Mammalian Hematopoiesis Research Articles

Identification of Nfel1a and Nfel3 as novel regulators for zebrafish thrombopoiesis

In mammalian hematopoiesis, megakaryocytes mature and become polyploid in the bone marrow before releasing platelets into circulation. In contrast, fish produce thrombocytes in kidney marrow, where young thrombocytes undergo maturation in circulation, akin to platelets. Despite morphological differences, our single-cell sequencing revealed significant gene expression similarities between fish thrombocytes and mammalian megakaryocytes, including Nfe2, which is crucial in platelet production. In addition to nfe2 expression, we found four nfe2-related genes, nfe2I1a, nfe2I1b, nfe2I2a, and nfe2I3, were expressed in mature thrombocytes. However, only nfe2, nfe2l2a, and nfe2l3 are expressed in young thrombocytes. Thus, we hypothesized that Nfe2-related factors may also be involved in thrombocyte production. To address this, we knocked down the four novel nfe2-related genes by the piggyback method and found both young and mature thrombocytes reduced when nfe2, nfe2l1a, and nfe2l3 were knocked down. They also exhibited greater gill bleeding and prolonged time to occlusion (TTO) compared to controls. In summary, our study shows Nfe2I1a and Nfe2l3 as novel transcription factors that are positive regulators influencing adult zebrafish thrombopoiesis.

Hematopoietic Stem Cell Fates and the Cellular Hierarchy of Mammalian Hematopoiesis: from Transplantation Models to New Insights from in Situ Analyses.

Hematopoiesis is the process that generates the cells of the blood and immune system from hematopoietic stem and progenitor cells (HSPCs) and represents the system with the most rapid cell turnover in a mammalian organism. HSPC differentiation trajectories, their underlying molecular mechanisms, and their dysfunctions in hematologic disorders are the focal research questions of experimental hematology. While HSPC transplantations in murine models are the traditional tool in this research field, recent advances in genome editing and next generation sequencing resulted in the development of many fundamentally new approaches for the analyses of mammalian hematopoiesis in situ and at single cell resolution. The current review will cover many recent developments in this field in murine models, from the bulk lineage tracing studies of HSPC differentiation to the barcoding of individual HSPCs with Cre-recombinase, Sleeping Beauty transposase, or CRISPR/Cas9 tools, to map hematopoietic cell fates, together with their transcriptional and epigenetic states. We also address studies of the clonal dynamics of human hematopoiesis, from the tracing of HSPC clonal behaviours based on viral integration sites in gene therapy patients to the recent analyses of unperturbed human hematopoiesis based on naturally accrued mutations in either nuclear or mitochondrial genomes. Such studies are revolutionizing our understanding of HSPC biology and hematopoiesis both under homeostatic conditions and in the response to various forms of physiological stress, reveal the mechanisms responsible for the decline of hematopoietic function with age, and in the future may advance the understanding and management of the diverse disorders of hematopoiesis.

Genome-Wide Methylation Profiling of Peripheral T-Cell Lymphomas Identifies TRIP13 as a Critical Driver of Tumor Proliferation and Survival.

Cytosine methylation contributes to the regulation of gene expression and normal hematopoiesis in mammals. It is catalyzed by the family of DNA methyltransferases that include DNMT1, DNMT3A, and DNMT3B. Peripheral T-cell lymphomas (PTCLs) represent aggressive mature T-cell malignancies exhibiting a broad spectrum of clinical features with poor prognosis and inadequately understood molecular pathobiology. To better understand the molecular landscape and identify candidate genes involved in disease maintenance, we profiled DNA methylation and gene expression of PTCLs. We found that the methylation patterns in PTCLs are deregulated and heterogeneous but share 767 hypo- and 567 hypermethylated differentially methylated regions (DMRs) along with 231 genes up- and 91 genes downregulated in all samples, suggesting a potential association with tumor development. We further identified 39 hypomethylated promoters associated with increased gene expression in the majority of PTCLs. This putative oncogenic signature included the TRIP13 (thyroid hormone receptor interactor 13) gene whose genetic and pharmacologic inactivation inhibited the proliferation of T-cell lines by inducing G2-M arrest and apoptosis. Our data thus show that human PTCLs have a significant number of recurrent methylation alterations that may affect the expression of genes critical for proliferation whose targeting might be beneficial in anti-lymphoma treatments.

