Haematopoietic stem and progenitor cells (HSPCs) are de novo generated within in the ventral aspects of the embryonic dorsal aorta (DA). Cells of this haemogenic endothelium (HE) will eventually undergo an endothelial to haematopoietic transition (EHT) that involves cell budding out of the aortic wall. Despite the detailed description of the cellular events, the exact haemogenic lineage path and the underlying molecular mechanism that establish full haematopoietic competence are still not entirely understood. The transcription factor Runx1 is critical for the emergence of HSPCs and shows expression in the zebrafish HE as early as 24 hpf. To facilitate a detailed analysis of the transient HE population I generated a TgBAC(runx1P2:Citrine) reporter line under the control of the endogenous runx1 promoter on a bacterial artificial chromosome (BAC). Double-transgenic reporter lines for runx1 and the endothelial marker kdrl allowed us to isolate specifically cells of the DA away from the whole endothelial population, which could be further sub-divided into HE and non-haemogenic cells. Genomewide expression analysis within the respective tissues and upon Runx1 loss of function enabled the identification of HE-specific Runx1-regulated genes. Hereby, the gfi1ab gene appeared as the functional homologue of the murine Gfi1. I show that in zebrafish, EHT is orchestrated through a conserved Runx1-Gfi1-Lsd1 axis. The cellular functions of the remaining Runx1 targets imply that maturation into fully functional HSCs depends on epigenetic regulation due to the up-regulation of de novo DNAmethyltransferases, as well as on factors that allow the developing HSCs to respond to extrinsic cues from haematopoietic niches. Lastly, it became evident that the early HE expresses dll4 at similar levels to the rest of the aortic endothelium, indicating a common lineage path. In the absence of RUNX1 the HE remains essentially arterial and persists as an integrated part of the DA.

