Regulation of Proboscipedia in Drosophila by Homeotic Selector Genes

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 specification 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 identified 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 first 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 Ja¨ckle 1993). In the speci- Hox genes labial (lab), proboscipedia (pb), Deformed (Dfd), fication 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 (Nu¨sslein-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 specific 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 Ja¨ckle 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 specification 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 specifi- 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@sunflower.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-specific 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 (Ro¨der et al. 1992; de Zulueta pressed in only a subset of the cells in which Dfd or Scr et al. 1994; Ku¨hnlein 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 identification 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 identified 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-defined 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) identified 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 reflect 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 identifi- 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 fixed and stained essentially as described scura (Randazzo et al. 1991). A 500-bp DNA fragment by Kapoun and Kaufman (1995a). For examination of Pb taken from the second intron has been identified as accumulation antisera directed against the peptide encoded the minimal regulatory element that, when placed in by the second exon of pb (anti-E2) and against the peptide a reporter construct containing the pb promoter, can encoded by the ninth exon of pb (anti-E9) were used (Cribbs et al. 1992). Antibodies raised against Scr (Gorman and Kauf- partially recapitulate the expression pattern of pb in man 1995), Dfd (Mahaffey et al. 1989), and Dll (Cohen et both the embryo and in the imaginal discs (Kapoun al. 1993) were also used. Staining of reporter expression was and Kaufman 1995a). In addition to the enhancer ele- detected using monoclonal mouse anti-␤-galactosidase from ments in the pb locus numerous pairing-sensitive sites Boehringer Mannheim (Indianapolis). have been identified (Kassis 1994; Kapoun and Kauf- Microscopy and photography: Embryos were mounted on slides in methyl salicylate. Subsequent examination was per- man 1995b). These sites are thought to be bound by formed on a Zeiss axiophot and photographed using ASA100 large multiprotein complexes made up of the products print film. of the PcG and trxG genes (Wolffe 1996; Strutt et al. Protein purification and gel mobility shift: Dfd protein puri- 1997). However, unlike the pairing-sensitive sites found fication and gel mobility shifts were performed essentially as in the BX-C, the sites from the pb locus have been found described by Dessain et al. (1992) with the following minor modifications. Binding was performed in 10 ␮l containing to be insensitive to several mutations in either the PcG 100 mm KCl, 20 mm HEPES, pH 7.4, 0.25 mm EDTA, 1 mm or trxG genes (Kapoun and Kaufman 1995b). Recently dithiothreitol, 10% glycerol, and 100 ␮g/ml calf thymus DNA it has been shown that the genes Dfd and Scr are re- (Boehringer Mannheim). Reactions were then run on a Bio- Regulation of proboscipedia 185

Rad Mini-PROTEAN II gel running apparatus at room temper- constructs and their embryonic expression has been ature for 1 hr at 120 V. assayed (Kapoun and Kaufman 1995a). One of these The DNA fragments used in the gel mobility shift were generated by annealing complementary synthetic oligonucleo- constructs that contains a 500-bp pb enhancer and the tides (Oligos Etc.), which generated ends that were comple- lacZ gene fused to the pb promoter is designated mentary to EcoRI and XbaI restriction sites. The annealed P{0.5ϩpbZR}. This construct recapitulates the embry- ϩ fragments were then cloned into Bluescript (Stratagene, onic and imaginal disc expression patterns of pb. Two La Jolla, CA) and transformed into Subcloning Efficiency of the P{0.5ϩpbZR} transgenic lines, referred to as re- DH5␣ cells (GIBCO-BRL, Gaithersburg, MD). Transformants were amplified and the DNA was isolated using Plasmid Maxi- porter #19.2 and reporter #7, located on the X chromo- prep (QIAGEN, Chatsworth, CA). The fragments were excised some and second chromosome, respectively, were used using