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First published online 15 October 2003
doi: 10.1242/dev.00832

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The role of buttonhead and Sp1 in the development of the ventral imaginal discs of Drosophila

Carlos Estella, Gabrielle Rieckhof^*, Manuel Calleja and Ginés Morata

Centro de Biología Molecular CSIC-UAM, Universidad Autónoma de Madrid, 28049 Madrid, Spain

View larger version (100K): [in a new window]   Fig. 1. Some aspects of embryonic expression of btd in relation to that of Dll, esg and hdc. Double immunofluorescent staining of in situ hybridisation for btd and anti-ß-gal antibodies for Dll, esg and hdc. (A) Stage 10 embryo showing btd expression (green) in two cephalic segments (maxillary and labial) and in three thoracic discs primordia (T1-T3). Note that at this stage Dll (red) is not active in the thoracic primordia. (B) Stage 13 embryos equally stained for btd and Dll to show that at this stage Dll is co-expressed with btd in the thorax. (C) Higher magnification of the thoracic disc primordia of a stage 13 embryo. Note that the btd domain is bigger than that of Dll. (D) Double staining for btd and esg in a stage 14 embryo. Higher magnification is shown in F. (E) Stage 14 embryo stained for btd and hdc. Higher magnification is shown in G.

View larger version (92K): [in a new window]   Fig. 2. Expression of btd (blue), hth (green) and Dll (red) in leg (A-C) and antennal (D-F) imaginal discs. (A) Triple staining for three genes. (B) btd and Dll. (C) btd and hth. Notice that the btd domain overlaps partially with those of Dll and hth, indicating that it is expressed both in the proximal and the distal leg domain. The arrow indicates the ventral region where btd and hth overlap. (D) Triple label. (E) btd and Dll. (F) btd and hth.

View larger version (84K): [in a new window]   Fig. 3. Effect of the loss of btd, Sp1and Dll on the formation of Keilin's organs (KO) and ventral pits (VP). The third thoracic and first abdominal denticle belts are shown. (A) Wild-type segment displaying the normal set of KOs and VPs, a pair of each per segment. (B) btdXG81 larva. Only one KO is present and it is defective. The two ventral pits are present in this case, but they are frequently lacking in the mutant larvae (see main text). (C) Df(1)C52 larva showing complete absence of KOs and ventral pits. (D) DllSA1 larva. The KOs are always missing but the ventral pits are present.

View larger version (84K): [in a new window]   Fig. 6. RNA interference experiments. The upper left panel shows the transcript levels for RP49, as an internal control, and of btd and Sp1. Lanes 1 and 3 show normal levels of btd and Sp1 in control btd-Gal4 larvae. Lane 2 shows decreased btd levels in a btd-Gal4>UAS-btdi larva (compare with lane 1). Lane 4 shows the very low Sp1 transcript levels in btd-Gal4>UAS-Sp1i in comparison with control (lane 3). (A) Wild-type antenna. Antennal segments I, II and III, and the arista (ar) are indicated. (B) Antenna of a fly of genotype btd-Gal4>UAS-btdi UAS-Sp1i. Note the reduction in size of the segments I and II. (C) Legs of a fly of the same genotype. All leg segments are reduced in size and often fuse.

View larger version (140K): [in a new window]   Fig. 4. Alteration of embryonic expression of Dll, hdc and esg in the absence of btd and Sp1function. (A) Wild-type Dll expression. The antennal (ant), maxillary (mx) and labial (la) head segments contain Dll activity, as well as the three thoracic (T1, T2, T3) primordia. (B) btdXG81 embryo. The expression in the antennal segment is lost and there is a marked reduction of Dll activity in the three thoracic primordia (arrows). (C) Df(1)C52 embryo showing complete loss of btd activity in the thoracic primordia. The expression in the maxillary and labial segments is not altered. (D) Ubx-Gal4/UAS-btd embryo exhibiting ectopic Dll expression in the abdominal segments, which do not normally contain Dll activity. Note the expression in the amnioserosa cells (arrow). (E) Ventral view of a wild-type embryo showing hdc expression restricted to the anterior region of the ventral cord and the thoracic discs primordia (arrows). (F) Df(1)C52 embryo with loss of hdc activity in the thoracic primordia. (G) Ventral view of ptc-Gal4/UAS-btd embryo showing high levels of hdc activity (compare with E). (H) Wild-type expression of esg in the thoracic discs primordia. Wing (w) and haltere (h) clusters are indicated. The three arrows point to the three leg primordia. (I) esg expression in a Df(1)C52 embryo. Expression in the wing and haltere primordia remain but no activity is seen in the region corresponding to the leg primordia.

