Alternatively spliced isoforms of Fibronectin, Tenascin-R and other potential players in early vasculogenesis

Abstract

The absence of both the EIIIA and EIIIB domains of fibronectin (FN) has been shown to negatively affect blood vessel formation and maintenance. Vascular defects have been observed in the yolk sacs of EIIIA/B double-null embryos by as early as embryonic day E9.5, and these defects are likely due to alterations in the extracellular matrix (ECM). Therefore, I have conducted this study to investigate the differences in the ECM composition of the yolk sac in the presence and absence of EIIIA and EIIIB. I first collected yolk sacs at E9.5 from wild type, EIIIA-null, EIIIB-null, EIIIA/B heterozygous and EIIIA/B double-null mouse embryos, enriched for ECM content, and used quantitative proteomics to analyze their ECM composition. From these data, we identified a set of matrisome proteins that had decreased abundance in EIIIA/B double-null yolk sacs but were relatively unchanged in single-null and heterozygous yolk sacs compared to wild type.

Some of these proteins could play a role in ECM remodeling or directly affect angiogenesis, and their reduced level in double-null yolk sacs might contribute to the vascular defects seen in the double-null tissue. Subsequently, I carried out further studies with tenascin-R (TN-R), one of proteins that was downregulated in the ECM of EIIIA/B double-null yolk sac. TN-R has been previously described to be restricted to the central nervous system, and our finding of TN-R in the yolk sac is novel. TN-R is localized to the mesoderm layer of yolk sacs. TN-R fibers partially overlap with FN, and TN-R area coverage in EIIIA/B double-null yolk sacs are decreased compared to wild type, suggesting that the presence of EIIIA/B promotes TN-R assembly in the yolk sac ECM. In addition, TN-R colocalizes with blood vessels in both the yolk sac and the retina, suggesting that TN-R might participate in vasculogenesis and angiogenesis at these locations.

Together, this study extends our understanding of yolk sac ECM, provides insight into the role of EIIIA and EIIIB domains, identifies novel expression patterns of ECM proteins, and opens up the possibility of a novel function for TN-R.