EcoRI and XbaI, gel purified, precipitated, and then in these experiments (Kapoun and Kaufman 1995a). labeled as described by Dessain et al. (1992). To facilitate certain crosses, reporter #19.2 was mobi- lized off the X chromosome and a line isolated with an insertion on the third chromosome referred to as RESULTS reporter #38. The lacZ expression pattern is shown for Expression pattern of proboscipedia and pb reporter both reporter #7 (Figure 1C) and reporter #38 (Figure constructs: The expression pattern of pb has been de- 1D). Occasionally these reporter constructs express lacZ scribed previously (Randazzo et al. 1991). Detectable outside of the normal domain of pb. Both lines display levels of Pb can first be identified in the mesoderm ectopic expression seen in the dorsal ridge and faintly of the mandibular segment at the onset of germband in a lateral striping pattern along the trunk of the em- extension (early stage 10; Campus-Ortega and Har- bryo (data not shown). Reporter #38 also consistently tenstein 1985). Ectodermal accumulation of Pb begins generates an additional domain of ectopic expression in the maxillary and labial lobes concurrent with their in the mandibular ectoderm (Figure 1D). formation just prior to segmentation. As segmentation pb is positively regulated by Dfd and Scr through an of the trunk is completed, a ventral stripe of Pb that intronic enhancer element: pb is positively regulated by overlaps the posterior mandible and anterior maxillary both the homeotic genes Dfd and Scr (D. Miller, S. segments becomes apparent. At the same time, the Holtzman, A. Kalkbrenner and T. C. Kaufman, un- mesodermal expression separates into three discrete published results). The P{0.5ϩpbZR} reporters contain patches, one of which becomes associated with the man- the smallest identified regulatory element of pb that is dible. At later stages, Pb accumulates in the brain and sufficient to recapitulate a pb-like pattern of lacZ expres- along the length of the ventral nerve cord. Lateral Pb sion in the maxillary and labial lobes (Figure 1, C and accumulation in a stage 12 embryo is shown in Figure D; Kapoun and Kaufman 1995a,b). Here we used im- 1A and ventral expression is shown in Figure 1B. munohistochemistry to assay and compare the expres- Regulatory elements from the second intron of pb sion patterns of the pb gene and pb reporter #7 in em- have been analyzed previously in P-element reporter bryos mutant for the genes Dfd and Scr.

Figure 1.—The expression pattern of endogenous Pb and pb reporter constructs. In all figures embryos are shown with anterior end to the left. (A and B) Composite images showing endogenous accumulation of Pb. Composite images are created through the superimposition of four different images of the same embryo, thereby showing all the different staining aspects that are not normally in the same plane of focus. (A) Lateral view of wild-type Pb. (B) Ventral view of wild- type Pb. (C and D) Expres- sion of ␤-galactosidase (␤-gal) protein in embryos containing pb reporter constructs. (C) Embryo containing pb reporter 7. (D) Embryo containing pb reporter 38. Note that reporter 38 displays the ectopic ␤-gal accumulation just anterior to the maxillary lobe in the ectoderm of the mandibular lobe. For all embryos in this figure, large solid arrows indicate staining of the labial lobe, large solid arrowheads indicate staining of maxillary lobe; large open arrows point out ventral ectodermal expression; large open arrowheads indicate mandibular mesoderm; small solid arrows point to expression in the brain; and finally, the small solid arrowhead shows prothoracic-associated mesodermal expression. 186 D. B. Rusch and T. C. Kaufman

Figure 2.—Comparison of Pb protein accumulation (A, C, and E) vs. ␤-gal accumulation (B, D, and F) in embryos mutant for the ANT-C genes Dfd and Scr. All embryos are homozygous for reporter 7. (A and B) Embryos carrying the Scr4 mutation. In the labial lobe (indicated by arrow), accumulation of Pb (A) is reduced while ␤-gal accumulation (B) is completely eliminated. (C and D) Dfd16 mutant embryos show reduced Pb accumulation (C) and elimination of ␤-gal (D) in the maxillary lobe (maxillary segment indicated by arrowheads). Note that ␤-gal is completely absent from the maxillary segment except in a few cells at the posterior edge of the lobe that are known to express Scr protein (Riley et al. 1987; Car- roll et al. 1988). (E and F) Dfd16 Scr4 double mutant embryos show a more severe reduction of Pb accumulation (E) and a complete absence of ␤-gal (F) accumulation in both the maxillary and labial lobes.

