Control of dynamic cell behaviors during angiogenesis and anastomosis by Rasip1

Abstract

Organ morphogenesis is driven by a wealth of tightly orchestrated cellular behaviors, which ensure proper organ assembly and function. Many of these cell activities involve cell-cell interactions and remodeling of the F-actin cytoskeleton. Here, we analyze the requirement for Rasip1 (Ras-interacting protein 1), an endothelial-specific regulator of junctional dynamics, during blood vessel formation. Phenotype analysis of rasip1 mutants in zebrafish embryos reveal distinct functions of Rasip1 during sprouting angiogenesis, anastomosis and lumen formation. During angiogenic sprouting, loss of Rasip1 causes cell pairing defects due to a destabilization of tricellular junctions, indicating that stable tri-cellular junctions are essential to maintain multicellular organization within the sprout. During anastomosis, Rasip1 is required to establish a stable apical membrane compartment; rasip1 mutants display ectopic, reticulated junctions and the apical compartment is frequently collapsed. Loss of Ccm1 and Heg1 function mimics junctional defects of rasip1 mutants. Furthermore, downregulation of ccm1 and heg1 leads to a delocalization of Rasip1 at cell junctions, indicating that junctional tethering of Rasip1 is required for its function during junction formation and stabilization during sprouting angiogenesis.

Initiation of Lumen Formation from Junctions via Differential Actomyosin Contractility Regulated by Dynamic Recruitment of Rasip1

“…We next explored the recruitment of apical proteins during the initial lumen formation of DLAV. Rasip1, a vascular-specific regulator of GTPases, has been implicated in the de novo formation of apical compartments during vasculogenesis and angiogenesis ( Barry et al, 2016; Lee et al, 2021 ). We therefore generated a fluorescent reporter line [ Tg(fli1a: Rasip1-scarlet-I) ubs59 to follow the distribution of Rasip during lumen formation in the DLAV.…”

“…Rasip1 has been implicated in apical junction clearance ( Barry et al, 2016; Lee et al, 2021 ). To explore the temporal and spatial junctional dynamics of this process in both wild-type embryos and rasip1 mutants, we utilized the rasip1 ubs28 allele, which harbors a 35 kb deletion from exon 3 to 16( Lee et al, 2021 ). We first examined the shape of junctions via antibody staining of ZO-1 and Cdh5 at the DLAV.…”

“…Luminal defects in vasculogenesis in rasip1 mutants have been proposed to be caused by the failure to remove junctional proteins from the apical membrane ( Barry et al, 2016; Lee et al, 2021 ). To elucidate the mechanisms underlying the mislocalized junctional complexes in zebrafish DLAV, we created a photoconvertible Cdh5 reporter, Cdh5-mClav [ Tg(cdh5: cdh5-mClavGR2) ubs58 ] (Fig.…”

“…In mouse mutant embryos, the angioblasts fail to localize junctional proteins to the cord periphery during vasculogenesis of the dorsal aorta (DA) ( Koo et al, 2016; Xu et al, 2009, 2011 ). Similarly, in zebrafish embryos undergoing DLAV formation, rasip1 mutants manifest ectopic and reticulated junctional patterns within the presumed apical compartments ( Lee et al, 2021 ).…”