Early-wave macrophages control late hematopoiesis.

Macrophages constitute the first defense line against the non-self, but their ability to remodel their environment in organ development/homeostasis is starting to be appreciated. Early-wave macrophages (EMs), produced from hematopoietic stem cell (HSC)-independent progenitors, seed the mammalian fetal liver niche wherein HSCs expand and differentiate. The involvement of niche defects in myeloid malignancies led us to identify the cues controlling HSCs. In Drosophila, HSC-independent EMs also colonize the larva when late hematopoiesis occurs. The evolutionarily conserved immune system allowed us to investigate whether/how EMs modulate late hematopoiesis in two models. We show that loss of EMs in Drosophila and mice accelerates late hematopoiesis, which does not correlate with inflammation and does not rely on macrophage phagocytic ability. Rather, EM-derived extracellular matrix components underlie late hematopoiesis acceleration. This demonstrates a developmental role for EMs.

Gcm counteracts Toll-induced inflammation and impacts hemocyte number through cholinergic signaling.

Hemocytes, the myeloid-like immune cells of Drosophila, fulfill a variety of functions that are not completely understood, ranging from phagocytosis to transduction of inflammatory signals. We here show that downregulating the hemocyte-specific Glial cell deficient/Glial cell missing (Glide/Gcm) transcription factor enhances the inflammatory response to the constitutive activation of the Toll pathway. This correlates with lower levels of glutathione S-transferase, suggesting an implication of Glide/Gcm in reactive oxygen species (ROS) signaling and calling for a widespread anti-inflammatory potential of Glide/Gcm. In addition, our data reveal the expression of acetylcholine receptors in hemocytes and that Toll activation affects their expressions, disclosing a novel aspect of the inflammatory response mediated by neurotransmitters. Finally, we provide evidence for acetylcholine receptor nicotinic acetylcholine receptor alpha 6 (nAchRalpha6) regulating hemocyte proliferation in a cell autonomous fashion and for non-cell autonomous cholinergic signaling regulating the number of hemocytes. Altogether, this study provides new insights on the molecular pathways involved in the inflammatory response.

Temporal specificity and heterogeneity of Drosophila immune cells.

Immune cells provide defense against non-self and have recently been shown to also play key roles in diverse processes such as development, metabolism, and tumor progression. The heterogeneity of Drosophila immune cells (hemocytes) remains an open question. Using bulk RNA sequencing, we find that the hemocytes display distinct features in the embryo, a closed and rapidly developing system, compared to the larva, which is exposed to environmental and metabolic challenges. Through single-cell RNA sequencing, we identify fourteen hemocyte clusters present in unchallenged larvae and associated with distinct processes, e.g., proliferation, phagocytosis, metabolic homeostasis, and humoral response. Finally, we characterize the changes occurring in the hemocyte clusters upon wasp infestation, which triggers the differentiation of a novel hemocyte type, the lamellocyte. This first molecular atlas of hemocytes provides insights and paves the way to study the biology of the Drosophila immune cells in physiological and pathological conditions.

Embryonic hematopoiesis modulates the inflammatory response and larval hematopoiesis in Drosophila.

Recent lineage tracing analyses have significantly improved our understanding of immune system development and highlighted the importance of the different hematopoietic waves. The current challenge is to understand whether these waves interact and whether this affects the function of the immune system. Here we report a molecular pathway regulating the immune response and involving the communication between embryonic and larval hematopoietic waves in Drosophila. Down-regulating the transcription factor Gcm specific to embryonic hematopoiesis enhances the larval phenotypes induced by over-expressing the pro-inflammatory Jak/Stat pathway or by wasp infestation. Gcm works by modulating the transduction of the Upd cytokines to the site of larval hematopoiesis and hence the response to chronic (Jak/Stat over-expression) and acute (wasp infestation) immune challenges. Thus, homeostatic interactions control the function of the immune system in physiology and pathology. Our data also indicate that a transiently expressed developmental pathway has a long-lasting effect on the immune response.

An evolutionary conserved interaction between the Gcm transcription factor and the SF1 nuclear receptor in the female reproductive system.

