -expressing cells in a dpp=>pb T3 leg disc. No overlap (yellow) between Dac and Scr is detected. (I) Ectopic expression of Scr (green) in a dpp=>pb wing disc. Again there is no co-expression of Scr and Dac. W, wing disc.

View larger version (65K): [in a new window]   Fig. 5. Distribution of Dac, Dll, Hth, Sal and Scr in pb4 mutant labial discs. (A-C) Wild-type eye-antennal disc stained with Dll (red), Hth (green) and Sal (blue). (B,C) The same disc showing the ring of overlap between Dll and Hth in yellow, and the separate blue channel of Sal alone in the antennal disc. (D-F) A labial disc from a pb4 mutant also stained with Dll (red), Hth (green) and Sal (blue). Note that where the expression of Dll and Hth overlap (yellow, E), Sal is expressed in a small band of cells (F). (G-I) Comparison of Dac, Sal and Scr in a wild-type eye-antennal disc and a pb4 labial disc. (G) In a wild-type eye-antennal disc, a distinct ring of Sal surrounds a crescent-shaped ring of Dac; Scr is not expressed in the antennal disc proper. (H) Scr is expressed in the proximal portion of the pb4 mutant labial disc but is expressed at lower levels at the most distal tip (also see Fig. 5E), coinciding and slightly overlapping with the region of Sal (overlap appears turquoise). (I) Same disc as in (H) showing only Dac and Sal expression. pb4 mutant labial discs express a small band of Sal-expressing cells just proximal to a small region of Dac-expressing cells, similar to antennae. E-A, eye-antennal disc.

View larger version (27K): [in a new window]   Fig. 6. The role of Scr in the regulation of appendage genes. (A) Expression of Pb (green) and Scr (red) in a labial disc from an Scr4/Scr5 mutant animal. (B,C) Images of the same disc as in A, showing individual expression patterns. Scr expression is clearly reduced; Pb expression remains unaltered. (D) A Scr4/Scr5 mutant labial disc stained to reveal expression of Dll (red), Exd (blue) and Sal (green). (E-G) Images of separate channels of the same disc as in D. Dll expression is slightly expanded when compared with wild-type (see Fig. 1B); low levels of Exd and little or no Sal (green) expression is detectable, similar to wild-type labial discs.

View larger version (51K): [in a new window]   Fig. 7. Patterns of Dll, Exd, Hth and Sal accumulation in pb5Scr5/pb1Scr4 mutants. (A) Scanning electron micrograph showing a transformation of the proboscis into an antenna in a pb5Scr5/pb1Scr4 mutant. Note the absence of claws or pulvillae. (B) Wild-type labial discs express low levels of Hth (green) and no Sal (blue). (C) pb5Scr5/pb1Scr4 mutant labial discs express domains of Dll (red), Exd (green) and Sal (blue) that resemble those in the antennae. (For reference, wild-type labial disc expression patterns of Dll and Exd are shown in Fig. 2B,C.) (D,E) In the labial discs of pb1Scr4/pb5Scr5 mutants, Sal expression (blue) is localized to cells expressing both Dll (red) and Hth (green, shown with arrows in D).

View larger version (36K): [in a new window]   Fig. 8. Ectopic expression of Pb or Scr represses Sal expression in the antennae. (A) dpp-lacZ reporter stained for ß-gal (green) and Sal (red). Note the region of overlap between the reporter and Sal. (B-D) There is no overlap between Scr-expressing cells and the broken ring of Sal in a dpp=>Scr eye-antennal disc. (E-G) Similarly, there is no overlap between Pb-expressing cells and Sal expression in dpp=>pb eye-antennal discs. (B,E) Merged images; (C,D,F,G) separated channels.

View larger version (24K): [in a new window]   Fig. 9. A summary diagram showing the expression domains in wild-type leg, wild-type antenna, wild-type labial discs and mutant labial discs of the appendage patterning genes analyzed in this study. Wild-type leg and antenna with their respective imaginal disc expression patterns are at the top for reference. The wild-type appendage of the labial disc, the proboscis and various mutant phenotypes are diagramed below and to the right. The genotype of the individual mutants is given above each appendage. The corresponding patterns of protein accumulation in the normal and mutant discs are shown to the right of the appendage phenotypes. Dark blue indicates relatively high levels of Hth/Exd protein accumulation and pale blue indicates low levels. The proboscis (asterisk) is displayed laterally to represent the same plane of view as the labial disc in this figure, and half is lightened to represent a single disc derivative.

View larger version (21K): [in a new window]   Fig. 10. A diagram showing the deduced regulatory interactions within the labial disc. The peripodial stalk is to the left and the anatomically distal portion of the disc to the right. The portion of the disc expressing both Pb and Scr is shaded in gray. The broken vertical line separates the most proximal region from the distal region in which most of the mutant effects of pb and Scr were observed. The lines with bars at the end indicate a negative or suppressive interaction, while arrows indicate a positive or inductive interaction. The broken lines indicate uncertainty about the indicated interaction. The width of the arrows indicates the strength of the interaction.

View larger version (43K): [in a new window]   Fig. 11. (A) Morphology of the adult Drosophila proboscis. Posterior (left) and anterior (right) views (from Kumar et al., 1979). (B) Comparison of the original fate map (left) and molecular maps of wild-type (center) and pb5 (right) labial discs. Note that the Dll (green) expression domain and its function in pseudotracheal formation are not consistent with the positions of the pseudotracheal and border hair anlagen on the original fate map. ALP, anterior labial plate; BH, border hairs; DP, distiproboscis; FU, furca; HS, horse-shoe sclerite; LB, labellar bolster; LC, labellar cap; MP, mediproboscis; PA, prestomal aperture; PM, prementum; PT, pseudotracheae; SB, sensilla basiconica; SS, sigmoid sclerite. The colored domains and shading are the same as those used in Fig. 9 and indicate the accumulation patterns of the appendage patterning genes in the labial discs.