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The Drosophila RhoA (Rho1) GTPase is essential for postembryonic morphogenesis of leg and wing imaginal discs. Mutations in RhoA enhance leg and wing defects associated with mutations in zipper, the gene encoding the heavy chain of nonmuscle myosin II. We demonstrate here that mutations affecting the RhoA signaling pathway also interact genetically with mutations in the Stubble-stubbloid (Sb-sbd) locus that encodes an unusual type II transmembrane serine protease required for normal leg and wing morphogenesis. In addition, a leg malformation phenotype associated with overexpression of Sb-sbd in prepupal leg discs is suppressed when RhoA gene dose is reduced, suggesting that RhoA and Sb-sbd act in a common pathway during leg morphogenesis. We also characterized six mutations identified as enhancers of zipper mutant leg defects. Three of these genes encode known members of the RhoA signaling pathway (RhoA, DRhoGEF2, and zipper). The remaining three enhancer of zipper mutations interact genetically with both RhoA and Sb-sbd mutations, suggesting that they encode additional components of the RhoA signaling pathway in imaginal discs. Our results provide evidence that the type II transmembrane serine proteases, a class of proteins linked to human developmental abnormalities and pathology, may be associated with intracellular signaling required for normal development.

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.sup.+]-dependent target genes. Our data lead us to conclude that [br.sup.+] function is only provided by the Z2 isoform. We find that Z1 transgenes provide full [rbp.sup.+] function, while Z4 provides partial function. Likewise, while Z3 provides full 2[Bc.sup.+] function, Z2 also provides partial function. These results indicate possible functional redundancy or regulatory dependence (via autoregulation) associated with the [rbp.sup.+] and 2[Bc.sup.+] 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.

The Broad-Complex, a 20-hydroxyecdysone-regulated gene, is essential for the development of many tissues during metamorphosis. In Broad-Complex mutants of the rbp complementation group, dorsoventral indirect flight muscles (DVM) are largely absent, and the dorsal longitudinal indirect flight muscles, tergotrochanteral muscles, and remaining DVM often select incorrect attachment sites. The Broad-Complex encodes a family of zinc-finger-containing transcription factors, and it is hypothesized that Broad Complex proteins containing the Z1 zinc-finger pair (BRC-Z1) mediate [rbp.sup.+] function. We provide additional strong support for this hypothesis by showing that heat-shock-induced BRC-Z1 expression rescues the thoracic muscle defects of rbp mutants completely. BRC-Z4 induction can also rescue the thoracic musculature, but BRC-Z2 and -Z3 can not. Thus, the effect is specific to BRC-Z1 and its closest relative, BRC-Z4. Formation of muscle primordia from imaginal myoblasts appears normal in rbp mutants. However, the myotendinous junctions linking the DVM to the dorsal epidermis are weak, and the muscles detach during pupal life and subsequently degenerate. The data indicate that rbp mutations disrupt the cell-cell interactions between developing muscles and epidermal tendon cells as they recognize and attach to one another. Using a BRC-Z1-specific monoclonal antibody, we show that both the developing muscles and epidermal tendon cells express BRC-Z1 at the time of pupation, before mutant muscles begin to detach. We conclude that 20-hydroxyecdysone acts through the Broad-Complex to control the development of thoracic myotendinous junctions.

The ensemble of tissue-specific changes that drives Drosophila metamorphosis is initiated by the steroid hormone ecdysone and proceeds through a transcriptional cascade comprised of primary response transcriptional regulators and secondary response structural genes. The Broad-Complex (BR-C) primary response early gene is composed of several distinct genetic functions and encodes a family of related transcription factor isoforms. Our objective in this study was to determine whether individual BR-C isoforms directly regulate secondary response target genes. A cluster of 10 salivary gland-specific secondary response L71 late genes are dependent on the BR-C [rbp.sup.+] genetic function. Transgenic animals expressing individual BR-C isoforms were tested for their ability to provide the BR-C [rbp.sup.+] genetic function by monitoring the transcriptional activation of the L71 genes. We found that the BR-C Z1 isoforms could complement the transcriptional defects seen in rbp mutants but the Z2, Z3, and Z4 isoforms could not. We conclude that the BR-C [rbp.sup.+] function is provided by the BR-C Z1 isoform in prepupal salivary glands. L71 gene rescue was restricted to the prepupal salivary gland, suggesting the involvement of additional factors in L71 gene regulation. Interestingly, we found that the overexpression of Z3 or Z4 isoforms in [BR-C.sup.+] salivary glands repressed L71 expression, indicating that BR-C proteins might also function as transcriptional repressors. Molecular mapping and characterization of the regulatory sequences that control L71-6 expression revealed several Z1 isoform binding sites. Mutagenesis of these Z1 binding sites resulted in the failure to activate late gene expression in vivo when measured by transgenic reporter genes. We conclude that the BR-C early gene directly activates late gene transcription by interacting with late gene cis-acting regulatory elements and that this interaction is responsible for the temporal linkage of early and late ecdysone-induced gene expression.

