INTRODUCTION Homeobox-containing genes encode transcriptional regulators involved in many developmental processes including specification of unpatterned tissues. These include the highly conserved Hox gene class (Gehring et al., 1994; Manak and Scott, 1994). The Hox genes that specify the segments reiterated along the antero-posterior axis of diverse animals (Lawrence and Morata, 1994; McGinnis and Krumlauf, 1992) are viewed as selectors that control morphogenetic processes through the coordinate regulation of batteries of downstream target genes (Garcia-Bellido, 1975; Garcia-Bellido, 1977). Indeed, the Drosophila Hox genes encode homeodomain transcription factors whose patterned and overlapping expression leads to differential activation of target genes in individual segments (Botas, 1993; Graba et al., 1997; Morata, 1993). Despite the identification of several target genes, important gaps remain in our understanding of the pathway from patterned Hox gene expression to the development of diverse segmental morphologies (Mann, 1995). For example, the poor DNA sequence specificity exhibited by Hox transcription factors in vitro contrasts with their highly specific effects in vivo. One source of enhanced Hox specificity derives from their joint action with cofactors such as extradenticle and its mammalian PBX family counterparts that enhance sequence selectivity and alter in vivo specificity (Chan et al., 1994; Passner et al., 1999; Pinsonneault et al., 1997; Rauskolb et al., 1993; van Dijk and Murre, 1994). An important role for stoichiometric combinations of homeotic proteins with functional partners is supported by observations suggesting that Hox protein levels can be crucial for their developmental effects (Cribbs et al., 1995; Greer et al., 2000; Roch and Akam, 2000; Smolik-Utlaut, 1990). A combinatorial coding model predicts that different Hox genes and their partners acting together can specify novel cell or segment identities, deploying developmental pathways that are different to those specified by one gene product acting alone. The Drosophila proboscipedia (pb) gene codes for a conserved homeodomain protein required for the correct development of the adult fly mouthparts (Cribbs et al., 1992) where two distinct appendages, the labial and maxillary palps, show a dose-sensitive requirement for pb gene function (Kaufman, 1978; Pultz et al., 1988). These appendages derive 575 Development 130, 575-586 © 2003 The Company of Biologists Ltd doi:10.1242/dev.00226 Diversification of Drosophila segmental and cellular identities both require the combinatorial function of homeodomain-containing transcription factors. Ectopic expression of the mouthparts selector proboscipedia (pb) directs a homeotic antenna-to-maxillary palp transformation. It also induces a dosage-sensitive eye loss that we used to screen for dominant Enhancer mutations. Four such Enhancer mutations were alleles of the eyeless (ey) gene that encode truncated EY proteins. Apart from eye loss, these new eyeless alleles lead to defects in the adult olfactory appendages: the maxillary palps and antennae. In support of these observations, both ey and pb are expressed in cell subsets of the prepupal maxillary primordium of the antennal imaginal disc, beginning early in pupal development. Transient co-expression is detected early after this onset, but is apparently resolved to yield exclusive groups of cells expressing either PB or EY proteins. A combination of in vivo and in vitro approaches indicates that PB suppresses EY transactivation activity via protein- protein contacts of the PB homeodomain and EY Paired domain. The direct functional antagonism between PB and EY proteins suggests a novel crosstalk mechanism integrating known selector functions in Drosophila head morphogenesis. Key words: Hox, Pax6, proboscipedia, Maxillary palps, Protein- protein, Differentiation SUMMARY Evidence for a direct functional antagonism of the selector genes proboscipedia and eyeless in Drosophila head development Corinne Benassayag 1 , Serge Plaza 2 , Patrick Callaerts 3 , Jason Clements 3 , Yves Romeo 4 , Walter J. Gehring 2 and David L. Cribbs 1, * 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 *Author for correspondence (e-mail: cribbs@cict.fr) Accepted 15 October 2002