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First published online 18 October 2006
doi: 10.1242/dev.02609

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The Ultrabithorax Hox gene of Drosophila controls haltere size by regulating the Dpp pathway

Centro de Biología Molecular Severo Ochoa (C.S.I.C.-U.A.M.), Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain.

View larger version (73K): [in a new window]   Fig. 1. Ubx regulates dpp transcription. (A) Wing (w) and haltere (h) imaginal discs hybridized with a dpp probe. In this and subsequent discs, anterior is to the left. (B-D') wing (w; C,C') and haltere (h; D,D') imaginal discs of dpp-lacZ10638 larvae stained with an anti-ß-galactosidase antibody. Note the different dpp expression in both discs. (E,E') A Ubx mutant clone, marked by the absence of GFP (in green) showing expression of dpp-lacZBS3.0 (in red). Within the clone, the dpp band of expression widens and is more intense. (F-G') Haltere discs of dpplacZ10638/+; bx3/TM2 (F,F') and dpp-lacZ10638/+; pbx/TM2 (G,G') larvae. In the bx mutant haltere disc the A compartment increases its size and the dpp expression is like that of the anterior wing whereas in pbx mutant haltere discs the dpp expression is slightly wider but the P compartment increases its size significantly. C'-G' are magnifications of the insets shown in C-G. The magnifications of the wing and haltere discs were done at exactly the same values.

View larger version (70K): [in a new window]   Fig. 2. Ubx controls the expression of Dpp targets. (A-B'') omb-lacZ (in red) and En (in green) expression in wing (A-A'') and haltere (B-B'') imaginal discs. A and P stand for anterior and posterior compartments, respectively. In bx3/TM2 (C-C'') and pbx/TM2 (D-D'') haltere discs the omb expression extends significantly in the anterior (C) and posterior (D) compartments, respectively. Arrowheads in A-D mark the A/P boundary. (E-F'') Ubx mutant clones, marked by the absence of GFP expression (in green) and showing omb-lacZ expression (in red). Note in E' the extended expression of omb in Ubx- cells (arrow). In F-F'' there are three types of clones: a clone far from the A/P boundary does not activate omb (asterisk in F); clones closer to this boundary show extended omb expression (arrowhead in F and F', posterior clone), and another clone (arrow in F, anterior clone) activates omb also outside the clone (the arrow in F' points to non-cell-autonomous omb expression). Merged images in E'' and F''.

View larger version (47K): [in a new window]   Fig. 3. Ubx governs Dpp spread. (A,B) Wing (A) and haltere (B) imaginal discs of tub-Gal80ts/ptc-Gal4; UAS-Dpp-GFP bx3/MKRS larvae, showing a more restricted spread of Dpp-GFP in the haltere disc. In tub-Gal80ts/ptc-Gal4; UAS-Dpp-GFP bx3/TM2 haltere discs (C) the Dpp-GFP expression and spread in the A compartment are similar to those of the wing disc, but spread in the P compartment is much reduced compared with that of the wing disc. (D) A plot representing the average value of the intensity of the Dpp-GFP dots along the A/P axis in tub-Gal80ts/ptc-Gal4; UAS-Dpp-GFP bx3/MKRS wing and haltere discs and in tub-Gal80ts/ptc-Gal4; UAS-Dpp-GFP bx3/TM2 haltere discs. Note the reduction in extent and the abrupt fall of the Dpp-GFP signal when Ubx is present. Numbers in the x-axis indicate distance in microns from the A/P boundary (0 value). The anterior compartment is to the left. w, wing disc: h, haltere disc.

