A Novel KH-Domain Protein Mediates Cell Adhesion Processes inDrosophila

DEVELOPMENTAL BIOLOGY 190, 241–256 (1997) ARTICLE NO. DB978699

A Novel KH-Domain Protein Mediates Cell Adhesion Processes in Drosophila Patrick C. H. Lo and Manfred Frasch Brookdale Center for Developmental and Molecular Biology, Mount Sinai School of Medicine, 1 Gustave Levy Place, New York, New York 10029

Adhesion of cells to one another and to extracellular matrices has major roles in morphogenetic processes during development. One important family of cell adhesion receptors are the integrins, which in Drosophila have crucial functions in at least two adhesion-mediated developmental events: embryonic muscle attachment and adhesion of the wing epithelia. We have cloned and characterized a gene (struthio) that is expressed in embryonic mesodermal and muscle cells, including cardioblasts, and epidermal muscle attachment sites in a pattern that is reminiscent of the expression pattern of the PS integrins. Maternal and zygotic transcripts are produced by this gene and encode similar proteins with two alternative carboxy tails. Both proteins contain identical KH domains, a protein sequence motif that is found in numerous proteins that interact with RNA. The struthio protein is highly homologous in a region including the KH domain to the mouse quaking and C. elegans gld-1 proteins, two developmentally important genes. Somatic homozygous clones of an embryonic lethal mutation in this gene (stru1A122) cause wing blisters and flight impairment, phenotypes which are associated with PS integrin subunit mutations. Thus, the struthio gene encodes a putative RNA-binding protein that appears to regulate some aspects of Drosophila integrin functioning. q 1997 Academic Press

INTRODUCTION Cell adhesion plays a critical role in the processes of cell proliferation, migration, attachment, and differentiation that occur during the morphogenesis of multicellular organisms. A number of different cell adhesion proteins (for a review see Gumbiner, 1996) mediate and regulate these morphogenetic processes. An important class of these proteins are the integrins, a family of conserved transmembrane receptor proteins that serve to transmit various cellto-cell and cell-to-matrix interactions through the plasma membrane to the cytoskeleton (for a review see Hynes, 1992). Integrins are heterodimers of an a and a b subunit and in vertebrates there are more than 20 different receptors that are generated from at least 16 a and 8 b chains. Such a wide diversity of integrin receptor types allows for temporal and cell-type specificities of cell adhesion function in various processes, including morphogenesis. In Drosophila development the importance of cell adhesion, especially that mediated by integrins, has been demonstrated in two specific processes: embryonic muscle attachment to the epidermis and the adhesion of the two wing epithelial layers to each other. A number of molecules have been shown to play roles during the attachment of the ends of myotubes to their final attachment sites in the epidermis. These include the stripe gene, which encodes an egr-like

zinc finger protein that is expressed in the subset of ectodermal cells that form these attachment sites (Frommer et al., 1996; Volk and VijayRaghavan, 1994). stripe function is required for proper specification of attachment sites, and mutations in this gene appear to disrupt both the guidance of myotubes to the epidermal attachment sites and their actual attachment to those sites. In the developing muscle cells, the spectrin family molecule MSP-300 is an actinbinding protein located at the leading edges of migrating myotubes and becomes associated with the myofibrillar network at the ends of muscles in the attachment sites (Volk, 1992). The function of integrins as cell adhesion molecules in the process of muscle attachment has been the best studied. The two major Drosophila integrin receptors (for reviews see Brown, 1993; Brown et al., 1993; Gotwals et al., 1994), named PS1 and PS2 based on their positionspecific expression patterns (Brower et al., 1984; Wilcox et al., 1981), are heterodimers of an a and a b subunit. PS1 consists of an aPS1 and a bPS subunit, while PS2 has an aPS2 and a bPS subunit. During embryonic development, these two integrins are expressed in complementary patterns such that PS1 is expressed mainly in the ectoderm and endoderm, while PS2 is found in the mesoderm (Bogaert et al., 1987; Leptin et al., 1989; Zusman et al., 1990). By the time the somatic musculature has developed, the PS integrins have become concentrated at the points of muscle attachment

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so that PS1 is expressed at the epidermal muscle attachment cells and PS2 is concentrated at the ends of the muscles that attach to these sites. The aPS1 , aPS2 , and bPS subunits are encoded by the multiple edematous wings (mew) (Brower et al., 1995; Wehrli et al., 1993), inflated (if) (Bogaert et al., 1987; Wilcox et al., 1989), and myospheroid (mys) (Leptin et al., 1989; MacKrell et al., 1988) genes, respectively. The importance of the PS2 integrin for muscle attachment is shown by embryonic lethal mutations in if and mys that result in the myospheroid phenotype of detached and rounded up somatic body wall muscles which is caused by the failure of the muscle attachment sites when these muscles first start to contract in late embryos (Brabant and Brower, 1993; Brown, 1994; Leptin et al., 1989; Newman and Wright, 1981; Roote and Zusman, 1995; Wright, 1960). In the developing wing disc, a complementary pattern of PS integrins is also observed where PS1 is expressed in the presumptive dorsal wing epithelium, while PS2 is expressed in the presumptive ventral wing epithelium (Brower et al., 1985). After eversion of the wing disc and the apposition of the dorsal and ventral epithelial layers, the PS integrins are found concentrated at the junctions which join these layers together (Fristrom et al., 1993). Both PS integrins are required for the proper adhesion of the two wing epithelial layers to each other since somatic wing clones homozygous for null mutations in all three integrin subunits result in separation of the wing layers and the formation of blisters (Brabant and Brower, 1993; Brower et al., 1995; Brower and Jaffe, 1989; Zusman et al., 1990). It is clear from these studies that integrin function is crucial for cell adhesion-mediated processes during Drosophila development. However, little is known about how integrin expression and functioning may be controlled and about the other elements of the integrin pathway required for its function, such as extracellular ligands, intracellular accessory proteins, cytoskeletal components, and proteins that act in integrin-mediated signaling pathways. In this report we describe the isolation of a novel gene, struthio, that appears to be required in some of these events. This gene was isolated on the basis of its expression in embryonic mesoderm and muscle cells and also in the epidermal muscle attachment sites. A mutation in this gene results in embryonic lethality and disrupts integrin-mediated cell adhesion in the wing blade and functioning of the flight muscles. The gene codes for a protein containing a KH domain, a protein sequence motif that is found in various proteins involved in RNA metabolism and which has RNA-binding activity. Based on this, we suggest that the struthio protein is an RNA-binding protein that may control some aspects of Drosophila integrin functioning.