Macrophages are present in every tissue, and have a central role in immune responses, development and homeostasis. Typically recognised as scavenger cells phagocytising pathogens and dead cells, macrophages also regulate the innate and adaptive immune responses via the secretion of cytokines. In mammals, the differentiation, proliferation, and survival of macrophages are controlled by macrophage colony-stimulating factor, or CSF1, which acts through the CSF1 receptor (CSF1R), a ligand-dependent protein tyrosine kinase. IL34, a more recently discovered cytokine with a differential expression, shares the CSF1R. Natural or artificial knock out of these genes in mice and rats depletes macrophage populations with consequent pleiotropic effects on development of multiple organs. The mammalian CSF1R is exclusively expressed on the cells of the macrophage lineage, and their progenitors. For this reason, the CSF1R promoter has been used to generate fluorescent reporter transgenic mice, to permit analysis of macrophage function in vivo. Macrophages are present in very large numbers from midgestation in mice, but dynamic studies of their biology are difficult in a mammal. The chick has been used extensively as a developmental biology model because of the ease of visualisation and manipulation in ovo. It has the added advantage of being economically important. At the start of this project, the factors that control avian myelopoiesis had not been identified. Indeed, CSF1 was not identified in the chicken genome. The primary objective of this research was to characterise the chicken mononuclear phagocyte system. To this end, the CSF1, IL34, and CSF1R genes in chicken and zebra finch were identified from respective genomic/cDNA sequence resources. Comparative analysis of the avian CSF1R loci revealed likely orthologs of mammalian macrophage-specific promoters and enhancers, and the CSF1R gene was shown to be expressed specifically in macrophages of the developing chick embryo. These observations formed the basis of the generation of a chicken CSF1R reporter transgenic by a colleague in the laboratory. Structure-based modelling, comparative amino acid sequence analysis and co-evolution study across all vertebrates demonstrated the conservation of the IL34/CSF1/CSF1R complex in birds. Modelling also suggested that IL34 was a four helix bundle factor, structurally related to CSF1, which was subsequently confirmed by published crystal structure. To show that these factors were active in birds, chicken CSF1 and IL34 were expressed in HEK293 cells. Although chicken CSF1 lacked the interchain disulphide present in the mammalian protein, it formed a dimer. Both factors were able to promote the generation of pure macrophage cultures when added to chicken bone marrow. The specificity of action of chCSF1 and chIL34 on chCSF1R was assessed using murine myeloid IL-3 dependent Ba/F3 cells stably transfected with chCSF1R. Either chCSF1 or chIL34 alone could substitute for IL3 in receptor-expressing cells and caused them to differentiate further into the monocytic lineage pathway and to undergo growth arrest. The avian factors were not active on mammalian CSF1R. The observed species specificity and inactivity of the CSF1R inhibitor GW2580 in chicken were linked to the dissimilarities between the avian and mammalian CSF1/IL34/CSF1R proteins. To enable functional studies in vivo, a project was initiated to produce a monoclonal antibody against chicken CSF1R. Binding of the monoclonal to cells demonstrated that CSF1R was, indeed, monocyte-macrophage restricted. Chicken CSF1 was expressed as a fusion protein with the domains 3 and 4 of the chicken immunoglobulin. This increased the half-life of the recombinant chCSF1 without impairing its activity. Injection of chCSF1-Fc in the neural tube of stage HH21 chick embryos stimulated the proliferation of embryonic macrophages. Similarly, four consecutive daily injections of chCSF1-Fc in chicken hatchlings resulted in an increase in tissue macrophage number, notably in the spleen, liver and lung. To investigate the pathway of development of macrophages during embryogenesis, bone marrow from chicken ubiquitously expressing EGFP was transplanted into the circulation of stage HH16-17 embryos. The results demonstrated effective colonisation of the hematopoietic organs, and highlighted the presence of large numbers of macrophages in embryonic tissues, similar to those seen in MacGreen mice. The results are discussed in the context of the proposed yolk sac origin of some macrophage subpopulations, such as microglia cells and Langerhans cells, and the presence of a clonogenic macrophage-committed progenitor in the bone marrow that is distinct from the pluripotent stem cell. Bone marrow-derived macrophages (BMDMs) grown in CSF1 have been used extensively as a model to understand gene regulation in mice. The cloning and expression of chicken CSF1 permitted the production of large numbers of BMDMs from chicken bone marrow. To enable the characterisation of chicken macrophages and comparison to mammalian BMDMs, the gene expression profile of these cells was examined using RNAseq. For comparison, mid incubation embryos and a fibroblast line were also profiled. These data could identify several novel chicken macrophage-specific transcripts that may assist in further dissection of macrophage differentiation in birds and contribute to chicken genome annotation. Overall, this project has demonstrated that the CSF1/IL34/CSF1R system is conserved in birds, and controls the generation of monocytes and tissue macrophages. It has provided the tools to enable detailed analysis of the function of this system in embryogenesis and immunity.

In 2106, there were 171,550 new cases of blood cancers and over one million people in the United States living with one of these disorders. Bone marrow transplants have good outcomes, but these procedures require a donor who is a perfect match, and thus many patients are unable to receive treatment. It is important to find patient-derived treatments, such as molecules which stimulate hematopoietic stem cell (HSC) formation without the need for a donor. Therefore, a study was initiated to use human-induced pluripotent stem cell (hiPSC) technology to make a patient-derived, personalized HSC. Epigenetic regulators are divided into readers, writers, and erasers, and each of these classes has shown some effect on HSC formation. Writers add functional groups to deoxyribonucleic acid (DNA) and histone proteins, whereas erasers remove them. Readers are groups on transcription factors which interpret these changes and increase or decrease the recruitment of transcriptional machinery accordingly. In this study, a screen of 12 different candidate molecules with distinct epigenetic targets in casper zebrafish was conducted at 36 hours postfertilization (hpf) to reveal increases or decreases in definitive HSC development. The two writer molecules, C646 (histone acetyltransferase, or HAT, inhibitor) and OICR9249 (WDR5 inhibitor), and the two eraser molecules, Ex-527 (Sirt1 inhibitor) and JIB-04 (bromodomain inhibitor), showed varying degrees of increasing HSC formation. Of these molecules, C646 created the most significant increase and was further tested in the zebrafish at 48 and 72 hpf and in a murine model using ex vivo technique and a colony-forming unit (CFU) assay. In contrast to these results, the two eraser molecules, entinostat (class I histone deacetylase, or HDAC, inhibitor) and vorinostat (general HDAC inhibitor), were found to decrease HSC formation in zebrafish. The overall findings of this study provide insight into specific epigenetic regulators in HSC development and identify particular epigenetic markers that could regulate HSC formation from endothelial cells. This discovery will be a stepping stone in utilizing patient-derived hemogenic endothelial cells as a novel source of bone marrow-independent HSCs to treat patients with leukemia, lymphoma, and bone marrow cancers. / 2019-07-11T00:00:00Z

