An in vivo RNA interference screen identifies gene networks controlling Drosophila melanogaster blood cell homeostasis

<p>Abstract</p> <p>Background</p> <p>In metazoans, the hematopoietic system plays a key role both in normal development and in defense of the organism. In Drosophila, the cellular immune response involves three types of blood cells: plasmatocytes, crystal cells and lamellocytes. This last cell type is barely present in healthy larvae, but its production is strongly induced upon wasp parasitization or in mutant contexts affecting larval blood cell homeostasis. Notably, several zygotic mutations leading to melanotic mass (or "tumor") formation in larvae have been associated to the deregulated differentiation of lamellocytes. To gain further insights into the gene regulatory network and the mechanisms controlling larval blood cell homeostasis, we conducted a tissue-specific loss of function screen using hemocyte-specific Gal4 drivers and <it>UAS-dsRNA </it>transgenic lines.</p> <p>Results</p> <p>By targeting around 10% of the Drosophila genes, this <it>in vivo </it>RNA interference screen allowed us to recover 59 melanotic tumor suppressor genes. In line with previous studies, we show that melanotic tumor formation is associated with the precocious differentiation of stem-cell like blood progenitors in the larval hematopoietic organ (the lymph gland) and the spurious differentiation of lamellocytes. We also find that melanotic tumor formation can be elicited by defects either in the fat body, the embryo-derived hemocytes or the lymph gland. In addition, we provide a definitive confirmation that lymph gland is not the only source of lamellocytes as embryo-derived plasmatocytes can differentiate into lamellocytes either upon wasp infection or upon loss of function of the Friend of GATA cofactor U-shaped.</p> <p>Conclusions</p> <p>In this study, we identify 55 genes whose function had not been linked to blood cell development or function before in Drosophila. Moreover our analyses reveal an unanticipated plasticity of embryo-derived plasmatocytes, thereby shedding new light on blood cell lineage relationship, and pinpoint the Friend of GATA transcription cofactor U-shaped as a key regulator of the plasmatocyte to lamellocyte transformation.</p

Adenine methylation is very scarce in the Drosophila genome and not erased by the ten-eleven translocation dioxygenase

N6-methyladenine (6mA) DNA modification has recently been described in metazoans, including in Drosophila, for which the erasure of this epigenetic mark has been ascribed to the ten-eleven translocation (TET) enzyme. Here, we re-evaluated 6mA presence and TET impact on the Drosophila genome. Using axenic or conventional breeding conditions, we found traces of 6mA by LC-MS/MS and no significant increase in 6mA levels in the absence of TET, suggesting that this modification is present at very low levels in the Drosophila genome but not regulated by TET. Consistent with this latter hypothesis, further molecular and genetic analyses showed that TET does not demethylate 6mA but acts essentially in an enzymatic-independent manner. Our results call for further caution concerning the role and regulation of 6mA DNA modification in metazoans and underline the importance of TET non-enzymatic activity for fly development

Drosophila TET acts with PRC1 to activate gene expression independently of its catalytic activity

Enzymes of the ten-eleven translocation (TET) family play a key role in the regulation of gene expression by oxidizing 5-methylcytosine (5mC), a prominent epigenetic mark in many species. Yet, TET proteins also have less characterized noncanonical modes of action, notably in Drosophila, whose genome is devoid of 5mC. Here, we show that Drosophila TET activates the expression of genes required for larval central nervous system (CNS) development mainly in a catalytic-independent manner. Genome-wide profiling shows that TET is recruited to enhancer and promoter regions bound by Polycomb group complex (PcG) proteins. We found that TET interacts and colocalizes on chromatin preferentially with Polycomb repressor complex 1 (PRC1) rather than PRC2. Furthermore, PRC1 but not PRC2 is required for the activation of TET target genes. Last, our results suggest that TET and PRC1 binding to activated genes is interdependent. These data highlight the importance of TET noncatalytic function and the role of PRC1 for gene activation in the Drosophila larval CNS

The Ly6 Protein Coiled Is Required for Septate Junction and Blood Brain Barrier Organisation in Drosophila

Background: Genetic analysis of the Drosophila septate junctions has greatly contributed to our understanding of the mechanisms controlling the assembly of these adhesion structures, which bear strong similarities with the vertebrate tight junctions and the paranodal septate junctions. These adhesion complexes share conserved molecular components and have a common function: the formation of paracellular barriers restraining the diffusion of solutes through epithelial and glial envelopes. Methodology/Principal Findings: In this work we characterise the function of the Drosophila cold gene, that codes for a protein belonging to the Ly6 superfamily of extracellular ligands. Analysis of cold mutants shows that this gene is specifically required for the organisation of the septate junctions in epithelial tissues and in the nervous system, where its contribution is essential for the maintenance of the blood-brain barrier. We show that cold acts in a cell autonomous way, and we present evidence indicating that this protein could act as a septate junction component. Conclusion/Significance: We discuss the specific roles of cold and three other Drosophila members of the Ly6 superfamily that have been shown to participate in a non-redundant way in the process of septate junction assembly. We propose tha

Macrophage Functions in Tissue Patterning and Disease:New Insights from the Fly

Macrophages are multifunctional innate immune cells that seed all tissues within the body and play disparate roles throughout development and in adult tissues, both in health and disease. Their complex developmental origins and many of their functions are being deciphered in mammalian tissues, but opportunities for live imaging and the genetic tractability of Drosophila are offering complementary insights into how these fascinating cells integrate a multitude of guidance cues to fulfill their many tasks and migrate to distant sites to either direct developmental patterning or raise an inflammatory response.Published versio

Characterization of the Drosophila Adult Hematopoietic System Reveals a Rare Cell Population With Differentiation and Proliferation Potential

International audienc

Mécanismes de régulation de l'hématopoïèse embryonnaire chez la Drosophile

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Les Cellules hématopoïétiques de la drosophile comme modèle d'étude in vivo de l'activité de l'oncogène humain AML1-ETO

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Teashirt is required for transcriptional repression mediated by high Wingless levels

During development, extracellular signals often act at multiple thresholds to specify distinct transcriptional and cellular responses. For example, in the embryonic midgut of Drosophila, low Wingless levels stimulate the transcription of homeotic genes whereas high Wingless levels repress these genes. Wingless- mediated transcriptional activation is conferred by Drosophila T-cell factor (dTCF) and its co-activator Armadillo, but the nuclear factors mediating transcriptional repression are unknown. Here we show that teashirt is required for Wingless-mediated repression of Ultrabithorax in the midgut. Teashirt is also a repressor of the homeotic gene labial in this tissue. Furthermore, the target sequence for Tsh within the Ultrabithorax midgut enhancer coincides with the response sequence for Wingless-mediated repression. Finally, we demonstrate that the zinc finger protein Teashirt behaves as a transcriptional repressor in transfected mammalian cells. It thus appears that the response to high Wingless levels in the Drosophila midgut is indirect and based on transcriptional activation of the Teashirt repressor