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No description available.

No description available.

No description available.

In the mid-gestation embryo, the first definitive transplantable hematopoietic stem cells (dHSCs) emerge by embryonic day E10.5-E11 in the aorta-gonadomesonephros (AGM) region, as a result of a step-wise maturation of precursors called pre-HSCs. The analysis of several Notch mutants suggests that Notch signalling is essential for the execution of the definitive hematopoietic programme in the AGM. Mouse embryos deficient for Notch1, RBP-Jk or Jagged1 cannot efficiently generate intra-embryonic hematopoeitic progenitors. It has also been reported that knockdown of Notch target genes (Hes1, Hes5) results in hematopoietic impairment. However a clear picture of the role of Notch pathway in HSC development is still missing. In this work we characterised precise stages and cell types during HSC development in which Notch signalling is involved. First we used a Hes1-dEGFP reporter mouse line that allowed us to monitor Notch pathway activity in a narrow window of time. The results suggest that the level of Notch activity fluctuates in HSC lineage in the AGM region and is down-regulated in dHSCs in the foetal liver (where dHSCs migrate after generation in the AGM region). By using transplantation assay, we further showed that fluctuations of Notch activity are essential for HSC development, and that this pattern in the HSC lineage might work as a switch between maturation and proliferation of PreHSC1, PreHSC2 and dHSC, in which temporary decrease might be required to mature from one type to another, both in vitro and in vivo. These findings might need to be taken into consideration for in vitro generation of haematopoietic stem cells, where a fine tuning of Notch signalling activity could greatly improve their emergence.

Haematopoiesis is the process to produce haematopoietic stem cells (HSCs), haematopoietic progenitors (HPCs) and terminally differentiated cell types. In the adult, HSCs resided in bone marrow while in the embryo, haematopoiesis occurred sequentially in several niches including yolk sac, aorta-gonad-mesonephros (AGM) region, placenta and fetal liver. The AGM region is the first place where HSCs arise in vivo and therefore should provide important factors to induce haematopoiesis. The mouse embryonic stem cells (mESC) system is a powerful platform to mimic the development process in vitro and is widely utilized to study the underlying mechanisms because they are pluripotent and can be genetically manipulated. A novel co-culture system has been established by culturing differentiating mESCs with primary E10.5 AGM explants and a panel of clonal stromal cell lines derived from dorsal aorta and surrounding mesenchyme (AM) in AGM region. Results of these co-culture studies suggested that the AM-derived stromal cell lines could be a potent resource of signals to enhance haematopoiesis. Molecular mechanism involved in haematopoiesis is a key research direction for understanding the regulation network of haematopoiesis and for further clinical research. A series of studies have demonstrated involvement of the Notch signalling pathway in haematopoiesis during development but with controversial conclusions because of the difference of models concerning various time windows and manipulating populations. This project aimed to investigate the role of Notch signalling pathway during haematopoiesis in the AGM environment. We analyzed the expression of Notch ligands in AGM-derived stromal cells with or without haematopoietic enhancing ability. No correlation was observed between ligand expression and haematopoietic enhancing ability in stromal cell lines or between Notch activity in EBs and haematopoietic enhancing ability. We demonstrated that inhibition of the Notch signalling pathway using the γ-secretase inhibitor could abrogate Notch activity in both mES-derived cells and the haematopoietic enhancing AM stromal cell line. To better understand the involvement of the Notch signalling pathway in a more specific spatial-temporal environment, we established a co-culture system of haemangioblast like cells (Flk1+) with one of AM region derived stromal cell lines with haematopoietic enhancing ability . We found that the AM stromal cell line could enhance Flk1+ derived haematopoiesis as assessed by haematopoietic colony formation activity and production of CD41+cKit+ progenitor cells. Based on the issue that the inhibitor could potentially affect both the ES cells and stromal cells, we carried out genetic approaches to overexpress or knock down Notch signalling pathway in this Flk1+/AM co-culture system. Interestingly, it was found that when Notch activity was enhanced in Flk1+ cells, the production of haematopoietic progenitors was inhibited and the number of cells expressing the pan-haematopoietic marker CD45 was reduced. By using the inducible dominant negative MAML1 system to knock down Notch activity, it was found that the haematopoiesis in the Flk1+/AM co-culture system was not affected, which could be accounted for the low Notch activity in this system. These results supported the hypothesis that the Notch signalling pathway plays a role in modulating Flk1+ derived haematopoietic differentiation within the AGM microenvironment.