NR5A1 is essential for the development and for the function of steroid producing glands of the reproductive system. Moreover, its misregulation is associated with endometriosis, which is the first cause of infertility in women. Hr39, the Drosophila ortholog of NR5A1, is expressed and required in the secretory cells of the spermatheca, the female exocrine gland that ensures fertility by secreting substances that attract and capacitate the spermatozoids. We here identify a direct regulator of Hr39 in the spermatheca: the Gcm transcription factor. Furthermore, lack of Gcm prevents the production of the secretory cells and leads to female sterility in Drosophila. Hr39 regulation by Gcm seems conserved in mammals and involves the modification of the DNA methylation profile of mNr5a1. This study identifies a new molecular pathway in female reproductive system development and suggests a role for hGCM in the progression of reproductive tract diseases in humans.

The Gcm/Glide molecular and cellular pathway: new actors and new lineages.

In Drosophila, the transcription factor Gcm/Glide plays a key role in cell fate determination and cellular differentiation. In light of its crucial biological impact, major efforts have been put for analyzing its properties as master regulator, from both structural and functional points of view. However, the lack of efficient antibodies specific to the Gcm/Glide protein precluded thorough analyses of its regulation and activity in vivo. In order to relieve such restraints, we designed an epitope-tagging approach to "FLAG"-recognize and analyze the functional protein both in vitro (exogenous Gcm/Glide) and in vivo (endogenous protein). We here (i) reveal a tight interconnection between the small RNA and the Gcm/Glide pathways. AGO1 and miR-1 are Gcm/Glide targets whereas miR-279 negatively controls Gcm/Glide expression (ii) identify a novel cell population, peritracheal cells, expressing and requiring Gcm/Glide. Peritracheal cells are non-neuronal neurosecretory cells that are essential in ecdysis. In addition to emphasizing the importance of following the distribution and the activity of endogenous proteins in vivo, this study provides new insights and a novel frame to understand the Gcm/Glide biology.

The ligand-binding domains of the three RXR-USP nuclear receptor types support distinct tissue and ligand specific hormonal responses in transgenic Drosophila.

In insects, 20-hydroxyecdysone acts by binding on a heterodimer constituted by the ecdysone receptor (EcR) and Ultraspiracle (USP), the homolog to the vertebrate retinoid X receptor (RXR). Two types of USP have been characterized based on their structure and function, Mecopterida USP (Diptera/Lepidoptera USP), in particular the fruitfly Drosophila melanogaster USP (DmUSP) and non Mecopterida USP, exemplified by the beetle Tribolium castaneum USP (TcUSP) both showing structural differences from the vertebrate RXR. Here, by combining in vivo and organ culture observations in Drosophila transgenic animals, we show that ectopic expression of GAL4-DmUSP, GAL4-TcUSP or GAL4-HsRXR results in tissue- and ligand-dependent activities. In parallel, we show that neither juvenile hormone (JH) nor the related methyl farnesoate has an effect on GAL4-USP activation although JH induces the expression of a factor inhibiting the receptor transcriptional activity in the presence of EcR or RXR agonists. This study suggests that not only is USP important for hormonal regulation, via heterodimer formation, but that tissue-specific expression of cofactors may represent a higher level of control of this regulation. This in vivo approach should lead to a better understanding of the modes of action of USP and the identification of transcriptional cofactors essential for its function.

pannier encodes two structurally related isoforms that are differentially expressed during Drosophila development and display distinct functions during thorax patterning.

Previous studies have shown that the pannier (pnr) gene of Drosophila encodes a GATA transcription factor which is involved in various biological processes, including heart development, dorsal closure during embryogenesis as well as neurogenesis and regulation of wingless (wg) expression during imaginal development. We demonstrate here that pnr encodes two highly related isoforms that share functional domains but are differentially expressed during development. Moreover, we describe two genomic regions of the pnr locus that drive expression of a reporter in transgenic flies in patterns that recapitulate essential features of the expression of the isoforms, suggesting that these regions encompass crucial regulatory elements. These elements contain, in particular, sequences mediating regulation of expression by Decapentaplegic (Dpp) signaling, during both embryogenesis and imaginal development. Analysis of pnr alleles reveals that the isoforms differentially regulate expression of both wg and proneural achaete/scute (as/sc) targets during imaginal development. Pnr function has been demonstrated to be necessary both for activation of wg and, together with U-shaped (Ush), for its repression in the dorsal-most region of the presumptive notum. Expression of the isoforms define distinct longitudinal domains and, in this regard, we importantly show that the dual function of pnr during regulation of wg is achieved by one isoform repressing expression of the morphogen in the dorsal-most region of the disc while the other laterally promotes activation of the notal wg expression. Our study provides novel insights into pnr function during Drosophila development and extends our knowledge of the roles of prepattern factors during thorax patterning.