INTRODUCTION Tremendous morphological diversity exists among animal appendages, both between species and within a single organism. Such variations facilitate the many functions of appendages, such as sensing their environments, swimming, feeding, crawling, walking and ﬂying. The Drosophila antenna, for example, has both auditory and olfactory functions, while the leg is used primarily for locomotion. Nonetheless, loss-of-function mutations in a variety of Drosophila genes lead to antenna to leg or leg to antenna transformations (Balkaschina, 1929; Struhl, 1981; Struhl, 1982; Sunkel and Whittle, 1987; Casares and Mann, 1998; Pai et al., 1998), supporting the idea that the antenna and leg are homologous structures. To elucidate the types of genetic changes that underlie morphological diversiﬁcation of appendages, we are investigating the hierarchies involved in subdividing the proximodistal (PD) axes of the antenna and the leg, and asking at what levels the differences are manifested. In the primordia of both Drosophila antenna and leg, gradients of secreted factors encoded by decapentaplegic (dpp) and wingless (wg) regulate formation of the PD axes. dpp and wg are activated similarly by Hedgehog, possess similar relative expression patterns and exhibit similar mutual antagonism in both appendage primordia (Diaz-Benjumea et al., 1994; Brook and Cohen, 1996; Jiang and Struhl, 1996; Morimura et al., 1996; Penton and Hoffmann, 1996; Theisen et al., 1996; Lecuit and Cohen, 1997). It therefore has been thought that the PD axes of the antenna and leg are similarly constructed. Indeed, until now their primordia have been used interchangeably to examine PD development (e.g. Diaz- Benjumea et al., 1994; Lecuit and Cohen, 1997). Nonetheless, as we report here, crucial differences between the antenna and leg exist at the level of three genes, Distal-less (Dll), dachshund (dac) and homothorax (hth), that are regulated by Dpp and Wg in the developing leg. Some of these differences are due to differential regulation by Hox genes such as Antennapedia (Antp) (Casares and Mann, 1998). However, the fact that a variety of mutations, including those in Dll and hth, can transform the antenna towards leg without activating Antp or other trunk Hox genes (Casares and Mann, 1998; R. Bolinger, personal communication) indicates that the presence or absence of Hox alone is insufficient to explain the morphological differences between the antenna and the leg. Furthermore, although Dpp and Wg are similarly expressed between the Drosophila antenna and leg, differences in the expression of Dpp in the appendages of other arthropods 2365 Development 128, 2365-2372 (2001) Printed in Great Britain © The Company of Biologists Limited 2001 DEV5907 The morphological diversiﬁcation of appendages represents a crucial aspect of animal body plan evolution. The arthropod antenna and leg are homologous appendages, thought to have arisen via duplication and divergence of an ancestral structure (Snodgrass, R. (1935) Book Principles of Insect Morphology. New York: McGraw- Hill). To gain insight into how variations between the antenna and the leg may have arisen, we have compared the epistatic relationships among three major proximodistal patterning genes, Distal-less, dachshund and homothorax, in the antenna and leg of the insect arthropod Drosophila melanogaster. We ﬁnd that Drosophila appendages are subdivided into different proximodistal domains speciﬁed by speciﬁc genes, and that limb-speciﬁc interactions between genes and the functions of these genes are crucial for antenna-leg differences. In particular, in the leg, but not in the antenna, mutually antagonistic interactions exist between the proximal and medial domains, as well as between medial and distal domains. The lack of such antagonism in the antenna leads to extensive coexpression of Distal-less and homothorax, which in turn is essential for differentiation of antennal morphology. Furthermore, we report that a fundamental difference between the two appendages is the presence in the leg and absence in the antenna of a functional medial domain speciﬁed by dachshund. Our results lead us to propose that the acquisition of particular proximodistal subdomains and the evolution of their interactions has been essential for the diversiﬁcation of limb morphology. Key words: Distal-less, homothorax, dachshund, teashirt, decapentaplegic, wingless, hedgehog, Antenna, Leg, Limb, Drosophila SUMMARY Proximodistal domain speciﬁcation and interactions in developing Drosophila appendages P. D. Si Dong, Jessie Chu and Grace Panganiban* Department of Anatomy, University of Wisconsin, Madison, WI 53706, USA *Author for correspondence (e-mail: gepangan@facstaff.wisc.edu) Accepted 9 April 2001