MATERIALS AND METHODS Molecular Characterization of the struthio Gene Genomic DNA flanking the P-lacW insertion of E7-3-4 was plasmid rescued by circularization of EcoRI-digested (for upstream

flanking DNA) or BamHI- and BglII-digested (for downstream flanking DNA) E7-3-4 genomic DNA to generate pGR-E-l and pGR-E-r, respectively. Downstream genomic DNA flanking the P-lArB insertion of 1A122 was plasmid rescued by circularization of HindIIIdigested 1A122 genomic DNA to generate pGR-A-r. The genomic DNA inserts from pGR-E-l and pGR-A-r, which showed identical in situ hybridization expression patterns as the b-gal stainings of the enhancer trap lines, were separately used as probes to isolate genomic clones from a lEMBL4 library and cDNA clones from a 4- to 8-hr (pGR-E-l probed) or 12- to 24-hr (pGR-A-r probed) embryonic plasmid library (Brown and Kafatos, 1988). Cosmid clones were later isolated using cDNA fragments as probes. The 4.0-kb stru cDNA insert was sequenced on both strands by double-stranded sequencing both automatically with a 373A DNA sequencer (Applied Biosystems) and manually with Sequenase, v.2 (USB). Northern hybridization was done as described in Azpiazu and Frasch (1993).

Antibody and in Situ Hybridization Staining These were done for embryos as described in Azpiazu and Frasch (1993), except that in situ hybridizations were carried out at 557C with DIG-labeled T7 RNA transcripts of the stru cDNA inserts. Wing discs were dissected out of wandering third instar larvae into 1.51 PBS on ice. The discs were fixed by the addition of formaldehyde to a final concentration of 8% and incubation for 20 min at RT, followed by 31 PBT (PBS / 0.1% Tween) washes and 21 70% ethanol washes. The discs were rehydrated by several washes in PBT before in situ hybridization was done as for the embryos, and wild-type embryos were included in the hybridization as an internal control. Mouse anti-b-gal antibody was purchased from Sigma. Muscle myosin antiserum was obtained from Dan Kiehart and monoclonal anti-groovin antibody from Talila Volk. Anti-Tinman antibody was raised in rabbit against a bacterially expressed His(6)-tagged Tinman polypeptide (amino acids 2–338).

Drosophila Stocks The E7-3-4 and 1A122 enhancer trap lines were obtained from Volker Hartenstein and Norbert Perrimon, respectively. The struthio1 stock was obtained from Mary Prout and Jim Fristrom. All other stocks and chromosomes described were obtained from the Bloomington stock center.

Generation of Germline and Somatic Homozygous Clones The FLP recombinase/dominant female sterile (FLP/DFS) technique (Chou et al., 1993; Chou and Perrimon, 1992) was used to generate germline clones homozygous for the stru1A122 mutation. Use of this technique will also generate somatic clones homozygous for stru1A122. The stru1A122 mutation was recombined onto a chromosome 3R bearing an FRT site at 82B by crossing w//; P{neoFRT}82B P{w/}90E/stru1A122 virgin females to y w; TM3, Sb/ TM6 males. The w; P{neoFRT}82B stru1A122/TM3, Sb recombinant progeny were recovered by isolating w and Sb flies from among the neoR progeny which had survived on G418 food (prepared as described in Xu and Rubin, 1993). Individual P{neoFRT}82B stru1A122/TM3, Sb flies were then used to establish several separate lines of the P{neoFRT}82B stru1A122 recombinant chromosome balanced over TM6B, D and one of them was crossed to Df(3R)e-BS2/ TM3, Sb flies to verify that the recombinant chromosome retained the stru1A122 mutation by its lethality over this deficiency. w;

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P{neoFRT}82B stru1A122/TM6B, D virgin females from this line were mated to y w P{hsFLP}22/Y; P{neoFRT}82B P{w/; ovoD}98A/TM3, Sb males that were obtained from a cross of w/Y; P{neoFRT}82B P{w/; ovoD}98A/TM3, Sb males with y w P{hsFLP}22; CxD/TM3, Sb virgin females. The mated female flies were serially transferred to a new group of fresh food vials every 24 hr. Each group of vials from the same day was then heat-shocked in a 377C water bath 48, 72, and 96 hr after the G0 flies had been transferred out those vials. As result of the heat shock-induced production of FLP recombinase during larval growth which catalyzes efficient recombination at the paired FRT sites, the y w P{hsFLP}22/w; P{neoFRT}82B stru1A122/ P{neoFRT}82B P{w/; ovoD}98A females are the only class of progeny that will have undergone recombination to produce homozygous stru1A122 germline and somatic cells. These homozygous stru1A122 germline cells will then develop into stru1A122 haploid oocytes lacking the maternal store of stru gene products. The fertile y w P{hsFLP}22/w; P{neoFRT}82B stru1A1 22 /P{neoFRT}82B P{w/; ovoD}98A females containing these oocytes were crossed to stru1A122/TM3, lacZ-hg males to obtain homozygous stru1A122 and heterozygous stru1A122/TM3, lacZ-hg embryos.

Reversion of stru1A122 to Viability The stru1A122 mutation on the P{neoFRT}82B stru1A122 chromosome was chosen for reversion to viability by excision of the P-lArB transposon in order to verify that the blister phenotype observed in this chromosome by somatic recombination and the embryonic lethal phenotype are linked to the 1A122 insertion. w; P{neoFRT}82B stru1A122/TM6B, D virgin females were crossed to Df(3R)C4/TM3, Sb D2-3 males and the w/Y; P{neoFRT}82B 1A122/TM3, Sb D2-3 males individually crossed to Df(3R)e-BS2/TM3, Sb virgin females. The chromosomes with viable excisions of the P-lArB transposon from stru1A122 (denoted as *) were isolated as */e-BS2 males which were then individually crossed to w; P{neoFRT}82B stru1A122/TM6B, D virgin females. The */P{neoFRT}82B stru1A122 progeny were recovered and examined for wing blisters, which were not observed in the progeny of 13 different G0 */e-BS2 males.

Eye Sectioning The heads of flies were first cut off and the unwanted eye was removed. The heads were fixed in 2% glutaraldehyde in PBS for 1 hr, washed 31 for 10 min each in PBS, dehydrated through an ethanol step series, and then incubated in acetone 21 for 10 min each before being prepared for Araldite sectioning as described in Leptin and Grunewald (1990).

Flight Ability Testing This is a modification of the method described in Drummond et al. (1991). Two- to five-day-old adult flies were sorted into food vials in groups of 8 to 10 and allowed to recover from CO2 anesthesization for at least 2 hr. The vials were placed in the center of a 9-cm petri dish cover under a strong light source located 8 cm away and the plug was then removed from the vial. Flies which flew straight up into the light source were classified as flight up. Those that flew in any other direction and landed outside the dish were classified as flight down. Any flies that fell or jumped into the dish were considered flightless.