In Scr null mutant embryos (Scr4; Reuter et al. 1990) is also eliminated in the double mutant embryos. How- pb expression is markedly reduced but not eliminated ever, ectopic trunk expression is unaffected (data not from the labial lobe (Figure 2A). Based on the pattern shown). pb expression in the double mutant is also of expression in embryos doubly stained with antibodies greatly reduced (Figure 2E), more so than would be against both Pb and the engrailed (en) gene product, Pb expected given the results for either of the single mu- is eliminated almost entirely from the posterior com- tants. These results are consistent with previous reports partment of the labial lobe (data not shown). The stain- that pb is regulated by Dfd and Scr (D. Miller, S. Holtz- ing intensity of the remaining expression in the anterior man, A. Kalkbrenner and T. C. Kaufman, unpublished compartment is markedly reduced when compared to results). From these results we also conclude that this pb expression in the neighboring maxillary lobe. In this regulation takes place in part through regulatory ele- mutant background, reporter #7 responds even more ments found in the P{0.5ϩpbZR} transgene. strongly than does the endogenous gene. lacZ expres- As both Dfd and Scr are known transcription factors, sion is completely eliminated from the labial lobe (Fig- they could act to directly regulate pb expression. It is ure 2B). Similarly, in embryos mutant for a null allele known that many of the Antennapedia-class homeotic of Dfd (Dfd16; Merrill et al. 1987b), pb expression is genes bind a core DNA consensus sequence containing signiﬁcantly lower in the maxillary lobe (Figure 2C). In the ATTA motif (Ekker et al. 1994). Examination of this case, however, residual expression of pb is found the sequence of the pb minimal regulatory element re- in the posterior compartment of the maxillary lobe. veals that it contains four ATTA motifs (Randazzo et Mutations in Dfd do not affect mesodermal expression al. 1991). Since it has been shown that Dfd can bind of pb in the mandible (Figure 2, C and E). lacZ expres- DNA independently of other cofactors in vitro (Regul- sion is completely eliminated from all but the most ski et al. 1991), we decided to examine whether Dfd posterior edge of the maxillary lobe (Figure 2D). Consis- protein binds to any of the ATTA-containing sequences tent with these results, lacZ expression is completely from the pb enhancer. Using partially puriﬁed Dfd, gel eliminated from both the maxillary and labial lobes of mobility shifts were performed on control DNA frag- Dfd16 Scr4 double-mutant embryos (Figure 2F). Interest- ments and on DNA fragments derived from the pb mini- ingly, the ectopic expression of lacZ in the dorsal ridge mal enhancer. The results of these experiments indicate Regulation of proboscipedia 187

Figure 3.—Gel mobility shift assay using Dfd protein performed on putative Dfd binding sites. (A) Gel mobility shift using a control Dfd binding site (Ctrl; Dessain et al. 1992) as well as three other sites taken from within the 500- bp regulatory element of pb. Presence of partially purified Dfd extract in the mobility shift is indicated by ϩ. The white arrowhead indicates unbound fragments while the black arrowhead indicates shifted fragments. Extracts prepared from cells without the Dfd-expressing plasmid failed to result in shifts (data not shown). (B) Sequence of the 47-bp DNA fragments used in the gel mobility shift. The ATTA homeotic core binding motifs in each fragment are underlined. that only one of the ATTA-containing fragments binds weak expression of endogenous pb (Figure 4C). This Dfd strongly (Figure 3, A and B). The fragment bound lacZ expression closely resembles the graded expression by Dfd contains a sequence that exactly matches the pattern of Scr normally seen in the first thoracic segment Pbx-Hox consensus binding site (Chan et al. 1997). This (Gorman and Kaufman 1995). result, in conjunction with the previously mentioned cnc is a leucine zipper (bZIP class)-containing tran- genetic interactions, argues that Dfd binds pb regulatory scription factor that is expressed in the clypeolabrum elements directly in vivo to regulate pb expression. and in the mandibular segment (Mohler et al. 1991). Restriction of pb expression by cap’n’collar and tea- Mutations in this gene cause a transformation of the shirt: Both Dfd and Scr are capable of driving pb expres- mandible toward a maxillary identity (Mohler et al. sion in the ectoderm of the embryo (D. Miller, S. 1995). In cnc PZ mutant embryos, pb is expressed ectopi- Holtzman, A. Kalkbrenner and T. C. Kaufman, un- cally in the posterior ectoderm of the mandibular lobe published results). However, there is no ectodermal ex- (Figure 4, D and E). Analysis of embryos mutant for both pression of pb in either the mandibular lobe, where Dfd Dfd16 and cnc PZ indicates that this ectopic expression of is normally expressed, or in the first thoracic segment pb is dependent on Dfd expression (data not shown). where Scr is expressed. This suggests that other genes Unexpectedly, faint ectopic pb can also be seen ventrally are involved in the regulation of pb expression and act in the intercalary lobes (Figure 4E). We chose to exam- to prevent activation