View larger version (108K): [in a new window]   Fig. 5. btd expression in disc primordia requires wg activity and is downregulated by Ubx in the first abdominal segment. (A) btd expression in a wild-type embryo. Arrows indicate the three thoracic imaginal primordia. (B) wg- embryo lacking btd expression in the thoracic imaginal primordia. Dots of expression observed probably correspond to the precursors of the CNS. (C) In an Ubx1 mutant embryo, there is an additional imaginal primordium in the first abdominal segment, which also expresses btd (arrows).

View larger version (150K): [in a new window]   Fig. 7. Ectopic btd expression induces transformation of dorsal disc patterns into the corresponding ventral discs. (A) Head of an ey-Gal4/UAS-btd fly completely lacking eyes and showing duplication of antennae. The duplication is clear on the left side but not on the right because duplicated antennae tend to fuse. (B) Clone of btd-expressing cells marked with f36 (arrow) showing transformation towards antenna. Compare with the normal antenna towards the left. (C) Thorax of a nub-Gal4/UAS-btd fly. Wings and halteres are totally replaced by leg structures. Although overall leg patterns observed in this genotype are abnormal, the individual pattern elements of leg identity can be recognised. The inset shows a high magnification of a region of a transformed wing: all the bristles present an associated bract, a typical feature of leg bristles (arrows). Note that the bracts are similarly orientated with respect to the bristles in all cases, indicating that the ectopic leg patterns have acquired normal polarity. (D) Side view of an omb-Gal4/UAS-btd fly illustrating the transformation towards leg of most of the wing (halteres are also transformed although they are not visible). Although there are supernumerary leg structures, they often form local arrangements that reproduce normal leg patterns. Arrows indicate tibia-like and tarsus-like patterns. (E) A large clone of btd-expressing cells in the wing about to sort themselves from the wing cells. (F) Higher magnification of the clone in E showing the autonomously generated leg pattern and the presence of an edge bristle, which is characteristic of the midleg (arrow).

View larger version (120K): [in a new window]   Fig. 8. Clones of btd-expressing cells in eye and wing discs induce activity of developmental markers of antenna and leg discs respectively. (A) The arrow indicates a clone of btd-expressing cells (blue) in the eye field, inducing Dll (red, B) and hth activity (green, C). Other clones scattered in the eye in the same picture also show similar effect. (D) Clone (arrow) of btd-expressing cells (marked by the loss of CD2, see Materials and methods) in the wing disc showing loss vg activity (red, H). (E) Another clone in the wing (arrow, blue) showing gain of hth function (green, I). (F) Clone inducing gain of dac activity (red, J) in the wing. (G) Several btd-expressing clones (some are indicated by arrows) inducing ectopic Dll activity (red, K).

View larger version (90K): [in a new window]   Fig. 9. Ectopic btd activity in wing induces spatially discriminated expression of leg developmental markers. (A) Large btd-expressing clone (blue) induces hth activity (green), but only on the periphery (B), resembling the disposition of the normal leg disc. (C) Another btd-expressing clone showing non-homogenous gain of Dll and dac activity: all cells express Dll (red) but only about half gain dac (green, D). (E) Another clone (arrow) showing a spatial deployment of dac and Dll resembling closely that along the PD axis in the normal leg (F): dac activity alone (green), dac and Dll (yellow), and Dll only (red). In the normal leg, these patterns of gene expression would correspond to the positions of the femur, tibia and tarsus, respectively.

View larger version (67K): [in a new window]   Fig. 10. Ectopic btd activity induces the production of morphogenetic signals in the wing and haltere discs. (A) Clone of btd-expressing cells in the wing disc showing en activity only in part of the clone (arrow, B). (C) Two clones in the haltere disc both showing en activity inside the clones but only in some cells (arrows, D). (E) Local gain of wg activity in a clone of btd-expressing cells. Only some cells show high wg levels (arrow, F). (G) Clone of btd-expressing cells in the hinge region of the posterior compartment showing gain of dpp activity in some cells (arrow, H).