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 [C.sub.2][H.sub.2] 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 regulation of the alcohol dehydrogenase (Adh) gene of Drosophila mojavesis was elucidated with respect to possible upstream regulatory sequences. Deletion studies showed that correct larval expression of Adh-1 is determined by a sequence 70 bp upstream of the transcriptional start site. Further localizationof this cis-acting element placed it downstream of -100 but upstream of -80. This region was found to contain a sequence neccessary for Adh-1 expression in the ovary.

Metamorphosis in Drosophila melanogaster is orchestrated by the steroid hormone ecdysone, which triggers a cascade of primary-response transcriptional regulators and secondary effector genes during the third larval instar and prepupal periods of development. The early ecdysone-response Broad. Complex (BR-C) gene, a key regulator of this cascade, is defined by three complementing functions (rbp, br, and 2Bc) and encodes several distinct zinc-finger-containing isoforms (Z1 to Z4). Using isoform-specific polyclonal antibodies we observe in the fat body a switch in BR-C isoform expression from the Z2 to the other three isoforms during the third instar. We show that the [2Bc.sup.+] function that corresponds presumably to the Z3 isoform is required for the larval fat body-specific expression of a transgenic construct (AE) in which the lacZ gene is under the control of the ecdysone-regulated enhancer and minimal promoter of the fat body protein 1 (Fbp1) gene. Using hs(BR-C) transgenes, we demonstrate that overexpression of Z1, Z3, or Z4, but not Z2, is able to rescue AE activity with faithful tissue specificity in a BR-C null (npr1) genetic context, demonstrating a partial functional redundancy between Z1, Z3, and Z4 isoforms. We also show that continuous overexpression of Z2 during the third instar represses AE, while conversely, expression of Z3 earlier than its normal onset induces precocious expression of the construct. This finding establishes a tight correlation between the dynamic pattern of expression of the BR-C isoforms and their individual repressive or inductive roles in AE regulation. Altogether our results demonstrate that the balance between BR-C protein isoforms in the fat body mediates, in part, the precise timing of the ecdysone activation of the AE construct but does not modulate its tissue specificity. [C] 2000 Academic Press Key Words: larval fat body; ecdysone; fat body protein 1 gene; temporal regulation; zinc fingers.

The Drosophila melanogaster broad locus is essential for normal metamorphic development. Broad encodes three genetically distinct functions (rbp, br, and 2Bc) and a family of four zinc-finger DNA-binding proteins (Z1-Z4). The Z1, Z2, and Z3 protein isoforms are primarily associated with the rbp, br, and 2Bc genetic functions respectively. The Z4 protein isoform also provides some rbp genetic function, however an essential function for the Z4 isoform in metamorphosis has not been identified. To determine the degree of conservation of Z4 function between the tobacco hornworm Manduca sexta and Drosophila we generated transgenic Drosophila expressing the Manduca broad Z4 isoform and used this transgene to rescue rbp mutant lethality during Drosophila metamorphosis. We find that the Manduca Z4 protein has significant biological activity in Drosophila with respect to rescue of rbp-associated lethality. There was also some overlap in effects on cuticle gene expression between the Manduca Z4 and Drosophila Z1 isoforms that was not shared with the Drosophila Z4 isoform. Our findings show that Z4 function has been conserved over the 260-million-year period since the divergence of Diptera and Lepidoptera, and are consistent with the hypothesis that the Drosophila Z4 and Manduca Z4 isoforms have essential roles in metamorphosis. [ABSTRACT FROM AUTHOR]

Polyamine transport is elevated in many tumor types, suggesting that toxic polyamine-drug conjugates could be targeted to cancer cells via the polyamine transporter (PAT). We have previously reported the use of Chinese hamster ovary (CHO) cells and its PAT-deficient mutant cell line, CHO-MG, to screen anthracene-polyamine conjugates for their PAT-selective targeting ability. We report here a novel Drosophila-based model for screening anthracene-polyamine conjugates in a developing and intact epithelium ( Drosophila imaginal discs), wherein cell-cell adhesion properties are maintained. Data from the Drosophila assay are consistent with previous results in CHO cells, indicating that the Drosophila epithelium has a PAT with vertebrate-like characteristics. This assay will be of use to medicinal chemists interested in screening drugs that use PAT for cellular entry, and it offers the possibility of genetic dissection of the polyamine transport process, including identification of a Drosophila PAT.