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Characterization of Mutant Alleles of Myospheroid, the Gene Encoding the @ Subunit of the Drosophila PS Integrins

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Characterization of Mutant Alleles of Myospheroid, the Gene Encoding the @ Subunit of the Drosophila PS Integrins Copyright 0 1992 by the Genetics Society of America Characterization of Mutant Alleles of myospheroid, the Gene Encoding the @ Subunit of the Drosophila PS Integrins Thomas A. Bunch, Richard Salatino,Marc C. Engelsgjerd, Leona Mukai, Robert F. West and Danny L. Brower Department of Molecular and CellularBiology and Department of Biochemistry, University of Arizona, Tucson, Arizona 85721 Manuscript received March 17, 1992 Accepted for publicationJune 24, 1992 ABSTRACT This paper presents the characterization of nine alleles of myospheroid, which encodes the Bps subunit of the DrosophilaPS integrins. On Southern blots, the my?B87, and my?Ro4 genes yield restriction digest patterns similar to that seen for wild-type chromosomes, howevermys' the and my#c43 genes contain detectable deletions. mys', my?'" and my?"43 make little or no stable protein product, andgenetically behave as stronglethal alleles. For the mutation,protein product is seen on immunoblots and a reduced amount of (Bps protein is seen at muscle attachment sites of embryos; this mutant protein retains some wild-type function,as revealed by complementation tests with weak alleles. Protein is also seen on immunoblots from my?Ro4 embryos, and this allele behaves as an antimorph, being more deleterious in some crosses than the complete deficiency for the locus. mysf" and mys"J4' are typically lethal in various combinations with other alleles at high temperatures only,but even at highphysiological temperatures,neither appears to eliminate gene function completely. The complementation behaviors of mys'"' and mys"' are quite unusual and suggest that these mutations involve regulatory phenomena. For mystS3,the data are mosteasily explained by postulating transvection effectsat the locus. The results for mysfs' are less straightforward, but point to the possibility of a chromosome pairing-dependent negative interaction. NTEGRINS area largefamily of cell surface recep- BROWNand WILCOX1987). With the identification of I tors,strongly conserved throughout the animal genes coding for the aps2 subunit and the common kingdom (for review see ALBELDAand BUCK 1990). Pps subunit, this suggestion has been confirmed in a Integrins areinvolved in a varietyof cell adhesion and number of cases (WRIGHT1960; BOGAERT,BROWN morphogenetic processes, and have also been dem- and WILCOX1987; MACKRELLet al. 1988; LEPTINet onstrated to be importantin cell signaling events.Each al. 1989; BROWERand JAFFE 1989; WILCOX,DI- functional integrin is composed of noncovalently as- ANTONIOand LEPTIN1989; VOLK,FESSLER and FES- sociated a and p subunits, both of which are large SLER 1990; WILCOX1990; ZUSMAN et al., 1990). (generally greater than 100 kD) transmembrane gly- The PPs subunit is common to both PS integrins, coproteins. Recently, integrin functions suchas ligand and is encoded by the myospheroid(mys) locus binding have been shown to be modulated by various (MACKRELLet al. 1988; LEPTIN et al. 1989). (This externaland internal effectors. Most, butnot all, locus was formerly named lethal(l)myospheroid, how- known integrin ligands are extracellular matrix com- ever it is now more commonly referred to simply as ponents; on the cytoplasmic side of the plasma mem- myospheroid, and listed as such by LINDSLEYand ZIMM brane, many integrins associate with the actin cyto- 1992.) The original mys' phenotype was described in skeleton. detail by WRIGHT(1960), and a number of alleles of Because the integrins are large, complex proteins, the locus had been isolated by the time of its identifi- studying their function in complicated processes like cation as the gene encoding the Bps subunit. Many of morphogenesis is greatly facilitated by genetic dissec- these mutations have been used in various develop- tion. Thus,the discovery thatthe PS antigens of mentalinvestigations, although a systematic exami- Drosophila are integrins (LEPTIN,AEBERSOLD and nation of the alleles has not been reported. In this WILCOX1987) has stimulated a widespread interestin paper, we present such a study, with the aim of iden- these proteins. The PS integrins were originally de- tifying particularly interesting characteristics,or prop- fined by monoclonal antibody screens, and the com- erties that will be useful in further work. We have plex expression patterns of the PSI and PS2 integrins focused on the commonly used lethal alleles of the X- immediately suggested that they were important for series (see below) and potentially useful viable alleles. various morphogeneticprocesses (BROWERet al. 1984; The genesis and phenotypic properties of the alleles BROWER,PIOVANT and REGER 1985; BOGAERT, examined