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Abstract

In higher vertebrates, the development of a functional circulatory system based on blood and lymphatic vessels is a basic step in order to generate a full organism. The formation of the vasculature involves the generation of mesodermal-derived angioblasts and their subsequent differentiation into blood endothelial or lymphatic endothelial cell lineages. The switch between the undifferentiated and cell-specific genetic programs during cell differentiation requires an orchestrated spatiotemporal coordination of gene expression. JUNB, a member of the AP-1 family, is a context-dependent transcription factor that exerts both positive and negative functions. Loss of function studies in mice revealed that JUNB is a key regulator of vascular development in embryos. Hence, JUNB transactivates pro-angiogenic molecules such as Vegfa, Cbfβ and Hmox1. Recently, Junb was described to regulate the development of lymphatic vessels in zebrafish via its target miR182. However, it remained unclear whether activator functions of JUNB are relevant for lymphangiogenesis. Therefore, I aimed to investigate whether JUNB is necessary for the formation of lymphatics and if so, to unravel in which specific step of the lymphatic vascular development JUNB is implicated. For this purpose, I used a dual approach of in vitro differentiation of mouse embryonic stem cells into lymphatic endothelial cells and an in vivo approach by generating junb mutant zebrafish in the background of the transgenic reporter line Tg(fli:EGFP)y1 to evaluate the development of the vasculature. The study of the JUNB kinetics during the LEC differentiation process revealed that JUNB is strongly induced at the RNA and protein level during the angioblast formation until the formation of the LEC-like cells. Strikingly, Junb-/- mESCs failed to form proper LECs due to considerable cell death during the angioblast formation. This increased apoptosis could be associated to a failure to initiate the induction of VEGFR1 and VEGFR2 expression. In the parallel in vivo approach, novel zebrafish mutants with ablated junba and junbb expression were generated using CRISPR-Cas9 technology. Successful mutations resulted in a premature end of the reading frame. Homozygous junba-/- embryos could not be identified and the Mendelian ration of wildtype and heterozygous offspring suggests a recessive lethal phenotype. By contrast, junbb mutants were detected according to expected Mendelian ratio, reached adulthood and were fertile. Albeit the mutant embryos exhibited an allele-dependent decrease in the number of parachordal cells present at 3 days post fertilization; almost all the analyzed embryos displayed a complete thoracic duct at 5 dpf. Surprisingly, the mutants developed ectopic sprouts at the dorsal side of the trunk from 3 dpf until 5 dpf recapitulating the phenotype previously observed upon neuronal loss of soluble vegfr1 in zebrafish. In summary, these data underscore the role of JUNB not only in lymphangiogenesis but also at an earlier developmental stage, namely the angioblast formation and suggest a JUNB-dependent regulation of vascular endothelial growth factor receptors during development.