RESULTS The E7-3-4 and 1A122 P-lacZ Enhancer Trap Lines Express lacZ in the Early Mesoderm and Muscles of Drosophila In order to identify novel genes that may be involved in the control of mesodermal and muscle development in Drosophila, two P-lacZ enhancer trap lines that showed expression of the lacZ marker in the embryonic mesoderm and its derivatives were selected for this study. These two lines were derived from separate screens utilizing two different P-lacZ elements. The E7-3-4 line is a homozygous viable line that was generated in a screen using the P-lacW transposon (Bier et al., 1989; Hartenstein and Jan, 1992), while the other line, 1A122, is homozygous lethal and resulted from the insertion of a P-lArB transposon at 93F (Bellen et al., 1989; Perrimon et al., 1991). A homozygous lethal mutation in a gene at 93B-94 named struthio (stru) has recently been isolated (Prout et al., 1997) and since we have found that the 1A122 insertion does not complement stru for lethality, we have named the mutation caused by this P-lArB insertion stru1A122. Early mesodermal and muscle expression of the lacZ marker gene in embryos was previously shown for 1A122 (Perrimon et al., 1991) and was also described for E7-3-4 (Hartenstein and Jan, 1992). Our examination of the precise spatial and temporal pattern of expression of the lacZ gene in the E7-3-4 and 1A122 lines by anti-b-gal staining indicated that their patterns were identical. Subsequent results (see below) have demonstrated that these two lines derive from the insertion of their respective P-lacZ elements into the same locus. Expression of b-gal (which is nuclearly localized) in these two lines is first strongly detected at the blastoderm stage during which it becomes localized to the prospective mesoderm on the ventral side of the embryo that will invaginate during gastrulation (Figs. 1A and 1B). During germ band elongation, the entire layer of the mesoderm, which lies internal to the ectoderm, expresses b-gal (Fig. 1C). By the completion of germ band elongation, staining is seen in both the visceral and the somatic mesoderm (Fig. 1D) that comprise the first major subdivision of the mesoderm. Toward the end of embryogenesis, b-gal expression is found in the visceral (Fig. 1E), somatic (Fig. 1F), and heart (Figs. 1F and 1G) musculature, the three major muscle types that arise from the mesoderm. In addition, strong expression is also detectable in muscle attachment sites (Fig. 1G), which are ectodermally derived cells required for the attachment of the body wall (somatic) muscles to the overlying hypoderm of the larva. Staining of third instar larvae shows strong expression in the nuclei of the somatic body wall muscles (Fig. 1H).

Molecular Cloning of the stru Gene Genomic DNA flanking both sides of the P-lacW transposon of E7-3-4 were recovered separately by plasmid rescue, as was the genomic DNA flanking one side of the P-lArB

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FIG. 1. Expression of b-gal in the E7-3-4/1A122 enhancer trap lines. Embryos (A–G) and third instar larvae (H) from the two enhancer lines were antibody stained for b-gal. Embryos are shown oriented with anterior to the left. (A) Lateral view of a cellular blastoderm embryo showing staining in the entire embryo (except the poles) that is stronger in the prospective mesodermal cells located ventrally. (B) Ventrolateral view of a stage 6 gastrulating embryo showing staining in the prospective mesodermal cells undergoing invagination through the ventral furrow. (C) Lateral view of an early germ-band-elongated embryo. Staining is seen in the entire mesoderm layer lying interior to the the ectoderm along the entire germ band, including the head mesoderm. (D) Dorsolateral view of a stage 13 germ-band-retracted embryo with staining in the visceral mesoderm (vm) and somatic mesoderm (sm). (E) Dorsolateral view of a late stage 15 embryo showing staining of the pharyngeal muscle (pm) and the visceral musculature (vm) surrounding the midgut. (F) The same embryo as in E showing staining of the somatic body wall muscles (sm) and the dorsal vessel (dv). (G) Dorsal view of a late stage 15 embryo showing staining of the dorsal vessel (dv) and the muscle attachment site (mas) cells, which are seen as rows of cells in the hypoderm extending along the dorsal/ventral axis. (H) Staining of the body wall of third instar larvae shows strong b-gal expression in the nuclei of body wall muscles.

transposon in 1A122. The presence of coding sequences in these genomic rescue plasmids was verified by in situ hybridizations of embryos with these plasmids. One of the

two E7-3-4 genomic rescue plasmids and the one 1A122 genomic rescue plasmid showed a pattern of in situ hybridization that was identical to that seen for the b-gal expres-

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sion (data not shown). Both of these genomic rescue plasmids were separately used as probes to obtain genomic and cDNA clones. Preliminary tests revealed that most of the clones identified by the E7-3-4 and 1A122 genomic rescue plasmids were identical, indicating that these two independently derived lines had ‘‘enhancer-trapped’’ the same gene. Two major classes of cDNAs of 4.0 and 4.5 kb were identified and in situ hybridizations to embryos were carried out with fragments of these cDNAs. The staining patterns observed (Fig. 2) were identical to each other and to the bgal expression pattern of the enhancer trap lines (Fig. 1), indicating that these cDNAs correspond to the gene defined by these lines. Developmental Northern analysis using the larger 4.5-kb cDNA as probe detected two mRNA species of 4.0 and 4.5 kb. (Fig. 3). Expression of the 4.5-kb mRNA is first seen at a low level in 2- to 4-hr embryos and then increases to a high level in 4- to 8-hr embryos that lasts until the end of embryogenesis. This pattern of expression of the 4.5-kb mRNA indicates that it is a zygotically transcribed message. Postembryonically, decreasing levels of this mRNA species are found during larval stages, and there is a strong increase during pupation. In adult flies, there is a very low level of the mRNA in females compared with males, which have a much higher level. In contrast to this expression pattern, the 4.0-kb species is first seen at a low level in 0- to 2-hr embryos that is maintained until the end of embryogenesis. No expression of this mRNA is seen postembryonically, except for adult females. Such an expression profile indicates that the 4.0-kb species is a maternally transcribed message.

