Copyright Q 1995 by the Genetics Society of America

A Screen for Modifiers of Dejin-med Function in

Katherine W. Hardjng,* Gabriel Gellon? Nadine McGinnis* and William M~Ginnis*~~ *Departwnt of Molecular Biophysics and Biochemistry and iDepartment of Biology, Yak University, New Haven Connecticut 06520-8114 Manuscript received February 16, 1995 Accepted for publication May 13, 1995

ABSTRACT Proteins produced by the homeotic of the Hox family assign different identities to cells on the anterior/posterior axis. Relatively little is known about the signalling pathways that modulate their activities or the factors with which they interact to assign specific segmental identities. To identify genes that might encode such functions, we performed a screen for second site that reduce the viability of carrying hypomorphic mutant of the Drosophila homeotic locus, Deformed. Genes mapping to six complementation groups on the third chromosome were isolatedas modifiers of Defolmd function. Productsof two of these genes, sallimus and moira, have been previously proposed as homeotic activators since they suppress the dominant adult of Polycomb mutants. Mutations in hedgehog, which encodes secreted signalling proteins, were also isolated as Deformed loss-of-function enhancers. Hedgehog mutant alleles also suppress the Polycomb phenotype. Mutations were also isolated in a few genes that interact with Deformed but not with Polycomb, indicating that the screen identified genes that are not general homeotic activators. Twoof these genes, cap ‘n’collarand defaced, have defects in embryonic head development that are similar to defects seen in loss of functionDefmed mutants.

OMEOTIC proteinsencoded in the Anten- in embryos, and some evidence exists to support this H napedia and bithorax complexes of Drosophila, view. One breakthrough has come from the character- like their Hoxhomologues in other animals, determine ization of a called extraohtick (ex4 , which seems anterior-posterior identities in developing the embry- to modulate homeotic protein specificity in a dose-de- onic (GARC~A-BELLIDO1977; LEWIS 1978; pendent manner(PEIFER and WIESCHAUS1990). Recent KAUFMAN et al. 1980,1990; BENDERet al. 1983;MCGINNIS results have shown that exd encodes a homeodomain and KRUMLAUF 1992). Homeotic proteins aretranscrip- proteinthat enhances the DNA binding affinity of tion factors that bindDNA through their 60 amino acid some, but not all, homeotic proteins (RAUSKOLB et al. homeodomain regions (SCOTT et al. 1989; MOLTER1993; CHANet al. 1994; VAN DIJK and MURRE1994). et al. 1990; HAYASHI and SCOTT 1990;LAUGHON 1991; Previous genetic screens for factors required for ho- DESSAINand MCGINNIS1993); their abilities to instruct meotic gene function have identified genes that can cells to develop as head, thorax, or abdomen are appar- be divided into two broad classes: the trithorax group ently mediated by the differential regulation of diverse (LEWIS1968; CAPDEWLA and GARC~A-BELLIDO ING1981; sets of target genes (GAR&-BELLIDO1977; BOTAS HAM 1981,1983; SHEARNet al. 1987; J~ENNISONand TAM- 1993). Little is known about the underlying mecha- KUN 1988; SHEARN 1989; TAMKUNet al. 1992; FARKAS et nisms responsible for differential target selection, but al. 1994) and the Polycomb group (LEWIS 1978; STRUHL the modest differences in in vitroDNA binding specific- 1981, 1983; DUNCAN andLEWIS 1982; DUNCAN1982; ity between different homeodomains do not appearto SATOet al. 1983; INGHAM1984; DURAet al. 1985; JUR- determine targetselection in vivo (HAYASHI and SCO~T GENS 1985; JONES and GELBART1990; PHILLIPS and 1990). SHEARN1990). Polycomb group functions are required Homeotic proteins are requiredin a diverse set of cell to maintain the normal boundaries of types at successive stages ofdevelopment; these proteins expression by repressing homeotic gene transcription must therefore be able to coordinate their anterior/ in regions outside these boundaries (STRUHLand AKAM posterior identity function with many different cellular 1985; WEDEENet al. 1986; KUZIORA and MCGINNIS regulatory pathways. The requirement to function in 1988b; MCKEON and BROCK1991; SIMONet al. 1992). many developmental environments makes it seem likely Few trithorax group functions are well understood, but that a variety of different cofactors contribute both to trithorax (trx)and hahma (h)activities are required to the activity and to the specificity of homeotic proteins maintain wild-type levels of homeotic gene expression (TAMKUNet al. 1992; BREENand HARTE 1993; SEDKOV Corresponding author: William McGinnis, Department of Molecular et al. 1994). The repressive function of the Polycomb Biophysics and Biochemistly, , 224 Bass Bldg., 266 Whitney Ave., New Haven, CT 065204114, group proteinsis believed to be exertedby their partici- E-mail: [email protected] pation in large multimeric complexes that alter chro-

