Clarification of mural cell coverage of vascular endothelial cells by live imaging of zebrafish

Abstract

Mural cells (MCs) consisting of vascular smooth muscle cells and pericytes cover the endothelial cells (ECs) to regulate vascular stability and homeostasis. Here, we clarified the mechanism by which MCs develop and cover ECs by generating transgenic zebrafish lines that allow live imaging of MCs and by lineage tracing in vivo. To cover cranial vessels, MCs derived from either neural crest cells or mesoderm emerged around the preformed EC tubes, proliferated and migrated along EC tubes. During their migration, the MCs moved forward by extending their processes along the inter-EC junctions, suggesting a role for inter-EC junctions as a scaffold for MC migration. In the trunk vasculature, MCs derived from mesoderm covered the ventral side of the dorsal aorta (DA), but not the posterior cardinal vein. Furthermore, the MCs migrating from the DA or emerging around intersegmental vessels (ISVs) preferentially covered arterial ISVs rather than venous ISVs, indicating that MCs mostly cover arteries during vascular development. Thus, live imaging and lineage tracing enabled us to clarify precisely how MCs cover the EC tubes and to identify the origins of MCs.

Mural-Endothelial cell-cell interactions stabilize the developing zebrafish dorsal aorta

“…Through use of transgenic reporters and nuclear tracking studies, we demonstrate that the majority of vSMCs that end up resident on the dorsal aorta in the zebrafish trunk are twist1 + cells derived from the sclerotome, not sox10 + neural crest-derived cells. Our findings are entirely consistent with previous mammalian reports and with a well-documented recent report in the zebrafish from Ando et al (2016) that identified the paraxial and lateral plate mesoderm as the source of mural cells in the trunk. Together with those of Ando et al (2016), our data suggest that most early dorsal aorta vSMC progenitors emerge from the immediately adjacent twist1 + sclerotomal compartment of the somites, and in most cases appear to move little distance before investing the dorsal aorta.…”

“…S5). The findings we describe above are entirely consistent with a study recently published by Ando et al (2016).…”

“…2A,B), with recent work in the zebrafish and other species suggesting that the majority of trunk vSMCs are mesothelial derived (Ando et al, 2016;Majesky, 2007;Pouget et al, 2008;Wiegreffe et al, 2007;Wasteson et al, 2008;Armulik et al, 2011a). Prior to the onset of vSMC recruitment to the dorsal aorta, vSMC markers tagln (sm22) and pdgfrb are expressed fairly broadly in the ventralmedial quadrant of the somites (Fig.…”

“…A number of previous studies have provided solid documentation that the zebrafish dorsal aorta acquires a vSMC-containing vascular wall similar to that found in other vertebrates, and have suggested a somitic origin for these cells (Santoro et al, 2009;Whitesell et al, 2014;Fortuna et al, 2015;Wang et al, 2014;Ando et al, 2016;Zaucker et al, 2013). We crossed a previously reported zebrafish Tg(tagln:egfp) smooth muscle cell transgenic reporter line (Seiler et al, 2010;Yang et al, 2003) to a Tg(kdrl:mCherryCAAX) red fluorescent vascular endothelial-specific transgenic reporter line (Fujita et al, 2011) and employed confocal microscopy to examine the time course of mural/vSMC recruitment to the dorsal aorta during early (Kimmel et al, 1995), with the magnified red boxed region shown in B and blue boxed region imaged in C-F. (B) Schematic ('cut-away') diagram showing the anatomy of the zebrafish trunk and its blood vessels at approximately 2 days post-fertilization.…”

“…Pericytes are associated with smaller-caliber vessels, in particular capillary beds and post-capillary venules. Pericytes associate with these smaller-caliber vessels in lower numbers, with pericyte/EC ratios between 1:1 and 1:10, localizing primarily along endothelial cell-cell junctions, branch points, and in areas that need to maintain high levels of barrier function [such as the blood-brain barrier (BBB)] (Ando et al, 2016;Owens, 1995;Daneman et al, 2010;Armulik et al, 2010Armulik et al, , 2011a. PDGF-BB/PDGFRβ signaling is required for recruitment of vSMCs and pericytes to vessels (Hellstrom et al, 1999;Fortuna et al, 2015;Lindblom et al, 2003;Lindahl et al, 1997;Hirschi et al, 1999;Lehti et al, 2005;Benjamin et al, 1998), although a variety of other signaling pathways have been shown to contribute to mural cell differentiation, recruitment and stabilization, in particular TGFβ, Notch and EGF signaling among others (Lee et al, 2007;Higashiyama et al, 1993;Iivanainen et al, 2003;Krymskaya et al, 1997;Wang et al, 2014Wang et al, , 2012Kofler et al, 2015;Fortuna et al, 2015;Kennard et al, 2008;Hirschi et al, 2003;Aplin et al, 2010;Stratman et al, 2010;Zaucker et al, 2013).…”