Hematopoiesis in the developing zebrafish embryo

Abstract

This thesis describes the use of zebrafish, Danio rerio, to unravel the complexity of hematopoiesis in the developing embryo with an emphasis on the role of two phosphatases. In chapter 1, we provide a background for the following four chapters. Chapter 2 focuses on the role of Pten during hematopoiesis. To analyse the role of Pten we used embryos lacking Pten expression with various transgenic markers to visualize hematopoiesis. Embryos lacking Pten expression display, among other things, increased PI3K activity. We found that embryos lacking Ptenshowed less hematopoietic stem/progenitor cells (HSPCs) compared to their siblings, with a striking phenotype when HSPCs emerge in the aorta. When we compensated the lack of Pten expression by either restoring Pten expression or by inhibiting PI3K activity, we observed normal emergence of HSPCs and normal numbers of HSPCs. To our surprise, when we inhibited PI3K activity in wild type embryos, we observed a similar phenotype as in embryos lacking Pten expression. These results suggests that a moderate level of PI3K activity is required for emerging HSPCs. When using single cell RNA sequencing (scRNA-seq), we observed two, subtly different, HSPCs clusters when HSPCs emerge from the aorta, one having more stem cell-like properties and the other having more progenitor-like characteristics. Our overall conclusions in this chapter are that PI3K signalling controls the survival and stemness of HSPCs. In chapter 3 we used the zebrafish to model a specific patient mutation commonly observed in Noonan Syndrome (NS). We generated a mutant zebrafish line carrying a Shp2a-D61G mutation using the CRISPR/Cas9-technique. This mutant zebrafish displayed several characteristics of NS patients. Focusing on the hematological phenotype, these fish showed an expansion of the myeloid lineage, an increased sensitivity to myeloid differentiation factors, mild anemia and thrombocytopenia. In chapter 4 we focused on the role of Shp2a and Shp2b during zebrafish hematopoiesis. To this end, we performed live imaging and whole mount in situ hybridization on ptpn11a and ptpn11b mutant zebrafish embryos. Surprisingly, we found that lack of Shp2a did not affect the ontogeny of HSPCs, but that lack of Shp2b did. Together, our results suggest that ptpn11b is required for normal emergence of HSPCs at the onset of the definitive wave of hematopoiesis and that ptpn11b mutants overcome these defects at the end of definitive hematopoiesis. In chapter 5 we unveil a subpopulation of HSPCs. These HSPCs do not only express the hematopoietic stem cell marker cd41low, but also express the endothelial marker kdrl. scRNA-seq in zebrafish embryos revealed transcriptomic differences between HSPCs that express both kdrl/cd41low and HSPCs that only express cd41low. In conclusion, the loss of kdrl expression marks the shift from embryonic HSPCs to adult HSPCs. Finally, chapter 6 provides a summarizing discussion of the work presented in each previous chapter in the context of the latest publications in the field and the implications of our findings for future research.