Genetics 140: 1339-1352 (August, 1995) 1340 K W. Harding et al. matin structure (PARO 1990; PAROand HOGNESS1991; To identify genes in the Dfd-dependent posterior FRANKEet al. 1992; MARTIN and ADLER 1993; RASTELLI head specification pathway, we screened for mutations et al. 1993). Trithorax group proteins, often defined that, when heterozygous, cause a striking reduction in by their ability to suppress the effects of Polycomb (PC) the viability of adults carrying two hypomorphic Dfd mutations (SHEARN et al. 1987; KENNISON 1993), are a alleles. Analogous genetic screens for haplo-insufficient heterogeneous group. Some promote transcription of enhancers have provided insight into signal transduc- homeotic genes by contributing to a multimeric com- tion pathways used in Drosophila development (GERT- plex that could antagonize the repressive effects ofPoly- LER et al. 1989; SIMONet al. 1991; DOYLEand BISHOP comb group proteins (TAMKUNet al. 1992; JONES and 1992; DICKSONand HAFEN 1993; RAFTERY et aZ. 1995). GELBART1993). Other trithorax group genes such as In this paper we report theDfd enhancers we have iden- TrZ (encoding GAGA factor) (BIGGINand TJIAN 1988; tified in a screen of third chromosome mutations. More FARKASet al. 1994) probably act more directly to influ- than 12,000 mutagenized chromosomes were tested for ence homeotic gene function, while the function of Dfd modifiers, and 49 chromosomes were found to carry most others is not understood. To generalize the cur- mutations thatenhance the lethality of Dfd hypo- rent understanding of their function, the trithoraxand morphs. We have concentrated on the loci for which Polycomb group proteins are required to maintain the we isolated multiple alleles; these mutations correspond wild-type levels and boundaries of homeotic selector to six lethal complementation groups. Analysis of these gene expression. groups indicates that we isolated new alleles of two tri- We were interested in using a genetic screen to iden- thorax group genes: sallimus (sls) and moira (mor). We tify factors required for the function of the homeotic also identified hedgehog (hh) as a gene critical for Dfd gene Deformed (Dfd), especially those influencing Dfd function; surprisingly, hh mutations also satisfythe crite- functional specificity. Such factors are expected to in- ria for inclusion in the trithorax group. Three alleles clude members of the trithorax/Polycomb groups, of a gene recently shown to modulate Dfd function, cap which are required for homeotic expression and may 'n' collar (cnc), were isolated, as were two alleles of a be involved in functional specificity. Such a screen novel gene, defaced (dfc), that might function as a Dfd could also identify other factors acting in parallel and/ coactivator. The and behavior of the muta- or more specifically with Dfd as well as those that act tions recovered in the screen suggest that a variety of downstream of Dfd in head developmental pathways. In mechanisms contribute to Dfd activity and targeting contrast to the Drosophila homeotic genes controlling specificity in developing Drosophila. abdominal development, Dfd has a relatively weak re- quirement for trithorax (trx) activity for maintenance of MATERIALSAND METHODS its transcription. Dfd relies on an autoregulation circuit that involves DFD protein persistently activating the Stocks: Flies were reared on standard yeast-agar-cornmeal- transcription of its own gene in the embryonic CNS and molasses medium at 25". In all experimental crosses, care was taken not to overcrowd the vials and food was periodically epidermis through upstream and intronic enhancers supplemented with dry yeast; unless otherwise indicated, ex- (KUZIORAand MCGINNIS 1988a;BERGSON and MCGINNIS perimental crosses wereperformed at 25". All mutations, aber- 1990; MCGINNISet al. 1990; REGULSKI et al. 1991; ZENG rations and abbreviations are described either in LINDSLEY et al. 1994). and ZIMM (1992) or as indicated. hh' was obtained from the Dfd is required for thespecification of posterior head Bowling Green stock center. PcS and Pc4 were provided by J. TAMKUN.tm', sls, moil and 1(3)87Ca" were provided by J. segment identity. Dfd null mutants die at the end of KENNISON. cncvLI" is a P-induced null cnc and was pro- embryogenesis due tothe lossof structures derived vided by J. MOHLER.Ki ppand a set of third chromosome from the maxillary and mandibular segments, dorsal deficiencies (Df(3)kit) were obtained from the Bloomington ridge and optic lobe (MERRILL et al. 1987; REGULSN et stock center. TUCUCU is the designation for a multiply marked al. 1987). Ectopic Dfd expression in embryos induces chromosome bearing the markers TU h th st cu ST d ca and was provided by E. WIESCHAUS.ve st ca flieswere obtained by the development of maxillarycuticular structures in the outcrossing a scj a1 b sp; vest ca stock provided by the Bowling labial and thoracic segments (KUZIORAand MCGINNIS Green stock center. 1988a). Animals carrying partial loss-of-function Dfd al- Two Dfd alleles in several different genetic backgrounds leles can survive to adulthood but often lack maxillary were used in this study. Dfd '"' (LINDSLEYand ZIMM, Dfd') is palps, have malformed rostral membranes, and show a weak hypomorphic allele and Dfd (LINDSLEYand ZIMM, Dfd'') is a temperature sensitive allele (MERRILLet al. 1987). apparent transformations of dorsal head to dorsal tho- Dfd'" red e, Dfd <'Ired e and the parental red e chromosome rax (MERRILL et al. 1987). Some of the mouse homo- were provided by T. KAUFMAN. The Ki DfdTv8red chromosome logues of Dfd have been shown to be required for the used throughout the screen was constructed from isogenic normal development of structures in thecervical region DfdrvR red e and Ki pP lines. Two separate Ki Dfdrv8 red lines of the embryos (RAM~REZ-SOL~Set 1993; KOSTICand were tested for viability over an isogenized Dfd <"red e chro- aZ. mosome at 25, 27 and 29.2", and the one with the higher GWECCHI 1994) and sufficient to induce cervical-like viability was used in the screen. vertebral structures in more anterior regions of the To identify the mutations resulting from the screen as en- head (LUFKINet al. 1992). hancers of the Dfd phenotype, it was necessary to test their Modifiers of Dfd in Drosophila 1341 interaction with Dfd in another genetic background (see be- *Ki DfdTv8red line (see below); for this analysis, the balancer low). The Dfd chromosomes used in these tests were made as chromosome was not removed. If the *Ki Dfdrv8red/*KiDfdTv8 follows. The ue st Dfd rcll ppca chromosome was obtained by red phenotype resembled that of a previously described muta- first making a Dfd rcll ppchromosome from Dfd red e and tion, we tested the *Ki Dfdrv8 red line for allelism with that Ki ppand then recombining Dfd rcll onto an isogenized ve locus. *Ki Dfdrv8 red lines with a hh or Polycomb phenotype st ca chromosome. The uest Dfdrcl ca chromosome is were tested against hh2 or PC'. homozygous viable at 25". The Dfd dpp chromosome was Previous screens for suppressors of PCresulted in the identi- constructed from isogenic Dfdrv8 red e and Ki pplines; it is fication of a number of loci required for homeotic gene func- homozygous viable. tion (KENNISON and RUSSELL1987; KENNISON and TAMKUN Sequencing of mutant alleles: DNA was isolated from he 1988). To see whether we had isolated mutations at any of mozygous Ki Dfd rv8 red larvae, homozygous ue st Dfd p ca these loci,each *Ki Dfd '"* red line not yet assigned toa comple- adults, or control adults (red e). The DNA sequence of all five mentation group was tested for lethal complementation behav- exons from the Dfd loci on these chromosomes was deter- ior against h5,ash16, md, osa', td, kt$, 1(3)87Ca'', ash2', mined by asymmetric PCR amplification and chain termina- dm2, sk, skd', urd' and utd3 (all provided by J. KENNISON). tion sequencing, as described in detail in ZENG et al. 1994. Separation of mutations from Ki DfdTVsred The mutations The screen: We wished to isolate mutations in isolated in the screen had to be separated from the parental genes whose products are required for the wild-type function Ki DfdTv8red background before further analysis was possible. of