Wnt signaling couples G2 phase control with differentiation during hematopoiesis in Drosophila

During homeostasis, a critical balance is maintained between myeloid-like progenitors and their differentiated progeny, which function to mitigate stress and innate immune challenges. The molecular mechanisms that help achieve this balance are not fully understood. Using genetic dissection in Drosophila, we show that a Wnt6/EGFR-signaling network simultaneously controls progenitor growth, proliferation, and differentiation. Unlike G1-quiescence of stem cells, hematopoietic progenitors are blocked in G2 phase by a β-catenin-independent (Wnt/STOP) Wnt6 pathway that restricts Cdc25 nuclear entry and promotes cell growth. Canonical β-catenin-dependent Wnt6 signaling is spatially confined to mature progenitors through localized activation of the tyrosine kinases EGFR and Abelson kinase (Abl), which promote nuclear entry of β-catenin and facilitate exit from G2. This strategy combines transcription-dependent and -independent forms of both Wnt6 and EGFR pathways to create a direct link between cell-cycle control and differentiation. This unique combinatorial strategy employing conserved components may underlie homeostatic balance and stress response in mammalian hematopoiesis.

Cell fate determinant Llgl1 is required for propagation of acute myeloid leukemia

Scribble complex proteins can influence cell fate decisions and self-renewal capacity of hematopoietic cells. While specific cellular functions of Scribble complex members are conserved in mammalian hematopoiesis, they appear to be highly context dependent. Using CRISPR/Cas9-based genetic screening, we have identified Scribble complex-related liabilities in AML including LLGL1. Despite its reported suppressive function in HSC self-renewal, inactivation of LLGL1 in AML confirms its relevant role for proliferative capacity and development of AML. Its function was conserved in human and murine models of AML and across various genetic backgrounds. Inactivation of LLGL1 results in loss of stemness-associated gene-expression including HoxA-genes and induces a GMP-like phenotype in the leukemia stem cell compartment. Re-expression of HoxA9 facilitates functional and phenotypic rescue. Collectively, these data establish LLGL1 as a specific dependency and putative target in AML and emphasizes its cell-type specific functions.

Inhibitor of DNA binding proteins revealed as orchestrators of steady state, stress and malignant hematopoiesis.

Adult mammalian hematopoiesis is a dynamic cellular process that provides a continuous supply of myeloid, lymphoid, erythroid/megakaryocyte cells for host survival. This process is sustained by regulating hematopoietic stem cells (HSCs) quiescence, proliferation and activation under homeostasis and stress, and regulating the proliferation and differentiation of downstream multipotent progenitor (MPP) and more committed progenitor cells. Inhibitor of DNA binding (ID) proteins are small helix-loop-helix (HLH) proteins that lack a basic (b) DNA binding domain present in other family members, and function as dominant-negative regulators of other bHLH proteins (E proteins) by inhibiting their transcriptional activity. ID proteins are required for normal T cell, B cell, NK and innate lymphoid cells, dendritic cell, and myeloid cell differentiation and development. However, recent evidence suggests that ID proteins are important regulators of normal and leukemic hematopoietic stem and progenitor cells (HSPCs). This chapter will review our current understanding of the function of ID proteins in HSPC development and highlight future areas of scientific investigation.

Solute Carrier Nucleoside Transporters in Hematopoiesis and Hematological Drug Toxicities: A Perspective.

Simple SummaryAnticancer nucleoside analogs are promising treatments that often result in damaging toxicities and therefore ineffective treatment. Mechanisms of this are not well-researched, but cellular nucleoside transport research in mice might provide additional insight given transport’s role in mammalian hematopoiesis. Cellular nucleoside transport is a notable component of mammalian hematopoiesis due to how mutations within it relate to hematological abnormities. This review encompasses nucleoside transporters, focusing on their inherent properties, hematopoietic role, and their interplay in nucleoside drug treatment side effects. We then propose potential mechanisms to explain nucleoside transport involvement in blood disorders. Finally, we point out and advocate for future research areas that would improve therapeutic outcomes for patients taking nucleoside analog therapies.Anticancer nucleoside analogs produce adverse, and at times, dose-limiting hematological toxicities that can compromise treatment efficacy, yet the mechanisms of such toxicities are poorly understood. Recently, cellular nucleoside transport has been implicated in normal blood cell formation with studies from nucleoside transporter-deficient mice providing additional insights into the regulation of mammalian hematopoiesis. Furthermore, several idiopathic human genetic disorders have revealed nucleoside transport as an important component of mammalian hematopoiesis because mutations in individual nucleoside transporter genes are linked to various hematological abnormalities, including anemia. Here, we review recent developments in nucleoside transporters, including their transport characteristics, their role in the regulation of hematopoiesis, and their potential involvement in the occurrence of adverse hematological side effects due to nucleoside drug treatment. Furthermore, we discuss the putative mechanisms by which aberrant nucleoside transport may contribute to hematological abnormalities and identify the knowledge gaps where future research may positively impact treatment outcomes for patients undergoing various nucleoside analog therapies.