The stru cDNAs Encode a KH-Domain Protein Based on restriction mapping, Southern hybridizations, and a comparison of cDNA sequences with the partial sequences of the genomic rescue plasmids, the genomic organization of the 5* end of this gene and the P-lacZ insertion sites was determined (Fig. 4). The P-lacW insertion of E73-4 is located 47 bp upstream of the 160-bp first intron consisting of 5* UTR, while the P-lArB insertion of 1A122 is located in the first intron, 354 bp downstream of the first exon. While its exact position has not been determined, the second exon is located within 700 bp downstream of the 1A122 insertion site. These upstream exons are separated from the remainder of the exons containing the majority of the open reading frame sequences by a large intron that is at least 20 kb long. The coding sequence of both cDNAs is contained within a genomic region of at least 40 kb that is encompassed by the lE4 genomic l clone and the overlapping F68-7 cosmid clone (Fig. 4). The 4.0-kb cDNA, presumably corresponding to the maternal mRNA, was sequenced in its entirety. It contains a long open reading frame coding for 407 amino acids (from nucleotides 318 to 1541) and an extremely long 3* UTR of approximately 2.5 kb (Fig. 5). A database search with this protein sequence revealed that it contains a KH domain, a protein motif that appears to be involved in RNA binding

and that is found in a large family of proteins that function in different aspects of RNA metabolism (Gibson et al., 1993; Musco et al., 1996; Siomi et al., 1993). The sequences of two proteins, the mouse quaking (qkI) gene product and the C. elegans gld-1 protein, were published during the course of this work and found to display high degrees of similarity to the stru protein in a region including the KH domain. The mouse quaking gene (Ebersole et al., 1996; Hardy et al., 1996) is required for survival during early embryogenesis as well as for myelination in the central nervous system, while the gld-1 gene is a tumor suppressor gene necessary for oocyte development in C. elegans (Jones et al., 1996; Jones and Schedl, 1995). Originally defined as an about 50amino-acid-long motif predicted to have a b-a-a-b-b secondary structure (Gibson et al., 1993), the KH domain has recently been redefined as the maxi-KH domain to include an additional a-helix of about 18 amino acids at the carboxy end that was discovered in structural studies (Musco et al., 1996). The location of this maxi-KH domain is shown in the amino acid sequence alignment of the stru protein with the quaking and gld-1 proteins in Fig. 6. Within this domain, the stru protein is 79 and 73% identical to the quaking and gld-1 proteins, respectively. These three proteins are members of a small subfamily of single KH-domain proteins that have a unique, strongly conserved loop between the b2 and b3 strands of the domain. Other members of this subfamily include the Artemia glycine-rich protein GRP33 (Cruz-Alvarez and Pellicer, 1987), the mammalian splicing factor SF1 (Arning et al., 1996), and the Src-associated during mitosis protein Sam68, both mouse (Fumagalli et al., 1994; Taylor and Shalloway, 1994; Weng et al., 1994) and human (Lock et al., 1996; Wong et al., 1992). An additional, smaller region of homology of about 25 amino acids carboxy-terminal to the maxi-KH domain called the CGA region (Jones and Schedl, 1995) is shared by the stru, quaking, gld-1, and SF1 proteins. Apart from these aforementioned regions of homology, the other prominent feature of this protein is an alanine (15 residues)- and glutamine (31 residues)-rich region from amino acids 1–75 that is characteristic of a number of Drosophila transcriptional factors. Sequencing of the first 360 bp of the 4.5-kb cDNA showed sequence identity to the 5* portion of the 4.0-kb cDNA except for 1 additional bp at the very 5* end. Comparative restriction mapping further indicated that the sequence identities apparently extended until just before the end of the open reading frame of the 4.0-kb cDNA. Additional sequencing of the 4.5-kb cDNA was then carried out with a primer immediately upstream of this sequence difference to determine the amino acid sequence of the C-terminal end of its open reading frame. As shown in Fig. 5, this portion of the 4.5-kb cDNA codes for an alternative 6-amino-acid Cterminal tail that replaces the final 36 amino acids of the open reading frame of the 4.0-kb cDNA. This type of alternative splicing to produce proteins with divergent C-termini is similar to that seen in the mouse quaking gene, where alternative splicing of the qkI transcript produces three different mRNAs, resulting in proteins with three alternative carboxy tails (Ebersole et al., 1996). The longest

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FIG. 2. Expression of the stru mRNAs in embryos. Wildtype embryos were in situ hybridized with the 4.5-kb stru cDNA. (A) Lateral view of a cellular blastoderm embryo showing staining in the entire embryo (except the poles) that is stronger in the prospective mesodermal cells located ventrally. (B) Ventral view of a stage 6 gastrulating embryo showing staining in the prospective mesodermal cell undergoing invagination through the ventral furrow. (C) Lateral view of an early germ-band-elongated embryo. Staining is seen in the entire mesoderm layer lying interior to the the ectoderm along the germ band, including the head mesoderm. (D) Dorsolateral view of a stage 13 germband-retracted embyro with staining in the visceral mesoderm (vm) and somatic mesoderm (sm). (E) Dorsal view of a stage 16 embryo showing staining of the visceral musculature (vm) surrounding the midgut. (F) Dorsal view of a stage 17 embryo showing strong staining of the pharyngeal muscle (pm), the dorsal vessel (dv), and the muscle attachment site (mas) cells. (G, H) Lateral views of late stage embryos showing strong staining in the muscle attachment site cells (mas). Note that the expression in the somatic body wall muscles (sm) is weaker during late stages.

QKI carboxy tail is 30 amino acids long, of which 10 are identical in the 36-amino-acid tail of the protein encoded by the 4.0-kb cDNA, while the two other QKI tails are 14

or 8 amino acids long and lack any sequence similarity to the short 6-amino-acid tail of the protein encoded by the 4.5-kb cDNA.

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Phenotypic Analysis of the stru1A122 Mutation Perrimon et al. (1991) have described the stru1A122 mutation as being a recessive embryonic lethal. This lethality is uncovered by the deficiencies Df(3R)e-BS2 (93C3-6; 93F1494A1) and Df(3R)e-N19 (93B; 94), but not Df(3R)e-D7 (93C36; 93F6-8). In order to determine if stru is expressed in homozygous stru1A122 mutant embryos, in situ hybridization with a stru cDNA probe was carried out on embryos from a stock of the stru1A122 mutation balanced over a TM3, lacZhg chromosome marked with a lacZ gene that is expressed in the hindgut. This allows for the identification of the homozygous stru1A122 mutant embryos by their lack of staining for b-gal in the hindgut. While expression of stru is clearly detectable in the heterozygous stru1A122/TM3, lacZ-hg embryos (Fig. 7A), it is not detectable in the homozygous stru1A122 embryos (Figs. 7B and 7C), thus indicating that the stru1A122 mutation either severely reduces or completely abolishes stru gene expression. Since the pattern of b-gal staining in homozygous stru1A122 embryos (which can be seen clearly in Figs. 7B and 7C) is virtually identical to that of the homozygous viable E7-3-4 line, it would appear that the lethality of stru1A122 does not result from any overt loss or disorganization of the cell types that express stru in

FIG. 3. Developmental Northern analysis of the stru mRNAs. Equal amounts of poly(A)/ RNA from embryos at 0– 2, 2–4, 4 –8, 8– 12, and 12–24 hr, from first (L1), second (L2), and third (L3) instar larvae, from pupae (P), and from adult females (F) and males (M) were blotted and probed with the 4.0-kb cDNA. The position of RNA size markers (obtained from a previous hybridization of the same blot) is indicated on the left in kilobases. The 4.5-kb zygotic (zyg) and 4.0-kb maternal (mat) mRNAs are indicated on the right. For the loading control of a similar Northern blot loaded with exactly the same amount of these poly(A)/ RNAs see Fig. 2 of Azpiazu and Frasch (1993).

