Maternal and Zygotic Phenotypes

The polycomb-group genes, a set of genes characterized by mutations that cause similar phenotypes and dosage-dependent interactions, are required for the normal expression of segment-specific homeotic loci. .Here we report that polycombeotic (formerly 1(3)1902), originally identified by a lethal mutation that causes a small-disc phenotype, is also a member of this group of essential genes. Adults homozygous for temperature-sensitive pco alleles that were exposed to the restrictive temperature during larval life display the second and third leg to first leg transformation characteristic of polycombgroup mutants. Adult females homozygous for temperature-sensitive alleles exposed to the restrictive temperature during oogenesis produce embryos that show anterior segments with structures normally unique to the eighth abdominal segment, another transformation characteristic of polycomb-group mutants. Mutations in the polycombeotic gene also cause defects not reported for mutations in other polycomb-group genes. Females homozygous for the most extreme temperature-sensitive allele are sterile, and larvae homozygous for null alleles have small imaginal discs and reduced frequencies of mitotic figures in the brain. Dominant mutations originally identified as enhancers or suppressors of zeste are gain-of-function alleles of polycombeotic. The type and variety of defects displayed by different mutations in this gene indicate that the product might be involved in chromosome structure and/or function. T HE polycomb-group of genes has become the subject of intense study recently, because of the variety of homeotic transformations caused by mutations in these genes, and because of their role in the maintenance of appropriate expression of the bithorax and Antennapedia gene complexes (BX-C and ANTC, respectively). These different loci are considered a ‘group’ because mutations in each of these genes cause similar phenotypes, because loss-of-function mutations in a gene can enhance the phenotype of loss-offunction mutations in another gene of this set (JURGENS 1985; KENNISON and TAMKUN 1988), and because duplications of a gene can suppress the phenotype of loss-of-function mutations in another gene of this set (KENNISON and RUSSELL 1987). The polycombgroup is thought to contain as many as 40 different genes (JURGENS 1985), including Polycomb (PC) (DENELL 1978; DUNCAN and LEWIS 1982), Polycomb-like ( P c l ) (DUNCAN 1982), Addit ional sex combs (Asx) ( JURGENS 1985), Posterior sex combs (Psc) ( JURGENS 1985), Sex combs on midleg (Scm) (JURGENS 1985), super sex combs ( xc) (INGHAM 1984), extra sex combs (esc) (STRUHL 1981), polyhomeotic ( p h ) (DURA, BROCK and SANTAMARIA 1985), Sex comb extra (&e) (BREEN and DUNCAN 1986), pleiohomeotic (pho) (DENELL, HUMMELS and GIRTON 1988), and extra dent ic les (exd) (WIESCHAUS, NUSSLEIN-VOLHARD andJuRGENs 1984). Mutants of this group display a transformation of the second and third legs to the first leg in adults and a transformation of the ventral setal belt patterns of thoracic and abdominal segments to patterns of more posterior segments in larvae. Such transformations, as well as the altered expression of RNAs and proteins from BX-C and ANT-C as shown by in s i tu hybridization to tissue sections and antibody staining of the CNS and imaginal discs, suggest that these complex loci are being inappropriately expressed in some tissues (BEACHY, HELFAND and HOGNESS 1985; STRUHL and WHITE 1985; WEDEEN, HARDING and LEVINE 1986; GLICKSMAN and BROWER 1988). Recent experiments have suggested that homeotic transformations caused by poly-comb-group mutations may be due to an instability in the segment-specific expression of ANT-C and BX-C (STRUHL and AKAM 1985; DURA et al. 1987). Another subject of intense study has been the zeste gene. zeste was discovered due to the effect of a gainof-function allele, z’, on the expression of the white locus (GANS 1953). This effect was shown to be dependent on the number and proximity of copies of the white locus. Later it was determined that zeste also had an influence on the complex loci bithorax (BX-C) and decapentaplegic (DPP-C) (KAUFMAN, TAKAZA and SUZUKI 1973; GELBART and Wu 1982). A common feature of these complex loci is the influence of close Genetics 125: 91-101 (May, 1990) 92 M. D. Phillips and A. Shearn proximity of gene copies on expression, an effect called transvection (LEWIS 1955). Our studies on the polycombeotic gene, and those of WU et al. (1989), suggest that the functions of the polycomb-group of genes and the zeste gene may be related at some fundamental level. polycombeotic (pco) , originally identified as letha1(3)1902 (1(3)1902) (SHEARN et al. 1972, 1978; SHEARN and GAREN 1974; SHEARN 1977; SHEARN, HERSPERGER and HERSPERGER 1978), displays a wide range of phenotypes, depending on the type of mutation observed. Leaky alleles, such as the temperature-sensitive alleles formations similar to those seen in other polycombgroup genes. Null alleles, such as cause a small disc phenotype and block mitosis. In addition, alleles of the Enhancer of zeste gene ( E ( z ) ) (KALISCH and RASMUSON 1974), which can either enhance or suppress the zeste-white interaction, have been identified as gain-of-function alleles of polycombeotic. The wide range of phenotypes caused by different alleles suggest that the primary role of polycombeotic is in a basic cellular process, such as chromosome structure or function. This gene may be one of many that are a link between cell proliferation and determination. PCOPC025hs ' PCOox73hhs , and p ~ ~ ~ y ~ ~ ~ ~ ~ cause homeotic transMATERIALS AND METHODS Fly strains and culture: All strains were maintained on standard culture medium at 20°, unless stated otherwise. The pco alleles used in this study were induced by N-methylA"-nitro-N-nitrosoguanidine (pco""') or by ethylmethanesulfonate ( p ~ ~ ~ y ~ ~ ~ ~ ~ , pconhoUh, pcoox73hhs , and pcop'""") (SHEARN, et al. 1971, 1978; SHEARN, HERSPERCER and HERSPERCER 1978). The E(z) ' mutation was induced by ethylmethanesulfonate (KALISCH and RASMUSON 1974), and the Su(z)?OZ mutation was induced by X-rays (W. GELBART, unpublished results); both the E ( z ) and Su(z)?OZ stocks were kindly provided to us by RICK JONES in the laboratory of WILLIAM GELBART. For the other stocks used, the Df(?L)vin2 stock was provided by the Mid-America Drosophila Stock Center, Bowling Green, Ohio, the Tp(3)PZO stock was provided by E. B. LEWIS, the Dp(?;3)S2a? stock was provided by MINX FULLER, and the Df(3L)lxd' and D f ( ? L ) l ~ d ' ~ stocks were provided by VICTORIA FINNERTY. The breakpoints associated with the above rearrangement stocks are listed in Table 1. Other stocks used are described in LINDSLEY and GRELL (1 968) or LINDSLEY and ZIMM (1 985, 1986, 1987). Adult phenotype analysis: Adult legs were examined at 10-50X magnification using a stereomicroscope. T o examine the adult homeotic phenotype, homozygous TM? or mwh ~ C O " ) " ~ red eITM3 males and their eggs were collectedat 20" for 24 hr, shifted to 20°, 24", or 27", and allowed to develop into adults. Viability of male progeny was calculated as the percent of mutant males eclosing relative to the number 0fpcoJ"~'~~~~red/TM3 control brothers. The "extra sex combs" phenotype was analyzed in two ways. Some males were observed live under a stereomicroscope and the total number of legs displaying sex comb teeth was scored. Other males were dissected and their ventral thoraces were mounted on slides. The number of sex comb teeth PCOPC"25hs red females were crossed to either p ~ 0 @ ~ ' ~ ~ ~ ~ red/ TABLE 1 Breakpoints of rearrangement chromosomes used