The molecular mechanism of Semaphorin 3E-Plexin-D1 signaling in shaping vascular topology during development

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The proper function of the cardiovascular system is dependent on its correct patterning during development. In addition to VEGF, a major regulator of vascular morphogenesis, also a class of molecules previously recognized as axon guidance cues (ephrins, semaphorins, slits, and netrins), is required for vascular patterning. A novel signaling cascade regulating angiogenesis via a reciprocal interaction between the VEGF pathway and the Semaphorin 3E (Sema3E)-Plexin-D1 pathway, was recently discovered. It was shown that in the retina, VEGF directly controls the expression of Plexin-D1 in endothelial cells at the front of actively sprouting blood vessels. Although Sema3E secreted by retinal neurons is evenly distributed, Sema3E-Plexin-D1 signaling is temporally and spatially regulated by VEGF through its regulation of Plexin-D1 expression. Sema3E-Plexin-D1 signaling then negatively regulates the activity of the VEGF-induced Dll4-Notch signaling pathway, which controls the balance of tip and stalk cell fate decisions. Thus, Sema3E-Plexin-D1 signaling is highly required for controling the topology of the retinal vascular network. This function is important in embryonic development, but also in pathlogical conditions like ischemic retinopathy. My proposal builds upon these recent findings and aims to fill the gap of knowledge in understanding the cell autonomous mechanism of this novel feedback mechanism in angiogenesis. My first aim is to examine the in vivo function of Sema3E-Plexin-D1 signaling during developmental angiogenesis. I hypothesize, that by regulating VEGF activity, Sema3E-Plexin-D1 signaling determines the timing of tip/stalk cell switching, which is critical for continuous vascular network formation. I will investigate how this negative feedback mechanism is translated into tip/stalk cell behavior using retinal explant and differentiated embryonic stem cell analysis in combination with time lapse imaging. I will trace cellular behavior in the absence of Sema3E-Plexin-D1 signaling and analyze its consequences on vascular topology. My second aim is to understand the molecular mechanisms of how Sema3E-Plexin-D1 signaling regulates VEGF activity during angiogenesis. I hypothesize that Sema3E-Plexin-D1 modulates VEGF activity and downstream Dll4 expression by directly regulating either VEGFR2 trafficking or VEGF-induced Erk phosphorylation. I will test these two hypotheses using biochemistry and cell biology assays as well as the in vivo retinal vasculature system. Together, my studies aim at furthering the understanding of the fundamental molecular and cellular mechanisms governing angiogenic processes and neurovascular interactions. They will also shed new light on potential therapeutic strategies for the treatment of cardiovascular disease, diseases involving angiogenesis and angiogenesis-dependent tumors.