Dfd. To do this, we performed an F2 screen for second To do this, it was necessary not only to remove the site mutations that, when heterozygous, dramatically decrease from the *Ki DfdrV8 red chromosome but also to show that the viability of Ki Dfd'"' red/DfdrcJ1red e individuals. The the mutation in question was responsible for the observed screen is diagrammed in Figure 1. Ki Dfdrv8 red/ TM3Sb males interaction with Dfd. We performed two series of crosses for were mutagenized with EMS as described in GRIGLIATTI each relevant *Ki Dfd'"' red line. In the first series,we recom- (1986). The mutagenized males were mass mated to TMI/ bined apparent Dfd enhancers (*") off *Ki DfdTv8red onto a TM3Servirgin females and removed after 3 days. The females Dfd lClZchromosome and tested each for the ability toenhance were transferred to fresh vials and allowed to lay eggs for 2- the Dfd 'v8/Dfd lC1" phenotype. Those having an effect similar 3 more days before being discarded. TMI is lethal in combi- to that seen with the original *Ki Dfd "* red line were balanced nation withTM3Sb, so all surviving F1 progeny carried the and retained as *E DfdeC" ppstocks. In the second series of mutagenized Ki Dfd rv8 red chromosome (* Ki Dfd rV8 red, where crosses, we recombined lethal mutations (*") off *Ki Dfd"" * denotes a mutation of potential interest) and were balanced red onto a Dfdi chromosome (*L+). We then tested the with either TMI or TM3Ser (TM). *"+ lines for failure to complement the *" DfdlC1' pplines. This *Ki Dfd Iv8 red/ TM males (or virgin females) were individu- procedure is outlined in Figure 2. ally crossed to Dfd "11 red e/ TM virgin females (or males), left at room temperature for 4-6 hr and then placed at 29.2'. Next, we wanted to determine whether a given lethal muta- Four to six females were used for each F1 male and three tion was, in fact, at a locus required for Dfd function. We males for each F1 female. Parents were removed from the reasoned that the product of a given locus was likely to be vials after 4-5 days. Vials were scored daily on the 11th- 14th required for Dfd function if we had isolated multiple alleles days after mating for the presence of *Ki Dfd'"* red/Dfd""' of that locus from our screen and if at least one of these red e adult progeny. *Ki Dfdrv8 red lines giving zero to two *Ki alleles failed to complement theappropriate *E Dfd pf' *LE+ Dfd'"' red/Dfd rcll red e adults per vial were recovered as *Ki line. To identify such loci, one or more lines derived Dfd rV8 red/ TM males and immediately rescreened. Sibling * Ki from a given *Ki Dfd'"' red chromosome were tested for lethal Dfd "* red/ TM males were outcrossed to TMI/TM3Ser and complementation behavior with allthe *Ki Dfd rV8 red lines not the progeny was used for the final rescreen. In this second yet assigned to complementation groups. While performing rescreen, *Ki Dfdrv8 red/ TMI males were crossed to DfdKIJ these tests, we found that a fraction (3'/s0) of the *Ki DfdrV8 red e/TMlvirgin females and 100-300 progeny counted red lines carried a lethal mutation in one of three complemen- per cross. Lines where *Ki DfdTv8 red/Dfd"" red e showed tation groups. After separation from the *Ki Dfd'"'red chro- a viability 533% that of Ki Dfdrv8 red/Dfd"" red e at 29.2" mosome, mutations in any of these three groups showed no were kept for further study. significant enhancement of the DfdTv8/Dfd phenotype. Calculation of ofd interactionstrength: The degree of These lines were set aside and not further considered. interaction with Dfd for a given *Ki Dfd"'red chromosome Cytogeneticmap positions: Mutations corresponding to was determined by expressing the viability of *Ki Dfd'"' red/ multiallelic complementation groups were meiotically mapped Dfd lCl1 red e as a fraction of the viability of Ki Dfd"' red/ using the multiply marked rucuca chromosome. Once a mei- Dfd red eat 29.2". *Ki Dfd'"'red chromosomes were placed otic map position had been determined, each complementa- into different categories of interaction strength based on tion group was tested for lethality against deficiencies in the their relativeviability: strong (relative viability = 0-O.ll), relevant region of the chromosome. moderate (relative viability = 0.12-0.33) or weak (relative Preparation of embryonic cuticles: Unless otherwise indi- viability >0.33), cated, balancer chromosomes were removed by outcrossing The degree of Dfd interaction for*E DfdlC"pPlines (where mutant stocks. Embryos were collected for 4-6 hr then aged *E represents an apparent Dfd enhancer, see below) was cal- for 24 hr at 25", harvested, dechorionated in 100% Clorox culated as the viability of *" DfdrcC"pp/Ki DfdTv8red relative and placed into 4 ml 1X PBS/4 ml heptane. Hatched larvae to that of ue st DfdKC"pp ca/Ki DfdTV8red at 29.2". sank to the bottom and were removed withthe aqueous phase. The degree of Dfd interaction for hhCso was calculated as The aqueous phase was replaced with 4 ml methanol, and the viability of st Dfd"" pphhCs0/DfdTV8 pP relative to that of embryos were devitillenized by shaking vigorously for 1 min. ue st Dfd ppca/DfdTV8 p P. Devitillenized embryos wereplaced into microfuge tubes and Identification of mutationsat known loci: We used two rinsed three times with methanol and twice with water. Cuti- methods to test whether the mutants isolated from our screen cles were fixed in 1:4 glycero1:acetic acid for 1 hr at 65" then corresponded to previously identified loci: embryonic cuticu- overnight at room temperature. Embryos were then mounted lar phenotype and lethal complementation behavior. in 1:2 lactic acid:Hoyer's medium (WIESCHAUSand NUSSLEIN- We analyzed the homozygous cuticular phenotype of each VOLHARD1986) and allowed to clear at 65" overnight. Prepa- 1342 R W. Harding et al. rations were flattened by placing a 35-g weight on the cov- EMS erslip for 24-48 hr. The embryonic cuticular phenotypes presented here are in fc a Dfd' back round mutations were removed from the paren- P Ki Dfdrv8red TMl tal *Ki Dfd"red chromosome as described above. The em- TM3,Sb x TM~,Ser bryos shown for sls, mor and hdln were obtained by crossing # flies heterozygous for the relevant mutant allele to flies het- erozygous for the appropriate deficiency. hh5A7emb os were "1 27 Select individual Ki d or . obtained from the cross shd hh5A7/+ + X shd2 hh /+ +; ! the embryo shown is presumably of the hh5A7/shd2 hhJA7. dfc mutant embryoswere obtained from the cross rV8 djiiA54/+ X dfcl/+; in this case, embryos were collected and F1 *Ki Dfd red aged at 29.2". cnc mutant embryos were obtained from three TMI (or TM3, Ser) different crosses: cncVL"'/TM6B X C~C~~~'/TMGB,C7/+ X 2E16/+ and 2E16/TMl x cncVL1I0/TM6B. Interaction with PC: Previous screens for genes required for JI Score Ki progeny. homeotic gene function resulted in theisolation of mutations that have been assigned to two broad groups: the trithmax group and the Polycomb group (KENNISON and TAMKUN1988). Mutants in the tm group suppress the T2 and T3 to T1 leg F2 * Ki Dfdrv8red LKi DfdrV8 red transformation of PC/+ males; mutants in the PoZycomb group Dfd rcIied e TM1(or TM3, Ser) enhance this phenotype. To see whether any ofthe loci identi- fied in our screen belong to either of these two groups, we If Ki, red flies pent.discard line. tested them for interaction with Pc4. If absent, take balanced sib* outcross and rescreen. Ki DfdTvx red/ TMl, sls/ TM4B, tm3/TM3, mor'/ TM6C, * FIGURE1.-Screen to isolate genes required for Dfd+ func- mm2/ TMGC, cncvL1lo/TMGB, Ki DfdTmred/ TMl, Ki Dfd red/ tion. Mutagenized Ki Dfd rV8 red chromosomes were screened TM3 and wild-type males were mated to Pc4/ TM3 Sb females for lethality in trans with Dfd red e at 29.2'. Dfd rw/Dfd rcll and cultured at 29.2". T2 legs were removedfrom */Pc4males adults were recognized in the F2 by the presence of Kinked and mounted directly in 1:l lactic acid:Hoyer's medium. The bristles and red eyes. Mutant lines producing few or no DfdTv8/ preparations were cleared by overnight incubation at 65",and Dfd"" adults in the F2 were recovered as balanced stocks, the number of sex-combs per leg counted at 150X magnifica- outcrossed and rescreened. See MATERIALS AND METHODS for tion. details.

