Fibronectin signals through integrin α5β1 to regulate cardiovascular development in a cell type-specific manner

Abstract

Fibronectin (Fn1) is an evolutionarily conserved extracellular matrix glycoprotein essential for embryonic development. Global deletion of Fn1 leads to mid-gestation lethality from cardiovascular defects. However, severe morphogenetic defects that occur early in embryogenesis in these embryos precluded assigning a direct role for Fn1 in cardiovascular development. We noticed that Fn1 is expressed in strikingly non-uniform patterns during mouse embryogenesis, and that its expression is particularly enriched in the pharyngeal region corresponding with the pharyngeal arches 3, 4, and 6. This region bears a special importance for the developing cardiovascular system, and we hypothesized that the localized enrichment of Fn1 in the pharyngeal region may be essential for cardiovascular morphogenesis. To test this hypothesis, we ablated Fn1 using the Isl1Cre knock-in strain of mice. Deletion of Fn1 using the Isl1Cre strain resulted in defective formation of the 4th pharyngeal arch arteries (PAAs), aberrant development of the cardiac outflow tract (OFT), and ventricular septum defects. To determine the cell types responding to Fn1 signaling during cardiovascular development, we deleted a major Fn1 receptor, integrin α5 using the Isl1Cre strain, and observed the same spectrum of abnormalities seen in the Fn1 conditional mutants. Additional conditional mutagenesis studies designed to ablate integrin α5 in distinct cell types within the Isl1+ tissues and their derivatives, suggested that the expression of integrin α5 in the pharyngeal arch mesoderm, endothelium, surface ectoderm and the neural crest were not required for PAA formation. Our studies suggest that an (as yet unknown) integrin α5-dependent signal extrinsic to the pharyngeal endothelium mediates the formation of the 4th PAAs.

Neural crest cell-autonomous roles of fibronectin in cardiovascular development

“…2F): 29-42% penetrance (Table S2). Cardiac NC contributes mesenchymal cells to the thymus, regulating morphogenesis of this organ (Bockman and Kirby, 1984); consistent with that, we found that 44% of mutant embryos had mild defects in thymus development (Chen et al, 2015). Taken together, these phenotypes are characteristic of many other mouse mutants with defective development or function of the cardiac NC (Hutson and Kirby, 2007).…”

“…The high evolutionary conservation of Fn1 and the essential role of Fn1 in cardiovascular development suggested that enriched expression of Fn1 in specific embryonic regions is biologically significant. The studies described in this paper and in our earlier publication (Chen et al, 2015) reveal requisite, cell typespecific functions of Fn1 in embryogenesis and cardiovascular development.…”

“…S2). The incidence of AAA defects varied from 57% to 82% among the three strains assayed (Table S2); (2) formation of the intracardiac cartilage, similar to that described in Gao et al (2010) occurred at 89-100% penetrance (Chen et al, 2015); and (3) the membranous ventricular septal defect (VSD; curved arrow in Fig. 2F): 29-42% penetrance (Table S2).…”

“…These defects are specific to abnormal development of the 4th pair of PAAs (Stoller and Epstein, 2005) and could be due to either defective PAA formation or defective remodeling of the well-formed PAAs into the AAAs (Conway et al, 2003). Injection of black India ink into the hearts of control and mutant embryos isolated at E10.5 showed that the three pairs of PAAs formed normally (n=3) (Chen et al, 2015). Similarly, examination of histological sections in n>10 mutants and controls 1 day later, at E11.5, indicated that all of the PAAs were well formed in the mutants (e.g.…”

“…Previous studies demonstrated that integrin α5β1 is a major Fn1 receptor in early and mid-gestation mouse embryos (Chen et al, 2015;George et al, 1997George et al, , 1993Mittal et al, 2013Mittal et al, , 2010Pulina et al, 2014Pulina et al, , 2011Takahashi et al, 2007;Yang et al, 1999Yang et al, , 1993. Therefore, we used conditional mutagenesis to ablate integrin α5 in the NC.…”