"Research conducted at the Department of Haematology, Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science."--T.p. Includes bibliographical references (leaves 170-214). Elevated levels of receptor tyrosine kinases have been implicated in carcinogenesis. It is possible that high expression of c-Kit by the leukaemic cell provides them with a growth advantage over their normal counterparts in the bone marrow microenvironment. Thus, a means of inhibiting the interaction of c-Kit on these cells with ligand Steel Factor may remove proliferation and survival signals. Main aim of the study was to produce a biological inhibitor of this interaction and evaluate its ability to prevent ligand Steel Factor from binding to c-Kit on live cells.

Normal tissue homeostasis and appropriate responses to injury and infection are dependent on cellular communication mediated by cell surface receptors that respond to extrinsic stimuli. The GM-CSF receptor was the major focus of this project. This receptor shares a common signalling subunit, β [subscript c], with the IL-3 and IL-5 receptors. The unique GM-CSF receptor α-subunit ( GMRα ) confers ligand binding specificity to the complex and is essential for GM-CSF receptor signalling, although the full complement of signalling events mediated by GMRα remains elusive. Through cloning of candidate interacting proteins, expression and co-immunoprecipitation studies, we have confirmed interactions for two proteins previously reported to interact with the GMRα, p85 and IKKβ. Additionally, we identified the Src family kinase, Lyn, as a novel direct interacting partner of GMRα and provide insights into possible roles of this kinase in initiating signalling from the GM-CSF receptor. In addition to GMRα associated events we aimed to further characterise the role of the common β [subscript c] subunit in GM-CSF mediated signalling. We utilised two classes of consitutively active β [subscript c] mutants ( extracellular or transmembrane ) which transform the bi-potential myeloid FDB1 cell line to either factor-independent growth and survival, or granulocyte-macrophage differentiation, respectively. Here we report a comprehensive biochemical analysis of signalling by these two classes of mutants in this cell line. The two activated GMR mutants displayed distinct and non-overlapping signalling capacity. In particular, expression of a mutant with a substitution in the transmembrane domain ( V449E ) selectively activated JAK / STAT5 and MAPK pathways resulting in a high level of sensitivity to JAK and MEK inhibitors. In contrast, expression of a mutant with a 37 amino acid duplication in its extracellular domain ( FI Δ ) selectively activates the PI3K / AKT and IKKβ / NFkB pathways. Cells responding to this mutant display a relative high level of sensitivity to two independent PI3K inhibitors and relative resistance to inhibition of MEK and JAK2. The non-overlapping nature of signalling by these two activated mutants suggests that there are alternative modes of receptor activation that differentially dependent on JAK2 and that act synergistically in the mature liganded cytokine receptor complex. Further detailed analysis of these mutants will facilitate the dissection of the signalling pathways involved in the GM-CSF response that mediate proliferation, survival and differentiation. / Thesis (Ph.D.)--School of Medicine, 2007.

Bibliography: leaves 154-185 / iv, 185 leaves, [15] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Science, 1987

"Research conducted at the Department of Haematology, Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science."--T.p. / Includes bibliographical references (leaves 170-214). / xiv, 221 leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Elevated levels of receptor tyrosine kinases have been implicated in carcinogenesis. It is possible that high expression of c-Kit by the leukaemic cell provides them with a growth advantage over their normal counterparts in the bone marrow microenvironment. Thus, a means of inhibiting the interaction of c-Kit on these cells with ligand Steel Factor may remove proliferation and survival signals. Main aim of the study was to produce a biological inhibitor of this interaction and evaluate its ability to prevent ligand Steel Factor from binding to c-Kit on live cells. / Thesis (Ph.D.)--University of Adelaide, Dept. of Molecular Biosciences, 2001

Thesis (Ph. D.)--University of Chicago, Dept. of Pathology, August 1999. / Includes bibliographical references. Also availabel on the Internet.

Thesis (M. Phil.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 96-103). Also available in print.

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy