Shark (SH2 domain ankyrin repeat kinase, Ferrante, 1995) is a Drosophila nonreceptor tyrosine kinase that contains from amino to carboxyl terminus, a Src homology 2 (SH2) domain (N-SH2), five ankyrin repeats, a second SH2 domain (C-SH2), a proline-rich and basic region, and a tyrosine kinase domain. Analysis of the phenotypes associated with a shark loss-of-function mutation demonstrates that Shark activity is essential for the migration of the dorsolateral epidermis of the embryo during dorsal closure (DC). Shark kinase functions in DC upstream of Dpp expression by leading edge (LE) cells (Fernandez, 2000).

shark is also required for dorsal-appendage (DA) morphogenesis in Drosophila oogenesis. shark function is required in follicle cells for cell migration and chorion deposition. bullwinkle (bwk) regulates, through a novel germline-to-soma signal, morphogenesis of the eggshell dorsal appendages. A screen was carried out for dominant modifiers of the bullwinkle mooseantler eggshell phenotype and shark was idenfied as a dominant modifier of bullwinkle. At the onset of dorsal-appendage formation, shark is expressed in a punctate pattern in the squamous stretch cells overlying the nurse cells. Confocal microscopy with cell-type-specific markers demonstrates that the stretch cells act as a substrate for the migrating dorsal-appendage-forming cells and extend cellular projections towards them. Mosaic analyses reveal that shark is required in follicle cells for cell migration and chorion deposition. Proper shark RNA expression in the stretch cells requires bwk activity, while restoration of shark expression in the stretch cells suppresses the bwk dorsal-appendage phenotype. These results suggest that shark plays an important downstream role in the bwk-signaling pathway. Candidate testing implicates Src42A in a similar role, suggesting conservation with a vertebrate signaling pathway involving non-receptor tyrosine kinases (Tran, 2003).

The folding and remodeling of epithelia into more complex structures is a recurrent phenomenon in metazoan development. Intercellular interactions are important regulatory components of these processes. Adjacent cells typically provide cues that direct morphogenesis or establish an extracellular milieu permissive for cell movements (Tran, 2003).

In Drosophila melanogaster, remodeling epithelia can interact with an adjacent epithelium. Two well-studied examples include the migration of the embryonic dorsal epithelium over the amnioserosa, and eversion of leg and wing primordia relative to the peripodial tissue that bounds the imaginal discs. These cell layers actively regulate the patterning and movements of neighboring epithelia. Ablation of the peripodial membrane results in growth and patterning defects in the eye and wing discs. In the embryo, the amnioserosa contributes signals and mechanical force to dorsal closure. During germband retraction, the amnioserosa also signals to and extends lamellipodia-like structures towards the retracting germband cells. This study elaborates on a novel extracellular pathway defined by bullwinkle (bwk) (Rittenhouse, 1995) that is essential for proper tubulogenesis of the follicular epithelium during synthesis of the dorsal appendages (DAs), specialized respiratory structures of the eggshell. Additionally, it is demonstrated that an adjacent squamous cell layer acts as a substrate for the migrating epithelium and expresses factors required for this morphogenetic process (Tran, 2003).

DA formation occurs within the context of the Drosophila egg chamber, which consists of ~650 somatically derived follicle cells surrounding a germline cyst composed of one oocyte and 15 nurse cells. The germ cells are interconnected via cytoplasmic bridges called ring canals, which provide access for the transfer of nurse-cell material into the developing oocyte. At stage 11, the nurse cells transport most of their cytoplasm into the oocyte, and then undergo programmed cell-death. DA morphogenesis begins at stage 11, coincident with nurse-cell apoptosis (Tran, 2003).

During DA formation, the somatic layer consists of two major populations with distinctive morphologies, the stretch cells and columnar cells. At the anterior, ~50 squamous stretch cells cover the nurse cells. These cells provide signals that pattern the anterior eggshell-forming cells and ensure proper nurse-cell cytoplasmic dumping. The columnar cells overlie the oocyte at the posterior and secrete the layers and specialized structures of the eggshell. The anterior-most columnar cells (the centripetal cells)migrate inwards, closing off the anterior end of the oocyte while synthesizing the operculum and micropyle. In addition, two subpopulations of ~65 dorsoanterior follicle cells form the two dorsal appendages through a complex reshaping and reorganization of a flat epithelium into three-dimensional tubes (Tran, 2003 and references therein).

These DA-forming cells apically constrict and evert outwards, changing from a flat layer into tubular structures that extend anteriorly. Secretion of chorion proteins into the tube lumens creates the appendages. This process occurs during the final stages of oogenesis, downstream of the events that pattern the eggshell and embryonic axes (Tran, 2003).

Although much is known about the induction and refinement of follicle-cell patterning, little is known about the factors that govern the cellular movements. One pathway that contributes to the morphogenesis is the Jun-kinase (JNK) pathway. The Drosophila Jun and Fos transcription factors are expressed highly in the stretch cells and in an anterior subset of the two DA-forming cell populations. Loss of JNK-pathway function results in two short paddleless DAs and defective nurse-cell cytoplasmic transport (Tran, 2003 and references therein).

The DA-forming cells require additional extracellular cues for normal tubulogenesis. Mosaic analyses demonstrate that bwk is required in the germline to regulate formation of the dorsal appendages (Rittenhouse, 1995). bwk encodes several SOX/TCF transcription factors with pleiotropic functions, regulating dorsal follicle-cell migration, anteroposterior (AP) patterning in the embryo, and transport of nurse-cell cytoplasm into the oocyte. In bwk mutants, the DA-forming cells not only fail to migrate anteriorly, but instead extend much more laterally (Dorman, 2004), as indicated by the wide DA paddle (Tran, 2003 and references therein).

