INTRODUCTION The conserved Hox genes function in laying out the body plan along the anterior-posterior axis of many, and maybe most, animal phyla (Carroll, 1995; Slack et al., 1993). All Hox genes encode a protein that contains the DNA-binding protein domain, the homeodomain (HD) (McGinnis and Krumlauf, 1992). The activity of the Hox genes were initially identified in Drosophila by the phenotypes produced by loss-of-function or gain-of-function alleles. A mutation in a Drosophila Hox gene results in the transformation of one body part into another (Lewis, 1978; Kaufman et al., 1990). Ironically, most of the information on how the HOX proteins work has come from the analysis of the role of Ultrabithorax (UBX) in gut development and not their role in the determination of segmental identity (Capovilla et al., 1994). The reason for this is the lack of knowledge about what genes are specifically regulated by HOX proteins during determination of segmental identity, and the lack of knowledge about what activities are required for deter- mination of segmental identity (Andrews and Scott, 1992). In gut development, UBX activity is required in the visceral mesoderm for the synthesis of Decapentaplegic (DPP) (Capovilla et al., 1994). Using a change of specificity mutation, UBX has been shown to act directly as a transcriptional activator of dpp expression (Capovilla et al., 1994). UBX binds the dpp regulatory region via the HD. However, UBX alone is unable to recognize with high affinity the cis-regulatory dpp sequences. UBX requires the cofactor Extradenticle (EXD) (Chan et al., 1994). Both EXD and UBX activities are required for determination of the correct segmental identity of paraseg- ments 5 and 6 (Peifer and Wieschaus, 1990). EXD interacts with UBX and other HOX proteins through a hexapeptide sequence of amino acids found in most HOX products (Johnson et al., 1995; Chan et al., 1996). This UBX-EXD inter- 5049 Development 124, 5049-5062 (1997) Printed in Great Britain © The Company of Biologists Limited 1997 DEV5134 Both Proboscipedia (PB) and Sex Combs Reduced (SCR) activities are required for determination of proboscis identity. Here we show that simultaneous removal of PB and SCR activity results in a proboscis-to-antenna trans- formation. Dominant negative PB molecules inhibit the activity of SCR indicating that PB and SCR interact in a multimeric protein complex in determination of proboscis identity. These data suggest that the expression pattern of PB and SCR and the ability of PB and SCR to interact in a multimeric complex control the determination of four adult structures. The absence of PB and SCR expression leads to antennal identity; expression of only PB leads to maxillary palp identity; expression of only SCR leads to tarsus identity; and expression of both PB and SCR, which results in the formation of a PB-SCR-containing complex, leads to proboscis identity. However, the PB-SCR interaction is not detectable in vitro and is not detectable genetically in the head region during embryogenesis, indicating the PB-SCR interac- tion may be regulated and indirect. This regulation may also explain why ectopic expression of SCR Q50K and SCR do not result in the expected transformation of the maxillary palp to an antennae and proboscis, respec- tively. Previous analysis of the requirements of SCR activity for adult pattern formation has shown that ectopic expression of SCR results in an antenna-to-tarsus transformation, but removal of SCR activity in a clone of cells does not result in a tarsus-to-arista transformation. Here we show in five inde- pendent assays the reason for this apparent contradictory requirement of SCR activity in tarsus determination. SCR activity is required cell nonautonomously for tarsus determi- nation. Specifically, we propose that SCR activity is required in the mesodermal adepithelial cells of all leg imaginal discs at late second/early third instar larval stage for the synthesis of a mesoderm-specific, tarsus-inducing, signaling factor, which after secretion from the adepithelial cells acts on the overlaying ectodermal cells determining tarsus identity. This study characterizes a combinatorial interaction between two HOX proteins; a mechanism that may have a major role in patterning the anterior-posterior axis of other animals. Key words: HOX function, proboscipedia, Sex combs reduced, induction, pattern formation, Drosophila, limb development SUMMARY Genetic characterization of the role of the two HOX proteins, Proboscipedia and Sex Combs Reduced, in determination of adult antennal, tarsal, maxillary palp and proboscis identities in Drosophila melanogaster Anthony Percival-Smith*, Jennifer Weber, Elaine Gilfoyle and Peggy Wilson Department of Zoology, University of Western Ontario, London, Ontario, Canada, N6A 5B7 *Author for correspondence (e-mail: aperciva@julian.uwo.ca)