The international exchange of Drosophila melanogaster strains.

Drosophila melanogaster has been a model organism for experimental research for more than a century, and the knowledge and associated genetic technologies accumulated around this species make it extremely important to contemporary biomedical research. A large international community of highly collaborative scientists investigate a remarkable diversity of biological problems using genetically characterised strains of Drosophila, and frequently exchange these strains across borders. Despite its importance to the study of fundamental biological processes and human disease-related cellular mechanisms, and the fact that it presents minimal health, agricultural or environmental risks, Drosophila can be difficult to import. The authors argue that streamlined regulations and practices would benefit biomedical research by lowering costs and increasing efficiencies.

Drosophila melanogaster sert d'organisme modèle pour la recherche expérimentale depuis plus d'un siècle, aboutissant à une somme de connaissances sur cette espèce et sur les technologies génétiques qui lui sont associées qui la rendent extrêmement importante pour la recherche biomédicale contemporaine. Aujourd'hui, une vaste communauté internationale de chercheurs dotés d'une culture élevée de la collaboration étudie un nombre impressionnant de problèmes biologiques en utilisant des souches de drosophile caractérisées sur le plan génétique, ce qui conduit à un partage fréquent de ces souches au-delà des frontières nationales. Or, il est parfois difficile d'importer des drosophiles, en dépit de l'importance de cette espèce pour l'étude des processus biologiques fondamentaux et des mécanismes cellulaires liés aux maladies humaines, et du risque minime qu'elle représente pour la santé, l'agriculture ou l'environnement. Les auteurs estiment qu'une rationalisation des réglementations et des pratiques serait bénéfique pour la recherche biomédicale car elle permettrait de réduire les coüts et de gagner en efficacité.

Drosophila melanogaster es un organismo modelo para las investigaciones experimentales desde hace más de un siglo. El acervo de conocimientos y las correspondientes tecnologías genéticas que se han ido acumulando en relación con esta especie hacen de ella un elemento de suma importancia para la actual investigación biomédica. Hay una gran comunidad internacional de científicos que, trabajando en estrecha colaboración, investigan muy diversos problemas biológicos sirviéndose de cepas de Drosophila genéticamente caracterizadas, para lo cual intercambian a menudo estas cepas de uno a otro país. Pese a su importancia para el estudio de procesos biológicos fundamentales y de mecanismos celulares ligados a enfermedades humanas, y pese a que los riesgos sanitarios, agrícolas o ambientales que entraña son mínimos, la importación de Drosophila puede resultar dificultosa. Los autores postulan que la simplificación de procesos y reglamentos sería beneficiosa para la investigación biomédica, pues reduciría costos y permitiría ganar en eficiencia.

Ligand endocytosis drives receptor dissociation and activation in the Notch pathway.

Endocytosis of the ligand delta; is required for activation of the receptor Notch during Drosophila development. The Notch extracellular domain (NotchECD) dissociates from the Notch intracellular domain (NotchICD) and is trans-endocytosed into delta;-expressing cells in wild-type imaginal discs. Reduction of dynamin-mediated endocytosis in developing eye and wing imaginal discs reduces Notch dissociation and Notch signalling. Furthermore, dynamin-mediated delta endocytosis is required for Notch trans-endocytosis in Drosophila cultured cell lines. Endocytosis-defective delta proteins fail to mediate trans-endocytosis of Notch in cultured cells, and exhibit aberrant subcellular trafficking and reduced signalling capacity in Drosophila. We suggest that endocytosis into delta-expressing cells of NotchECD bound to delta plays a critical role during activation of the Notch receptor and is required to achieve processing and dissociation of the Notch protein.

Engineered truncations in the Drosophila mastermind protein disrupt Notch pathway function.

The phenotypes and genetic interactions associated with mutations in the Drosophila mastermind (mam) gene have implicated it as a component of the Notch signaling pathway. However, its function and site of action within many tissues requiring Notch signaling have not been thoroughly investigated. To address these questions, we have constructed truncated versions of the Mam protein that elicit dominant phenotypes when expressed in imaginal tissues under GAL4-UAS regulation. By several criteria, these effects appear to phenocopy loss of function for the Notch pathway. When expressed in the notum, truncated Mam results in failure of lateral inhibition within proneural clusters and perturbations in cell fate specification within the sensory organ precursor cell lineage. Expression in the wing is associated with vein thickening and margin defects, including nicking and bristle loss. The truncation-associated wing margin phenotypes are modified by mutations in Notch and Wg pathway genes and are correlated with depressed expression of wg, cut, and vg. These data support the idea that Mam truncations have lost key effector domains and therefore behave as dominant-negative proteins. Coexpression of Delta or an activated form of Notch suppresses the effects of the Mam truncation, suggesting that Mam can function upstream of ligand-receptor interaction in the Notch pathway. This system should prove useful for the investigation of the role of Mam within the Notch pathway.

The dynamics of neurogenic signalling underlying bristle development in Drosophila melanogaster.