FIG. 4. Genomic organization of the 5* end of the stru gene. A 22-kb portion of the genome at 93F containing the upstream exons of the stru gene is shown with the 160-bp first exon (black box) and approximate location of the second exon (gray stippled box) indicated immediately underneath. Shown below this are the two overlapping l genomic clones that contain this portion of the stru gene and the two cosmid clones that overlap with clone lE4. These cosmid clones both possess a polymorphic NotI site not found in the overlapping part of the lE4 clone. The F68-7 cosmid clone contains all of the remaining coding sequences of both the 4.0- and 4.5-kb cDNAs within its 35-kb-long insert, as judged by Southern hybridizations with cDNA probes. The locations and relative orientations of the P-lacZ elements of the 1A122 and E7-3-4 lines are shown above. Abbreviations: B, BamHI; N, NotI.

the embryo. However, subtle changes in these particular cell types would be difficult to observe by the b-gal staining since the b-gal expressed by the P-lacW transposons is nuclearly localized. In order to carefully examine the phenotype of embryos lacking stru gene activity, stainings of homozygous stru1A122 embryos with antibodies to various molecular markers of the cell types expressing stru were performed. In order to generate the severest possible embryonic phenotype, the maternal contribution of the stru gene was eliminated by creating homozygous germline stru1A122 clones in females through germline recombination using the FLP/DFS technique (see Materials and Methods). The stru1A122 homozygous embryos lacking the maternal store of stru gene products that were thus obtained were antibody-stained for myosin to detect muscles, for Tinman (Azpiazu and Frasch, 1993; Bodmer, 1993) to detect heart cells, and for groovin (Volk and VijayRaghavan, 1994) to detect the muscle attachment site cells. For all three markers, there were no noticeable alterations in the patterns of their expression in those particular cell types. The pattern of somatic body wall muscles was not visibly affected in the anti-myosin staining of these embryos (Fig. 7D) and neither was that of the visceral musculature surrounding the gut (data not shown). Staining with the anti-Tinman antibody revealed a normal pattern

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FIG. 5. Nucleotide and deduced amino acid sequence of the stru 4.0-kb cDNA. The heptanucleotide matching the Drosophila consensus sequence for translation start sites is shown boxed, while the polyadenylation consensus sequence is underlined. The sequence of the maxi-KH domain in the conceptual translation product is shown underlined. The partial nucleotide sequence of the 4.5-kb cDNA containing the alternative carboxy tail is shown in italics. The complete 4.0-kb and partial 4.5-kb cDNA sequences have been respectively given the GenBank Accession numbers AF003106 and AF003107. 248

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FIG. 6. Amino acid sequence alignment of the stru, quaking viable, and gld-1 proteins. The stru (ORF of the 4.0-kb cDNA), quaking qkI-5 (translation provided in GenBank accession no. U44940), and gld-1 (translation provided in GenBank accession no. U20535) protein sequences were aligned to each other using the Clustal W program (Thompson et al., 1994). The residues that are identical between Stru and either QKI-5 or GLD-1 are shown highlighted in black. The maxi-KH domains of all three proteins is outlined in black, while the long alternative carboxy tails of Stru (4.0-kb cDNA) and QKI-5 are outlined in gray.

of Tinman expression and of cardial and pericardial cells in these mutant embryos (Fig. 7E). The pattern of groovin, which is a cell surface protein expressed by the ectodermal cells of the muscle attachment sites (Volk and VijayRaghavan, 1994) was also not overtly affected.

Homozygous stru1A122 Clones in the Wing Result in Blisters During the generation of the female flies that were germline-recombined for the stru1A122 mutation, it was observed that these flies had wing blisters (Figs. 8A and 8B). These blisters were found in about 80% of the flies and were fluidfilled in the wings of newly eclosed flies and eventually collapsed as the flies matured. The blisters could be large (Figs. 8A and 8B) or fairly small (Fig. 8B) and were located anywhere on the wing blade. Veins and interveins within these blisters appeared to be normal. As a result of the presence of the collapsed blisters, especially the larger ones, the wings of these flies tended to be deformed. Wings with a larger blister were often curled or twisted to varying degrees

and were smaller in size compared to the other wing on the same fly that happened to have either a very small blister or no blister at all. Large blisters abutting the wing margin also frequently resulted in tearing of the wing in that region. Presumably, the wing blisters arise from homozygous stru1A122 clones generated by mitotic recombination in the wings of the female flies that were induced to undergo the germline recombination. Such wing blisters were not observed in the autosomally equivalent male flies that lack the hsFLP gene and thus are incapable of undergoing the mitotic recombination necessary to produce homozygous stru1A122 clones. We verified that the blister phenotype of the homozygous stru1A122 wing clones was due to the PlArB insertion into the stru gene and not an unrelated but tightly linked mutation by excising the P transposon from the stru1A122 allele on the FRT chromosome used to generate these clones. Precise excisions were recovered as revertants to viability over Df(3R)e-BS2 (see Materials and Methods). Heterozygotes of these revertants over stru1A122 had wildtype wings without blisters, confirming that the wing blister phenotype is caused by the insertion of the P-

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FIG. 7. stru expression pattern and phenotypic analysis of stru1A122 embryos. (A–C) Anti-b-gal (brown) and stru in situ (purple) double staining of late embryos collected from the stru1A122/TM3,lacZ-hg (TM3 balancer chromosome with a hindgut-expressing lacZ gene) stock. (A) Lateral view of a heterozygous stru1A122/TM3,lacZ-hg embryo showing stru mRNA expression and also b-gal expression from the PlArB insertion in stru1A122. The hindgut b-gal expression is not visible in this focal plane. (B) Lateral view of a homozygous stru1A122 embryo showing strong b-gal expression in the body wall muscle and muscle attachment site cells due to the P-lArB insertion in stru1A122 but no expression of the stru mRNA in those same cells as detected by in situ hybridization. (C) Dorsal view of a homozygous stru1A122 embryo showing strong b-gal expression in the dorsal vessel, pharyngeal muscle, dorsal somatic body wall muscle, and muscle attachment site cells due to the P-lArB insertion in stru1A122 but again no expression of the stru mRNA in those same cells as detected by in situ hybridization. (D–F) Homozygous stru1A122 late embryos lacking the maternal store of stru gene products were double-antibody-stained for b-gal (brown) and molecular markers (purple) normally found in the various tissues derived from stru-expressing cells. (D) Lateral view of an anti-myosin-stained embryo reveals an unaltered pattern of the body wall muscles. (E) Dorsal view of an anti-Tinman-stained embryo showing normal tinman expression and structure of the normal dorsal vessel. (F) Lateral view of an anti-groovin-stained embryo showing a normal pattern of groovin expression in the muscle attachment site cells (arrowheads).