of pb in the segments anterior and ine whether lab was responsible for the intercalary ex- posterior to its normal domain of ectodermal expres- pression of pb for several reasons. First, mutations in cnc sion. In an attempt to identify additional regulators of do not significantly alter either the expression of Dfd pb, a survey of Pb accumulation in various mutant back- or Scr (Mohler et al. 1995). Second, Dfd and Scr are grounds was performed. The genes included in this the only genes to date that are known to activate pb survey were picked for their potential to regulate pb on expression (D. Miller, S. Holtzman, A. Kalkbrenner the basis of their timing and pattern of expression. Table and T. C. Kaufman, unpublished results). Perhaps lab, 1 shows a summary of the results of this survey. As a being a fellow homeotic gene, can also activate pb under consequence of this survey, the genes cnc and tsh were the proper circumstances. lab is expressed in the in- clearly identified as negative regulators of pb expression tercalary segment and is required for embryonic head in the mandible and first thoracic segments, respec- morphogenesis (Merrill et al. 1987a; Diederich et al. tively. 1989). Pb accumulation still occurs in the intercalary tsh encodes a zinc finger-containing transcription fac- lobes in a cnc PZ labVD1 double-mutant background (data tor that is expressed throughout the trunk of the embryo not shown). This indicates that lab is not responsible and is involved in the specification of the thoracic and for pb expression in the intercalary lobe. When reporter abdominal segments (de Zulueta et al. 1994). Embryos #7 is placed in a cnc PZ mutant background, strong ec- homozygous for a strong allele tsh8 display weak ectopic topic lacZ expression can be detected only in the man- expression of pb in the first thoracic segment (Figure dibular lobe (Figure 4F). 4, A and B). To test whether this ectopic expression was Effects of gap, pair rule, segment polarity, and other due to Scr, pb expression was examined in Scr4 tsh8 double genes on pb expression: As shown in Table 1, many of mutants. In the double mutant there is no ectopic ex- the genes that are members of either the gap, pair pression of pb (data not shown). When lacZ expression rule, or segment polarity genes have some effect on the from reporter #38 was examined in a tsh8 mutant back- pattern of pb accumulation. For the most part, mutations ground, surprisingly strong levels of lacZ were found in in genes of these classes reduce the number of cells the first thoracic segment compared to the relatively expressing pb but do not eliminate Pb entirely from the 188 D. B. Rusch and T. C. Kaufman

TABLE 1 Summary of results from a survey of pb expression in various developmental mutant backgrounds excluding PcG and TrxG mutants

Gene class Mutant locus Alleles Severity Comments Gap hunchback hb12 a LB lobe missing; LB expression absent Kruppel Kr2 — No effect knirps kni1 — No effect giant gtX11 b LB lobe missing; LB expression severely reduced or absent buttonhead btd1aMN lobe missing and mesodermal expression absent Empty spiracles ems1 — No effect orthodenticle ocYH — No effect Pair rule Paired prd9bLB lobe absent; LB expression severely reduced or absent even-skipped eve1, eve15H6a — No effect runt runE9 b MX lobe absent; LB expression severely reduced or absent odd-paired opa1bMX lobe absent; LB expression severely reduced or absent hairy h25 b LB lobe absent; LB expression severely reduced or absent sloppy-paired slp1bLB lobe absent; LB expression severely reduced or absent fushi-tarazu ftzw20 — Fused MX and LB lobe display normal levels of PB accumulation odd-skipped odd5cMX lobe fused with reduced LB lobe; pb expression appears normal in fused lobe Segment wingless wgCX4 a GN lobes reduced; LB expression polarity abscent early then severely reduced engrailed enX31, en4bGN lobes reduced; expression corespondingly reduced hedgehog hhAC b GN lobes reduced; expression correspondingly reduced armadillo arm1bGN lobes reduced; expression correspondingly reduced patched ptcIN b GN lobes reduced; expression correspondingly reduced naked nkd2cMisformed GN lobes show nearly normal expression Miscellaneous Sex combs reduced Scr4aPartial to complete loss of LB lobe expression Deformed Dfd16, DfdW21 a Complete loss of ventral ectoderm and partial to complete loss of MX lobe expression teashirt tsh8cWeak ectopic PB in T1 cap’n’collar cncPZ b Weak ectopic PB in IC lobes and strong ectopic PB in posterior MN spalt salm1 — No effect extradenticle exd1 — No effect decapentaplegic dppH46 a GN morphology severely disrupted; expression lost from GN lobes; remnants of ventral and mesodermal expression occasionally visible Distalless DllMP — No effect

IC, intercallary; MN, mandibular; MX, maxillary; LB, labial; T1, ﬁrst thoracic segment; GN, gnathal. a Large number of cells affected, sometimes resulting in absence of pb expression in the affected segment. b Large number of cells affected. c Small number of cells affected. Regulation of proboscipedia 189

