Vegfc Regulates Bipotential Precursor Division and Prox1 Expression to Promote Lymphatic Identity in Zebrafish

Abstract

Lymphatic vessels arise chiefly from preexisting embryonic veins. Genetic regulators of lymphatic fate are known, but how dynamic cellular changes contribute during the acquisition of lymphatic identity is not understood. We report the visualization of zebrafish lymphatic precursor cell dynamics during fate restriction. In the cardinal vein, cellular commitment is linked with the division of bipotential Prox1-positive precursor cells, which occurs immediately prior to sprouting angiogenesis. Following precursor division, identities are established asymmetrically in daughter cells; one daughter cell becomes lymphatic and progressively upregulates Prox1, and the other downregulates Prox1 and remains in the vein. Vegfc drives cell division and Prox1 expression in lymphatic daughter cells, coupling signaling dynamics with daughter cell fate restriction and precursor division.

Vegfc acts through ERK to induce sprouting and differentiation of trunk lymphatic progenitors

“…In the trunk, Flt4 signals through ERK to drive progenitor differentiation, expansion and sprouting at the earliest stages of lymphatic development. These observations are consistent with recent findings that demonstrate an important role for Vegfc in the direct induction of Prox1 expression in lymphatic progenitors within the posterior cardinal vein, as well as their subsequent expansion (Koltowska et al, 2015). Our results further show that ERK plays an essential role downstream of Vegfc and Flt4 in this crucial early step in lymphatic development.…”

“…TD formation entails a complex migratory process in which Prox1-positive endothelial cells sprout from the posterior cardinal vein (PCV) beginning at 30 hpf (Koltowska et al, 2015;Nicenboim et al, 2015;Yaniv et al, 2006). After sprouting dorsally, Prox1-positive lymphatic endothelial cells branch laterally and sprout along the horizontal myoseptum as parachordal cells (PACs).…”

Vegfd modulates both angiogenesis and lymphangiogenesis during zebrafish embryonic development

“…Overall, our data suggest that zebrafish Vegfd can signal through Kdr (Vegfr2) during primary angiogenesis and most likely through Flt4 during secondary angiogenesis based on genetic interactions. Interestingly, a recent study by Shin et al shows that Prox1 induction in zebrafish facial lymphatics can occur in the absence of Flt4 signaling (Shin et al, 2016b), but in the trunk the induction of Prox1 expression is Vegfc/Flt4 dependent (Koltowska et al, 2015;Shin et al, 2016b). These studies combined suggest that differential signaling events are at play in facial and trunk lymphangiogenesis.…”

“…Sprouts emerging from the cardinal vein can become vISVs or LECs when they either anastomose with existing aISVs to form vISVs, or fail to anastomose and migrate dorsally to form parachordal LECs at the horizontal myoseptum (HM) (Isogai et al, 2003;Koltowska et al, 2013Koltowska et al, , 2015. Parachordal LECs in the HM proliferate and migrate dorsally and ventrally to form the major trunk lymphatics: the thoracic duct (TD), dorsal longitudinal lymphatic vessel (DLLV) and the intersegmental lymphatic vessels (ISLVs) (Cha et al, 2012;Hogan et al, 2009b;Küchler et al, 2006;Yaniv et al, 2006).…”

“…Vegfd has been shown by MO knockdown to compensate for loss of Vegfc during facial lymphatic development but not elsewhere in the embryo . Growth factor-induced signaling downstream of Flt4 occurs through the activation of Erk, which is necessary and sufficient in the trunk to induce the normal sprouting of LECs and expression of the LEC marker Prox1 (Koltowska et al, 2015;Shin et al, 2016b). During facial lymphangiogenesis from the CCV, Erk signaling controls LEC sprouting but not Prox1 expression, suggesting spatially distinct mechanisms that remain to be fully understood (Shin et al, 2016b).…”