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.

Notch Signaling in the Bone Marrow Lymphopoietic Niche.

Lifelong mammalian hematopoiesis requires continuous generation of mature blood cells that originate from Hematopoietic Stem and Progenitor Cells (HSPCs) situated in the post-natal Bone Marrow (BM). The BM microenvironment is inherently complex and extensive studies have been devoted to identifying the niche that maintains HSPC homeostasis and supports hematopoietic potential. The Notch signaling pathway is required for the emergence of the definitive Hematopoietic Stem Cell (HSC) during embryonic development, but its role in BM HSC homeostasis is convoluted. Recent work has begun to explore novel roles for the Notch signaling pathway in downstream progenitor populations. In this review, we will focus an important role for Notch signaling in the establishment of a T cell primed sub-population of Common Lymphoid Progenitors (CLPs). Given that its activation mechanism relies primarily on cell-to-cell contact, Notch signaling is an ideal means to investigate and define a novel BM lymphopoietic niche. We will discuss how new genetic model systems indicate a pre-thymic, BM-specific role for Notch activation in early T cell development and what this means to the paradigm of lymphoid lineage commitment. Lastly, we will examine how leukemic T-cell acute lymphoblastic leukemia (T-ALL) blasts take advantage of Notch and downstream lymphoid signals in the pathological BM niche.

Robust Autonomous Generation of Definitive Hematopoietic Stem Cells from Pre-Circulation Murine Embryos

Introduction:The ontogeny of mammalian hematopoiesis is organized in at least three embryonic hematopoietic waves. The first wave is characterized by the generation of primitive erythrocytes emerging from the yolk sac (YS); a second one produces bipotent erythro-myeloid progenitors also in the YS. Finally, multipotent definitive Hematopoietic Stem and Progenitor Cells (HSPCs) with the ability to reconstitute all mature adult blood lineages and sustain life long hematopoiesis emerge from a subset of endothelial cells named hemogenic endothelium (HE). The embryonic anatomical site of origin of these HSPCs is still under debate in the field. An intra-embryonic origin is well accepted, which primarily encompasses the Aorta-Gonad-Mesonephros region (AGM) and umbilical and vitelline arteries. In contrast, a YS contribution to adult hematopoiesis remains controversial. The same developmental origin (e.g. endothelial cells with a mesodermal ancestor) makes it difficult to identify specific markers to discern between an AGM- vs. YS-origin using a lineage trace approach. This together with the highly migratory nature of blood cells and the inability of cells isolated from pre-circulatory embryos to robustly engraft in transplantation assays, even after ex vivo culture, has precluded scientists from properly answering these questions. For example, cultured YS and para-aortic splanchnopleura (PSP, the precursor of the AGM) dissected from embryos prior to the onset of circulation resulted in the generation of HSPCs with multilineage engraftment potential only from the embryonic PSP and not the YS (Cumano A. et al., Immunity 2002). However, blood chimerism was low (<5%) and engraftment was achieved only after transplantation into severely immuno-compromised mice (i.e. Rag2gc-/-). This low engraftment baseline makes difficult any comparison between intraembryonic tissues (IET) and Yolk Sac because a smaller frequency of YS-derived transplantable HSPCs would be difficult to detect, yielding a false negative result.Thus, here we sought to interrogate if we could generate robust engraftment from pre-circulation embryos that would allow to rigorously compare the ability to derive definitive HSC in the IET and YS.Results:We have established ex vivo culture conditions to allow for the derivation of robust multi-lineage engraftment activity from pre-circulation murine embryos. CD45.2+ embryos at the 2-3 somite pairs (s.p.) stage and 5-7 s.p. embryos were dissected from their YS, cultured as explants in our optimized conditions and transplanted into lethally irradiated CD45.1+/CD45.2+ recipients. Here, we observed multilineage reconstitution of the peripheral blood (PB) for >16 weeks post-transplant in 5/7 and 9/11 recipients, respectively. Moreover these cells were serially transplantable, confirming the generation of self-renewing HSCs. In primary recipients, PB chimerism was >50% in 8/18 total recipients. This robust PB chimerism allowed us to rigorously interrogate the corresponding CD45.2+ YS (cultured under the same conditions). In these transplants, we observed PB engraftment in 0/7 and 0/11 recipients of cells isolated from 2-3 s.p. and 5-7 s.p. cultured YSs, respectively. These data suggest that either YS do not autonomously generate HSPCs or that they can not support their maturation ex vivo . Moreover we found that the engraftment ability from the IET is confined to an emerging CD31+CD45+c-Kit+CD41- population. Importantly, this population also develops in the YS explants, but lacks engraftment ability. We have repeated these studies with cultured 19-22 s.p. embryos and YSs, a post-circulation time point and still observed a failure of YS-derived cells to engraft irradiated recipients (0/9) while embryo-derived cells (9/9) engraft robustly. These data suggest that YS likely cannot support the maturation of HSPCs that might have migrated from other sites. Only when we transplanted 32-36 s.p. or 46-47 s.p. YS-derived cells we observed robust PB engraftment under our culture conditions (3/7 and 4/6 recipients engrafted, respectively).Conclusions:We observed robust generation of HSCs with long-term and serially transplantable multilineage potential from pre-circulation murine embryos. Our study demonstrates that the YS is not able to support the maturation of HSPCs. Further studies would be required to determine if the YS is an actual source of HSPCs. DisclosuresNo relevant conflicts of interest to declare.