RESULTS F2 screen to identify loci required for of& func- tion: At 29.2", Ki Dfdrv8 red/Dfd red e adults show an The ofd mutant background In our genetic screen intermediate Dfd hypomorphic adult phenotype and for Dfd modifiers, we tested for an enhancementof the have a viability of-50% that expected.Using this back- reduced viability of Dfd partial loss-of-function alleles. ground, we screened for mutations that further de- The rationale is to have Dfd function at such a low level crease the viability ofthe Dfd mutants to near zero (Fig- that the elimination of one of the two doses of gene ure 1).We tested 12,263 mutagenized F1 chromosomes product from a modifylng locus is sufficient to result (*KiDfd rV8 red, where * represents a mutation of poten- in lethality. We used two Dfd hypomorphic alleles in tial interest) fora significant reduction in viability when our screen: Dfd m and Dfd K1l, rV8 is a weak hypomorph in trans with Dfd red e. A total of49 *Ki Dfdrv8 red and rCll is a temperature-sensitive allele (MERRILL et lines showed a significant interaction with the Dfdsl' al. 1987). Dfdrv8has a G to A transition at position 3 of red e chromosome and were selected for further study. the first codon, mutating the codon for the initiating The strength of interaction with Dfd for each *Ki methionine; presumably translation is initiated at the DfdyV*red chromosome was determined and is summa- next available methionine codon (wild-type codon 6). rized in Table 1. Thirty-three were classified as strong It is worth noting that thefive N-terminal residues elimi- enhancers, 11 as moderate and five as weak. The weak nated from the presumptive rV8 protein are evolution- interactions are associated with a strong decrease in the arily conserved between Drosophila and vertebrate viability of both *Ki Dfd rV8 red/Dfd &I1 red e and *Ki DFD-like proteins (REGULSKI et al. 1987). rC11 has a T Dfd rV8 red/ TMl. to A transversion at codon 49 of the homeo domain, Assignment to lethal complementation groups: We resulting in thesubstitution of isoleucine for phenylala- were able toassign 15 mutant alleles to seven previously nine. This change results in a smaller hydrophobic identified loci. Five *Ki Dfdrv8 red lines had embryonic group in the core of the homeodomain, which is likely cuticular phenotypes resembling those of known mu- to disrupt its stability and lead to the heat sensitivity of tants and were tested for allelism with the appropriate dl1 homozygotes. Both Dfd "'and Dfd homozygous stocks (see MATERIALS AND METHODS). Inthis way,we embryos accumulate Dfd transcripts and protein in the identified four hh alleles and one PC allele. The re- normal maxillary/mandibular pattern, though the lev- maining 44 *KZ Dfdrv8 red lines were tested for lethal els of protein expression appear to be lower and more complementation with mutations in 13 tn'thurax group variable than in wild-type embryos (data not shown). loci (see MATERIALS AND METHODS).We found that we Modifiers of Dfd in Drosophila 1343

