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.

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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.

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.[Supplementary material available online at http://www.genome.org. The following individuals kindly provided reagents, samples, or unpublished information as indicated in the paper: N. Brown]Pattern formation represents an initial event in the development of multicellular organisms. Subsequent organismal complexity is generated from translation of the early embryonic pattern into differentiated cell types and tissues. Our current understanding of the molecular mechanisms of patterning in Drosophila embryogenesis derives in large part from an analysis of genes corresponding to embryonic lethal mutants that display defects in larval cuticular morphology Nusslein-Volhard et al. 1984;Wieschaus et al. 1984;Schupbach and Wieschaus 1989). Molecular characterization of genes essential for embryonic patterning has revealed that these genes code almost exclusively for components of signal transduction cascades and their associated transcription factors. Taken together, molecular and genetic studies have furnished us with a comprehensive understanding of how patterned transcription can arise in Drosophila embryogenesis. From a developmental perspective, the importance of patterned transcription is clear: Placement of gene products in a subset of embryonic cells causes these cells to assume fates different from their nonexpressing neighbors.In contrast to our comprehensive understanding of how embryonic patterns are established in Drosophila, our understanding of the molecular mechanisms that are employed in translating pattern into differentiated tissues and cell types is more limited. It is notable that many of the transcriptionally regulated targets of the pattern-establishing signaling cascades have escaped detection using standard methods of genetic surveillance. It has been suggested that patterned expression of single genes belonging to subgroups that are e...

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