Lymphatics in Noonan Syndrome

In this thesis we describe the use of zebrafish (Danio rerio), to study lymphangiogenesis in health and in the presence of patient associated Noonan syndrome mutations. In chapter 1 we give a general overview of the subjects discussed in this thesis, namely the function of the lymphatic vasculature, lymphangiogenesis in the zebrafish and the congenital disorder Noonan syndrome. In chapter 2 we review the current knowledge of the role of SHP2 variants in rare diseases and advances in the understanding of its pathogenesis using model systems. These two chapters provide the background for the scientific questions we pursue in the subsequent experimental chapters. In chapter 3 we describe the consequences of the absence of the ptpn11a and ptpn11b genes in zebrafish embryos. We show that all double mutant zebrafish embryos completely lack their lymphatic vasculature including the lymphatics in the head. In addition, we show that the absence of ptpn11a and ptpn11b targets the lymphatic vasculature, but not the venous connections established by the same cell population. In chapter 4 we describe how we generated zebrafish knockouts for both sos1 and sos2. We show that loss of sos1 is viable but knockout of sos2 is embryonically lethal. We show that all double mutant zebrafish embryos lack an intact lymphatic vasculature including the lymphatics in the head. Additionally, sos double mutants also displayed defects in the establishment of venous connections in the zebrafish trunk. In chapter 5 we describe the establishment of a zebrafish line with a patient-associated Noonan syndrome mutation in Sos2 and compare this line with a previously characterized zebrafish model for Noonan Syndrome. We show a lymphatic phenotype, including vasodilation of the thoracic duct (TD), whereas lymphangiogenesis was normal in ptpn11a+/D61G embryos. We demonstrate that treatment with the MEK inhibitor trametinib, which is currently being used in the clinic, effectively rescued the dilated TD associated with the Sos2+/M264R mutation, highlighting the potential of our model to study treatment options in zebrafish. In chapter 6 the clinical outcomes of trametinib therapy in eight patients with Noonan syndrome-like RASopathies and central conducting lymphatic anomaly was assessed, each offering unique insights into the therapeutic efficacy of MEK inhibition. Finally, chapter 7 provides a summarizing discussion of the observations and findings of the previous chapters in the context of implications for further research. To conclude, the hope is that an integrated understanding of Noonan syndrome and lymphangiogenesis will not only elucidate fundamental developmental biological questions, but also pave the way for better management of lymphatic diseases in patients with Noonan syndrome.