TABU 1 mentation with appropriate deficiencies. The map posi- Loci potentially required for Z@d function tions of these complementation groups are shown in Table 1. For dfi, only the meiotic map position was Cytology determined. The location of 2E16 group alleles sug- Strength of Dfd No. of (map gested this group might correspond to the previously interaction alleles Locus position) identified cap 'n' collar locus. Two of the three alleles Strong (0-0.11) sallimus 5 62B8F2" were tested forcomplementation withcncml"; both headline 4 64G65C failed to complement cnclethality and so this group was cap 'n'colhr 3 94Eb provisionally assigned to the cnc locus (however, see &faced 2 (3-4.0) below). trithorax 1 88A12B5" Embryonic cuticular phenotypes:Dfd+ function is re- Polycomb 1 78D7-8d quired during embryogenesis for the development of 1(3)87Ca 1 87C" Unassigned 16 - structures arising from the maxillary and mandibular Moderate (0.12-0.33) hedgehog 4 94D 10-E5c segments, the dorsal ridge, and the optic lobe(MERRILL moira 2 89A11-B4" et al. 1987; REGULSKI et al. 1987). These include cuticu- Unassigned 5 - lar structuressuch as the cirri, mouth hooks, ectostomal Weak (>0.33) deuenir 1 70C2-D3" sclerites, H-piece (all principally derived from the max- Unassigned 4 - illary segment), and the lateralgraten (principally de- Strength of Dfd interaction was calculated as described in rived from the mandibular segment). To see whether the text. Loci listed as unassigned have not yet been assigned any of the mutations isolated from our screen cause to complementation groups. Meiotic map positions are con- disruptions in these Dfd-dependent structures, we exam- tained within parentheses. ined the cuticular phenotypes of mutant embryos. The a KENNISON and TAMKUN(1988). bMo~~~~et al. (1991). phenotypes fall into two general classes: those with mor- MOZERand DAWID (1989). phological defects in every segment, e.g., hh, and those PARO et al. (1984). with head specific defects, e.g., cnc and dfc. The mutant

