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doi: 10.1242/10.1242/dev.00226

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Evidence for a direct functional antagonism of the selector genes proboscipedia and eyeless in Drosophila head development

Corinne Benassayag1, Serge Plaza2, Patrick Callaerts3, Jason Clements3, Yves Romeo4, Walter J. Gehring2 and David L. Cribbs1,*

1 Centre de Biologie du Développement-CNRS and Institut d'Exploration Fonctionnelle du Génome, 118 route de Narbonne, Bâtiment 4R3, F-31062 Toulouse Cedex 04, France
2 Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland
3 Department of Biology and Biochemistry, University of Houston, 369 Science and Research Bldg. 2, Houston TX 77204-5001, USA
4 IBCG-CNRS, Université Paul Sabatier, 118 route de Narbonne, F-31062 Toulouse Cedex, France



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  		Fig. 1. ey alleles isolated as dominant Enhancers of eye loss induced by ectopic expression from the HSPBsy transgene. Dose-sensitive eye loss induced by HSPBsy. (A-C) Heads of flies carrying one, two or four HSPBsy copies, respectively. (B) Two copies; slightly reduced eye (arrowhead). (C) Four copies; complete eye deletion (arrowhead). The genetic screen selected for female progeny carrying two HSPBsy copies (B) or an interacting Enhancer locus, which yields an eye loss resembling four copies (C). (D) Strength and specificity of the pb-ey genetic interaction. Four new alleles (eyJD, eyD1Da, ey11, eyEH), two previously isolated ey loss-of-function alleles [ey2 and Df(4)BA], and several alleles of other genes implicated in eye differentiation, were tested for dose-sensitive interactions with HSPBsy. Males harboring one HSPBsy copy and heterozygous for mutant alleles of the eye development genes sine oculis (so), eyes absent (eya), eye gone (eyg) or ey were crossed with homozygous HSPBsy females. Maximal eye loss expected is 50% in the resulting female progeny (harboring two copies of HSPBsy) because half should carry the eye gene mutation. Several allele names are shortened: eyac1 is eyaclift1 (from L. Zipursky), eygM is eygM3-12 (from H. Sun) and eyD1 is eyD1Da (this paper). (E,F). All four newly isolated ey alleles should yield truncated forms of the EY protein. (E) Representations of wild-type EY protein and of the proteins encoded by eyJD, eyD1Da (Callaerts et al., 2001Go), or predicted based on the sequences of ey11 and eyEH (this paper). HD, homeodomain; PD, paired domain. (F) Sequences of wild-type ey and of the mutant lesions in ey11 and eyEH. Modifications in the mutant sequences are underlined, exon or intron boundaries are indicated by arrows, and a new stop codon by `-'.

 


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  		Fig. 2. ey and pb collaborate in maxillary palp development. (A-C) Interaction of HSPBsy and eyJD in head development. (A) This frontal view of the head of an eyJD heterozygote shows no marked defects [as for a female harboring two copies of HSPBsy (see Fig. 1B)], whereas the combination 2xHSPBsy; eyJD/+ often leads to complete eye loss resembling the head in Fig. 1C. (B) Frontal view, head of a HSPBsy/HSPBsy; eyJD/eyJD female showing complete eye loss and the fusion of an antenna and maxillary palp (boxed). (C) Detail of B. This female lacks a normally placed maxillary palp associated with the labial palps (lb) (arrow); the maxillary appendage (with sensilla trichodea, arrowheads) is fused to the antenna. (D-F) Profile view of the head of an eyJD heterozygote. (D) This head possesses normal antennae (ant), maxillary palps (mx) and labial palps (lb). (E) Enlargement of the maxillary palps. Arrowheads indicate the two maxillary sensilla trichodea. (F) Enlargement of the antennal segment A3 (ant) and arista (ar). (G-I) Profile view of the head of an eyJD homozygote. (G) Overall reduction in head size, full eye loss, altered antennae (ant) and maxillary palps (mx), but normal labial palps (lb). (H) Higher magnification of the maxillary palps. Arrowheads indicate the two maxillary-specific sensilla trichodea signaling maxillary identity, but the appendage is strongly reduced (compared with E, same magnification). (I) The antennal segment A3 (ant) is malformed, slightly enlarged and extended from the head. The arista (ar) is shortened, curved and thickened at the base, malformed and extended from the head. This image is from a different individual from that shown in G, but the phenotype is equivalent.