Figure 4.—Mutations in the genes tsh and cnc result in ectopic expression of both endogenous pb and pb reporters. (A and B) tsh8 mutant embryos showing patches of ectopic accumulation of Pb in the T1 segment (arrows) from lateral (A) and ventral (B) views. (C) Reporter 38 shows strong ectopic ␤-gal expression throughout T1 in a tsh8 mutant embryo. Note that ␤-gal accumulation in the mandibular lobe and dorsal ridge is normal for this reporter line. (D) Ectopic Pb appears in the posterior ectoderm of the mandible in cnc PZ mutant (arrowhead). (E) A close-up view of the head of a cnc PZ mutant embryo reveals Pb accumulation in the posterior mandible (arrowhead) and also faint accumulation in the intercalary lobes (open arrows). The Pb expression visible but out of the plane of focus in the ventral mandible is in the mesoderm. (F) Reporter 7 shows strong ectopic ␤-gal expression in the mandible (arrowhead) in a cnc PZ mutant background. affected segments (data not shown). In no case do they ing degrees perturb pb accumulation. In general, muta- cause pb to accumulate ectopically. The most striking tions in the pair rule genes eliminate either the maxil- results were caused by zygotic mutations in the genes lary or labial lobe, as well as reduce the width of the buttonhead (btd), giant (gt), and hunchback (hb). btd is a respective segment. Despite these effects on morphol- head gap gene required for formation of the mandibu- ogy, pb expression can often be seen in the affected lar segment (Cohen and Jurgens 1990). In btd mutants, segments. Mutations in the segment polarity genes af- no mandibular structures are seen and no pb accumula- fect the morphology of both the maxillary and labial tion occurs anterior of the maxillary segment (data not lobes. The overall effect is a reduction in the size of these shown). pb accumulation is normal in the other gnathal lobes, resulting in a correspondingly reduced number of segments. Mutations in both gt and hb disrupt the forma- pb-expressing cells. Of the segment polarity genes tested, tion of the labial lobe (Lehmann and Nu¨sslein-Vol- wingless (wg) has the strongest effect on pb expression. hard 1987; Petschek et al. 1987) and result in concomi- At early stages in wg CX4 mutants, no pb expression is tant loss of pb expression therein (data not shown). apparent in the presumptive labial lobe, though later, We have also tested the pb reporter #7 in a hb mutant as head involution commences, some of these cells do background and found that there is no lacZ expression begin to express pb. in the presumptive labial segment (data not shown). In A number of other developmentally important genes the case of gt, pb expression is not entirely extinguished. that we examined in the survey are noteworthy for their Weak pb accumulation can sometimes be seen in the apparent lack of effect on pb accumulation. The first most dorsal and posterior cells of the presumptive labial of these genes is extradenticle (exd), known as pbx in segment (data not shown), overlapping with the few vertebrates, which encodes a homeobox-containing pro- remaining cells of the engrailed stripe in the labial seg- tein and a known cofactor of several vertebrate and ment (Petschek and Mahowald 1990). For both gt invertebrate Hox genes (van Dijk and Murre 1994; and hb, this reduction or loss of pb expression in the Ryoo and Mann 1999). The Dfd binding site we identi- labial lobe cannot be attributed to alterations in the Scr fied presumably would also bind Exd in conjuction with pattern as Scr accumulates in the cells posterior to the Dfd (Chan et al. 1997). Surprisingly, exd1 zygotic mutants maxillary segment (data not shown; Petschek and showed no effect on pb accumulation. Distal-less (Dll)is Mahowald 1990). It is possible that tsh expression has another homeobox-containing transcription factor and moved anteriorly to block pb expression. To test this is required for the formation of limbs (Cohen et al. we examined pb expression in tsh8 hb12 double-mutant 1989). Dll is expressed in both the maxillary and labial embryos. Because we find no restoration of pb expres- lobes in a fashion analogous to pb, but mutations in Dll sion in these embryos, we conclude that tsh is not respon- have no effect on pb accumulation. Another intriguing sible for loss of pb expression in hb mutants. gene is salm, which encodes a zinc-finger transcription Nearly all the pair rule and segment polarity genes factor expressed in the gnathal segments and is thought affect morphology of the gnathal segments and to vary- to be required for proper specification of gnathal iden- 190 D. B. Rusch and T. C. Kaufman

and Brock 1991). Interestingly, neither ph nor Psc causes ectopic accumulation of ␤-galactosidase from the P{0.5ϩpbZR} reporters (data not shown). However, using an eye color assay (Kapoun and Kaufman 1995b) we see derepression of the P-element white mini-gene in PscIIN48 heterozygous adult ﬂies carrying the pb reporter constructs (data not shown). This suggests that regulatory elements in the reporter used here can mediate the activity of the PcG genes but apparently only in the imaginal discs. Moreover, since the pattern of expression of the resident pb locus is changed in Psc and ph mutant backgrounds there must be cis-acting sequences in the native gene that reside outside the tested fragment that are responsible for the observed interaction. Further investigation will be necessary to map these components.