We have examined expression of the neurogenic gene, Delta (Dl), and the regulatory relationships between the Delta-Notch signalling pathway and the proneural gene, achaete, during microchaeta development in Drosophila. Delta is expressed in all microchaeta proneural cells and microchaeta sensory organ precursors (SOPs) and is expressed dynamically in SOP progeny. We find that Delta expression in microchaeta proneural cells is detected prior to the onset of achaete expression and arises normally in the absence of achaete/scute function, indicating that initial Delta expression in the notum is not dependent on proneural gene function. Activation of the Delta-Notch pathway results in loss of Delta protein accumulation, suggesting that Delta expression is regulated, in part, by Delta-Notch signalling activity. We find that Delta signalling is required for correct delineation of early proneural gene expression in developing nota. Within microchaeta proneural stripes, we demonstrate that Delta-Notch signalling prohibits adoption of the SOP fate by repressing expression of proneural genes.

Relationship between age of commercial broiler chickens and response to photostimulation.

A study was conducted to determine the relationship between age of commercial broiler chickens and response to photostimulation. The chickens were brooded collectively for 1 wk and then separated into five light treatment groups with each group replicated three times using a completely randomized block design. After Week 1, the five light treatments applied were: 6, 5, 4, 3, and 2 wk of continuous supplementary lighting, respectively. Chickens were fed a corn-soybean meal basal diet containing 22% CP and 3,300 kcal ME/ kg of feed from day old to 7 wk. At 7 wk of age, chickens in Group 5 treated with supplementary light during the last 2 wk of growth had the highest mean BW when the data on sexes were combined. When the data were separated by sex and analyzed, only male chickens showed a significant response to photostimulation and this was observed at 4 wk of age. Chickens in Group 5 had the best feed conversion with less mortality and no leg disorders. There were no significant differences among the relative organ weights among the treatment groups except for the pancreas. The pancreas taken from Group 5 had the smallest weight relative to BW. There were no significant differences in breast, thigh, drumstick, and wing weights when expressed as relative weight. However, there were significant differences when expressed as absolute weight. The breasts taken from birds in Group 5 were significantly heavier than the control breasts.

Relationships between complex Delta expression and the specification of retinal cell fates during Drosophila eye development.

Analysis of retinal development in Delta (Dl) temperature-sensitive mutants reveals requirements for Delta function in the specification of all retinal cells, including photoreceptors, cone cells, pigment cells and cells that make up interommatidial bristles. In situ hybridization and immunohistochemistry indicate that Delta is expressed dynamically during the specification of different cell types. Comparisons of Delta expression patterns with developmental defects in Dl mutants implies that Delta functions in a cell-nonautonomous manner in the specification of photoreceptors. Delta protein resides predominantly in subcellular vesicles located primarily at the apical ends of developing retinal cells. Localization of Delta protein in Dl and shibire tsl mutants implies that Delta is targeted to the cell surface, but is efficiently removed via endocytosis, resulting in vesicular accumulation.

Combined effects of tissue plasminogen activator and carboxymethylcellulose on adhesion reformation in rabbits.

OBJECTIVE: To test the effectiveness, in combination, of recombinant tissue plasminogen activator (rt-PA) and carboxymethylcellulose (CMC), which have both been investigated as adjuncts in the prevention of postsurgical adhesion reformation. METHODS: A total of 45 mature New Zealand rabbits underwent laparotomy and bilateral uterine horn abrasion using a scalpel for scarification. Adhesions were scored according to the extent of uterine horn involvement and quality of the adhesions. Three weeks later, after the adhesions were scored and lysed using microsurgical technique, the animals were randomized to receive 20 mL of one of four adjunctive treatments: (1) abnormal saline; (2) 2% sodium CMC, (3) 10 mg rt-PA mixed in 2% sodium CMC, or (4) 32% dextran 70. The adhesions were scored again 3 weeks later at necropsy. RESULTS: Although adhesion scores decreased slightly in all groups, there was no statistically significant difference in adhesion reformation between the four groups. Four rabbits died of intra-abdominal hemorrhage following lysis of adhesions, including three in the rt-PA/CMC group. The combination of intraperitoneal rt-PA and CMC did not reduce postsurgical adhesion reformation, and was associated with hemorrhagic complications.

Calpain-induced downregulation of activated protein kinase C-alpha affects lung epithelial cell morphology.

A few minutes after mouse lung epithelial cell lines were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA), the cells rounded up and pulled away from their neighbors. Several hours later, the cells flattened out to resume their original morphology. To begin to characterize the enzymology underlying these changes, the subcellular distribution and intracellular content of the TPA receptor, protein kinase C (PKC), and its putative endogenous regulator, the Ca(2+)-dependent protease, calpain, were investigated. Of eight PKC isozymes examined in several tumorigenic and nontumorigenic cell lines, all cells contained PKC-alpha, PKC-delta, and PKC-zeta. TPA rapidly (5 min) translocated PKC-alpha from the cytosol to the particulate fraction; PKC-alpha concentrations then decreased with continued TPA exposure. PKC-zeta levels and intracellular location were not affected. An inhibitor of PKC activity, GF 109203X, prevented the initial morphological change. The calpain II isozyme was also found in all cell lines, and its cellular content increased as a result of TPA treatment. Calpain inhibitor I did not affect the initial shape change but prevented subsequent flattening of the cells. We therefore conclude that PKC activation is required for the TPA-induced alterations in lung cell morphology and that calpain mediates the return to a flattened epithelial appearance.

