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Newly eclosed flies have wings that are highly folded and compact. Within an hour, each wing has expanded, the dorsal and ventral cuticular surfaces bonding to one another to form the mature wing. To initiate a dissection of this process, we present studies of two mutant phenotypes. First, the batone mutant blocks wing expansion, a behavior that is shown to have a mutant focus anterior to the wing in the embryonic fate map. Second, ectopic expression of protein kinase A catalytic subunit (PKAc) using certain GAL4 enhancer detector strains mimics the batone wing phenotype and also induces melanotic "tumors." Surprisingly, these GAL4 strains express GAL4 in cells, which seem to be hemocytes, found between the dorsal and ventral surfaces of newly opened wings. Ectopic expression of Ricin A in these cells reduces their number and prevents bonding of the wing surfaces without preventing wing expansion. We propose that hemocytes are present in the wing to phagocytose apoptotic epithelial cells and to synthesize an extracellular matrix that bonds the two wing surfaces together. Hemocytes are known to form melanotic tumors either as part of an innate immune response or under other abnormal conditions, including evidently ectopic PKAc expression. Ectopic expression of PKAc in the presence of the batone mutant causes dominant lethality, suggesting a functional relationship. We propose that batone is required for the release of a hormone necessary for wing expansion and tissue remodeling by hemocytes in the wing.

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Ecdysteroids regulate a wide variety of cellular processes during arthropod development, yet little is known about the genes involved in the biosynthesis of these hormones. Previous studies have suggested that production of 20-hydroxyecdysone in Drosophila and other arthropods involves a series of cytochrome P450 catalyzed hydroxylations of cholesterol. In this report, we show that the disembodied (dib) locus of Drosophila codes for a P450-like sequence. In addition, we find that dib mutant embryos have very low titers of ecdysone and 20-hydroxyecdysone (20E) and fail to express IMP-E1 and L1, two 20E-inducible genes, in certain tissues of the embryo. In situ hybridization studies reveal that dib is expressed in a complex pattern in the early embryo, which eventually gives way to restricted expression in the prothoracic portion of the ring gland. In larval and adult tissues, dib expression is observed in the prothoracic gland and follicle cells of the ovaries respectively, two tissues known to synthesize ecdysteroids. Phenotypic analysis reveals that dib mutant embryos produce little or no cuticle and exhibit severe defects in many late morphogenetic processes such as head involution, dorsal closure and gut development. In addition, we examined the phenotypes of several other mutants that produce defective embryonic cuticles. Like dib, mutations in the spook (spo) locus result in low embryonic ecdysteroid titers, severe late embryonic morphological defects, and a failure to induce IMP-E1. From these data, we conclude that dib and spo likely code for essential components in the ecdysone biosynthetic pathway and that ecdysteroids regulate many late embryonic morphogenetic processes such as cell movement and cuticle deposition.

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The forkhead gene FH1 encodes a HNF-3beta protein required for gastrulation and development of chordate features in the ascidian tadpole larva. Although most ascidian species develop via a tadpole larva, the conventional larva has regressed into an anural (tailless) larva in some species. Molgula oculata (the tailed species) exhibits a tadpole larva with chordate features (a dorsal neural sensory organ or otolith, a notochord, striated muscle cells, and a tail), whereas its sister species Molgula occulta (the tailless species) has evolved an anural larva, which has lost these features. Here we examine the role of FH1 in modifying the larval body plan in the tailless species. We also examine FH1 function in tailless speciesxtailed species hybrids, in which the otolith, notochord, and tail are restored. The FH1 gene is expressed primarily in the presumptive endoderm and notochord cells during gastrulation, neurulation, and larval axis formation in both species and hybrids. In the tailless species, FH1 expression is down-regulated after neurulation in concert with arrested otolith, notochord, and tail development. The FH1 expression pattern characteristic of the tailed species is restored in hybrid embryos prior to the development of chordate larval features. Antisense oligodeoxynucleotides (ODNs) shown previously to disrupt FH1 function were used to compare the developmental roles of this gene in both species and hybrids. As described previously, antisense FH1 ODNs inhibited endoderm invagination during gastrulation, notochord extension, and larval tail formation in the tailed species. Antisense FH1 ODNs also affected gastrulation in the tailless species, although the effects were less severe than in the tailed species, and an anural larva was formed. In hybrid embryos, antisense FH1 ODNs blocked restoration of the otolith, notochord, and tail, reverting the larva back to the anural state. The results suggest that changes in FH1 expression are involved in re-organizing the tadpole larva during the evolution of anural development.