lArB transposon into stru. In situ hybridization demonstrated that stru is widely expressed in the wing imaginal discs of third instar larvae (Fig. 8C), which is consistent with a function for this gene in controlling wing epithelial adhesion. Wing blisters that result from a disruption in the process of cell adhesion between the two wing epithelial layers are also seen in mutant alleles of the Drosophila PS integrin subunit genes. Certain viable alleles of these genes display a wing blister phenotype, as do clones in the wing that are homozygous for strong or null alleles of these three integrin subunit genes (Brabant and Brower, 1993; Brower et al., 1995; Brower and Jaffe, 1989; Wilcox et al., 1989; Zusman et al., 1990). Due to this similarity in the wing blister phenotype, the flies which had been induced to produce the homozygous stru1A122 clones were examined for two other postembryonic phenotypes associated with mutations in the PS integrin subunit genes, namely, disruption of eye morphogenesis and the impairment of flight ability. Clones of cells in the eye that are homozygous for null mutations of the mew (aPS1) (Brower et al., 1995; Roote and Zusman, 1996) and mys (bPS) (Zusman et al., 1993; Zusman et al., 1990) genes have disorganized photoreceptor arrays that are characterized by disoriented rhabdomere bundles and holes along the basal surface of the retina, although such clones of if null mutants appear to be normal (Brower et al., 1995; Roote and Zusman, 1996). Examination of the

stru1A122 homozygous clones in sections of the eye revealed no disruption of the organization of the photoreceptor arrays in those clones (Fig. 8D). The rhabdomere bundles are properly oriented and there are no visible holes along the basal surface of the retina in these clones of eye cells, thus indicating that the stru1A122 mutation has no effect on eye morphogenesis.

Homozygous stru1A122 Clones in the Adult Result in the Impairment of Flight Abililty Since flies that are hemizygous or homozygous for the mysnj42 allele are incapable of flight (Costello and Thomas, 1981; de la Pompa et al., 1989), an examination of the flight ability of the flies with the induced homozygous stru1A122 clones was undertaken. In order to rule out the possibility that any flight impairment was due to the deformation of the wings that resulted from the presence of the collapsed blisters found in most of these flies, the adult female flies were sorted into two groups prior to testing for flight ability. The first were flies with wings that had either no blisters at all or very small blisters that did not cause any deformation or reduction in wing size. The second group consisted of flies with wings that had small- or medium-sized (cf. Fig. 8B, upper wing) collapsed wing blisters but which were still flat and minimally reduced in size. The autosomally equivalent male flies that lacked the hsFLP gene were used as a

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FIG. 8. Effect of the stru1A122 mutation on integrin-dependent adhesion processes. (A, B) Dorsal view of recently eclosed female flies that have undergone FLP/FRT-induced mitotic recombination to produce homozygous stru1A122 clones and which possess fluid-filled wing blisters. The blisters were large (A, B; large arrowheads) or small (B; small arrowheads). (A) A fly with one large blister on each wing. Note that the blister could be located proximally (top wing) or distally (bottom wing) on the wing. (B) A fly with one large blister on one wing (bottom wing) and several small blisters on the other (top wing). (C) In situ hybridization reveals wide expression of stru in the wing disc of third instar larvae. (D) Parasagittal section of an eye with a homozygous stru1A122 clone (the w0 clone shown bracketed) that has no apparent abnormalities in the rhabdomere bundles or along the basal surface of the retina.

control. Utilizing their positive phototropic light response, the flies were tested for their ability to fly upward towards a strong light source located overhead (see Materials and Methods). The flight ability of flies tested in this manner was divided into three categories: (1) flight up toward the light source, indicating normal flight ability; (2) flight downward, indicating some impairment of flight ability; and (3) flightless, indicating a severe impairment in flight ability. When compared to the control male flies, the female flies with small wing blisters are clearly impaired in their flight ability, with approximately 75% of them being either flightless or flight down (Fig. 9). This impairment cannot be due to the presence of the small wing blisters since about 25% of these flies are able to fly normally. In order to further exclude the possibility that any minor wing deformations caused by the small blisters were responsible for flight impairment, the flies with no wing blisters were also tested.

The proportion of these flies that was flight impaired was 15%, compared to 4% for the control male flies. It was expected that the level of flight impairment for these flies would be much lower than that of the first group since the absence of wing blisters indicated a reduced number and size of somatic homozygous stru1A122 clones also in other tissues. Taken together, the flight impairment indicates that the loss of stru gene products in somatic homozygous stru1A122 clones impairs the proper functioning of the flight muscles.

DISCUSSION Based on its embryonic expression pattern, we have cloned and characterized the novel Drosophila mesoderm- and muscle-specific gene struthio, which encodes a KH-domain pro-

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FIG. 9. Flight ability of the flies with induced homozygous stru1A122 clones. The hsFLP/ female flies with small wing blisters (n Å 62) or no wing blisters (n Å 81) and the control hsFLP0 male flies (n Å 132) were tested for flight ability and the results shown on this graph as the percentage of flightless, flight down, and flight up (see text) for each group.

tein. Our analysis of homozygous somatic cell clones reveals that the stru1A122 allele of this gene has two prominent phenotypes: wing blisters and flight impairment. Notably, these phenotypes are also associated with certain mutant alleles of the Drosophila PS integrin subunit genes and the struthio gene is expressed in muscles and their ectodermally derived attachment sites, much like the PS integrin subunit genes. These similarities suggest that Stru acts through the control or functioning of integrins in Drosophila.