Delta function is required for bristle organ determination and morphogenesis in Drosophila.

Analyses of Delta (Dl) temperature-sensitive mutants reveal that reductions in Dl function during metamorphosis of Drosophila melanogaster can lead to multiplication or loss of bristle organs on the adult notum, depending on the developmental interval during which such mutants are pulsed at restrictive temperature. Site-dependent macrochaeta multiplication or loss results from pulses 0-21 hr after puparium formation (APF). Microchaeta multiplication results from pulses 7-30 hr APF, while microchaeta loss results from pulses 24-47 hr APF. Supernumerary bristle organs that develop in Dl mutants consist of the normal complement of trichogen, tormogen, neuron, and thecogen cells, and appear to arise due to the specification of super-numerary bristle first-order precursor (pI) cells within proneural groups in the developing notum. Loss of complete bristle organs in Dl mutants is correlated with the differentiation of all four progeny of the pI cell into neurons. We conclude that Dl function is required for cellular interactions central to the correct specification of pI cells within proneural groups, as well as for morphogenesis of the bristle organ itself.

Promoter structure and protein sequence of msp130, a lipid-anchored sea urchin glycoprotein.

The early fate specification of primary mesenchyme cells in sea urchin embryos makes them an attractive system for studying alterations in gene expression and protein synthesis during cell lineage determination and differentiation. To analyze the developmental regulation of gene expression in Strongylocentrotus purpuratus, we have isolated and sequenced genomic and cDNA clones encoding msp 130, a mesenchyme-specific cell surface glycoprotein. We have located the transcription initiation site of the msp130 gene and sequenced several kilobases of the promoter region. The region of the gene that encodes the protein is divided into numerous small (less than 500 base pairs) exons. The msp130 protein possesses two novel glycine-rich domains and a signal peptide, but apparently lacks a transmembrane domain. The carboxyl-terminal sequence suggests that msp130 may be phosphatidylinositol-linked to the cell membrane, and experiments with phospholipases support this conclusion. The implications of the msp130 sequence for its possible functions are discussed.

Immunocytochemical evidence suggesting heterogeneity in the population of sea urchin egg cortical granules.

Unfertilized eggs of many species of animals contain cortical granules, which are specialized secretory granules that upon fertilization release their contents from the egg. The unfertilized eggs of the sea urchin, Strongylocentrotus purpuratus, contain cortical granules that all display an identical and elaborate internal morphology. It has been assumed that they all contain identical components. In this report we present immunocytochemical data which indicate that the cortical granule population of S. purpuratus eggs is heterogeneous. Two monoclonal antibodies are shown to react to the spiral lamellae region of approximately 20% of the cortical granules, implying that the contents of the reactive granules differ from the contents of the majority of the population. An egg protein of greater than 320 kDa is recognized by the antibody. These antibodies also stain a 130-kDa protein expressed on the surface of primary mesenchyme cells in later development. Both antibodies recognize a post-translational modification of this protein. This suggests that an antigenically similar epitope is present both on the 130-kDa primary mesenchyme cell-specific protein and in the cortical granules. To determine if the primary mesenchyme and cortical granule proteins are related, a fusion protein antibody specific for a region of the 130-kDa protein was used to stain unfertilized eggs. This antibody did not stain cortical granules. Thus, 20% of the cortical granules contain a molecule that has an epitope antigenically similar to the post-translational modification recognized in primary mesenchyme cells by the monoclonal antibodies.

Localization and expression of msp130, a primary mesenchyme lineage-specific cell surface protein in the sea urchin embryo.

In this report, we use a monoclonal antibody (B2C2) and antibodies against a fusion protein (Leaf et al. 1987) to characterize msp130, a cell surface protein specific to the primary mesenchyme cells of the sea urchin embryo. This protein first appears on the surface of these cells upon ingression into the blastocoel. Immunoelectronmicroscopy shows that msp130 is present in the trans side of the Golgi apparatus and on the extracellular surface of primary mesenchyme cells. Four precursor proteins to msp130 are identified and we show that B2C2 recognizes only the mature form of msp130. We demonstrate that msp130 contains N-linked carbohydrate groups and that the B2C2 epitope is sensitive to endoglycosidase F digestion. Evidence that msp130 is apparently a sulphated glycoprotein is presented. The recognition of the B2C2 epitope of msp130 is disrupted when embryos are cultured in sulphate-free sea water. In addition, two-dimensional immunoblots show that msp130 is an acidic protein that becomes substantially less acidic in the absence of sulphate. We also show that two other independently derived monoclonal antibodies, IG8 (McClay et al. 1983; McClay, Matranga & Wessel, 1985) and 1223 (Carson et al. 1985), recognize msp130, and suggest this protein to be a major cell surface antigen of primary mesenchyme cells.