DISCUSSION We have examined pb expression during embryogenesis. While pb plays no known functional role during Figure 5.—PcG mutants result in ectopic accumulation of embryogenesis, its pattern of expression is clearly re- Pb throughout the embryo. (A) Ventral surface of a Psc IIN48 stricted to the gnathal region where it is known to func- embryo showing ectopic accumulation of Pb in the leg anlagen tion during adult development. Our results provide a IIN48 (arrow). (B) Psc mutant showing ectopic Pb accumulation mechanism by which pb expression is established and in cells of the lateral ectoderm along the entire length of the embryo. Arrowhead indicates ectopic Pb in the fourth that may also serve to determine pb expression subse- abdominal segment. (C) Lateral view of Pb expression in a quently in adult tissues. ph503 mutant embryo. Besides showing highly abnormal mor- Temporal and spatial regulation of pb: On the basis phology, Pb accumulates in the antennal segment (white of these results and previous work, we have developed arrow), laterally along the length of the embryo (arrowhead), a working model for the regulation of pb. This model and in cells in the center of the leg anlagen (arrow). accounts for both temporal and spatial aspects of pb expression (Figure 6). In effect, the regulation of pb tity (Ku¨hnlein et al. 1994). However, salm1 mutants had can be broken down into early, middle, and late phases. no effect on pb expression. The early phase represents the period prior to the onset Polycomb group interactions with pb: After the pat- of pb expression, during which the gap genes define the tern of Hox gene expression has been established, main- domain of pb expression through a presumably indirect tenance of this pattern is dependent on the function mechanism. During the middle phase, the genes cnc, of the PcG and trxG genes (McKeon and Brock 1991; Dfd, Scr, and tsh act to establish the initial expression Soto et al. 1995; Kingston et al. 1996). Here, we assayed pattern of pb along the A/P axis. During the late phase accumulation of Pb in embryos mutant for various PcG we propose that the PcG and trxG genes assume respon- and trxG genes (see materials and methods for sibility for maintaining the pattern of pb expression stocks). The PcG genes Psc and ph were the only genes through the later stages of embryogenesis. from either group that showed a detectable interaction The early phase reflects a requirement for gap gene with pb. In Psc IIN48 mutants, ectopic pb accumulates at function for normal expression of pb to occur during high levels in the antennal segment and in a compli- later stages. Specifically, btd, gt, and hb have been identi- cated pattern throughout the trunk segments (Figure fied as being required for proper gnathal expression of 5, A and B). In the abdomen, the ectopic accumulation pb. The function of the head gap gene btd has been of pb occurs as a pair of stripes in each segment. Addi- shown to be required only during early stages of em- tionally, ectopic pb accumulates in the leg anlagen. Simi- bryogenesis (Wimmer et al. 1997). The expression pat- larly, ph503 mutants result in ectopic accumulation of pb terns of gt and hb are such that they are no longer in the antennal segment and in the trunk segments; the expressed in the labial segment at the time when pb pattern is not as clearly defined as in Psc mutants and expression begins (Bender et al. 1988; Eldon and Pir- there is a lower accumulation of pb in the leg anlagen rotta 1991). We take this as a strong indication that (Figure 5C). While both Dfd and Scr are ectopically the gap genes influence pb indirectly. Consistent with expressed in zygotically mutant Psc embryos, neither is this hypothesis, we are unable to identify any gt or hb expressed in the antennal segment, in the leg anlagen, binding sites (Stanojevic et al. 1989; Zhang et al. 1991; or in the abdominal segments (data not shown; McKeon Zhang and Bienz 1992) in the regulatory elements of Regulation of proboscipedia 191

Figure 6.—A model depicting the temporal and spatial regulation of pb during embryogenesis. The regulation of pb has been di- vided into three phases referred to as early, middle, and late. The early phase corresponds to the time during development when the gap genes are necessary to permit expression of pb during the middle and late phases. The broken lines, depicting regulation of pb by the gap genes, are shown to suggest that these interactions are indirect in nature. During the middle phase, the initial expression pattern of pb is established. The known regulatory interactions, which may be mediated directly or indirectly, are represented by unbroken black lines. The nonautonomous influences, possibly mediated through wg signaling, are represented by dashed gray lines. Finally, during the late phase, the PcG and trxG genes act to prevent further changes in the pb