View larger version (126K): [in a new window]   Fig. 4. Ubx prevents downregulation of tkv in the haltere pouch mediated by mtv expression and Dpp signaling. (A) In the wing pouch, P-Mad signal is strongly reduced in AB cells (arrow) (Tanimoto et al., 2000). (B-C'') In the haltere disc, P-Mad signal (in red in B) is narrower, but strong, in these cells (abutting the En expression domain, in green in B'). Merged image in B''. The boxed region is magnified in C,C'; the arrows point to the posterior P-Mad signal and the white line marks the A/P boundary. (D-E'') ptc-Gal4 UAS-GFP wing (D-D'') and haltere (E-E'') pouches, showing that high levels of Hh signaling (GFP signal, in green in D and E) and Dpp signaling (P-Mad, in red in D' and E') coincide in the haltere but not the wing disc (arrow in D'). Merged images in D'' and E''. (F,G) tkv-lacZ expression in the wing and haltere discs. The arrow in F marks the reduced expression in AB cells of the wing pouch. The expression detected with an anti-Tkv antibody is similar but does not show the downregulation in AB cells of the wing disc so neatly as the P-lacZ insertion. (H-H'') A big Ubx- clone in the haltere disc, marked by the absence of GFP (H, in green), shows downregulation of Tkv protein expression (H', in red). Merged image in H''. (I-I''). Mutant Ubx clones (marked by the absence of GFP, in green in I) present reduced tkv-lacZ expression (I', in red) in medial (arrow) but not in lateral (arrowhead) regions of the haltere disc. Merged image in I''. (J,J') The ectopic expression of Ubx in CbxTwt mutants (J, in green) increases tkv-lacZ signal (J', in red). (K) The expression of mtv (mtv-lacZ, in green) in the wing disc is strong at the A/P boundary and in two lateral spots (arrowheads). In the haltere pouch (L), just these spots are observed. (M) In a mtv-lacZ/+; bx3/TM2 haltere disc there is mtv expression at the A/P boundary (arrow). (N,N') mtv (in red) is also derepressed (arrow) in haltere disc Ubx mutant clones (arrow), marked by the absence of GFP (in green in N). The arrowheads in K-N mark the lateral spots, and En expression, marking the posterior compartment, is shown in K-M in red. (O,O') Ectopic Ubx expression in CbxTwt wing discs (in red in O) strongly reduces mtv signal (in green) in medial regions (O,O'). (P) In MS1096-Gal4; tkv-lacZ/+; UAS-mtv/+ haltere discs the expression of tkv is downregulated in the dorsal (d) region, except in the periphery (arrows); compare with the expression in a tkv-lacZ haltere disc (G); v, ventral region. (Q) MS1096-Gal4; tkv-lacZ/+; UAS-tkvQD/+ wing disc showing repression of tkv in the dorsal pouch (d). (R) No such repression is observed in haltere discs.

View larger version (125K): [in a new window]   Fig. 5. tkv reverts the extended Dpp activity caused by Ubx loss. (A-A'') Two clones merged at the A/P boundary of the haltere disc (as revealed by hh-lacZ expression, not shown), and marked by the absence of GFP expression (in green in A), show wider P-Mad signal (A') and non-cell-autonomous expression both anteriorly and posteriorly to the clones (arrowheads in A'). A'', merged image. (B-B''') Clones mutant for Ubx and that simultaneously express tkv. The clones are marked by GFP expression (in green in B). Note that P-Mad (in red in B') and omb (in blue in B'') signals are restricted to a few cells within the clones (compare with Fig. 2E' and Fig. 5A'). Merged image in B'''. (C,D) P-Mad expression in dpp-Gal4/UAS-tkv (C) and dpp-Gal4/UAS-Ubx (D) wing discs. In both cases the extent of P-Mad signal is reduced, but the level of expression in AB cells is increased, compared with that of wild-type wing discs (Fig. 4A).

View larger version (119K): [in a new window]   Fig. 6. Ubx repression of dally restricts the extent of Dpp activity in the haltere disc. (A) dally (dally-lacZ) expression in the wing disc, showing stronger signal in the dorsal-ventral and A/P boundaries (arrow) and in the lateral regions. (B) In the haltere disc, dally is not transcribed in AB cells and the signal in the dorsal-ventral boundary is restricted to the anterior compartment. There is also lower signal throughout the pouch. (C) An omb-gal4; Df109 UAS-dsRNA>Ubx/+ haltere disc, showing a dally pattern similar to that of the wing disc. The arrow marks the A/P stripe. (D,D') Ubx-expressing clones in the wing disc, marked by the GFP expression (D, in green), eliminate dally signal (D,D', in red; arrows). (E) The expression of dally under the control of the ap-Gal4 line extends the P-Mad signal in the dorsal domain (d, arrows), where the line drives expression; v, ventral region. (F) The ectopic expression of activated Tkv in the dorsal (d) domain of the wing pouch (MS1096-Gal4 driver) downregulates mtv transcription; v, ventral region. (G,H) In the dorsal haltere pouch of MS1096; UAS-tkvDN/+ larvae, the expression of a dominant-negative form of Tkv does not activate mtv (G) or dally (H) expression at the A/P boundary.