The finely tuned regulation of haematopoietic stem and progenitor cells (HSPCs) is crucial to sustain normal haematopoiesis. The disruption of the balance between the quiescence state of haematopoietic stem cells (HSCs) and the proliferation/differentiation programs that are necessary to meet daily haematopoietic demands and respond to external insults, can lead to malignant transformation, such as acute myeloid leukaemia (AML). Therefore, it is essential to investigate the players that are responsible to maintain haematopoietic homeostasis, so that novel therapeutic targets can be identified. HSCs reside in a hypoxic environment that is crucial for their maintenance, as it protects them from over-proliferation and exhaustion. The response to a limited availability of oxygen is critically mediated by a transcription factor - hypoxia inducible factor (HIF). HIF is predominantly regulated by prolyl hydroxylases (PHDs) that are 2-oxoglutarate (2OG) dependent oxygenases. This superfamily of oxygen-sensing enzymes has been assigned important roles ranging from hypoxia signaling, DNA repair, chromatin modifications and oncogenesis Following the data published by our group attesting that HIF is dispensable for HSC survival and maintenance, we focused our investigation on HIF-independent pathways. This manuscript describes the study of the role of an oxygen-sensor enzyme, member of the 2OG oxygenases and HIF negative regulator, jumonji domain-containing protein 6 (Jmjd6), in normal and malignant haematopoiesis. Our knockout studies deleting Jmjd6 specifically within the haematopoietic system (Jmjd6fl/fl;Vav-iCre) demonstrate that the homeostasis of HSPC pool was compromised and lymphopoiesis was attenuated in Jmjd6-deficient cohorts. Upon transplantation, HSCs lacking Jmjd6 exhibited a defective chimerism and impaired capacity to fully reconstitute haematopoiesis of recipient mice. Thus, Jmjd6 is essential for HSC self-renewal and maintenance. Our assessment of the impact of Jmjd6 deletion in the context of inflammatory response and recovery from treatment with a myelotoxic agent treatment revealed that Jmjd6 is a positive regulator of HSC homeostasis and recovery from cytotoxic stress. There are accumulating data on the importance of epigenetics in the development of haematological malignancies. Being an epigenetic regulator, clearly involved in RNA splicing, we investigated Jmjd6 as possible player in leukaemogenesis. The results from our leukaemic studies unravelled a new biological function for Jmjd6 as a tumour suppressor in Meis1/Hoxa9 murine model. Altogether, our findings offer important novel insights into the biological functions of Jmjd6 and pave the way for further studies to discover on the mechanism of action of this complex enzyme. Our observations add value to the idea that Jmjd6 might constitute a good candidate for cancer diagnosis, that can be use to ameliorate patient’s prognosis and that it can be used to help patient prognosis in the future.

Point mutations found in human breast cancer samples introduced into the Drosophila dco gene affect the Drosophila haematopoiesis. I described different haematopoietic phenotypes in such mutants and tested their connection with the Toll and the JAK/STAT signalling pathways.