The stru1A122 Mutation and Drosophila Integrins The two major integrin receptors in Drosophila, PS1 and PS2, are heterodimers of a unique a subunit (aPS1 or aPS2) and a common b subunit that are expressed in complementary patterns. In embryos, PS1 is expressed mainly in the ectoderm and endoderm, while PS2 is found in the mesoderm (Bogaert et al., 1987; Leptin et al., 1989; Zusman et al., 1990). By the time the somatic musculature has developed, PS1 expression is restricted to the epidermal muscle attachment cells and PS2 is concentrated at the ends of the muscles that attach to these sites. This complementarity is also seen in the imaginal discs where PS1 is detected in the prospective dorsal portion of the wing disc and PS2 in the ventral portion (Brower et al., 1985). Previous work has demonstrated that a failure in cell adhesion by the two epithelial layers of the wing due to mutations in the integrin subunit genes results in wing blisters. This was observed in flies mutated for certain viable alleles of inflated (coding for aPS2) and myospheroid (coding for bPS), as well as by generating somatic clones in the wing that were homozygous for null alleles of multiple edema-

tous wings (coding for aPS1), inflated, or myospheroid (Brabant and Brower, 1993; Brower et al., 1995; Brower and Jaffe, 1989; Wilcox et al., 1989; Zusman et al., 1990). By examining marked wing clones, it was shown that these clones exhibited a dorsal/ventral specificity that correlated with their patterns of expression (Brabant and Brower, 1993; Brower and Jaffe, 1989; Brower et al., 1995). The size of large blisters was usually greater than that of the clone which caused the blister (Brabant and Brower, 1993; Brower and Jaffe, 1989; Zusman et al., 1990). Since the homozygous stru1A122 wing clones generated in our study were not marked, it is not possible to ascertain whether dorsal or ventral or both types of wing clones induce blisters. However, since some of the large wing blisters caused by the stru1A122 clones extend over the anterior/posterior compartment boundary which is not crossed by the wing clones, this would suggest that the stru1A122-induced blisters are also larger than the wing clones which cause them. A recent study has identified new genes functioning in the adhesion of the wing surfaces by screening for X-rayinduced autosomal mutations which cause blisters in homozygous wing clones (Prout et al., 1997). Among the 21 complementation groups representing new genes that were recovered, there is 1 group on chromosome 3R which was named struthio (stru) that has a single embryonic lethal allele stru1. We have determined that the lethal mutation associated with the P-lArB insertion of the 1A122 line fails to complement the lethality of stru1 and have accordingly renamed the mutation as stru1A122. The phenotypes of these two stru alleles are similar since both are recessive late embryonic lethals with no obvious abnormalities, and in somatic homozygous clones they both produce the same type of wing blisters, which are discrete and variably sized with normal venation found anywhere on the wing blade. Besides the wing blister phenotype, the flight impairment seen in the flies with somatic stru1A122 homozygous clones is an additional phenotype associated with an integrin mutation. Flies carrying the EMS-induced mysnj42 allele are incapable of flight and also lack the escape jump response (Costello and Thomas, 1981; de la Pompa et al., 1989). Histological examination reveals that the primary defect in these mutants is atrophy of the tergotrochanteral muscle, a mesothoracic indirect flight muscle (de la Pompa et al., 1989). Presumably as a consequence of this defect, the wings of mysnj42 flies are held out from the body, an effect which is temperature-sensitive (Wilcox et al., 1989; Zusman et al., 1993). This latter phenotype has not been observed in the flies with somatic stru1A122 homozygous clones, which could indicate that the held-out and blistered phenotypes may be differentially sensitive to the reduction of stru gene products. Because the nuclei of individual syncytial muscle fibers are not clonally related, each fiber would have nuclei originating from both wildtype and mutant myoblasts, making it unlikely that any significant number of the fibers in a muscle would be completely depleted of stru gene products. Thus, the reduction in levels of stru gene product in these fibers with mixed nuclei may be enough to cause flight impairment but not the held-out wing phenotype.

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We have, however, observed that held-out wing phenotypes occur in a fraction of flies homozygous for semilethal mutations derived by the imprecise excision of the P-lacW transposon of the E7-3-4 line (P.L. and M.F., unpublished results). This partially penetrant phenotype is also seen for these alleles in trans to either stru1A122 or stru1. During the submission of this paper, two other reports characterizing this gene were published (Baehrecke, 1997; Zaffran et al., 1997). Viable hypomorphic mutant alleles generated by both groups exhibit a held-out wing phenotype, which has led these groups to name this gene wings held-out (Baehrecke, 1997) or held out wings (Zaffran et al., 1997). Many of these mutant flies also exhibit wing blisters (Baehrecke, 1997). Flies with these hypomorphic alleles in trans to stru1A122 have the held-out wing phenotype and are flightless (Zaffran et al., 1997). Thus, hypomorphic alleles of the struthio gene possess the held-out wing, flightless, and wing blister phenotypes that are also associated with viable mutant alleles of the PS integrin subunit genes. There are several phenotypes associated with mutations in certain integrin subunit genes that were not observed in the stru1A122 mutation (Brabant and Brower, 1993; Brower et al., 1995; Brown, 1994; Leptin et al., 1989; Newman and Wright, 1981; Roote and Zusman, 1995; Wright, 1960). The first is the embryonic myospheroid phenotype seen in if0 and mys0 homozygotes of somatic body wall muscles which have balled up and detached from the hypoderm and from each other, due to the failure of the muscle attachments when these muscles first start to contract in late embryos. Abnormalities in midgut morphology and the presence of a dorsal hole in the cuticle which result from defective adhesion processes in mutants for certain integrin subunits were also not observed in embryos lacking both the maternal and zygotic stru gene products. Nevertheless, the lethality of these embryos may well be due to more subtle effects on adhesion or attachment processes or perhaps on muscle cytoarchitecture (Volk et al., 1990). Postembryonically, a disruption of photoreceptor organization in homozygous clones in the adult retina is a strong phenotype for mys0 alleles, weaker for mew0 alleles, and not seen at all for if0 alleles (Brower et al., 1995; Roote and Zusman, 1996; Zusman et al., 1990). Examination of homozygous stru1A122 clones in the eye demonstrate a lack of any effect of this mutation on photoreceptor organization. It is possible that a strict correspondence between the phenotype of stru1A122 with the phenotype of a mutation in one of the PS integrin subunits does not exist because the function of these integrins are modulated by but not completely dependent on stru or because the stru1A122 mutation may not be amorphic for the function of stru in controlling PS integrin function. Alternatively, since previous studies have indicated the existence of additional integrin subunits (Brower et al., 1995; Gotwals et al., 1994; Roote and Zusman, 1995; Roote and Zusman, 1996; Yee and Hynes, 1993), it is possible that this mutation is affecting a non-PS integrin. Lastly, the stru1A122 mutation may be affecting a parallel pathway involved in cell adhesion not mediated by integrins.

