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Myocardial infarctions are a major cause of death in the Western world. During such an event the heart loses millions of cardiomyocytes resulting from a lack of oxygen and nutrients. Since adult human cardiomyocytes are considered post-mitotic and replace lost tissue, the damaged tissue is replaced by a permanent scar. This scar does not contribute to contractions and as, a consequence, myocardial infarction patients frequently suffer from heart failure. To combat this, there is a need for new ways to replace the lost cardiac tissue. Opposite to humans, the zebrafish holds the unique capacity to replace the lost cardiomyocytes after cardiac damage. To achieve this the zebrafish makes use of a layer of surviving cardiomyocytes directly adjacent to the injury. We will call this region the border zone from now. The cardiomyocytes in this border zone start to proliferate after injury and produce new myocardial tissue that will eventually repopulate the injury area and restore cardiac function. In this thesis we used to zebrafish to study this special population of proliferating cardiomyocytes based on the premise that knowledge obtained from this cell population holds the potential to boost human cardiomyocyte proliferation as well. For this, we made use of microscopy and sequencing techniques to study the changes these cells undergo leading to proliferation at a single cell level. Doing so, we were the first to describe changes in sarcomeres and metabolism during the process of adult cardiomyocyte proliferation. Moreover, we generated a transcriptomic roadmap towards cardiomyocyte proliferation and identified novel targets that hold the potential to boost this process. For one of these factors we additionally confirmed its importance for successful heart regeneration. In conclusion, we used a live imaging and transcriptomic techniques at the single cell level to study border zone cardiomyocytes prior to and during proliferation. The knowledge we obtain in this thesis on these cells holds promise to stimulate cardiomyocyte proliferation in humans as well and boost heart regeneration after myocardial damage.

Cardiovascular disease is a leading cause of death worldwide. Currently, there is an increasing need to understand cardiac disease mechanisms, identify their pathological progression and find therapeutics to tackle triggers of the disease. Pathological conditions can occur due to genetic mutations leading to cardiac organ dysfunctions, caused by defective mechanical contraction and perturbed electrical signals. These dysfunctions result in structural remodeling and eventual heart failure. In order to understand human genetic diseases of the heart, we show case the zebrafish as an animal model to study these diseases. In our work, sarcomeric and arrhythmogenic forms of cardiomyopathy are investigated. Troponin T (TNNT2), is an important sarcomeric protein that interacts with the contractile proteins upon the presence of calcium (Ca2+) to initiate heart contraction. Adult fish with RK94del heterozygous mutation in Troponin T result in cardiac structural remodeling, as well as slower contractility and changes in Ca2+ dynamics at the embryonic stages. Moreover, phospholamban (PLN) is a sarcoplasmic reticulum (SR) protein that regulate the activity of Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) to control the amount of Ca2+ entry into the SR. Mutants of cardiomyopathy-specific PLN R14del mutation display increased fibrofatty replacement and inflammation around the muscle region of the zebrafish ventricle, similarly to human situations of PLN R14del arrhythmogenic cardiomyopathy. Both mutant adults and embryos show a decrease in Ca2+ levels and dysregulation in the electrical signaling of the heart prior to the structural changes. Istaroxime, which is an enhancer of SERCA activity, causes PLN-SERCA interaction to be relieved. This in turn results in more Ca2+ entry to the mutant heart cells and rescue of the electrical signaling defects. Bulk RNA-sequencing and spatial transcriptomics show that genes involved in immunity and fat binding were increased in levels, while genes involved in contraction of the heart is decreased. Furthermore, immunity and fat binding genes were concentrated at the area with most structural changes. Mitochondria, the energy source of the cell, was also dysfunctional in PLN R14del, which may play a role in the fat buildup and the eventual structural changes. In the last part of this thesis, we investigate cohesinopathies. These are diseases caused by genetic mutations in cohesins, the components of the cell that play a role in the process of cell division. Chronic atrial and intestinal dysrhythmia (CAID) is caused by mutation in Shugoshin-1 (SGO1), whereby patients have a genetic mutation that causes failure in the pace making ability of the cell in the heart and the gut. By targeting SGO1 in zebrafish, mutants show decrease in heart rate and reduction in function of several contraction parameters. Eye function in these mutants were disrupted due to structural damage in a layer of photoreceptors. These dysfunctions lead to early mortality in these fish as the heart does not function optimally and the fish is unable to visualize their food and environment. Therefore, the zebrafish is a promising model to use for understanding human heart genetic diseases and potentially identify therapies to treat the symptoms or alleviate them.

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.