Cdc42, orchestrator of vascular morphogenesis in the retina

University dissertation from Uppsala : Acta Universitatis Upsaliensis

Abstract: Cdc42 is a small GTPase that controls many cellular functions related to cytoskeletal dynamics, such as migration, polarity, and proliferation. Despite what we know of Cdc42 in other cell types, not much research has been done on the vasculature. This thesis describes the consequences of Cdc42 deletion in two vascular cell types—endothelial and mural cells—during developmental angiogenesis.In paper I, we demonstrate through a combination of in vitro, in silico, and in vivo assays, that Cdc42-deficient endothelial cells migrate less and fail to distribute normally in areas of naturally occurring high proliferation during angiogenesis, causing vascular malformations with enlarged lumens. In addition, these cells present impaired filopodia formation, a disadvantage for the tip cell position, disturbed axial polarity and altered junctions.With an in vivo approach, in paper III we demonstrate that the deletion of Cdc42 in mural cells has consequences on the morphogenesis of the retinal vasculature. Cdc42-deficient mural cells proliferate less and cannot keep up with the nascent angiogenic vasculature, which results in a complete pericyte loss at the sprouting front. Furthermore, we describe that mural cells contribute to the remodeling of the vasculature, also after the initial phases of angiogenesis.The CreERT2 system is frequently used for conditional gene deletion and lineage tracing. Tamoxifen administration allows spatiotemporally controlled recombination of fluorescent reporters, and tracing of the labeled cells. However, in the course of our studies, we observed tamoxifen-independent recombination. In paper II, we describe this phenomenon in detail, using different combinations of CreERT2 and fluorescent reporter lines. We conclude that tamoxifen-independent recombination is a widespread occurrence, and that fluorescent reporter lines present varying levels of susceptibility to it.In summary, the work presented here sheds new light on the role of Cdc42 in the vasculature. Additionally, this thesis describes in detail an important feature of CreERT2 and reporter lines that should be taken into account when performing lineage-tracing experiments.

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