Towards an integrated understanding of inflammatory pathway influence on hematopoietic stem and progenitor cell differentiation.

Recent research highlights that inflammatory signaling pathways such as pattern recognition receptor (PRR) signaling and inflammatory cytokine signaling play an important role in both on-demand hematopoiesis as well as steady-state hematopoiesis. Knockout studies have demonstrated the necessity of several distinct pathways in these processes, but often lack information about the contribution of specific cell types to the phenotypes in question. Transplantation studies have increased the resolution to the level of specific cell types by testing the necessity of inflammatory pathways specifically in donor hematopoietic stem and progenitor cells (HSPCs) or in recipient niche cells. Here, we argue that for an integrated understanding of how these processes occur in vivo and to inform the development of therapies that modulate hematopoietic responses, we need studies that knockout inflammatory signaling receptors in a cell-specific manner and compare the phenotypes caused by knockout in individual niche cells versus HSPCs.

Injury-induced inflammatory signaling and hematopoiesis in Drosophila.

Inflammatory response in Drosophila to sterile (axenic) injury in embryos and adults has received some attention in recent years, and most concentrate on the events at the injury site. Here we focus on the effect sterile injury has on the hematopoietic organ, the lymph gland, and the circulating blood cells in the larva, the developmental stage at which major events of hematopoiesis are evident. In mammals, injury activates Toll-like receptor/NF-κB signaling in macrophages, which then express and secrete secondary, proinflammatory cytokines. In Drosophila larvae, distal puncture injury of the body wall epidermis causes a rapid activation of Toll and Jun kinase (JNK) signaling throughout the hematopoietic system and the differentiation of a unique blood cell type, the lamellocyte. Furthermore, we find that Toll and JNK signaling are coupled in their activation. Secondary to this Toll/JNK response, a cytokine, Upd3, is induced as a Toll pathway transcriptional target, which then promotes JAK/STAT signaling within the blood cells. Toll and JAK/STAT signaling are required for the emergence of the injury-induced lamellocytes. This is akin to the derivation of specialized macrophages in mammalian systems. Upstream, at the injury site, a Duox- and peroxide-dependent signal causes the activation of the proteases Grass and SPE, needed for the activation of the Toll-ligand Spz, but microbial sensors or the proteases most closely associated with them during septic injury are not involved in the axenic inflammatory response.

Intermediate progenitor cells provide a transition between hematopoietic progenitors and their differentiated descendants.

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 owing 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 are a distinct cell type with a unique cell-cycle profile and they remain multipotent for all blood cell fates. In addition, 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. This study suggests that it would be useful to characterize such intermediate populations of cells in mammalian hematopoietic systems.

Paths and pathways that generate cell-type heterogeneity and developmental progression in hematopoiesis.

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.

Drosophila as a Genetic Model for Hematopoiesis.

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.

Quantitative framework for ordered degradation of APC/C substrates.

BACKGROUND: During 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/C(Cdc20), a key regulator of chromosome segregation in mitosis. RESULTS: We show experimentally that the rate of catalysis varies with different substrates of APC/C(Cdc20). Using a computational model based on multi-step ubiquitination, we then show how changes in the interaction between a single substrate and APC/C(Cdc20) can alter the timing of degradation onset relative to APC/C(Cdc20) 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/C(Cdc20), their relative enzyme affinities and rates of catalysis influence the partitioning of APC/C(Cdc20) among substrates, resulting in substrate competition. Depending on how APC/C(Cdc20) 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/C(Cdc20) 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. CONCLUSIONS: The degradation timing of APC/C(Cdc20) substrates depends on the multi-step nature of ubiquitination, differences in substrate-APC/C(Cdc20) 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.

An E2 accessory domain increases affinity for the anaphase-promoting complex and ensures E2 competition.

The anaphase-promoting complex/cyclosome (APC/C) is a member of the RING family of E3 ubiquitin ligases, which promote ubiquitin transfer from an E2 ubiquitin-conjugating enzyme to a substrate. In budding yeast, the APC/C collaborates with two E2s, Ubc4 and Ubc1, to promote the initiation and elongation, respectively, of polyubiquitin chains on the substrate. Ubc4 and Ubc1 are thought to compete for the same site on the APC/C, but it is not clear how their affinities are balanced. Here, we demonstrate that a C-terminal ubiquitin-associated (UBA) domain enhances the affinity of Ubc1 for the APC/C. Deletion of the UBA domain reduced apparent APC/C affinity for Ubc1 and decreased polyubiquitin chain length. Surprisingly, the positive effect of the UBA domain was not due to an interaction with the acceptor ubiquitin attached to the APC/C substrate or the donor ubiquitin attached to Ubc1 itself. Instead, our evidence suggests that the UBA domain binds to a site on the APC/C core, thereby increasing Ubc1 affinity and enhancing its ability to compete with Ubc4. The UBA domain is required for normal Ubc1 function and E2 competition in vivo. Thus, the UBA domain of Ubc1 ensures efficient polyubiquitination of substrate by balancing Ubc1 affinity with that of Ubc4.