 


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  		Fig. 3. EY and PB expression in the pre-pupal eye antennal disc. (A) Eye-antennal imaginal discs (DIC microscopy) after dissection from pre-pupae 8 hours after puparium formation. Eyes (ey), antennae (ant) and maxillary palps (mx) indicate the three adult structures derived from this compound disc. (B) PB expression revealed with a primary rabbit anti-E9 antibody and a HRP-coupled secondary antibody. Arrow indicates expression in the maxillary primordium. Higher magnification of the maxillary primordium is shown in the inset. (C) EY expression detected with a rabbit anti-EY. Arrowhead indicates EY expression in the eye disc. Arrow shows nuclear expression in the maxillary primordium. The inset shows higher magnification of the maxillary primordium. (D-G) pbGAL4>UASlacZ and EY expression in eye antenna disc. The lacZ immunodetection reflects PB GAL4 expression. Double immunostaining employed secondary antibodies revealing EY (red) and ß-galactosidase (green), and images were analyzed by confocal microscopy. (D,E) Maxillary primordium (boxed) is shown in more detail in F and G, respectively. In F, a small number of cells co-expressing PB and EY are indicated by the yellow arrowheads. The white open arrowheads show cells expressing only PB or EY. E and G are at a later stage, as judged by increased cell number. Cells express exclusively pb lacZ (green arrow) or EY (red arrow). The strong expression of lacZ seen in the antennal disc in D is ectopic expression in the distal antenna (see Kapoun and Kaufman, 1995Go). This expression is not visible in E.

 


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  		Fig. 4. PB inhibits EY function post-transcriptionally. (A) Targeted expression of PB in eyes represses the EY regulatory pathway, but does not affect ey transcription. In situ hybridization (parts 2,7), ß-galactosidase staining (parts 3,8) or immunostaining (parts 4,5,9,10) were performed on wild-type (parts 2-5) or dpp-Gal4; UAS-PB (parts 7-10) third instar eye antenna imaginal discs. Adult heads of wild-type or dpp-Gal4; UAS-PB are shown in (parts 1 and 6). (B) Model proposed to explain PB-dependent EY inhibition. Ectopic eye induction, and consequently activation mediated by ey, is inhibited by PB (C). Targeted expression of PB, EY or both proteins with dpp-Gal4 driver. PB led to eye inhibition and leg and wing defects (C1), whereas ectopic eyes were induced by EY (C4, arrowhead). On co-expressing EY and PB, ectopic eye formation was abolished (C7). Expression of the so-lacZ enhancer trap line was examined in eye antenna (C2,5,8) or wing discs (C3,6,9). ß-Galactosidase was induced by EY (C5,6 arrow) but not by PB (C2,3). Co-expression of PB with EY inhibits so activation in all tissues examined (C8,9, arrow).

 


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  		Fig. 5. PB interacts with EY in vitro and inhibits EY trans-activation activity. (A) The PB and EY proteins. HD, homeodomain; PD, paired domain. Grey boxes represent the conserved motifs (N, YPWM,C) of mammalian HOX-A2/B2 and PB (Cribbs et al., 1992Go). The region between amino acids 126-306 was fused to GST. (B) GST interaction assays. Aliquots (10 µl) of in vitro translated 35S-methionine-labeled EY (lanes 1,5,12,16), EXD (lanes 3,7,9,13), EYA (lanes 4,8,10,14) and SO (lanes 2,6,11,15) were incubated with glutathione agarose beads containing bound GST (lanes 5-8), GST-PB (126-306; lanes 9-12) or GST-EN (lanes 13-16). An aliquot (5 µl) of in vitro translation products is shown in lanes (1-4). In this experiment, GST-EN bound 13% of the input 35S-labeled EXD protein, whereas GST-PB bound 10% of input 35S-labeled EY. (C) The interaction requires the EY PD and PB HD. GST `pull-down' assays were performed in the same conditions as in B, with 35S-labeled full-length EY proteins (lanes 5,9) and truncated forms of EY lacking the HD (lanes 6,10), PD (lanes 7,11), or HD and PD (lanes 8,12), incubated with GST-PB (lanes 5-8) or GST-PBsy (lanes 9-12), carrying the homeodomain R5H mutation indicated in A. Aliquots (5 µl) of in vitro translation products are shown in lanes 1-4. (D) Yeast one-and-a-half hybrid experiment. A strain carrying the integrated reporter vector pLacZi containing one copy of the so10 enhancer upstream of the minimal promoter of the yeast iso-1-cytochromeC gene. Vectors were pAS and pACT. pAS-EY, EY encoding vector. pACT-PB (126-306, see in A) is fused to the GAL4 activation domain. Lane 1, control with empty vector; lane 2, transactivation of the enhancer so10 with EY; lane 3, co-expression of EY and PB inhibits EY-dependent so10 transactivation, whereas PB does not activate so10 enhancer.

 

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