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