TABLE 2 Phenotypes of mutations in multiallelic complementation groups

PC interaction

~~ ~ ~~ ~ ~~ ~~ No. of Suppression or M utant Phenotype Mutant TS,,," TSrangr alleles tested enhancement

sallimus (sls) Ectopictooth median 0.45 0.04-0.82 suppressorModerate 6 headline (hdln) Proventriculus and gut See text See text 4 See text abnormal; extra fold in head hedgehog (hh) Segment polarity gene; 0.43 0.06-0.83 5 suppressorModerate denticle lawn cap 'n' collar (cnc) Ectopic mouth hooks and cirri; 0.95 0.85-1.1 3 No effect labral structures unaffected moira (mor) T rib and hypopharyngeal 0.03 0.01-0.05 4 suppressorStrong defects defaced (dfc) Mouth hooks reduced; 0.74 0.63-0.84 2 No effect lateralgraten short and thick; dorsal bridge abnormal Phenotype refers to the embryonic cuticular phenotype. PC interaction was calculated as described in the text. TS,,,, is the average transformation strength of all the alleles tested. TSrangeis the range of transformation strengths observed for the alleles tested. A low TS value indicates a strong transformation. In the case of sls, hh, cnc and mm, previously isolated alleles were tested for PC interaction as well as the alleles isolated from our screen. At least 40 legs were scored for each genotype. optics. However,it is also possiblethat this median tooth- Dfd rc"embryos. Both dfc and Dfd Icl'mutants have re- like structure results from the fusion of enlarged and duced mouth-hooks and truncated lateralgraten; in malformed ectostomal sclerites insls mutants. The ectos- both cases, the dorsal bridge is displaced anteriorly so tomal sclerites are missing in Dfd null mutants. that it is adjacent to the base of the median tooth (com- mor and defaced(dfc) mutant embryos show defects pare Figure 4, E and F). The frontalsac is often sclero- similar to those seen in Dfd hypomorphs. In both mor tized, accounting for the extension of the dorsal bridge. and Dfd<""mutant embryos, the lateralgraten are trun- The dfc phenotype is temperature sensitive and some- cated and the T-ribs of the pharynx appear to be split what variable; some mutant embryos die before secret- medially (Figure 4D); in addition, mormutant embryos ing cuticle, others show more general patterning de- display defects in the hypopharyngeal region (not fects (not shown). However, dfc mutants that develop shown). dfc mutant embryoshave a phenotype that a properly segmented cuticle consistently display the closely resembles that of more severely disrupted phenotype described above.

Recombine *E onto Dfd&"chromosome:Recombine *L ontovesrca chromosome: -red x * Ki Dfdrwred -red x * Ki DfdrVsred FIGURE2.-Separation of Dfd enhancers red ve st Df8"p"ca red ve st ca from the Dfdrv8 mutant background. The scheme to recover Dfd enhancers (*E) from vi! ".1 vi! the mutagenized chromosomes is shown "1 on the left side of the figure; the scheme Select not-Ki, not-red Select not-Ki. not-red alr" and test for to recover lethal mutations (*L) is shown and test for Dfd interaction: lethality over original chromosome: on the right. Parentheses indicate that the gene in question is present in either wild- (&MveMst)(ca) x * Ki DfdrVBred type ormutant copy. Potential Dfd en- red TM3,Ser red hancers were identified by recombining re- a" gions of the original *KiDfdTv8 red chromo- 2YCI w w some onto a DfdC1' chromosome and v $4 screening for reductionof Dfd function. Le- Test Dfd interacting lines for Test lethal limes for complementation thal mutations were recombined away from complementation againstd- hs. against *E lines. Dfd rV8 into a Dfdc background. Any *" line carrying a single lethal mutationand failing d to complement the corresponding *E lines was considered likely to contain a Dfd en- hancer in a Dfd+ background (*"). lines were tested for lethal com lementa- tion against the other *Ki Dfd ''red lines isolated in the screen. Identify 9lines andtest for allelism with other mutations isolated from screen. Modifiers of Dfd in Drosophila 1345

-F-*. - ," ,..4y"-:Y.q

c- .. c..- , # - +.x.. .

z.. 1. - ; b* "- e 4 "

FIGURE3.-Embryonic cuticular phenotypes of hdln and hh. Embryos are oriented with anterior to the left and dorsal up. A- C are darkfield photomicrographs of whole embryos. D and E are phase contrast photomicrographs of the proventriculus. (A) Wild type. Eight abdominal and three thoracic segments are clearly visible. Arrow indicates esophagus; the gut is not visible in wild type. (B) hdin""'/Df(3L)zn-47. The gut is oversclerotized (arrow) and there is an extra fold in the head (arrowhead). (C) hh,5:\7/hh5Ai. Embryo is short and the ventral denticles are fused to make a continuous lawn. (D) Proventriculus of wild-type embryo (arrow); it has the characteristic inverted T shape. (E) Proventriculus of hdlnqAR/Df(3L)zn47embryo (arrow); it is disordered and crumpled.

In contrast to morand dfi, which are both required for be unaffected in 2E16 and C7 mutants. The most likely the formation of Dfiklependent structures, two putative explanation for thedifference in phenotype is that 2E16 mutant alleles of cnc (2E16 and CT) result in the loss and C7 are hypomorphic alleles of cnc. If so, the pheno- of some Dfiklependent structures and the ectopic pro- type of2E16/cn~~'~~~ is expected tobe more severe than duction of others. cnc null mutants lack structures de- that of either 2E16 homozygotes or 2E16/C7. It is not. rived from the labral segment and show a mandibular 2E16/cn~~-"~embryos have a phenotype nearly identi- to maxillary homeotic transformation (MOHLER et al. cal to that of 2El6/2E16or 2E16/C7embryos (compare 1995). The most obvious features of the cnc null pheno- Figures 5C and6D); the median tooth and dorsal type are the absence of the median tooth and dorsal bridge are nearly normal, but the mandibular region bridge, the presence of ectopic mouth-hooks and cirri has been transformed towards a maxillary identity. De- in the mandibular segment, and the truncation of the spite the differences in their loss of function pheno- lateralgriiten (MOHLERet al. 1995; Figure 5B). The pres- types, we have provisionally classified the 2E16 group ence of ectopic maxillary structures in more anterior as cnc based on meiotic map position and failure to regions is consistent with an increase in Dfd activity, complement cncw*ttO. while the truncation of the lateralgfiten suggests a de- Interaction with Pc4: Previous genetic analyses have crease. resulted in the identification of two opposing classes The phenotype of the 2E16 and C7 mutant alleles, of mutations that affect homeotic gene function: the in either homozygous or trans-heterozygous condition, Polycomh group and the tn'thmax group (LEWIS1978; resembles that of cncnull mutants. 2E16and C7mutant DUNCANand LEWIS 1982; INGHAM1983; SATO et al. embryos, like cnc"t,t'o, display ectopic mouth hooks and 1983;JuRc~~S1985; KENNlSON and TAMKUN1988; KEN- cirri in the mandibular region,indicating a mandibular NISON 1993). Polycomh group mutants enhance the PC to maxillary transformation (Figure 5). However, in mutant phenotype, while tn'thmux group mutants sup contrast to cnc nulls, the median tooth, dorsal bridge press it. Complementation analysis indicated we had and other moreanteriorly derived structures appear to isolated new alleles for several tn'thmax group genes and 1346 K. W. Harding et al.

