Genome-wide phenotypic RNAi screen in the Drosophila wing: phenotypic description of functional classes.

The Drosophila genome contains approximately 14,000 protein-coding genes encoding all the necessary information to sustain cellular physiology, tissue organization, organism development, and behavior. In this manuscript, we describe in some detail the phenotypes in the adult fly wing generated after knockdown of approximately 80% of Drosophila genes. We combined this phenotypic description with a comprehensive molecular classification of the Drosophila proteins into classes that summarize the main expected or known biochemical/functional aspect of each protein. This information, combined with mRNA expression levels and in situ expression patterns, provides a simplified atlas of the Drosophila genome, from housekeeping proteins to the components of the signaling pathways directing wing development, that might help to further understand the contribution of each gene group to wing formation.

Genome-wide phenotypic RNAi screen in the Drosophila wing: global parameters.

We have screened a collection of UAS-RNAi lines targeting 10,920 Drosophila protein-coding genes for phenotypes in the adult wing. We identified 3653 genes (33%) whose knockdown causes either larval/pupal lethality or a mutant phenotype affecting the formation of a normal wing. The most frequent phenotypes consist of changes in wing size, vein differentiation, and patterning, defects in the wing margin and in the apposition of the dorsal and ventral wing surfaces. We also defined 16 functional categories encompassing the most relevant aspect of each protein function and assigned each Drosophila gene to one of these functional groups. This allowed us to identify which mutant phenotypes are enriched within each functional group. Finally, we used previously published gene expression datasets to determine which genes are or are not expressed in the wing disc. Integrating expression, phenotypic and molecular information offers considerable precision to identify the relevant genes affecting wing formation and the biological processes regulated by them.

Impaired stem cell differentiation and somatic cell reprogramming in DIDO3 mutants with altered RNA processing and increased R-loop levels.

Embryonic stem cell (ESC) differentiation and somatic cell reprogramming are biological processes governed by antagonistic expression or repression of a largely common set of genes. Accurate regulation of gene expression is thus essential for both processes, and alterations in RNA processing are predicted to negatively affect both. We show that truncation of the DIDO gene alters RNA splicing and transcription termination in ESC and mouse embryo fibroblasts (MEF), which affects genes involved in both differentiation and reprogramming. We combined transcriptomic, protein interaction, and cellular studies to identify the underlying molecular mechanism. We found that DIDO3 interacts with the helicase DHX9, which is involved in R-loop processing and transcription termination, and that DIDO3-exon16 deletion increases nuclear R-loop content and causes DNA replication stress. Overall, these defects result in failure of ESC to differentiate and of MEF to be reprogrammed. MEF immortalization restored impaired reprogramming capacity. We conclude that DIDO3 has essential functions in ESC differentiation and somatic cell reprogramming by supporting accurate RNA metabolism, with its exon16-encoded domain playing the main role.

DIDO as a Switchboard that Regulates Self-Renewal and Differentiation in Embryonic Stem Cells.

Transition from symmetric to asymmetric cell division requires precise coordination of differential gene expression. We show that embryonic stem cells (ESCs) mainly express DIDO3 and that their differentiation after leukemia inhibitory factor withdrawal requires DIDO1 expression. C-terminal truncation of DIDO3 (Dido3ΔCT) impedes ESC differentiation while retaining self-renewal; small hairpin RNA-Dido1 ESCs have the same phenotype. Dido3ΔCT ESC differentiation is rescued by ectopic expression of DIDO3, which binds the Dido locus via H3K4me3 and RNA POL II and induces DIDO1 expression. DIDO1, which is exported to cytoplasm, associates with, and is N-terminally phosphorylated by PKCiota. It binds the E3 ubiquitin ligase WWP2, which contributes to cell fate by OCT4 degradation, to allow expression of primitive endoderm (PE) markers. PE formation also depends on phosphorylated DIDO3 localization to centrosomes, which ensures their correct positioning for PE cell polarization. We propose that DIDO isoforms act as a switchboard that regulates genetic programs for ESC transition from pluripotency maintenance to promotion of differentiation.

The Spalt Transcription Factors Generate the Transcriptional Landscape of the Drosophila melanogaster Wing Pouch Central Region.

The Drosophila genes spalt major (salm) and spalt-related (salr) encode Zn-finger transcription factors regulated by the Decapentaplegic (Dpp) signalling pathway in the wing imaginal disc. The function of these genes is required for cell survival and proliferation in the central region of the wing disc, and also for vein patterning in the lateral regions. The identification of direct Salm and Salr target genes, and the analysis of their functions, are critical steps towards understanding the genetic control of growth and patterning of the Drosophila wing imaginal disc by the Dpp pathway. To identify candidate Salm/Salr target genes, we have compared the expression profile of salm/salr knockdown wing discs with control discs in microarray experiments. We studied by in situ hybridization the expression pattern of the genes whose mRNA levels varied significantly, and uncovered a complex transcription landscape regulated by the Spalt proteins in the wing disc. Interestingly, candidate Salm/Salr targets include genes which expression is turned off and genes which expression is positively regulated by Salm/Salr. Furthermore, loss-of-function phenotypic analysis of these genes indicates, for a fraction of them, a requirement for wing growth and patterning. The identification and analysis of candidate Salm/Salr target genes opens a new avenue to reconstruct the genetic structure of the wing, linking the activity of the Dpp pathway to the development of this epithelial tissue.