Copyright 2000 by the Genetics Society of America Regulation of proboscipedia in Drosophila by Homeotic Selector Genes Douglas B. Rusch and Thomas C. Kaufman Howard Hughes Medical Institute, Department of Biology, Indiana University, Bloomington, Indiana 47405 Manuscript received February 28, 2000 Accepted for publication May 1, 2000 ABSTRACT The gene proboscipedia (pb) is a member of the Antennapedia complex in Drosophila and is required for the proper speci®cation of the adult mouthparts. In the embryo, pb expression serves no known function despite having an accumulation pattern in the mouthpart anlagen that is conserved across several insect orders. We have identi®ed several of the genes necessary to generate this embryonic pattern of expression. These genes can be roughly split into three categories based on their time of action during development. First, prior to the expression of pb, the gap genes are required to specify the domains where pb may be expressed. Second, the initial expression pattern of pb is controlled by the combined action of the genes Deformed (Dfd), Sex combs reduced (Scr), cap'n'collar (cnc), and teashirt (tsh). Lastly, maintenance of this expression pattern later in development is dependent on the action of a subset of the Polycomb group genes. These interactions are mediated in part through a 500-bp regulatory element in the second intron of pb. We further show that Dfd protein binds in vitro to sequences found in this fragment. This is the ®rst clear demonstration of autonomous positive cross-regulation of one Hox gene by another in Drosophila melanogaster and the binding of Dfd to a cis-acting regulatory element indicates that this control might be direct. HE metameric expression of the homeotic (Hox) genes (Ingham 1988). This hierarchy begins with mater- Tgenes of the Antennapedia complex (ANT-C) and nally provided factors that are differentially localized bithorax complex (BX-C) is crucial to the proper devel- within the oocyte. Upon fertilization, these factors then opment of Drosophila melanogaster (Kaufman et al. 1990; act to establish the primary axes of the embryo (Law- Morata 1993). The ANT-C contains the traditional rence 1992; Pankratz and JaÈckle 1993). In the speci- Hox genes labial (lab), proboscipedia (pb), Deformed (Dfd), ®cation of the A/P axis, gradients of these maternal Sex combs reduced (Scr), and Antennapedia (Antp), each factors act to establish the expression patterns of the of which encodes a homeodomain containing transcrip- gap and terminal genes that subdivide the embryo into tion factor (Kaufman et al. 1990). Generally speaking, discreet domains (NuÈsslein-Volhard and Weischaus the homeotics confer cellular identity to all of the cells 1980; Struhl et al. 1992). In turn, the gap and maternal within their domain of expression. The identity that any genes establish the periodic expression patterns of the speci®c cell adopts is highly dependent on the timing pair rule genes, which further subdivide the embryo of the Hox gene expression and on the presence of and determine the expression pattern of the segment other developmental factors (Rogers et al. 1997; Rog- polarity genes (Goto et al. 1989; Pankratz et al. 1990; ers and Kaufman 1997). Consequently, misexpression Small et al. 1992; Pankratz and JaÈckle 1993; Gross- or loss of expression of the Hox genes can result in niklaus et al. 1994). The initial expression pattern of homeotic transformations, which may greatly affect the the Hox genes has been attributed to genes at every viability of either the adult or larva (Lewis 1978). The level of this cascade (Jack and McGinnis 1990). In homeotics are primarily organized along the anterior/ particular, it has been shown that the Dfd expression posterior (A/P) axis of the embryos. The speci®cation pattern is dependent on the maternal, gap, and pair of the A/P axis is crucial to development and serves to rule genes (Jack et al. 1988; Jack and McGinnis 1990). subdivide the embryo into smaller and smaller units Ubx is regulated in a similar fashion (Zhang et al. 1991; wherein the homeotics are expressed and confer iden- Zhang and Bienz 1992). Once the expression pattern tity. The primary and most recognized units of the insect of the Hox genes has been established, later expression embryo are the segment and parasegment. The speci®- becomes, at least in part, dependent on the action of cation of the A/P axis, including the expression of the the trithorax group (trxG) and Polycomb group (PcG) Hox genes, is controlled by a hierarchy or cascade of genes. The trxG and PcG genes are thought to function in the maintenance of stable expression and repression of Hox gene expression, respectively (McKeon and Brock 1991; Gindhart and Kaufman 1995; Soto et al. Corresponding author: Thomas C. Kaufman, HHMI, Department of Biology, Indiana University, Bloomington, IN 47405. 