Deletion of TLR2+ erythro-myeloid progenitors leads to embryonic lethality in mice.

Early embryonic hematopoiesis in mammals is defined by three successive waves of hematopoietic progenitors which exhibit a distinct hematopoietic potential and provide continuous support for the development of the embryo and adult organism. Although the functional importance of each of these waves has been analyzed, their spatio-temporal overlap and the lack of wave-specific markers hinders the accurate separation and assessment of their functional roles during early embryogenesis. We have recently shown that TLR2, in combination with c-kit, represents the earliest signature of emerging precursors of the second hematopoietic wave, erythro-myeloid precursors (EMPs). Since the onset of Tlr2 expression distinguishes EMPs from primitive progenitors which coexist in the yolk sac from E7.5, we generated a novel transgenic "knock in" mouse model, Tlr2Dtr , suitable for inducible targeted depletion of TLR2+ EMPs. In this model, the red fluorescent protein and diphtheria toxin receptor sequences are linked via a P2A sequence and inserted into the Tlr2 locus before its stop codon. We show that a timely controlled deletion of TLR2+ EMPs in Tlr2Dtr embryos results in a marked decrease in both erythroid as well as myeloid lineages and, consequently, in embryonic lethality peaking before E13.5. These findings validate the importance of EMPs in embryonic development.

Unexpected redundancy of Gpr56 and Gpr97 during hematopoietic cell development and differentiation

AbstractIntegrated molecular signals regulate cell fate decisions in the embryonic aortic endothelium to drive hematopoietic stem cell (HSC) generation during development. The G-protein–coupled receptor 56 (Gpr56, also called Adgrg1) is the most highly upregulated receptor gene in cells that take on hematopoietic fate and is expressed by adult bone marrow HSCs. Despite the requirement for Gpr56 in hematopoietic stem/progenitor cell (HS/PC) generation in zebrafish embryos and the highly upregulated expression of GPR56 in treatment-resistant leukemic patients, its function in normal mammalian hematopoiesis remains unclear. Here, we examine the role of Gpr56 in HS/PC development in Gpr56 conditional knockout (cKO) mouse embryos and Gpr knockout (KO) embryonic stem cell (ESC) hematopoietic differentiation cultures. Our results show a bias toward myeloid differentiation of Gpr56 cKO fetal liver HSCs and an increased definitive myeloid progenitor cell frequency in Gpr56KO ESC differentiation cultures. Surprisingly, we find that mouse Gpr97 can rescue Gpr56 morphant zebrafish hematopoietic generation, and that Gpr97 expression is upregulated in mouse Gpr56 deletion models. When both Gpr56 and Gpr97 are deleted in ESCs, no or few hematopoietic PCs (HPCs) are generated upon ESC differentiation. Together, our results reveal novel and redundant functions for these 2 G-protein coupled receptors in normal mammalian hematopoietic cell development and differentiation.