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The release of 20-hydroxyecdysone at the end of the third larval instar provides a temporal signal that triggers specific developmental programs in hormone target tissues in Drosophila at metamorphosis. Imaginal discs respond to the steroid hormone by initiating morphogenesis leading to the formation of the adult head structures, appendages, and thoracic epidermis. The cellular events of morphogenesis are preceded and accompanied by 20-hydroxyecdysone-dependent activation of a set of genes encoding Inducible Membrane-bound Polysomal transcripts, the IMP-genes. Analysis of expression characteristics in imaginal discs cultured in vitro reveals that the IMP-E1 gene is expressed within 15-30 min after exposure to 20-hydroxyecdysone while the expression of the IMP-L1 gene is delayed 6-8 hr. Induction studies in the presence of cycloheximide establish that IMP-E1 is a primary response locus while IMP-L1 transcription is a secondary response. These genes are regulated at the level of transcription initiation. Differences between the induction characteristics of IMP-E1 and the early 20-hydroxyecdysone-responsive gene E74 lead us to propose an addition to the Ashburner model for the 20-hydroxyecdysone regulatory hierarchy. We suggest that the sequential temporal expression of steroid hormone-responsive genes in imaginal discs is important in organizing cellular mechanisms involved in morphogenesis of the epithelium.

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Lethal (1) discs-large-1 [l(1)dlg-1] is a non-epithelial overgrowth or neoplastic mutant of Drosophila, which results in tumor-like imaginal discs and enlarged larvae that never pupariate. In an ultrastructural analysis we found that the wing discs develop convoluted monolayers of epithelial cells characterized by well-defined apical-basal polarity and that these layered cells secrete large amounts of basement membrane material. Immuno-EM indicates that Drosophila laminin and collagen are components of this matrix. Late in development clusters or 'rosettes' of separated cells lacking cell-cell junctions and apical-basal polarity form. In in vitro culture experiments l(1)dlg-1 wing discs did not respond to a pulse of exogenous ecdysone by secreting cuticle or losing basement membrane as normal discs do. Our observations are consistent with the hypothesis that cell-cell interaction and communication is required for termination of disc cell proliferation, which must occur prior to a cellular response to ecdysone.

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Drosophila imaginal discs are induced by the steroid hormone 20-hydroxyecdysone to initiate morphogenesis leading to formation of the adult appendages and thoracic epidermis at the end of the third larval instar. Ecdysone-dependent transcriptional activation of a set of genes that encode imaginal disc transcripts found on membrane-bound polysomes precedes and may be responsible for some aspects of the cellular changes that mediate epithelial morphogenesis in this system. A 1.35 kb transcript from one of these genes, IMP-L1, is first observed in vivo at or just prior to pupariation, as ecdysone titers are peaking and beginning to decline. Expression is initiated in proximal areas of the antennal disc, later spreading to a more widespread but nonuniform distribution throughout other thoracic imaginal discs. IMP-L1 is not, however, expressed in other ecdysone target tissues such as salivary glands or fat body. The IMP-L1 gene encodes a novel protein product containing a signal peptide, a possible transmembrane domain, two highly charged domains and a proline rich C-terminal domain. We suggest that the delayed timing of expression of this secondary response gene is necessary for proper ordering of cellular events associated with disc morphogenesis.

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We describe our analysis of IMP-L2, one of a set of six ecdysone-inducible genes in imaginal discs of Drosophila whose transcripts are associated with membrane-bound polysomes. The spatial and temporal patterns of expression of the IMP-L2 transcript were analyzed. This transcript is first expressed in imaginal discs in areas that are precursors of head and thoracic epithelium, particularly the peripodial epithelia. It is later expressed in the imaginal histoblasts, precursors of the adult abdomen. The appearance of IMP-L2 transcript in each of these tissues precedes the spreading and fusion of the separate imaginal anlagen to form the continuous adult epidermis.

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We have isolated and initially characterized a novel set of four genes expressed during the prepupal differentiation of imaginal discs of Drosophila melanogaster. These four ecdysone-dependent genes are named EDG-42A, EDG-64CD, EDG-78E and EDG-84A-1 based on their respective chromosomal locations. Their expression is like that expected for genes encoding proteins that participate in the formation of the pupal cuticle. Transcripts complementary to these genes accumulate in imaginal discs during an 18-hr in vitro culture period that begins with a 6-hr pulse of 20-hydroxyecdysone (20-HE). Transcripts for three of these genes were not detected in imaginal discs following culture in the absence or the continuous presence of 20-HE (1 microgram/ml). Transcripts corresponding to EDG-64CD exhibit delayed accumulation in the continuous presence of 20-HE. Transcripts corresponding to three of the genes are only detected in the prepupal stage of development. Only EDG-64CD is complementary to transcripts present at other stages of development. One of the genes, EDG-78E, encodes a pupal cuticle protein. This is the first reported isolation of a set of steroid hormone-responsive genes that require first the presence, then removal of hormone for transcript accumulation.

