RESUMEN
We employed robotic methods and the whole-genome sequence of Drosophila melanogaster to facilitate a large-scale expression screen for spatially restricted transcripts in Drosophila embryos. In this screen, we identified a pair of genes, scylla (scyl) and charybde (chrb), that code for dorsal transcripts in early Drosophila embryos and are homologous to the human apoptotic gene RTP801. In Drosophila, both gene products are transcriptionally regulated targets of Dpp/Zen-mediated signal transduction and appear more generally to be downstream targets of homeobox regulation. Gene disruption studies revealed the functional redundancy of scyl and chrb, as well as their requirement for embryonic head involution. From the perspective of functional genomics, our studies demonstrate that global surveys of gene expression can complement traditional genetic screening methods for the identification of genes essential for development: beginning from their spatio-temporal expression profiles and extending to their downstream placement relative to dpp and zen, our studies reveal roles for the scyl and chrb gene products as links between patterning and cell death.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Tipificación del Cuerpo , Muerte Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Regulación del Desarrollo de la Expresión Génica , Cabeza/embriología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Proteínas de Homeodominio/fisiología , Humanos , Datos de Secuencia Molecular , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Transducción de SeñalRESUMEN
Dorsal closure in the fruit fly Drosophila melanogaster is a complex morphogenetic process, driven by sequential signaling cascades and involving multiple forces, which contribute to cell movements and rearrangements as well as to changes in cell shape. During closure, lateral epidermal cells elongate along the dorsoventral axis and subsequently spread dorsally to cover the embryonic dorsal surface. Amnioserosal cells, which are the original occupants of the most dorsal position in the developing embryo, constrict during closure; thus, the increase in epidermal surface area is accommodated by a reduction in the amnioserosal surface area. Several of the epidermal requirements for closure have been established in functional assays. In contrast, amnioserosal requirements for closure have remained elusive, in part because laser ablation and clonal approaches are limited to only subsets of amnioserosal cells. Here, we report our use of the UAS-GAL4 system to target expression of the cell autonomous toxin Ricin-A to all cells of the amnioserosa. We show that ablation of the amnioserosa leads to clear defects in dorsal closure and, thus, directly demonstrate a role for the amnioserosa in dorsal closure. We also show that DJNK (Drosophila Jun N-terminal kinase) signaling, an epidermal trigger of closure, is unaffected by amnioserosal ablation. These data, together with our demonstration that amnioserosal ablated and Dpp signaling mutant embryos exhibit shared loss-of-function phenotypes, point to a requirement for the amnioserosa in dorsal closure that is downstream of Dpp, perhaps as part of a paracrine response to this signaling cascade.
Asunto(s)
Amnios/embriología , Tipificación del Cuerpo , Drosophila/embriología , Drosophila/genética , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Amnios/efectos de los fármacos , Amnios/enzimología , Animales , Animales Modificados Genéticamente , Movimiento Celular , Forma de la Célula , Drosophila/citología , Drosophila/enzimología , Proteínas de Drosophila/genética , Embrión no Mamífero , Células Epidérmicas , Epidermis/efectos de los fármacos , Epidermis/embriología , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Modelos Biológicos , Mutación , Ricina/genética , Ricina/metabolismo , Ricina/farmacología , Transducción de Señal , Transcripción Genética , Transgenes , beta-Galactosidasa/metabolismoRESUMEN
Here we describe a high-throughput screen to isolate transcripts with spatially restricted patterns of expression in early embryos. Our approach utilizes robotic automation for rapid analysis of sequence-selected cDNAs in a whole-mount in situ hybridization assay. We determined the spatial distribution of a random collection of 778 different genes from an embryonic cDNA library and show that a significant fraction of these exhibit patterned profiles of expression. In addition, gene ontology studies revealed groups of gene products exhibiting shared expression patterns, providing new insights into the largely overlooked effector molecules that function in development. As described in this paper, automated hybridization to whole-mount embryos in situ proved to be straightforward and provided us with a very powerful method for the global survey of gene expression in early embryos. From the perspective of biological significance, our finding that many spatially restricted transcripts correspond to loci encoding novel transcripts that have not been previously identified in nearly saturating genetic screens for maternal effect and zygotic lethals is particularly notable.
Asunto(s)
Drosophila/embriología , Drosophila/genética , Perfilación de la Expresión Génica/métodos , ARN Mensajero/genética , Animales , ADN Complementario/clasificación , ADN Complementario/genética , ADN Complementario/fisiología , Embrión no Mamífero/química , Genes de Insecto/genética , Análisis de Secuencia de ADN/métodos , Transcripción GenéticaRESUMEN
Protein phosphorylation and specific protein kinases can initiate signal transduction pathways leading to programmed cell death. The specific protein phosphatases regulating apoptosis have been more elusive. Using double-stranded RNA-mediated interference (RNAi), the role of protein phosphatase 2A (PP2A) in cellular signaling was investigated. Knockdown of A or C subunits individually or of combined B subunits led to concurrent loss of nontargeted PP2A subunits, suggesting that PP2A is an obligate heterotrimer in vivo. Global knockdown of PP2A activity or specific loss of redundant B56 regulatory subunits caused cell death with the morphological and biochemical changes characteristic of apoptosis in cultured S2 cells. B56:PP2A-regulated apoptosis required caspases and the upstream regulators dark, reaper, head involution defective, and dp53. In Drosophila embryos, knockdown of B56-regulated PP2A activity resulted in apoptosis and failure of gastrulation, an effect that was blocked by concurrent RNAi of the caspase DRICE: B56-regulated PP2A activity appears to be required upstream of dp53 to maintain a critical proapoptotic substrate in a dephosphorylated, inactive state, thereby preventing apoptosis in Drosophila S2 cells.