Heterozygous mutation SLFN14 K208N in mice mediates species-specific differences in platelet and erythroid lineage commitment

AbstractSchlafen 14 (SLFN14) has recently been identified as an endoribonuclease responsible for cleaving RNA to regulate and inhibit protein synthesis. Early studies revealed that members of the SLFN family are capable of altering lineage commitment during T-cell differentiation by using cell-cycle arrest as a means of translational control by RNase activity. SLFN14 has been reported as a novel gene causing an inherited macrothrombocytopenia and bleeding in human patients; however, the role of this endoribonuclease in megakaryopoiesis and thrombopoiesis remains unknown. To investigate this, we report a CRISPR knock-in mouse model of SLFN14 K208N homologous to the K219N mutation observed in our previous patient studies. We used hematological analysis, in vitro and in vivo studies of platelet and erythrocyte function, and analysis of spleen and bone marrow progenitors. Mice homozygous for this mutation do not survive to weaning age, whereas heterozygotes exhibit microcytic erythrocytosis, hemolytic anemia, splenomegaly, and abnormal thrombus formation, as revealed by intravital microscopy, although platelet function and morphology remain unchanged. We also show that there are differences in erythroid progenitors in the spleens and bone marrow of these mice, indicative of an upregulation of erythropoiesis. This SLFN14 mutation presents distinct species-specific phenotypes, with a platelet defect reported in humans and a severe microcytic erythrocytosis in mice. Thus, we conclude that SLFN14 is a key regulator in mammalian hematopoiesis and a species-specific mediator of platelet and erythroid lineage commitment.

Characteristic Distribution of Hematopoietic Cells in Bone Marrow of Xenopus Laevis.

Bone marrow is the principal site of hematopoiesis in mammals. Amphibians were the first phylogenetic group in vertebrates to acquire bone marrow, but the distribution of hematopoietic cells in the bone marrow of the primitive frog, Xenopus laevis (X. laevis) has not been well documented. The purpose of this study was to perform a histological investigation of the distribution of hematopoietic cells in femoral bone marrow at various stages of development in X. laevis. Hematopoietic cells showed preferential distribution on the endosteal surface of cortical bone throughout all stages of development, from tadpole to aged frog. In mature frogs, hematopoietic cells appeared at the boundary between the epiphysis and the bone marrow. The distribution of hematopoietic cells around the blood vessels was limited to a small number of vessels in the bone marrow. Abundant adipose tissue was observed in the bone marrow cavity from the tadpole stage to the mature frog stage. Hematopoietic cells showed preferential distribution in a belt-like fashion on the surface of newly-formed bones in a bone regeneration model in the diaphysis of X. laevis. These results indicate that the distribution of hematopoietic cells in bone marrow in X. laevis differs from that in mammals, and that the bone marrow of X. laevis constitutes a useful model for exploring the mechanism underlying the phylogenetic differentiation of bone marrow hematopoiesis.

SSSPTA is essential for serine palmitoyltransferase function during development and hematopoiesis

Serine palmitoyltransferase complex (SPT) mediates the first and rate-limiting step in the de novo sphingolipid biosynthetic pathway. The larger subunits SPTLC1 and SPTLC2/SPTLC3 together form the catalytic core while a smaller third subunit either SSSPTA or SSSPTB has been shown to increase the catalytic efficiency and provide substrate specificity for the fatty acyl-CoA substrates. The in vivo biological significance of these smaller subunits in mammals is still unknown. Here, using two null mutants, a conditional null for ssSPTa and a null mutant for ssSPTb, we show that SSSPTA is essential for embryogenesis and mediates much of the known functions of the SPT complex in mammalian hematopoiesis. The ssSPTa null mutants are embryonic lethal at E6.5 much like the Sptlc1 and Sptlc2 null alleles. Mx1-Cre induced deletion of ssSPTa leads to lethality and myelopoietic defect. Chimeric and competitive bone marrow transplantation experiments show that the defect in myelopoiesis is accompanied by an expansion of the Lin−Sca1+c-Kit+ stem and progenitor compartment. Progenitor cells that fail to differentiate along the myeloid lineage display evidence of endoplasmic reticulum stress. On the other hand, ssSPTb null mice are homozygous viable, and analyses of the bone marrow cells show no significant difference in the proliferation and differentiation of the adult hematopoietic compartment. SPTLC1 is an obligatory subunit for the SPT function, and because Sptlc1−/− and ssSPTa−/− mice display similar defects during development and hematopoiesis, we conclude that an SPT complex that includes SSSPTA mediates much of its developmental and hematopoietic functions in a mammalian model.

