Nephronectin regulates atrioventricular canal differentiation via Bmp4-Has2 signaling in zebrafish

Abstract

The extracellular matrix is crucial for organogenesis. It is a complex and dynamic component that regulates cell behavior by modulating the activity, bioavailability and presentation of growth factors to cell surface receptors. Here, we determined the role of the extracellular matrix protein Nephronectin (Npnt) in heart development using the zebrafish model system. The vertebrate heart is formed as a linear tube in which myocardium and endocardium are separated by a layer of extracellular matrix termed the cardiac jelly. During heart development, the cardiac jelly swells at the atrioventricular (AV) canal, which precedes valve formation. Here, we show that Npnt expression correlates with this process. Morpholino-mediated knockdown of Npnt prevents proper valve leaflet formation and trabeculation and results in greater than 85% lethality at 7 days post-fertilization. The earliest observed phenotype is an extended tube-like structure at the AV boundary. In addition, the expression of myocardial genes involved in cardiac valve formation (cspg2, fibulin 1, tbx2b, bmp4) is expanded and endocardial cells along the extended tube-like structure exhibit characteristics of AV cells (has2, notch1b and Alcam expression, cuboidal cell shape). Inhibition of has2 in npnt morphants rescues the endocardial, but not the myocardial, expansion. By contrast, reduction of BMP signaling in npnt morphants reduces the ectopic expression of myocardial and endocardial AV markers. Taken together, our results identify Npnt as a novel upstream regulator of Bmp4-Has2 signaling that plays a crucial role in AV canal differentiation.

Kctd10 regulates heart morphogenesis by repressing the transcriptional activity of Tbx5a in zebrafish

“…Has2 catalyses the synthesis of hyaluronic acid, the major component of cardiac jelly, thus the cardiac jelly swells at the AVC. Has2 also plays a pivotal role in the epithelium-to-mesenchyme transformation of the endocardium at the AV boundary, which gives rise to the endocardial cushions and valves 22,23,27 . In the forming heart of the kctd10 mutant, Tbx5 is hyperactive due to the absence of functional Kctd10.…”

“…The ectopic expression of has2 results in excess hyaluronic acid between the myocardium and endocardium, causing the cardiac jelly to swell throughout the entire heart tube. The excessive cardiac jelly affects the cross-talk between the myocardium and endocardium to cause the heart malformations 23,27,41 .…”

“…This mutant exhibited AVC malformations and heart looping failure. The mutants were indistinguishable from their siblings until B36 hpf (hours post fertilization), when heart looping starts 8,27 . Thereafter, approximately one-fourth of the embryos displayed heart looping defects, then weakened heart contractility, weakening circulation, an accumulation of blood cells in the venous sinus, and pericardial oedema at 48 hpf (Fig.…”

Strategies for the chemical and biological functionalization of scaffolds for cardiac tissue engineering: a review

“…It contains an N-terminal signal peptide followed by EGF-like repeats, an RGD sequence and a C-terminal MAM domain [66]. Nephronectin is expressed in cardiomyocytes throughout the heart and it is secreted into the cardiac jelly, which is in direct contact with cardiomyocytes and endocardial cells [67]. Neonatal rat cardiomyocytes were seeded on nephronectin-coated glass coverslips, demonstrating the excellent properties of nephronectin for cardiomyocyte adhesion and function [68].…”

“…Cardiomyocytes have been shown to attach and spread markedly faster on Npnt than on gelatin [67]. This was in part due to the presence of the integrin-binding RGD sequence in Npnt [69].…”