1995; Kingston et al. 1996; Wolffe 1996). The Hox E-mail: kaufman@sun¯ower.bio.indiana.edu genes themselves are known to act in conjunction with Genetics 156: 183±194 (September 2000) 184 D. B. Rusch and T. C. Kaufman the region-speci®c homeotics cap'n'collar (cnc), spalt quired for expression of pb in the Drosophila embryo (salm), and teashirt (tsh), thereby specifying segmental (D. Miller, S. Holtzman, A. Kalkbrenner and T. C. identity by regulating the expression of various combi- Kaufman, unpublished results). However, pb is ex- nations of target genes (RoÈder et al. 1992; de Zulueta pressed in only a subset of the cells in which Dfd or Scr et al. 1994; KuÈhnlein et al. 1994; Mohler et al. 1995; is expressed. In light of this, we have undertaken a for a review see Rogers and Kaufman 1997). systematic analysis of pb expression in various develop- The Hox gene pb is unusual in that it does not confer mental mutants. Here we report the identi®cation of identity at the level of the segment, but instead acts to cnc and tsh as the genes responsible for the restriction modify structures on segments (i.e., limbs) to become of pb expression at its anterior and posterior boundaries, specialized for feeding. Adult Drosophila that are homo- respectively. Additionally, we have identi®ed the PcG zygous for pb null alleles have their labial palps trans- genes Posterior sex combs (Psc; Martin and Adler 1993) formed into legs (Kaufman 1978). Consistent with this and polyhomeotic (ph; Dura et al. 1985; DeCamillis et al. transformation, pb is expressed in the labial discs and 1992) as being required to maintain repression of pb central nervous systems of third instar larvae. However, expression. Using the expression of pb reporter con- in the Drosophila embryo, which gives rise to a limbless structs in these mutant backgrounds, we show that all larva, pb serves no known function. Nevertheless, it is of these genes function through the 500-bp conserved expressed in a well-de®ned pattern during embryogene- regulatory element taken from the second intron of pb. sis (Randazzo et al. 1991). In recent years, homeotic Additionally, we show that Dfd can bind in vitro to a mutations in the beetle Tribolium castanaeum have been Dfd consensus binding sequence (Chan et al. 1997) identi®ed and characterized. Mutations in the found in this regulatory element. These results describe Tribolium Hox gene maxillopedia (mxp), the beetle ho- a regulatory paradigm for pb that is unlike that of the molog of pb, result in transformation of the larval other Hox genes and that may have been conserved mouthparts into legs (Beeman et al. 1993). This result during the evolution of the insects. can be interpreted to mean that pb homologs play a functional role in the development of insect embryos MATERIALS AND METHODS outside the higher Diptera (Beeman et al. 1993). Indeed, comparison of embryonic expression of pb in insect Fly stocks and protein detection: Embryos from the follow- orders other than the Diptera indicates that the expres- ing stocks were collected and assayed using immunohisto- 12 1 X11 2 1 1 sion pattern of pb has been largely conserved over evolu- chemistry: gap gene mutants: hb , kni , gt , Kr , btd , ems , ocYH; pair rule mutants: ftzW20, h25, opa1, odd5, slp1, eve1, eve15H6a, tionary time (Denell et al. 1996; Rogers and Kaufman prd9, runE9; segment polarity mutants: wg CX4, enX31, en4, hhAC, 1997). Taken together, these results suggest that pb ex- arm1, ptc IN, nkd2; homeotic mutants: cncPZ, labVD1 cnc PZ, Dfd16, pression in Drosophila may re¯ect the existence of an- Dfd16 cnc PZ, exd1, salm1, tsh8, tsh8 Scr4, Scr4, Df(2R)Dll MP; polycomb cient regulatory mechanisms that endure despite the group mutants: ash1B1, pho1(l(4)29), Pcl10, E(Z)S1, E(Z)S6, E(Pc)*, D1 2 1 1 D1 1 5 503 IIN48 apparent nonfunctional nature of the protein in the Asx , esc , kto , Pc , Scm , sxc , vtd ph , Psc ; trithorax group mutants: trx3, dev2, osa1, brm2, skd1, urd2, sls1, ash22, mor 2, kis2. Drosophila embryo. Recombination was used to generate stocks doubly mutant Previous analysis of the pb locus has led to the identi®- for cnc PZ and the ANT-C homeotics. The P-element reporter cation of several important regulatory elements (Ran- construct P{0.5ϩpbZR} has been described previously (Kap- dazzo et al. 1991; Kapoun and Kaufman 1995a). Some oun and Kaufman 1995a,b). Recombinants between Psc IIN48 ϩ of these show a high degree of sequence conservation and P{0.5 pbZR} were isolated based on derepression of the white gene located in the P-element construct. when compared with similar regions from D. pseudoob- Embryos were ®xed and stained essentially as described scura (Randazzo et al. 1991). A 500-bp DNA fragment by Kapoun and Kaufman (1995a).