? n D AntSO /- m-. MxSO

u

F J /'

FIGURE4.-Embryonic cuticular preparations of wild-type and mutant heads. Heads are oriented with anterior to the left and dorsal up. (A) Wild type. Arrow indicates wild-type region between the antennal and maxillary sense organs that develops a extra fold in headline mutants. (B) hdh"'/Df(SL)zn~#7.Arrow indicates extra fold. (C) ~ls':'~'/Df(3L)RG7.Arrow indicates ectopic median tooth.(D) m~f~"~/Df(3R)sbd.Lateralgriiten are truncated; arrow indicatessplit in T-ribs. (E) dfcc'/df5.454.Mouth- hooks are reduced andthe lateralgfiten truncated;the frontal sac (arrow) appears sclerotized andthe dorsal bridge is anteriorly displaced. This phenotype resembles that of Dfd mutants. (F) Dfdd"'/Dfdr("'raised at 29". Mouth hooks are absent, the later- algfiten truncated and the dorsal bridge displaced. Comparewith E. Abbreviations: AntSO, antennal sense ; ci, cirri; DB, dorsal bridEe: ecs. ectostomal sclerites; eps,s. epistomal sclerite; H, H-piece; hys, hypostomal sclerites; LC, lateralgriten; MH, mouth hoovk; MT,.median tooth; P, pharynx. one new allele of PC, so we wished to test if any of the formation strengths forall the alleles tested. A TS value other mutants identified in our screen also affect PC of 1.0 indicates no suppression, and a TS value of 0 function. indicates complete suppression of the PCphenotype. We Pc4 heterozygotes show a partial transformation of T2 also tested previously isolated alleles of sls, mor, hh and and T3 legs toward T1 (HANNAH-~VA1958; DUNCAN cnc. These results are summarized in Table 2. 1982). This transformation is easily detected in males Mutant alleles for three of the loci identified in our by the presence of ectopic sex-combs on theT2 and T3 screen clearly suppress PC.mor acts as a strongsuppres- legs. Since sex-combs are normally found only on the sor (TS,,,, = 0.03, TSmnRC= 0.01 -0.05), while the effect T1 legs, the number of ectopic sex-combs per leg is a of sls is more moderate (TS,,,, = 0.45, TSmngc= 0.04- rough measure of the degree of transformation. 0.82). To our surprise, hh mutations also acted as mod- To determine whether agiven locus belonged to the erate supressors of PC (TS,,,,, = 0.43, TS,,, = 0.06- Polycomb group, thetrithoraxgroup or to neither,we first 0.83). The hdln groupcould notbe classified: two calculated the mean number of sex-combsper T2 legof alleles (4A8and 3AI) had no effect on thePcphenotype *KiDfd '"'red/Pc4 males for each allele of the multiallelic while two (6Al and 6A52) appeared to strongly suppress complementation groups. To find the relative strength it. Mutations in dfi and cnc had no discernable effect of the T2 to T1 transformation, we then divided this on PC. number by the mean number of sex-combs per T2 leg of the appropriate control to give a value called the DISCUSSION transformation strength, TS. We assessed the PCinterac- tion of a given complementation group by determining To identify trans-acting factors responsible for confer- the mean (TSmCan)and the range (TS,,,) of the trans- ring functional specificity onto theDfd protein, we per- Modifiers of Dfd in Drosophila 1347

FIGURE5.-cup 'n' collar mutant phenotype. Panels are high magnification phase contrast photomicrographs of the head structures of wild-type and mc mutant embryos. Embryos are oriented with anterior to the left and dorsal up. (A) Wild type. (B)cnc~.//f~/cncl'/./f~~ . A pair of ectopic mouth-hooks (MH') can be seen in the mandibular region; the lateralgriten are severely reduced. Note the absence of the median tooth and dorsal bridge. (C) rn~?~/cnc"'. Ectopic mouth-hooks are detected in the mandibular region and the lateralgriten appear truncated.Both the dorsal bridge and median tooth appear relatively unaffected. (D) cncD,.j6/cn~"I-~~*. The phenotype of this embryo is nearly identical to that of the one shown in C. Abbreviations: DB, dorsal bridge; ecs, ectostomal sclerites; hys, hypostomal sclerites; LC, lateralpten; MH, mouth hook; MT, median tooth. formed a genetic screen to isolate mutations in genes our screen are required for the normal development required forDfd+ function. Such genes couldaffect Dfd of these cuticular structures. dfc, sls, mor and cnc zygotic function at several levels. Upstream genes control Dfd mutant embryos display relatively specificdefects in pos- activity by regulating the pattern and/or levels of Dfd terior head structures, whereas mutations in hh cause expression. Genes acting in parallel in an additive or more general defects. In addition, we obtained one al- synergistic manner might influence Dfd target choice lele of tm, which has been shown to be required for and/or the level of activity of DFD on posterior head wild-type levels ofDfd expression in the embryo ( BREEN regulatory elements in a variety of ways. This could be and HARTE1993). The embryonic phenotype of at least done by transient or stable binding to DFD protein two *Ki Dfd"' red lines strongly suggests that, as ex- itself (e.g., kinases or coactivators), by the activation pected, we also obtained mutant allelesin Dfd itself of signalling pathways that modify the activity of DFD (data not shown).At this level of analysisit is not possi- cofactors, or by regulating target accessibility (e.g., re- ble to distinguish between upstream, sidestream, or pressors). Mutations might also be isolated in target downstream genes; however, we have isolated mutations genes that areresponsible for carrying out theprogram in a known upstream gene required for maintenance specified by Dfd (e.g., Distal-kss, paired) (E.O'HARA and of Dfd expression levels (tm), anda known gene acting W. MCGINNIS,unpublished results). These three levels in parallel with Dfd (cnc). are not mutually exclusive; for example, a Dfd target Tiiuunruf group genes that interact withL$d: The loci could also be a Dfd activator, resulting in a positive identified in our screen can be divided into two types feedback loop. of Dfd modifiers: those that interact with other HOM Mutations in genesthat interact with Dfd are ex- genes and those that are more specifically required for pected to result in the disruption of Dfddependent Dfd. We have isolated new alleles ofseveral tn'thorax structures in the posterior head. In the embryo, these group genes and one allele of PC; however, our screen structures include the mouth-hooks, cirri, H-piece, ec- appears to be biased toward a subset of trithorax group tostomal sclerites and lateralgrsten (MERRILLet al. 1987; genes that are differentfrom those isolated in previous REGULSKI et al. 1987). Most of the mutations isolated in screens. 1348 K W. Harding et al.