are as follows. Genetics 132: 519-528 (October, 1992) 520 T. A. Bunch et al. my' was generated by '*P (WRIGHT1960). This suggest that genetic interactions that depend on pair- mutation is embryonic lethal, and its phenotype has ingof homologous chromosomes, or transvection, been described in detail (WRIGHT1960; NEWMAN and may be operating at themys locus. WRIGHT 1981). The name myospheroid derives from the observation that the somaticmuscles pull away MATERIALS AND METHODS from thebody wall in mutant embryos, and round up into myospheroid bodies. Consistent with this role in Southern analysisof DNA. Genomic DNA was extracted from adult flies as described in DANIELSand STRAUSBAUGH holding muscles totendon cells, PS integrinsare (1986). Thereafter, standard protocols were used for the found localized in high concentrations at the muscle following steps (SAMBROOK,FRITSCH and MANIATIS 1989, attachment sites (BOGAERT, BROWN and WILCOX and suppliers' protocols). GenomicDNA was digested with 1987; LEPTINet al. 1989). Other obvious aspects of the indicated restriction endonucleases (Promega), electro- the lethal phenotype include dorsal herniation of the phoresed on an agarose gel and vacuum blotted using a Transvac vacuum blotter (Hoefer Scientific, TE80) to a embryo and malformationof the visceral musculature nylon membrane (Magna). The DNA was UV cross-linked (WRIGHT 1960). At the ultrastructural level, there is to the membrane using a Stratalinker (Stratagene). Prehy- a delayed accumulation of extracellular matrix mate- bridization, hybridization and washes were performed as rial (NEWMAN andWRIGHT 1981). described in CHURCHand GILBERT(1 984). The radiolabeled WRIGHT generated (with EMS) three mutations as probe was prepared by the random primer method (SAM- BROOK,FRITSCH and MANIATIS1989). Following hybridiza- temperature-sensitive noncomplementers of my', and tion and washing, the blot was exposed on preflashed Kodak these alleles havebeen described as having lethal X-OMAT diagnostic film. phenotypes similar to mys' (WRIGHT 1968; NEWMAN Immunoblot analysis of proteins: Protein gel samples and WRIGHT 198 1). Since a good temperature-sensi- were prepared as described in LEPTINet al. (1989), from hand picked homozygousmys embryos (8 embryos per sam- tive (ts) mutant would be very useful for a variety of ple, identified by the dorsal herniation phenotype at 24 & 1 developmental studies, we have characterized these hr after egg laying, 25"). Wild-type and my.~"J'~embryos alleles extensively. Another EMS mutation, originally were collectedat 20 hours after egg laying (25"), justbefore identified by defectsin muscle development (COS- hatching. Embryoextracts were electrophoresed on an SDS TELLO and THOMAS1981), is now known to be a polyacrylamide gel (7.5%), blotted and probed using puri- fied anti-Pps monoclonal antibody DX.4C8 (WILCOXet al. viable allele of mys (DE LA POMPA,GARCIA and FERR~S 1984) as described elsewhere(BUNCH and BROWER1992a). 1989; WILCOX,DIANTONIO and LEPTIN 1989). This Immunofluorescence of embryos: Embryos were aged nonjumper-42 (nj42) allele has particularly severe ef- 14 f 2 hr at 25", and then prepared essentially by the fects on the development of the tergal depressor of method of T. KARR,as described in ASHBURNER(1989a, Protocol 95), except that vitelline membrane removal was the trochanter (TDT)muscle, although other integrin as described by MITCHISONand SEDAT(1983). Briefly, em- functionsare clearly compromised (DE LA POMPA, bryos were dechorionated, fixed in heptane-formaldehyde, GARCIAand FERR~S 1989;WILCOX, DIANTONIO and and had their vitelline membranes removed in coldheptane- LEPTIN1989). For example, these mutants candisplay methanol. Primary antibody stainingwas overnight, second- significant defects in wing morphogenesis, particularly ary antibody was incubated for 1-4 hr, both at 4" in 10% goat serum in PBS. Stained embryos were observed using incombination with otherintegrin mutants. (This standard epifluorescence optics, or with an MRC 600 con- allele is also known asmy8; Lindsley and ZIMM 1992.) focal microscope. Primary antibodies were mouse mono- Finally, four strong mys alleles resulted from the clonalsagainst Bps (CF.6G11, BROWERet al. 1984) and large scale EMS mutagenesis screen of WIESCHAUS, against the Sex combs reduced protein (6H4, GLICKSMAN and BROWER1988). The latter was included as a positive NUSSLEIN-VOLHARDand JURGENS (1984). These al- control to ensure that embryos failing to stain for PS inte- leles, my.?G43,my?N101 and my.?R04 are all em- grinswere properly fixed and permeablized.Secondary bryonic Lethal and will be referred to generically as antibody was fluorescein-conjugated goat anti-mouse (Anti- the X- alleles. All of these alleles were reported to be bodies Incorporated). Genetic studies:Flies were reared in well yeasted vials on defective in dorsal closure,a hallmark of the original the
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