Logo: to the web site of Uppsala University

The lymphatic vascular network is composed of a series of blind ended vessels, and is involved in physiological functions such as fluid homeostasis, lipid metabolism and immune trafficking. 

Despite lymphatic endothelial cells being derived from multiple organs, they share common molecular denominators, including the expression of the transcription factor Prox1. However, many of the molecular mechanisms involved in the acquisition and maintenance of lymphatic identity remains to be uncovered.

The aim of my thesis is to investigate how transcription factors and chromatin re-arrangements are involved in the acquisition and maintenance of lymphatic identity. In Paper I, the role of the transcription factor mafbb and its ability to partially compensate for the loss of its paralog mafba is investigated, leading to the description of topologically distinct requitements for LECs development in zebrafish. In Paper II, an evolutionary conserved prox1a enhancer is shown to be necessary for correct lymphangiogenesis in mouse, and its ablation is shown to cause the transition of LECs to hematopoietic-like cells. In Paper III, the effects of partial and total prox1 loss in zebrafish are investigated from a transcriptomic and epigenetic perspective, revealing an important role for prox1 in establishing LEC identity by suppressing genes associated with VEC fate. In Paper IV, the cis-regulatory landscape of prox1a is characterised identifying a number of lymphatic enhancers driving expression in either all LECs in the embryo or in anatomically distinct subsets of lymphatic vasculature. Finally, in Paper V, the chromatin organisation signature of LECs is explored, finding new candidate genes and enhancers active in the lymphatic endothelium.

In summary, my doctoral studies investigated the different levels of regulation of cell identity involved in LECs differentiation. My work focused on chromatin organisation, enhancer activity, transcription factors and differential gene expression to uncover the complex interactions between these separate mechanisms. 

Lymphatic endothelial cells (LECs) migrate across body to form a branched network, which is crucial for fluid drainage and immune cell trafficking of the tissues. However, the molecular mechanisms behind the spatiotemporal regulation and fine-tuning of LEC migration remain largely unknown. The rapid development and the available transgenic tools in zebrafish allow to study these processes in unprecedented resolution and provide new insights into cellular and molecular dynamics regulating lymphangiogenesis. This study aims to dissect the lymphatic development of zebrafish, with a focus of LEC migration and its regulation in the trunk. 

Proper cell migration requires signalling and guidance cue from the environment and tissue-tissue interactions. In paper I, we identified arterial mural cells as a novel source of growth factor and chemokine essential for the migration and robust formation of the lymphatic network. This is important as understanding the sources of guiding molecules can help optimizing the formation and repair of lymphatic vessels in pathological conditions.

VEGFC-VEGFR3 is an essential signalling required throughout lymphangiogenesis. However, it still remains unclear how pro-lymphangiogenic cues are interpreted by LECs to induce differential behaviours. In paper II, we investigated the secondary sprouting which is dependent on VEGFC-VEGFR3 signalling. We characterized the cell migrating behaviours and analysed the molecular signatures of the sprouts. Furthermore, we identified Ca²⁺ activities are required for proper sprouting and potentially serves as modulator for VEGFC-VEGFR3 signalling. 

In paper III, we investigated LEC proliferation driven by VEGFC-VEGFR3 signalling at different development stages. We identified three key timepoints of LEC expansion as well as the novel molecular factors regulating the proliferation. Together we demonstrated the how cell cycle machinery is driven by VEGFC-VEGFR3 signalling. 

In paper IV, we identified enhancers surrounding prox1a which drives the expression in different subsets of the developing lymphatic vasculature, suggesting the tissue specific regulation of prox1a. In addition, we identified enhancer element driving valve expression and it is required for valve development, highlighting the important roles of enhancers in development of lymphatic vasculature. 

Taken together, this work provides novel insight of the molecular dynamics regulating lymphangiogenesis. Our in vivo analysis uncovered new cell types guiding lymphatic vessel formation as well as signal dynamics during embryonic lymphangiogenesis. We believe that the novel insights from this thesis in the future will help establishing pipelines to rebuilding the lymphatic vascular network.