Struthio as a KH-Domain Protein Originally defined as a triple repeat in the hnRNP K protein (Siomi et al., 1993), the KH domain was subsequently found in single or multiple copies in numerous proteins that were known to associate with RNA in different cellular processes (for reviews see Gibson et al., 1993; Musco et al., 1996). That mutations in the KH domain of a protein could have severe physiological consequences was first shown for the product of the fragile X mental retardation gene (FMR1), where a missense mutation in a conserved position of the second KH domain results in severe fragile X syndrome (DeBoulle et al., 1993). The stru protein is most closely related to the mouse quaking gene product and the C. elegans gld-1 protein, with the highest degree of sequence similarity in the maxi-KH domain and CGA region, and all three are members of the Sam68 (or GSG) subfamily of KH-domain proteins that have a unique conserved loop between the b2 and b3 strands of their single KH domain. The quaking viable mutation was discovered as a spontaneous recessive mutation that results in a rapid tremor phenotype in homozygotes (Sidman et al., 1964). This phenotype appears to be caused by severe dysmyelination in the CNS with some milder effects in the PNS. ENU-induced alleles (referred to as qke) are embryonic lethals with an arrested growth phenotype that has not been well characterized but occurs well before myelination (Justice and Bode, 1988). The quaking transcripts are expressed in the developing brain and neural tube of embryos, as well as in adult heart, lung, and testis (Ebersole et al., 1996). Examination of the pattern of expression of the different protein isoforms with antibodies raised specifically against their unique carboxy tails demonstrates that all three are abundantly expressed in myelinating cells of the central and peripheral nervous systems, but not in neurons (Hardy et al., 1996). In the qkv mutants, levels of all three isoforms are severely reduced in both CNS and PNS myelinating cells. These results underscore the importance of the quaking gene as a KH-domain gene with a developmentally important role. Interestingly, there is evidence that myelination requires integrin functions (Malek-Hedayat and Rome, 1994; Milner et al., 1997). Thus, it is conceivable that Stru and QKI act in related cellular pathways and have a similar molecular mode of function. In this regard, it should be noted that one of two sequenced qke alleles has a single glutamic acid to glycine change at amino acid 48, which is located N-terminal to the KH domain in a region of homology called QUA1 that is also found in the stru and gld-1 proteins (Ebersole et al., 1996). This glutamic acid is conserved in five of the six members of the Sam68 subfamily of KH-domain proteins, indicating some functional importance for this specific amino acid and the QUA-1 domain. A number of missense mutations in different gld-1 alleles are found at highly conserved positions in the KH domain (Jones and Schedl, 1995), clearly demonstrating the importance of the KH domain for the function of GLD-1. The gld-1 gene is a germline-specific tumor suppressor gene necessary for oogenesis and the development of the male germline. Based on the

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cytoplasmic localization of GLD-1 (Jones et al., 1996), this protein may regulate mRNA translation, stability, or localization. The properties of two other members of the Sam68 subfamily, the Sam68 protein and the mammalian splicing factor SF1, provide evidence that this type of KH domain mediates interactions with RNAs. The SF1 protein is required for spliceosome formation and direct binding of SF1 protein to RNA in a sequence-independent manner has been demonstrated (Arning et al., 1996). Nonspecific binding of the Sam68 protein to RNA has also been demonstrated but it is unknown if there are any specific RNA targets (Taylor and Shalloway, 1994; Wong et al., 1992). Besides the KH domain, this protein also contains SH2and SH3-binding sites to which Src kinases can bind (Fumagalli et al., 1994; Taylor and Shalloway, 1994; Weng et al., 1994). The Sam68 protein is Src-associated during mitosis and can be tyrosine-phosphorylated by members of the Src family of protein tyrosine kinases (Fumagalli et al., 1994; Taylor and Shalloway, 1994). As with the quaking protein, it appears that Sam68 may integrate signal transduction and RNA regulation. Besides Struthio, there are two other KH-domain proteins in Drosophila that have multiple copies of the KH domain and are not members of the Sam68 subfamily. The first is the PSI protein, a somatic inhibitor of splicing of the third intron of the P element primary transcript that has three copies of the KH domain and is known to bind a specific inhibitory element in the P element pre-mRNA (Siebel et al., 1995). Second is the Bicaudal-C protein, which has five copies of the KH domain and is necessary for the proper migration of the somatic follicle cells during oogenesis and for patterning the anterior region of the embryos (Mahone et al., 1995). The phenotype of anterior segmentation defects seen in Bicaudal-C mutant embryos seems to be due to the abnormal persistence of oskar RNA at the anterior pole of the embryo, thus indicating a role for Bicaudal-C in RNA localization. The importance of the KH domains to Bicaudal-C function is demonstrated by a strong missense allele that results from the substitution of an arginine for a highly conserved glycine in the third KH domain. Taken together, these data provide strong support for a role of Stru in the metabolism or localization of RNAs. There are two general ways in which the stru protein could be envisioned to have an effect on integrin-mediated cellular processes through the RNA-binding function of its KH domain. In an upstream control model, the stru gene product could regulate the levels and/or functions of the integrin subunits through alternative splicing of their premRNAs or the localization or translational control of their mRNAs. Alternative splicing of Drosophila integrin subunits is known to occur (Brown et al., 1989; Zusman et al., 1993) and appears to be functionally significant (Roote and Zusman, 1996), and the PS2 integrin has been shown to be concentrated at somatic muscle ends (Bogaert et al., 1987; Leptin et al., 1989; Martin-Bermudo and Brown, 1996). In addition, precedents exist in other systems for the translational control of mRNAs by KH-domain proteins (Kiledjian

et al., 1995; Ostareck et al., 1997). Alternatively, in the downstream control model the stru protein could mediate or regulate aspects of integrin-mediated signal transduction pathways which control adhesion-induced changes in cell physiology (for reviews, see Clark and Brugge, 1995; Schwarz, 1992). Such a pathway involving the PS integrins has been suggested by a study of pupal wing development (Brabant et al., 1996). Further genetic and biochemical analyses are required to firmly establish that Stru does indeed control some aspect of Drosophila integrin function, what its molecular targets are, and which of these models explains the mechanism of this control.

ACKNOWLEDGMENTS We thank Sungjin Kim for carrying out the plasmid rescue and initial genomic cloning; Miyuki Yussa, Rosemary Reincke, Johanna Ohlmeyer, and Jennifer Suggs for advice and protocols; Dan Kiehart and Talila Volk for antibodies; and Volker Hartenstein, Norbert Perrimon, Mary Prout, and Kathy Matthews for fly stocks. This work was supported by an NIH postdoctoral training fellowship to P.L. and a Pew fellowship to M.F.

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A KH-Domain Protein Mediating Cell Adhesion

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