expression pattern despite alterations in the expression patterns of the initial regulators of pb (Scr expression in T1). The overriding activity of the PcG and trxG genes is represented by the gray shading of the squares. IC, intercallary segment; MN, mandibular segment; MX, maxillary segment; LB, labial segment; T1, first thoracic segment; T2, second thoracic segment. the pb reporter. In the case of hb, we have investigated that Dfd can bind to pb regulatory elements in vitro. We the role that various trans-acting factors might play in think it likely that Scr also regulates pb directly based mediating loss of pb expression in the labial segment. on the similarity with which mutations in Dfd and Scr We find that expression of Scr, the positive regulator of affect expression of pb and the pb reporter. In addition pb in the labial segment, is not eliminated. Further, we to the Hox genes, the region-specific homeotics cnc and have shown that repression of pb is not attributable to tsh have been identified as negative regulators of pb and expansion of tsh expression. One possibility is that an- serve to restrict pb expression to the gnathos. It is not other negative regulator is being expressed such that Scr clear whether these genes regulate pb directly, though can no longer activate pb. Given the negative regulatory in the case of tsh we have identified the sequence interactions that occur between the gap genes (Capo- TGGAAAAGT in the 500-bp regulatory fragment used villa et al. 1992; Struhl et al. 1992), it is possible that in the pb reporter; this sequence is very similar to the one of the other gap genes might be misexpressed and identified tsh binding site (Alexandre et al. 1996). downregulate pb. On the other hand, it may be misex- While this regulatory paradigm does not completely de- pression of cnc or some other gene that has yet to be scribe the regulation of the endogenous gene, based identified. Alternatively, the “hit-and-run” hypothesis, on the presence of pb residual expression, it is sufficient proposed by Zhang and Bienz (1992) to explain the to explain the behavior of the 500-bp pb reporter. This long-term repression of Ultrabithorax (Ubx)byhb, may mechanism of regulation places pb downstream of the describe how transient expression of the gap genes is Hox genes and is the first instance in Drosophila where required very early in development to permit later ex- one Hox gene is positively and directly regulated by pression of pb. In their hypothesis, heritable changes in another, a distinction previously accorded only to ver- chromatin structure, mediated by the PcG genes, were tebrate Hox genes (Gould et al. 1997). Others (D. Miller, invoked to explain how hb regulates Ubx long after hb S. Holtzman, A. Kalkbrenner and T. C. Kaufman, un- expression has ceased. In the case of pb regulation, one published results) have suggested that wg may be medi- or more of these gap genes may be required to alter ating the nonautonomous residual expression of pb that chromatin structure in and around the pb locus, thereby is uncovered by mutations in Dfd and/or Scr. With the allowing the various trans-acting factors access to the pb exception that wg has the strongest effect on pb expres- cis-acting regulatory elements. sion of the segment polarity genes tested, our results During the middle phase, the initial expression pat- shed little light on the mechanism that underlies this tern of pb is set by a variety of trans-acting factors. Our phenomenon. However, signalling has been implicated focus has been on the identification of those factors to explain regulation of ectodermal pb function by meso- that determine the ectodermal pattern of pb expression dermal expression of Scr; perhaps the residual expres- along the A/P axis of the embryo. We have confirmed sion in the embryo is an example of this pathway (Perci- that the Hox genes Dfd and Scr act as positive regulators val-Smith et al. 1997). Further experiments, including of pb (D. Miller, S. Holtzman, A. Kalkbrenner and identification of an enhancer that mediates this residual T. C. Kaufman, unpublished results) and demonstrated expression, are needed. 192 D. B. Rusch and T. C. Kaufman

