We show that the Drosophila gene rhea, isolated because its wing blister phenotype is typical of mutants affecting integrin function, encodes talin. Embryos deficient in talin have very similar phenotypes to integrin (betaPS) null embryos, including failure in germ band retraction and muscle detachment. We demonstrate that talin is not required for the presence of integrins on the cell surface or their localization at muscle termini. However, talin is required for formation of focal adhesion-like clusters of integrins on the basal surface of imaginal disc epithelia and junctional plaques between muscle and tendon cells. These results indicate that talin is essential for integrin function and acts by stably linking clusters of ECM-linked integrins to the cytoskeleton.

The Broad-Complex (BR-C) is a key member of the 20-hydroxyecdysone regulatory hierarchy that coordinates changes in gene expression during Drosophila metamorphosis. The family of transcription factors encoded by the BR-C share a common amino-terminal domain which is fused by alternative splicing to one of four pairs of C2H2 zinc-finger domains (Z1, Z2, Z3, and Z4). In this study, we examine the temporal expression of transcripts encoding each BR-C zinc-finger isoform-including the newly discovered fourth zinc-finger domain-during the metamorphosis of imaginal discs which form the integumental structures of the adult head and thorax. We find that all BR-C zinc-finger RNA isoforms are induced as a primary response to 20-hydroxyecdysone. However, induced BR-C RNA isoforms exhibit two divergent expression profiles. The Z2, Z3, and Z4 RNA isoforms accumulate to high levels at the beginning of the ecdysone response and abruptly disappear after several hours. In contrast, the Z1 RNA isoform continues to accumulate while the others decline, resulting in a switch in relative isoform levels. Using probes specific to different regions of the BR-C, we show that the switch in BR-C RNA isoform expression appears to be posttranscriptionally regulated, presumably by ecdysone-responsive factors. We propose that this switch results from a change in splice acceptor site choice. Finally, we present a model describing how this temporal switch in isoform expression could mediate changes in BR-C function, from transcriptional activation to repression and vice versa, that are critical for coordinate downstream target gene expression.

The Stubble-stubbloid (Sb-sbd) gene is required for hormone-dependent epithelial morphogenesis of imaginal discs of Drosophila, including the formation of bristles, legs, and wings. The gene has been cloned by using Sb-sbd-associated DNA lesions in a 20-kilobase (kb) region of a 263-kb genomic walk. The region specifies an -3.8-kb transcript that is induced by the steroid hormone 20-hydroxyecdysone in imaginal discs cultured in vitro. The conceptually translated protein is an apparent 786-residue type II transmembrane protein (N terminus in, C terminus out), including an intracellular N-terminal domain of at least 35 residues and an extracellular C-terminal trypsin-like serine protease domain of 244 residues. Sequence analyses indicate that the Sb-sbd-encoded protease could activate itself by proteolytic cleavage. Consistent with the cell-autonomous nature of the Sb-sbd bristle phenotype, a disulfide bond between cysteine residues in the noncatalytic N-terminal fragment and the C-terminal catalytic fragment could tether the protease to the membrane after activation. Both dominant Sb and recessive sbd mutations affect the organization of microfilament bundles during bristle morphogenesis. We propose that the Sb-sbd product has a dual function. (i) It acts through its proteolytic extraceliular domain to detach imaginal disc cells from extracellular matrices, and (it) it transmits an outside-to-inside signal to its intracellular domain to modify the cytoskeleton and facilitate cell shape changes underlying morphogenesis.The attachment of cells to extracellular substrates-for example, by integrins (1)-plays an important role in determining cell shape and the intracellular organization of the cytoskeleton. Likewise, detachment of cells from substrates also leads to profound changes in cell shape and cytoskeletal organization. In particular, cell surface-associated proteases have been shown to mediate cell shape changes by local degradation of extracellular matrices and by signaling the reorganization of the actin cytoskeleton (2, 3). Proteases are also implicated in morphogenesis of imaginal discs to form adult appendages in Drosophila (4-7). The formation of legs and wings from discs results in part from actin-and myosindependent cell shape changes in the disc epithelium elicited by the steroid hormone 20-hydroxyecdysone (20HE) (8). Later in development, specific disc cells undergo actinmediated cell shape changes to form bristles (9, 10). Stubblestubbloid (Sb-sbd) mutants cause failures in cell-shape changes required for both disc and bristle morphogenesis. Gain-of-function Sb mutations affect bristle morphogenesis in a dominant manner. Both Sb and allelic loss-of-function sbd mutations act recessively to affect disc morphogenesis, producing characteristically malformed legs and wings (11).The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.We show here...

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Metamorphosis in holometabolous insects is an ecdysone-dependent process by which the larval form is replaced by a reproductive, adult form. At the onset of metamorphosis ecdysone induces a set of early genes which coordinate tissue-specific responses to hormone. The Broad-Complex (BR-C) early gene, which acts as a global regulator of tissue-specific responses to ecdysone, encodes a family of zinc-finger DNA binding proteins known as Z1, Z2, Z3, and Z4. Genetically the BR-C encodes three complementing functions, br, rbp, and 2Bc, and a class of npr1 alleles that fail to complement any of the other genetic functions. The effects of BR-C mutations on metamorphic development are highly pleiotropic, yet little is known about the roles of individual BR-C proteins in directing the required responses to ecdysone. Because the BR-C is a vital regulator of metamorphosis it is essential to establish the relationships between BR-C genetic functions and protein products. We present here the first general and definitive study of these relationships. Using heat-inducible transgenes we have rescued lethality associated with each of the complementing genetic functions and have restored transcriptional activity of tissue-specific BR-C(+)-dependent target genes. Our data lead us to conclude that br+ function is only provided by the Z2 isoform. We find that Z1 transgenes provide full rbp+ function, while Z4 provides partial function. Likewise, while Z3 provides full 2Bc+ function, Z2 also provides partial function. These results indicate possible functional redundancy or regulatory dependence (via autoregulation) associated with the rbp+ and 2Bc+ functions. The establishment of these relationships between BR-C genetic functions and protein isoforms is an important step toward understanding the roles of BR-C proteins in directing metamorphic responses to ecdysone.