In this FlyBook chapter, we present a survey of the current literature on the development of the hematopoietic system in Drosophila. The Drosophila blood system consists entirely of cells that function in innate immunity, tissue integrity, wound healing, and various forms of stress response, and are therefore functionally similar to myeloid cells in mammals. The primary cell types are specialized for phagocytic, melanization, and encapsulation functions. As in mammalian systems, multiple sites of hematopoiesis are evident in Drosophila and the mechanisms involved in this process employ many of the same molecular strategies that exemplify blood development in humans. Drosophila blood progenitors respond to internal and external stress by coopting developmental pathways that involve both local and systemic signals. An important goal of these Drosophila studies is to develop the tools and mechanisms critical to further our understanding of human hematopoiesis during homeostasis and dysfunction.

Mechanistic studies of Drosophila lymph gland hematopoiesis are limited by the availability of cell-type-specific markers. Using a combination of bulk RNA-Seq of FACS-sorted cells, single-cell RNA-Seq, and genetic dissection, we identify new blood cell subpopulations along a developmental trajectory with multiple paths to mature cell types. This provides functional insights into key developmental processes and signaling pathways. We highlight metabolism as a driver of development, show that graded Pointed expression allows distinct roles in successive developmental steps, and that mature crystal cells specifically express an alternate isoform of Hypoxia-inducible factor (Hif/Sima). Mechanistically, the Musashi-regulated protein Numb facilitates Sima-dependent non-canonical, and inhibits canonical, Notch signaling. Broadly, we find that prior to making a fate choice, a progenitor selects between alternative, biologically relevant, transitory states allowing smooth transitions reflective of combinatorial expressions rather than stepwise binary decisions. Increasingly, this view is gaining support in mammalian hematopoiesis.

BackgroundDuring cell-cycle progression, substrates of a single master regulatory enzyme can be modified in a specific order. Here, we used experimental and computational approaches to dissect the quantitative mechanisms underlying the ordered degradation of the substrates of the ubiquitin ligase APC/CCdc20, a key regulator of chromosome segregation in mitosis.ResultsWe show experimentally that the rate of catalysis varies with different substrates of APC/CCdc20. Using a computational model based on multi-step ubiquitination, we then show how changes in the interaction between a single substrate and APC/CCdc20 can alter the timing of degradation onset relative to APC/CCdc20 activation, while ensuring a fast degradation rate. Degradation timing and dynamics depend on substrate affinity for the enzyme as well as the catalytic rate at which the substrate is modified. When two substrates share the same pool of APC/CCdc20, their relative enzyme affinities and rates of catalysis influence the partitioning of APC/CCdc20 among substrates, resulting in substrate competition. Depending on how APC/CCdc20 is partitioned among its substrates, competition can have minor or major effects on the degradation of certain substrates. We show experimentally that increased expression of the early APC/CCdc20 substrate Clb5 does not delay the degradation of the later substrate securin, arguing against a role for competition with Clb5 in establishing securin degradation timing.ConclusionsThe degradation timing of APC/CCdc20 substrates depends on the multi-step nature of ubiquitination, differences in substrate-APC/CCdc20 interactions, and competition among substrates. Our studies provide a conceptual framework for understanding how ordered modification can be established among substrates of the same regulatory enzyme, and facilitate our understanding of how precise temporal control is achieved by a small number of master regulators to ensure a successful cell division cycle.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-015-0205-6) contains supplementary material, which is available to authorized users.

Significance We explore mechanisms by which stress caused by acute injury affects blood cell development and inflammatory response in Drosophila . Similar to their mammalian myeloid counterparts, these cells are predisposed to sense and react to sterile injury at distant sites. Upon sterile injury, a breach of epidermis sets up a reactive oxygen species-based signal that bypasses the pathogen-sensing apparatus of septic immune challenge, but merges downstream to activate Toll. A number of autonomous and nonautonomous signaling pathways follow in a sequence and are mapped temporally by the appearance of their corresponding molecular phenotypes. A cell-type that fights deposited parasitic wasp eggs appears with sterile injury without the immune challenge, perhaps in anticipation, because in nature injury is usually followed by infection.

Genetic and genomic analysis in Drosophila suggests that hematopoietic progenitors likely transition into terminal fates via intermediate progenitors (IPs) with some characteristics of either, but perhaps maintaining IP-specific markers. In the past, IPs have not been directly visualized and investigated due to lack of appropriate genetic tools. Here we report a split-GAL4 construct, CHIZ-GAL4, that identifies IPs as cells physically juxtaposed between true progenitors and differentiating hemocytes. IPs comprise a distinct cell type with a unique cell-cycle profile and they remain multipotent for all blood cell fates. Additionally, through their dynamic control of the Notch ligand, Serrate, IPs specify the fate of direct neighbors. The Ras pathway controls the number of IP cells and promotes their transition into differentiating cells. The split-GAL4 strategy is amenable for adoption in mammalian systems and would be invaluable in assigning trajectories that stem and progenitor populations follow as they develop into mature blood cells.