To elucidate the role of bwk in DA formation, other components of this germline-to-soma signaling pathway were sought. Second-chromosome deficiencies were screened for regions that genetically interact with bwk. Tests of candidate mutations identified shark as a strong Enhancer of bwk. shark encodes an SH2-ankyrin-repeat, tyrosine-kinase protein (Ferrante, 1995) that functions upstream of the JNK pathway (Fernandez, 2000) during dorsal closure of the embryo (Tran, 2003 and references therein).

shark is shown to act downstream of bwk in the squamous stretch cells, and shark mediates the regulation of DA formation by bwk. Furthermore, detailed cellular analyses with stretch-cell markers show that the stretch cells provide a substrate for the DA-forming cells and appear morphogenetically active (Tran, 2003).

Shark non-receptor kinase is conserved, with homologs in Hydra (Chan, 1994) and sponge (Suga, 1999). The mammalian counterparts contain homologous SH2 and tyrosine-kinase domains but lack the ankyrin repeats (Chan, 1991; Taniguchi, 1991). These mammalian proteins, Zap70 and Syk, are recruited to immunoreceptor complexes upon ligand binding and regulate immune-cell activation and differentiation, functioning alongside Src kinases (reviewed by Chu, 1998). In T-cells, Zap70 also mediates signaling downstream of integrin-receptor complexes that feature in T-cell motility (Bearz, 1999; Soede, 1998: Tran, 2003 and references therein).

Mosaic analyses with loss-of-function shark alleles have established two somatic functions in DA formation. First, shark is required in the DA cells for proper DA-chorion deposition, a complex process regulated at many levels. Mutations that disrupt chorion-gene amplification or chorion-protein synthesis result in thin, collapsed DAs and main-body eggshell. Unlike those mutations, loss of shark in the main-body follicle cells does not cause defects in follicular imprints, alter the appearance of the eggshell under darkfield optics, or produce thin chorion and collapsed eggs. Although the methods used in this study may miss subtle defects in main-body chorion, shark may play a DA-cell-specific role in the production/formation of chorion. Although regulatory sequences and a putative binding protein drive specific spatial expression of chorion-reporter constructs, no reported mutants disrupt DA-specific chorion expression (Tran, 2003).

The second function of shark lies in the stretch cells and affects the migration of the DA cells. Large stretch-cell clones resulted in shortened DAs that vary in their morphology and penetrance. This variability could result from residual activity of these mutant alleles, non-cell autonomy, or functional redundancy. Although no Shark paralogs are encoded in the genome, several non-receptor tyrosine kinases share homology in the SH2 and kinase domains, including Src42A (Tran, 2003).

In addition, stretch-cell expression of shark strongly suppresses the bwk-mutant DA phenotype, in concurrence with a direct role for bwk in regulating shark expression in this tissue. These results indicate that shark is key in regulating DA migration downstream of bwk. Full rescue was not likely achieved because of insufficient expression levels, the need to localize shark RNA, or the existence of shark-independent branches downstream of bwk (Tran, 2003).

These data suggest a model in which Bwk regulates factors in the germline that are required for proper shark expression in the stretch cells. Shark then regulates the activity of targets required for DA-cell movement across the stretch-cell layer. Another factor that could be regulated by bwk is the Src42A kinase, which behaves similarly to shark. Loss of Src42A enhances bwk mutants, while stretch-cell expression of activated Src42A suppresses bwk. Mammalian homologs of Shark function together with Src kinases, suggesting a conserved signaling cascade (Tran, 2003).

Two other stretch-cell signaling pathways, JNK and DPP, regulate DA morphogenesis. Tests with bwk and shark, however, fail to reveal strong or definitive interactions. Loss of JNK activity in oogenesis results in shortened and paddleless DAs, yet expression of UAS-basket⁺ and reduction of bsk dose do not alter the morphology of bwk eggshells. Furthermore, expression of the AP-1 components is unaffected in bwk mutants and shark clones. These data support the hypothesis that the bwk/shark pathway does not primarily act through JNK signaling (Tran, 2003).

Moderate overexpression of dpp and loss of the type I receptors, tkv and sax, can lead to shortened and somewhat broadened DAs, resembling bwk mutants. The expression of dpp RNA and a dpp enhancer trap, however, are unaffected in bwk mutants. Both hypomorphic dpp alleles and loss of type I receptors fail to interact with a strong bwk mutant. These data suggest that bwk does not directly regulate dpp4 expression or activity but rather may modulate downstream targets (Tran, 2003).

DA-cell movement over the stretch cells may require expression of stretch-cell factors that guide or facilitate migration. Mammalian proteins that share homology with Shark can bind to and regulate integrin complexes. Shark may bind these and/or other adhesion receptors to regulate cell migration either through signaling cues or by modulating the extracellular matrix (Tran, 2003).

Shark could also regulate stretch cell behaviors, controlling the small cellular projections that extend toward the DA cells during their anterior movement. These extensions may guide or signal the DA-forming cells, as occurs in imaginal discs (Tran, 2003).

Extracellular signals and interactions are key components of morphogenetic processes. Two downstream components of the bwk pathway have been identified that act in the stretch-cell layer to relay a novel germline signal required for the movement of a third tissue, the remodeling epithelium of the dorsal appendage cells (Tran, 2003).

GENE STRUCTURE

cDNA clone length - 3169

Exons - 5

Bases in 3' UTR - 317

PROTEIN STRUCTURE

Amino Acids - 939

Structural Domains

Shark is a nonreceptor tyrosine kinase that contains from amino to carboxyl terminus, a Src homology 2 (SH2) domain (N-SH2), five ankyrin repeats, a second SH2 domain (C-SH2), a proline-rich and basic region, and a tyrosine kinase domain (Ferrante, 1995).

date revised: 1 September 2004

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