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A unique set of genes that encodes hormone-inducible transcripts found on membrane-bound polysomes is expressed during ecdysone-dependent morphogenesis of Drosophila imaginal discs cultured in vitro. The pattern of expression of one of these genes, IMP-E1, was analyzed in tissues from late third instar larvae and white prepupae by hybridization of asymmetric RNA probes to tissue sections. The IMP-E1 transcript was detected in all anterior and posterior imaginal discs except the ommatidial region of the imaginal eye disc. Within the imaginal leg disc, the IMP-E1 transcript is expressed more abundantly in the proximal than in the distal portions of the epithelium. The distribution of transcripts is consistent with the hypothesis that the IMP-E1 gene product is involved in the cell rearrangements associated with morphogenesis of the disc epithelium. The IMP-E1 transcript is also expressed at pupariation in glial cell layers that ensheath the brain. This glial cell transcript is the same size (7.5 kb) and polarity as the imaginal disc transcript, and is also transcribed in response to 20-hydroxyecdysone. Similarities between the morphogenetic changes in imaginal disc and glial cell layers during metamorphosis are discussed.

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Interaction with the extracellular matrix is important for the proliferation and differentiation of cells during development. A specialized extracellular matrix, basement membrane, is built around a scaffold of procollagen IV molecules. We report the sequence of a 2.5-kilobase cDNA which contains the carboxyl end of a Drosophila melanogaster procollagen IV. The amino acid sequence of the carboxyl-terminal domain, which forms an essential intermolecular linkage between procollagen IV molecules, is 59% identical in Drosophila and vertebrate procollagens IV, and an additional 17% of residues are conservatively substituted. This implies that the nature of the linkage is also conserved. We suggest that intermolecular junctions through procollagen IV carboxyl domains are fundamental elements of the molecular architecture of Metazoan basement membranes and have been conserved during evolution. The isolation and identification of this basement membrane collagen gene of Drosophila will help in deducing the function of procollagen IV in basement membranes.

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Mass-isolated Drosophila imaginal discs cultured in vitro undergo morphogenesis (evagination) in response to the insect steroid hormone 20-hydroxyecdysone. In vitro translation of mRNA isolated from evaginating discs shows accumulation of at least five new mRNA transcripts that are present only in membrane-bound polysomal RNA and presumably encode imaginal disc membrane or secreted proteins. Using a modified differential hybridization screen employing a competition step, six different hormone-inducible membrane protein gene sequences were isolated. These genes constitute a new set of 20-hydroxyecdysone responsive loci that may encode gene products specifically required for imaginal disc morphogenesis.

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Both the alpha and the beta subunit of tubulin in Drosophila melanogaster are encoded by small multigene families. Using heterologous hybridization probes representing chicken alpha- and beta-tubulin genes, four complementary alpha- and four beta-tubulin gene sequences from Drosophila have been isolated. Each gene has been individually mapped cytogenically by in situ hybridization of nucleic acid probes to polytene chromosomes. It is clear that the genes in each family are dispersed rather than arranged in tandem arrays or clusters. Furthermore, alpha- and beta-tubulin genes are not physically linked as alpha-beta pairs. Transcripts from individual genes are differentially accumulated at particular stages of Drosophila development. This differential gene activity may provide expression of functionally specialized tubulin subunits. Alternatively, differences in expression may reflect tissue specific patterns of gene utilization.

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We have used cloned chicken cDNA sequences for alpha- and beta-tubulin to investigate tubulin gene organization in Drosophila melanogaster. Experiments using genomic Drosophila DNA from several sources indicate that there are at least four copies each of the alpha-tubulin gene and the beta-tubulin gene. In situ hybridization experiments show that both the alpha- and beta-tubulin multigene families have dispersed arrangements on the chromosome. Genes for alpha-tubulin have been localized at chromosomal bands 67C, 84B/C, 84D and 85E, while genes for beta-tubulin have been detected at bands 60A/B and 85D. alpha-Tubulin and beta-tubulin chicken cDNA sequences can be used to select a specific mRNA species from a complex mixture which translates in vitro into alpha- or beta-tubulin protein. RNA blot hybridization using the cloned chicken cDNA sequences as probes shows that the alpha- and beta-tubulin messages detected are clearly different in length, with the message for alpha-tubulin measuring approximately 2000 bases and the message for beta-tubulin containing approximately 1800 bases.

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