PSVIII-4 Bone marrow architectonics of turkeys hybrid cross «Converter»

Abstract The aim of this research was to study bone marrow architectonics and the peculiarities of hematopoiesis of turkeys cross «Converter». To study the hemopoiesis at the age of 50 days, the samples of the red bone marrow from the femur were selected. Samples were prepared according to standard methods for histological and cytological examination. Four lineages of hematopoiesis (erythrocytic, monocytic, granulocytic and platelet) were isolated from the bone marrow of turkeys. The erythrocytic lineage was represented by erythroblasts, rubricytes and mature erythrocytes. In the process of maturation, the erythroblast initially uniformly decreased, turning into rubricyte. Then the cytoplasm and nucleus enlarged and acquired an oval shape. A mature erythrocyte was formed. Hematopoiesis of eosinophils and basophils in turkeys was similar to hematopoiesis in mammals. They also formed from myeloblasts. At the stage of promyelocyte, granularity of the cytoplasm appeared. At the stage of myelocyte, the number of granules increased, and the granularity acquired a typical shape and color. Then the nucleus acquired a rod-shaped form, and after it was segmented into 2–5 segments. Heterophiles were divided into two types (with rounded and elongated granules). Heterophile granules were about 2 times bigger than eosinophils, and cytoplasm granularity was significantly sparser. Monocytes in turkeys had a rounded cytoplasm, in which small granules were sometimes present, and the nucleus was shifted to the periphery. Mature monocytes differed from promonocytes in a smaller nuclear-cytoplasmic ratio. Platelets had a centrally located nucleus and an oval cytoplasm with granules inside. Prothrombocytes were approximately 1.5 times larger than mature platelets. The lymphoid lineage is not represented, which indicates an extramedullary lymphopoiesis. A significant difference was the absence of megakaryocytes and the development of platelets from thromboblasts. Granulocytic and monocytic lineages were similar to those in mammals, and erythroid one was represented only by nucleated erythrocytes.

Context-specific functions of Notch in Drosophila blood cell progenitors

Drosophila Larval hematopoiesis takes place at the lymph gland, where myeloid-like progenitors differentiate into Plasmatocytes and Crystal Cells, under regulation of conserved signaling pathways. It has been established that the Notch pathway plays a specific role in Crystal Cell differentiation and maintenance. In mammalian hematopoiesis, the Notch pathway has been proposed to fulfill broader functions, including Hematopoietic Stem Cell maintenance and cell fate decision in progenitors. In this work we describe different roles that Notch plays in the lymph gland. We show that Notch, activated by its ligand Serrate, expressed at the Posterior Signaling Center, is required to restrain Core Progenitor differentiation. We define a novel population of blood cell progenitors that we name Distal Progenitors, where Notch, activated by Serrate expressed in Lineage Specifying Cells at the Medullary Zone/Cortical Zone boundary, regulates a binary decision between Plasmatocyte and Crystal Cell fates. Thus, Notch plays context-specific functions in different blood cell progenitor populations of the Drosophila lymph gland.