View larger version (103K): [in a new window]   Fig. 7. The dpp pathway and Ubx regulate haltere size. (A) Thorax of a Drosophila wild-type adult, showing the different size of the wing (W) and the haltere (H). (B) A Ubx mutant clone in the haltere disc of a third instar larva showing that the size of the clone (absence of GFP expression, in green) and that of the twin spot (more stained due to the two doses of the Ubi-GFP construct) are similar. (C) Wild-type (left) and dpp-Gal4/UAS-dpp (right) haltere discs showing the bigger pouch of the latter. (D-F) The P compartments of en-Gal4 UAS-GFP/UAS-dally (E) and en-Gal4 UAS-GFP/UAS-mtv (F) haltere discs are enlarged with respect to en-Gal4 UAS-GFP controls (D). The three compartments are marked with GFP and delimited by arrows. (G-I) In dppd12/dppd5 adults the size of the distal part of the haltere (the capitellum, c) is reduced (H) compared with the wild type (G), and in dppd12/dppd5; TM2/+ flies (I) this reduction is alleviated. (J-L) Wings of wild-type (J), dpp-Gal4/UAS-Ubx (K) and dpp-gal4/UAS-tkv (L) adults, showing the reduction in size of the latter two. (M) Haltere of a Ubx6.28/+ adult, showing a slightly bigger haltere than in wild-type flies (G) due to the haploinsufficient phenotype of the Ubx locus. (N) In Df tkv/+; Ubx6.28/+ siblings the size of the haltere is increased. (O-R) In pbx/Ubx6.28 (O) and ptc-Gal4/+; bx3/Ubx6.28 (Q) haltere discs there is an increase in the size of the P and A compartments, respectively (delimited by arrows). These increments are reduced if tkv is expressed in the posterior (en-gal4/+; pbx/UAS-tkv Ubx6.28 larvae; P) or anterior (ptc-Gal4/+; bx3/UAS-tkv Ubx6.28 larvae; R) compartments. The P compartment is marked by anti-En antibody (in red in O), by GFP (in green in P), and by anti-Ubx (in red in Q and R). A summary of the results is shown in U. (S) A ptc-Gal4/+; Df109 UAS-dsRNA>Ubx/tub-Gal80ts fly, showing a patch of haltere tissue (delimited by the discontinuous line in S') bigger than the anterior compartment of a wild-type haltere. Detail of the boxed region is shown in S'; w, wing territory; P, posterior compartment. (T) A capitellum of a ptc-Gal4/UAS-flp; FRT Ubx6.28/FRT GFP adult with Ubx mutant clones (cells transformed into wing, w) showing more haltere tissue (h) than in the wild type (compare with G). (U) Histograms showing the P/A (left) or A/P (right) ratios of different genotypes. P/A ratios: wild type (wt), 0.34 (n=16), pbx/Ubx6.28, 1 (n=14), en-gal4/+; pbx/UAS-tkv Ubx6.28, 0.63 (n=11), en-Gal4/UAS-mtv, 0.53 (n=8), and en-gal4/UAS-dally, 0. 41 (n=13). A/P ratios: wt, 2.9 (n=16), ptc-Gal4/+; bx3/Ubx6.28, 4.6 (n=14) and ptc-Gal4/+; bx3/UAS-tkv Ubx6.28, 3,9 (n=12).

View larger version (30K): [in a new window]   Fig. 8. Model of action of Ubx on the Dpp pathway. (A) Scheme of the relationship between Ubx and different elements of this pathway in the central domains of wing (W) and haltere (H) discs. In the haltere disc, Ubx reduces dpp transcription, eliminates dally and mtv expression in most of the pouch and elevates Tkv levels, thus reducing the extent of Dpp spread and activity (but increasing it in AB cells). (B,C) Cartoons representing the distribution of Dpp (blue balls) in wing (B) and haltere (C) discs, showing less Dpp and less Dpp spread in the latter. The Tkv expression is indicated by the size of the Y symbol and the colours in the nuclei represent Dpp activity. (D,E) Effect of Ubx- (D) and Ubx- tkv+ (E) mutant clones in Dpp signaling. In Ubx- clones the levels of Tkv are reduced so that Dpp can travel through the clone and reach the wild-type cells in more peripheral positions (D). If tkv is expressed in these clones, it retains Dpp and reduces the extent of Dpp activity (E).

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