Genetic screens designed to isolate mutations in ei- and TAMKUN actsas a strong PC suppressor (TS,,,, = ther Polycomb or trithmax group genes have principally 0.11) in our tests,whereas the sls alleles isolated in relied on the enhancementor suppression of either the our screen show anywhere from strong to very weak extra sex-combs or antenna to leg phenotype of adult suppression. A recent study (FELSENFELDand KENNISON PC heterozygotes (DUNCAN1982; SATOet al. 1983; KEN- 1995) has shown that a phenotype conferred by a domi- NISON and RUSSELL1987; KENNISON and TAMKUN nant allele of hh ( hhMd)can be suppressed by mutations 1988). Since these two phenotypes depend on ectopic in some trithuraxgroup genes. This observation is consis- expression of Scr and/or Antp (DUNCAN andLEWIS tent with our identification of hh as a trithurax group 1982; PATTATUCCIand KAUFMAN 1991; TAMKUNet al. gene; however, it is also possiblethat theectopic expres- 1992), the mutations isolated from PC suppressor/en- sion of hh seen in Mrt mutants results from the insertion hancer screens are expected to correspond to loci re- of novel cis regulatory sequences in the hh locus and quired forScr+ and Ant$+ activity. In contrast our screen that these non-hh sequences are regulated by the trithc- was designed to identify loci required for Dfdi activity. rax group. Based on previous results and evolutionary histories The predominance of sls alleles may indicate that Dfd (BOTAS 1993;KENNISON 1993), theregulation and func- has a stronger requirement for sls function than do tional activity of Antp, Scr and Dfd are expected to be other homeotic genes; that is, a wider range of defects influenced by some shared and some different trans in sls function affect Dfd activity. This possibility is sup- acting factors, which is consistent with the spectrum of ported by the apparent maxillary (ectosomal sclerites) mutations that we isolated. to labral (median tooth) transformation, in the absence It is interesting to compare the results of the KENNI- of any other obvious homeotic phenotype, seen in sls SON and TAMKUN(1988) screen for enhancers/suppres- mutant embryos.How this effect is mediated is not sors of PCwith the results of our screen for Dfd modifi- clear. Only two members of the trx group areknown to ers. Their screen resulted in the identification of 11 act by regulating the transcription of homeotic selectors tn’thorax group genes on the third chromosome. We (TAMKUNet al. 1992; BREENand WTE1993; SEDKOV obtained multiple alleles for only two of these groups et al. 1994), so many mechanisms are possible. Some (sls, mor) and single alleles forthree more (trx, dm, may affect HOM protein activity by contributing to dif- 1(3)87Ca).The trithorax group genes identified in both ferential regulation of target genes. screens are likely to correspond to factors commonly It is already known that different homeotic selector required for Antp, Scr and Dfd function. However, the genes have different requirements for trx function. For distribution of mutations in these trithurax group loci example, in embryos homozygous for a presumed null was different between the two screens. For example, allele, td”, bithorax complex gene expression is de- KENNISON and TAMKUNrecovered eight EMSinduced creased as early asstage 10-1 l, but com- trx alleles and one sls allele from 11,765 chromosomes, plex gene expression is unaffected until late stages of while we recovered only one trx allele but five sls alleles embryogenesis (stage 16-17) (BREENand HARTE 1993; from 12,263 chromosomes. Additionally, we identified SEDKOVet al. 1994); however, embryos homozygousfor a trithorax group gene not recovered in their screen the hypomorphic allele, tr~”~,display normal bithorax (hh).It is also possible that the four mutations that fail complex expression throughout embryogenesis but to complement brm and/or vtd correspond to other show decreased Antennapedia complex expression at trithorax group loci. The apparent mapposition of one stage 16-17 (SEDKOVet al. 1994). The requirements of of these mutations (5A29) indicates that it corresponds HOM genes for other trx group genes may have similar to neither brm nor vtd; however, when in a Dfd+ back- specificities. Different hypomorphic alleles of ash-1, ash- ground, 5A29/brm5 adults have held-out wings withserr- 2 and brm show different homeotic transformations ations along themargins (data not shown).This pheno- (SHEARN1989; BRIZUELAet al. 1994; TRIPOULASet al. type suggests that 5A29 interacts with brm and so may 1994). These differences could be due either to differ- correspond to a trithoraxgroup gene. The map positions ences in allele strengths or to different degrees of inter- of the other three mutations were not determined; it actions with different HOM genes or proteins. is not clear whether their failure to complement brm The mechanisms of two trithorax groupproteins sug- and/or vtd is associated with their effects on Dfd func- gest they are involved in regulating target accessibility tion or is due to thepresence of other lethal mutations in chromatin. The trithorax group protein brahma has on the chromosome. to the yeast transcriptional activator SNF2/ The different sets of trithurax group genes recovered SWI2 (TAMKUNet al. 1992). SNF2/SWI2has been in the two screens may be explained by the different shown to act in a multimeric complex comprised of homeotic genes involved. It is alsopossible that the several proteins includingSNF5, SNF6, SWIl and SWI3 criteria of the PC suppressor screens may have been (COTEet al. 1994). This complex assists in the binding too stringent to allow the isolation of any but strong of transactivator proteins like GAL4 to regulatory ele- suppressors. sls and hh both actas moderate PCsuppres- ments (PETERSONand HERSKOWITZ1992). The GAGA sors. In fact, the single sls allele isolated by KENNISON binding protein is encoded in the Trithorax-like (Trl) M odifiers of Modifiers Drosophila Dfd in 1349 gene (FARKASet al. 1994). The GAGA factor has been duced. cnc is also required for structures anteriorto the shown to act as an antirepressor in vitro (KERRIGAN et Dfd domain such as the median tooth and the dorsal al. 1991), capable of antagonizing theformation of bridge. Two of the putative cnc alleles isolated from our nucleosomes in anATPdependent manner (TSUKI- screen, 2E16and C7, appear to affect only the mandibu- YAMA et al. 1994). lar function of cnc; the third was not analyzed. Although we did not isolate any lethal alleles of brm, 2E16 and C7 mutant embryos show truncated later- we did identify one locus, hdln, that could correspond algraten and a strong mandibularto maxillary transfor- to a subunit of a multimeric complex. All four alleles mation. This latter phenotype suggests increased, of hdln failto complement thelethality of several alleles ratherthan decreased, Dfd activity. Such functions of a geneidentified in a screen forsecond chromosome would not be expected to be identified in our screen, enhancers of Dfd hypomorphs (G. GELLON,K. HAR- as it was designed to isolate mutants that decrease Dfd DING, M. MARTIN, N. MCGINNISand W. MCGINNIS,un- activity. One possible explanation is that in 2E16 and published results). Extragenic lethal noncomplementa- C7 mutants, ectopic maxillary Dfd function occurs at tion ofrecessive mutations has been associatedwith the same time as a loss of mandibular Dfd function. components of multimeric complexes. For example, This explanation is supported by the truncation of later- specific mutations in nonallelic members of the PC algraten seen in both DfdC" and 2E16/C7 mutants. group fail to complement one another (CHENGet al. Thus, 2E16 and C7, like cnc, appear to be positively 1994). Although only two of the four hdln alleles were required for mandibularDfd function but have a nega- capable of suppressing PC,it is possible that hdln repre- tive effect on maxillary Dfd function. However, unlike sents another member of the trithorax group. cncvI'I IO, these mutations do not severely disrupt the In addition to the mutations in tn'thorax group genes, more anterior cnc dependent structures. It seems likely we also isolated one allele of PC.This was unexpected that 2E16 and C7 are hypomorphic cnc alleles that dis- since PC" function represses ANT-C and BX-C gene ex- rupt the cnc mandibular function while having little pression; mutations in PCshould thereforesuppress, not effect on the cnc labral function. Themeiotic map posi- enhance, the Dfd hypomorphic phenotype. However, tion and complementation behavior support this possi- if other homeotic genes are weakly derepressed in PC bility, so we have assigned the E16 group to the cnc heterozygotes (DUNCAN andLEWIS 1982), then the ec- locus. topic expression of thehomeodomain proteins en- hh is a limiting component in a pathway required for coded by these genes might compete with mutant DFD Dfd function: hh is a signalling molecule involved in proteins and enhance thelethality of Dfd '"/Dfd &I1. the segment polarity pathway (NUSSLEIN-VOLHARDand Djiiiteracting genes required for posterior head de- WIESCHAUS1980; MOHLER1988; LEEet al. 1992; FORBES velopment: In addition to thetrithmax group genes, we 1993; HEEMSKERKand DINARDO1994; SIEGFRIEDet al. have isolated mutations in loci that appear to be more 1994). Segment polarity genes are required to specify specifically required for Dfd embryonic activity. The anterior-posteriorinformation within each segment phenotype of dfc mutant embryos resembles that of (NUSSLEIN-VOLHARDand WIESCHAUS 1980). This pro- strong Dfd mutants. The mouth-hooks arereduced, cess occurs at the same time asthe refinementof home- the lateralgraten truncated and the dorsal bridge dis- otic selector geneexpression patterns. If the positional placed anteriorly. The similarity between the dfc and information specified by the segment polarity genes is Dfd mutant phenotypes suggests that dfc is required for disrupted, the homeoticselectors are unable to specify Dfd activity. dfi mutants do not show any other obvious the correct cell fate. For example, if en is ectopically homeotic phenotype and do not have a detectable ef- expressed in the anterior compartmentof parasegment fect on PCfunction. Therefore, the interactionbetween 6, Ubx expression is repressed and the anterior com- Dfd and dfc appears to berelatively specific.At this level partment cells inappropriately express the Ubx target of analysisit is not possible to offer a mechanistic expla- gene Dl1 (MANN 1994). The concentrationof hhprotein nation for how the dfc product affects Dfd function; in midstage embryos has been shown to be critical in however, the specific positive requirement of Dfd for specifying anterior-posteriorinformation within at dfc is what one would expect for a coactivator. least the dorsal regions of segments (HEEMSKERKand In contrast to dfc, cnc has been molecularly and genet- DINARDO1994). It is possible that this concentration ically characterized (MOHLERet al. 1991, 1995). The cnc dependence allowed us to identify hh as a dosage sensi- protein belongs to the bZIP class of transcription fac- tive activator of Dfd function. However, whether hh pat- tors; cnc has been proposed both to repress Dfd activity terning functions in the posterior head are similar to, in the anterior part of the mandibular segment and to or different from, hh functions in the trunk segments act in a combinatorial fashion with Dfd in more poste- is unknown. rior mandibular cells. In the absence of cnc function, Conclusions: The mutations isolated from our screen some mandibular cells are transformed to maxillary suggest several waysof conferring functional activity identities, elaborating Dfd-dependent maxillary struc- and specificity onto Dfd. First, the action of segment tures; in addition, some mandibular structures are re- polarity signalling pathways may limit or modify home- 1350 K. W. Harding et al. otic gene expression and/or activity in specific anterior- exerts different tissue-specific,parasegrnent-specific and pro- moter-specific effects on each. Development 117: 119-134. posterior positions within a segment. The concentra- BRIZUELA,B. J., L. ELFRING,J. BALLARD,J. W. TAMKUNand J. A. tion ofhedgehog may be of great importance in this KENNISON,1994 Genetic analysis of the brahma gene of Drosoph- manner for regulating Dfd function. ilamlanogaster and polytene chromosome subdivisions 72AB. Genetics 137: 803-813. Second, more globally activefunctions from tn’thorux CAPDEVILA,M. P., and A. GARC~A-BELLIDO,1981 Genes involved in group genes like sls and mor, provide another pathway the activation of the bithorax complex of Drosophila. Wilhelm through which homeotic gene expression is known to Row’s Arch. Dev. Biol. 195: 417-432. CW, S.-K., L. JAFFE,M. CAPOVILLA,J. BOTAS and R MANN, 1994 be regulated and through which homeotic gene activity The DNA binding specificity of Ultrabithwax is modulated by might be differentially modulated. Targetchoice might cooperative interactions with extradentick, another homeoprc- depend on appropriate combinations of HOM and tri- tein. Cell 78 603-615. thorax groupproteins. These combinations could affect CHENG,N. N., D. A. R. SINCWR,R. B. CAMPBELL and H. W. BROCK, 1994 Interaction of polyhomeotic with Polycomb group genes of HOM gene activity on at least three levels: (1) direct Drosophila mlanogaster. Genetics 138: 1151-1162. regulation of the expression of the HOM gene, (2) di- COTE, J.,J. QUINN,J. L. WORKMAN C.and L. PETERSON,1994 Stimula- rect interaction with the HOM protein to act on a tar- tion of GAL4 derivative binding to nucleosomal DNAby the yeast SWI/SNF complex. Science 265: 53-60. get, and (3) regulation of target accessibility indepen- DESSAIN,S., and W. MCGINNIS,1993 Drosophila genes, dent of HOM gene activity. Although we have no direct pp. 1-55 in Advances in Developmental Biochemistly, edited by P. evidence for such a combinatorial mechanism, the large WASSARMAN.JAI Press Inc., Greenwich, CT. DICKSON,B., and E. 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