Finally, during the late phase, we have identified two tion may be conserved. A deficiency that deletes many PcG genes that are involved in maintaining repression of the Tribolium Hox genes except for the lab, pb, and of pb outside its normal domain of expression. This Abd-B homologues has been isolated. Embryos homozy- result supersedes a previous report that the PcG genes gous for this deficiency display a mutant phenotype in do not regulate pb (Kapoun and Kaufman 1995b). We which every segment of the Tribolium larva is trans- have yet to identify any trxG genes that are required formed toward an antennal segment and bears a pair for the maintenance of pb expression. To function, the of antennae (Stuart et al. 1991). These larvae have no PcG genes are thought to assemble on DNA in large mouthparts or walking legs. Further, gain-of-function multimeric complexes (Franke et al. 1992). Unlike the mutations in Tribolium pb, which result in ectopic ex- genes of the BX-C, which are regulated, to greater and pression of Tribolium pb in the antennal segment, are lesser extent, by all the PcG genes that have been tested known to transform the antennae into generic feeding (McKeon and Brock 1991; Simon et al. 1992), pb is not palps (Denell et al. 1996). Given these two results, we regulated by the majority of known PcG genes. Assum- can infer that pb is not being expressed in embryos ing that the PcG genes function similarly at the pb locus, containing this deficiency because the antennae are not the implication is that not all multimeric complexes can transformed into feeding palps. If pb is not being ex- be equal. However, it is not clear how these differences pressed, this implies that the positive regulators of pb are established. One possibility is that complexes com- have been removed by this deficiency. The simplest in- posed of different combinations of PcG genes are terpretation that is consistent with these results and the formed at different times during development, thereby results presented here is that the Dfd and Scr homologs regulating different loci (Wolffe 1996). Interestingly, in Tribolium may be required to positively regulate the a vertebrate homolog of Psc has been shown to bind a Tribolium pb much like their counterparts do in Dro- specific DNA sequence. This exact sequence is also sophila. Actual proof of this possibility requires in situ found in the regulatory elements of the pb reporter data on the expression pattern of the pb homolog in construct, indicating that Psc may bind directly, though various Tribolium Hox mutants. this remains to be shown (Kanno et al. 1995). We thank W. McGinnis for the Dfd-expressing bacterial cell line In addition to forming quantitatively different com- and for his helpful advice concerning the gel mobility shifts; M. plexes, the timing of complex formation may be crucial Peterson, A. Popadic, and B. Rogers for their helpful discussions and to the proper expression of pb. Normally, the PcG genes reviews of this manuscript; D. Noecker for his help with the antibody are required after the expression pattern of pb has been staining of the embryos; D. Verostko for administrative assistance; K. Matthews and the Bloomington Stock Center, and the Kaufman lab established to prevent ectopic expression. Presumably, as a whole for their support and assistance without which this paper this ectopic expression would result from newly ex- would not be possible. This work was supported by the Howard Hughes pressed transcription factors acting inappropriately at Medical Institute (HHMI) and by a National Institutes of Health the pb locus. This hypothesis may offer an explanation Predoctoral Fellowship (GM07757) to D.B.R.; T.C.K. is an Investigator for the differences seen between the expression pattern of the HHMI. of endogenous pb and the pb reporter in tsh mutant backgrounds. In this scenario, Scr expression in T1 begins concurrently with the initiation of PcG-mediated LITERATURE CITED repression at the pb locus. In the absence of tsh, competi- Alexandre, E., Y. Graba, L. Fasano, A. Gallet, L. Perrin et al., tion between activation by Scr and repression by the 1996 The Drosophila Teashirt homeotic protein is a DNA-binding protein and modulo, a HOM-C regulated modifier of variega- PcG genes results in the weak and variable pb expression. tion, is a likely candidate for being a direct target gene. Mech. Because the PcG genes do not regulate ␤-galactosidase Dev. 59: 191–204. expression from the pb reporter, it is free to respond Beeman, R. W., J. J. Stuart, S. J. Brown and R. E. Denell, 1993 Structure and function of the homeotic gene complex (HOM-C) strongly to Scr accumulation in T1. Further experiments in the beetle, Tribolium castaneum. Bioessays 15: 439–444. are required to support this hypothesis. Bender, M., S. Horikami, D. Cribbs and T. C. Kaufman, 1988 Iden- The evolution of regulation of pb in other insects: In tification and expression of the gap segmentation gene hunchback Drosophila, pb plays a role in specifying limbs to become in Drosophila. Dev. Genet. 9: 715–732. Campus-Ortega, J., and V. Hartenstein, 1985 The Embryonic Devel- specialized for feeding. In other insects where it has opment of Drosophila melanogaster. Springer-Verlag, New York. been examined, pb is expressed in and required for the Capovilla, M., E. D. Eldon and V. Pirrota, 1992 The giant gene formation of the larval mouthparts (Denell et al. 1996; of Drosophila encodes a b-ZIP DNA-binding protein that regulates the expression of other segmentation gap genes. 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