Functional analysis of the Drosophila embryonic germ cell transcriptome by RNA interference

In Drosophila melanogaster, primordial germ cells are specified at the posterior pole of the very early embryo. This process is regulated by the posterior localized germ plasm that contains a large number of RNAs of maternal origin. Transcription in the primordial germ cells is actively down-regulated until germ cell fate is established. Bulk expression of the zygotic genes commences concomitantly with the degradation of the maternal transcripts. Thus, during embryogenesis, maternally provided and zygotically transcribed mRNAs determine germ cell development collectively. In an effort to identify novel genes involved in the regulation of germ cell behavior, we carried out a large-scale RNAi screen targeting both maternal and zygotic components of the embryonic germ line transcriptome. We identified 48 genes necessary for distinct stages in germ cell development. We found pebble and fascetto to be essential for germ cell migration and germ cell division, respectively. Our data uncover a previously unanticipated role of mei-P26 in maintenance of embryonic germ cell fate. We also performed systematic co-RNAi experiments, through which we found a low rate of functional redundancy among homologous gene pairs. As our data indicate a high degree of evolutionary conservation in genetic regulation of germ cell development, they are likely to provide valuable insights into the biology of the germ line in general

dUTPase expression correlates with cell division potential in Drosophila melanogaster

dUTPase is a dNTP sanitizing enzyme that prevents the appearance of the potentially harmful uracil bases in DNA by hydrolyzing cellular dUTP. This function of dUTPase is found to be essential in many organisms including Drosophila melanogaster. Previously we showed that the expression pattern of dUTPase determines the extent of uracil accumulation in the genome of different tissues. We wished to reveal the regulatory mechanism that eventually leaves a set of tissues to have uracil-free and intact genome. We found that the expression pattern established by the promoter of Drosophila dUTPase overlaps with mRNA and protein expression pattern, excluding the involvement of other posttranscriptional contribution. This promoter was found to be active in primordial tissues, such as in imaginal discs of the larvae, in the larval brain and in reproductive organs. In the case of brain and imaginal tissues, we observed that the promoter activity depends on DRE motifs, the docking site of DREF, which is known as a transcriptional activator of genes involved in replication and proliferation. These results suggest that dUTPase expression is fine-tuned to meet the requirements of DNA synthesis, in tissues where the maintenance of genome integrity is of high importance. This article is protected by copyright. All rights reserved

Alternative Linker Histone Permits Fast Paced Nuclear Divisions in Early Drosophila Embryo

In most animals, the start of embryogenesis requires specific histones. In Drosophila linker histone variant BigH1 is present in early embryos. To uncover the specific role of this alternative linker histone at early embryogenesis, we established fly lines in which domains of BigH1 have been replaced partially or completely with that of H1. Analysis of the resulting Drosophila lines revealed that at normal temperature somatic H1 can substitute the alternative linker histone, but at low temperature the globular and C-terminal domains of BigH1 are essential for embryogenesis. In the presence of BigH1 nucleosome stability increases and core histone incorporation into nucleosomes is more rapid, while nucleosome spacing is unchanged. Chromatin formation in the presence of BigH1 permits the fast-paced nuclear divisions of the early embryo. We propose a model which explains how this specific linker histone ensures the rapid nucleosome reassembly required during quick replication cycles at the start of embryogenesis

Despite its sequence identity with canonical H4, Drosophila H4r product is enriched at specific chromatin regions

Histone variants are different from their canonical counterparts in structure and are encoded by solitary genes with unique regulation to fulfill tissue or differentiation specific functions. A single H4 variant gene (His4r or H4r) that is located outside of the histone cluster and gives rise to a polyA tailed messenger RNA via replication-independent expression is preserved in Drosophila strains despite that its protein product is identical with canonical H4. In order to reveal information on the possible role of this alternative H4 we epitope tagged endogenous H4r and studied its spatial and temporal expression, and revealed its genome-wide localization to chromatin at the nucleosomal level. RNA and immunohistochemistry analysis of H4r expressed under its cognate regulation indicate expression of the gene throughout zygotic and larval development and presence of the protein product is evident already in the pronuclei of fertilized eggs. In the developing nervous system a slight disequibrium in H4r distribution is observable, cholinergic neurons are the most abundant among H4r-expressing cells. ChIP-seq experiments revealed H4r association with regulatory regions of genes involved in cellular stress response. The data presented here indicate that H4r has a variant histone function

A Functional Genomic Screen Combined with Time-Lapse Microscopy Uncovers a Novel Set of Genes Involved in Dorsal Closure of Drosophila Embryos

Morphogenesis, the establishment of the animal body, requires the coordinated rearrangement of cells and tissues regulated by a very strictly-determined genetic program. Dorsal closure of the epithelium in the Drosophila melanogaster embryo is one of the best models for such a complex morphogenetic event. To explore the genetic regulation of dorsal closure, we carried out a large-scale RNA interference-based screen in combination with in vivo time-lapse microscopy and identified several genes essential for the closure or affecting its dynamics. One of the novel dorsal closure genes, the small GTPase activator pebble (pbl), was selected for detailed analysis. We show that pbl regulates actin accumulation and protrusion dynamics in the leading edge of the migrating epithelial cells. In addition, pbl affects dorsal closure dynamics by regulating head involution, a morphogenetic process mechanically coupled with dorsal closure. Finally, we provide evidence that pbl is involved in closure of the adult thorax, suggesting its general requirement in epithelial closure processes

A novel role for the Drosophila epsin (lqf): involvement in autophagy

Screening P-element-induced mutant collections, 52 lines were selected as potentially defected ones in endocytosis or autophagy. After excluding those which were rescued by 20-hydroxyecdysone treatment, the exact position of the inserted P-element was determined in the remaining lines. In the case of l(3)S011027 stock, the liquid facets (lqf) gene was affected which codes an epsin-homolog protein in Drosophila. We reveal that Lqf is essential to the receptor-mediated endocytosis of larval serum proteins (LSPs) in the larval fat body cells of Drosophila. In l(3)S011027 line, lack of Lqf fails the formation of autophagosomes thus leading to the arrest of destroying of trophocytes. Transgenic larvae carrying Lqf-RNAi construct were unable to generate endocytic and autophagic vacuoles and led to a prolonged larval stage. On the other hand, Lqf protein showed an exclusive colocalization with the Lyso Tracker Red- or GFP-Atg8a labeled autophagosomes. By using the antiserum generated against the fifth exon of lqf, we demonstrated that prior to the onset of developmental autophagy the Lqf protein was present in the nucleus of fat body cell, but thereafter the protein was localized in the territory of endocytic and autophagic vacuoles. The fact that the inhibition of the target of rapamycin (TOR) did not restore the autophagic process and the normal development in the case of lqf mutant larvae points to that the Lqf is downstream to the TOR, the central kinase of the autophagy pathway