Hematopoietic progenitors in Drosophila. Micro-environment and fate studies in normal and following parasitism. – prohemodroso

Background: The innate immune response confers all multicellular organisms broad protection against pathogens. Drosophila has become a major model organism to study hematopoiesis and cellular aspects of the innate immune response. As in vertebrates, Drosophila hematopoiesis occurs in two phases. A first population of hemocyte precursors (pro-hemocytes) is specified in embryos. A second 'definitive' population originates from a specialised hematopoietic organ, the lymph gland (LG). Both populations give rise to plasmatocytes/macrophages which are involved in phagocytosis and share morphological and functional properties with the mammalian monocyte/macrophage lineage and crystal cells which are involved in melanisation. Hematopoietic progenitors in the LG can also give rise to a third type of hemocytes, the lamellocytes which are devoted to the encapsulation of foreign bodies too large to be phagocytosed. The lamellocyte is only 'revealed' in specific conditions of immune challenge, such as parasitism by wasps, a common threat to many insects. We initially found that expression of Collier (Col), the Drosophila ortholog of mammalian Early B-Cell Factor (EBF), defined a small group of posterior cells of the embrionic LG. These cells prefigure the so called PSC, identified in 3rd instar larvae through the expression of the Notch-ligand Serrate (Lebestky, Jung et al. 2003; Crozatier, Ubeda et al. 2004) but whose function remained essentially unknown. We then showed that Col activity in the PSC is required to control the balance between multipotent pro-hemocytes and differentiating hemocytes in the LG. PSC cells act, in a non cell autonomous manner, to maintain JAK/STAT signalling activity in prohemocytes, preventing their premature differentiation and preserving a multipotent character necessary for these cells to adopt a lamellocyte fate in response to parasitism. This key role of the PSC thus revealed that larval hemocyte homeostasis is dependent upon interactions between hematopoietic progenitors and their micro-environment in insects, as is the case in vertebrates (Krzemien 2007). Of particular interest was the recent finding that mouse EBF2 is expressed in immature osteoblasts that contribute to the HSC niche, the specific microenvironment in which stem cells are housed and maintained by allowing self-renewal in the absence of differentiation (Calvi, Adams et al. 2003; Kieslinger, Folberth et al. 2005; Wilson and Trumpp 2006) and M. Kieslinger, personal communication). This novel parallel between the Drosophila PSC and the mammalian HSC niche further highlights the value of Drosophila as a model system to define the cellular micro-environment and signals that support hematopoietic progenitor cells. The objectives of our proposed research program include : a) Determine whether hematopoietic stem cells exist in the Drosophila LG and if so, whether secondary 'niches' exist in the adult. b) Identify which components of the JAK-STAT signalling pathway are involved in the maintainance of hemocyte homeostasis and establish the molecular details of this regulation. c) Identify new genes specifically expressed in the PSC, the hematopoietic progenitors, and the differentiating lamellocytes using both transcriptome analyses and a computational approach. d) Characterise some of the new genes identified in our different screens. Selection of the most promising candidates for a role in contributing the micro-environment of hematopoietic progenitors will take into account the latest data on EBF2 function. e) Identify genes directly regulated by Col by adapting a new affinity purification procedure for selecting Col-containing chromatin fragments. Methodology: We will use a combination of standard and novel genetic and transgenic approaches, including homologous recombination, dsRNA gene extinction in cultured cells and in vivo, and recombinase-mediated trangenesis, in vivo imaging and cell lineage analyses. Our experimental design includes systematic integration of datasets from micro-arrays and computational predictions (in collaboration). We will undertake an in vivo profiling of Col binding sites by adapting a newly developed Biotin-Tag affinity purification protocol for isolating chromatin fragments (3-years salary for an assistant-engineer is requested). Conclusion Deciphering in more detail the role of Col and its direct targets in the control of Drosophila hematopoiesis should provide important new knowledge on a primary aspect of innate immunity that is the control of quiescence/proliferation of hematopoietic cells in physiological conditions and the cellular response to specific immune threats. Together with ongoing studies on the role of EBF2 in vertebrate hematopoiesis, our research program should shed novel light on the emergence and diversification of microenvironments for hematopoietic cells in the course of metazoan evolution.