ZBTB11 IS REQUIRED FOR HEMATOPOIETIC STEM CELL FUNCTION

Hematopoiesis orchestrates the daily requirement of producing and maintaining the various blood cell lineages in the right quantities. It must also respond rapidly to injury and infection to expand specific lineages on demand then return them to steady state levels. Understanding the sophisticated mechanisms regulating this balancing act can uncover therapeutic targets for manipulating blood cell production in disease treatment. In a forward genetic screen for zebrafish myeloid mutants, we identified a requirement for ZBTB11, a ZBTB (BTB/POZ) transcriptional repressor, in definitive hematopoiesis. To understand its relevance to mammalian systems, we extended these studies to mouse. When Zbtb11 is deleted in the hematopoietic compartment embryos do not survive past E18.5 likely due to insufficient hematopoiesis. Phenotypic HSCs (SLAM) are specified in over-abundance at E14.5 through E17.5 compared to controls. Functional analysis, however, shows they are unable to effectively differentiate into committed progenitors and mature lineages in fetal liver. Hematopoietic content in fetal bone marrow is mostly absent at E16.5 – E17.5 pointing to bone marrow failure. Further analysis showed Zbtb11-/- HSCs fail to proliferate in an in vitro proliferation assay and are constrained in their progression through the cell cycle demonstrating a role for Zbtb11 in proliferation and cell cycle regulation in mammalian hematopoiesis. We have undertaken scRNAseq in Zbtb11 /- and control HSC populations to understand heterogeneity within and between these populations, and what genetic programs have been altered. Mechanistic evaluation is under way. In summary, our findings establish a conserved cell autonomous role for Zbtb11 in mammalian definitive hematopoiesis, specifically in HSC function, opening up new avenues for understanding and manipulating HSCs.

GPR56 AND GPR97 PLAY REDUNDANT ROLES IN REGULATING HEMATOPOIESIS

Hematopoietic stem cells (HSCs) are responsible for the lifelong production and maintenance of adult blood cell types. They arise from the aorta at the embryonic day (E) 10.5 from a subset of endothelial cells that undergo to endothelial-to-hematopoietic transition (EHT). Our lab discovered that a G-coupled receptor protein, Gpr56, is required for the generation of HSCs in zebrafish. However, its role in mammalian hematopoiesis remains controversial. We investigate the functional role of Gpr56 in vitro using our mouse Gata2Venus (G2V) embryonic stem cell (ESC) line which allows us to highly enrich for hematopoietic progenitor cells (HPCs). G2V.G56-/-. We tested the Gpr56 loss of function by generating a full knock out (KO) Gpr56 mouse G2V (G2V.G56-/-) ESC line. The in vitro examination of phenotypic hematopoietic cell production by FACS and colony forming unit-culture (CFU-C) assay reported an increase in hematopoietic cells (HC) and HPCs production from G2V.G56-/- ESC as compared to G2V ESC. Further analyses performed on cell-sorted HPC population showed that another GPR protein, Gpr97 (located in the same locus), is highly expressed when Gpr56 is deleted. This led us to generate a novel G2V Gpr56/Gpr97 double knockout (G2V.G56/97-/-) ESC line. The in vitro differentiation of the G2V.G56/97-/- revealed a significant decrease in HPCs, supporting the idea that a compensatory mechanism by Gpr97 is taking place when Gpr56 is absent. Preliminary in vivo data using a Gpr56 conditional KO mouse model corroborates our hypothesis. The data demonstrate that the Gpr97 performs a redundant function upon deletion of Gpr56 in the HS/PC population and the deletion of both Gpr56 and Gpr97 is sufficient to impair the production of HS/PC and HC in vitro. We conclude that GPR signalling through Gpr56 and/or Gpr97 is required for HPC production.

Detecting proliferation of adult hemocytes in Drosophila by BrdU incorporation.

Drosophila and mammalian hematopoiesis share several similarities that ranges from phases to the battery of transcription factors and signaling molecules that execute this process. These resounding similarities along with the rich genetic tools available in fruitfly makes it a popular invertebrate model to study blood cell development both during normal and aberrant conditions. The larval system is the most extensively studied to date. Several studies have shown that these hemocytes just like mammalian counterpart proliferate and get routinely regenerated upon infection. However, employing the same protocol it was concluded that blood cell proliferation although abundant in larval stages is absent in adult fruitfly. The current protocol describes the strategies that can be employed to document the hemocyte proliferation in adulthood. The fact that a subset of blood cells tucked away in the hematopoietic hub are not locked in senescence, rather they still harbour the proliferative capacity to tide over challenges was successfully demonstrated by this method. Although we have adopted bacterial infection as a bait to evoke this proliferative capacity of the hemocytes, we envision that it can also efficiently characterize the proliferative responses of hemocytes in tumorigenic conditions as well as scenarios of environmental and metabolic stresses during adulthood.