The haematopoietic stem cell (HSC) is found in the adult human bone marrow, where it gives rise to all the circulating blood cells throughout adulthood. Understanding the signalling events that programme these cells during development will improve HSC in vitro culture, their generation from embryonic stem cells or induced pluripotent stem cells, and their potential therapeutic application. HSCs bud from the floor of the dorsal aorta and seed the bone marrow via circulation. The precursors to the dorsal aorta and HSCs are called haemangioblasts, which are found in the dorsal lateral plate mesoderm in Xenopus. The knowledge of the location of these precursors allows their programming to be studied in detail during embryonic development. A key pathway implicated in the programming of HSCs is the BMP signalling pathway. Here, using both a small molecule inhibitor and a transgenic Xenopus line, BMP signalling has been inhibited post-gastrulation without perturbing the gross morphology of the embryo. This has shown that BMP signalling is required for HSC programming in the dorsal lateral plate mesoderm via the expression of a critical haematopoietic transcription factor, gata2. Morpholino knockdown of evi3has revealed it to be essential for HSC programming in the dorsal lateral plate mesoderm, where it is required for the expression of gata2. Furthermore, as evi3 is known to bind to the active BMP signalling complex, and as evi3 knockdown phenocopies post-gastrulation BMP inhibition, evi3 appears to be required for BMP signalling to initiate gata2 expression in the DLP. Taken together, the findings presented here demonstrate an essential post-gastrulation role of BMP signalling and Evi3 for programming HSCs in Xenopus.

High turnover in the haematopoietic system is sustained by stem and progenitor cells, which divide and mature to produce the range of cell types present in the blood. This complex system has long served as a model of differentiation in adult stem cell systems and its study has important clinical relevance. Maintaining a healthy blood system requires regulation of haematopoietic cell fate decisions, with severe dysregulation of these fate choices observed in diseases such as leukaemia. As transcriptional regulation is known to play a role in this regulation, the gene expression of many haematopoietic progenitors has been measured. However, many of the classic populations are actually extremely heterogeneous in both expression and function, highlighting the need for characterising the haematopoietic progenitor compartment at the level of individual cells. The first aim of this work was to chart the single-cell transcriptional landscape of the haematopoietic stem and progenitor cell (HSPC) compartment. To build a comprehensive map of this landscape, 1,654 HSPCs from mouse bone marrow were profiled using single-cell RNA-sequencing. Analysis of these data generated a useful resource, and reconstructed changes in gene expression, cell cycle and RNA content along differentiation trajectories to three blood lineages. To investigate how single-cell gene expression can be used to learn about regulatory relationships, data measuring the expression of 41 genes (including 31 transcription factors) in 2,167 stem and progenitor cells were used to construct Boolean gene regulatory network models describing the regulation of differentiation from stem cells to two different progenitor populations. The inferred relationships revealed positive regulation of Nfe2 and Cbfa2t3h by Gata2 that was unique to differentiation towards megakaryocyte-erythroid progenitors, which was subsequently experimentally validated. The next study focused on investigating the link between transcriptional and functional heterogeneity within blood progenitor populations. Single-cell profiles of human cord blood progenitors revealed a continuum of lympho-myeloid gene expression. Culture assays performed to assess the functional output of single cells found both unilineage and bilineage output and, by investigating the link between surface marker expression and function, a new sorting strategy was devised that was able to enrich for function within conventional lympho-myeloid progenitor sorting gates. The final project aimed to study changes to the HSPC compartment in a perturbed state. A droplet-based single-cell RNA-sequencing dataset of 44,802 cells was analysed to identify entry points to eight blood lineages and to characterise gene expression changes in this transcriptional landscape. Mapping single-cell data from W41/W41 Kit mutant mice highlighted quantitative shifts in progenitor populations such as a reduction in mast cell progenitors and an increase towards more mature progenitors along the erythroid trajectory. Differential gene expression identified upregulation of stress response and a reduction of apoptosis during erythropoiesis as potential compensatory mechanisms in the Kit mutant progenitors. Together this body of work characterises the HSPC compartment at single-cell level and provides methods for how single-cell data can be used to discover regulatory relationships, link expression heterogeneity to function, and investigate changes in the transcriptional landscape in a perturbed environment.