klf2a couples mechanotransduction and zebrafish valve morphogenesis through fibronectin synthesis

Abstract

The heartbeat and blood flow signal to endocardial cell progenitors through mechanosensitive proteins that modulate the genetic program controlling heart valve morphogenesis. To date, the mechanism by which mechanical forces coordinate tissue morphogenesis is poorly understood. Here we use high-resolution imaging to uncover the coordinated cell behaviours leading to heart valve formation. We find that heart valves originate from progenitors located in the ventricle and atrium that generate the valve leaflets through a coordinated set of endocardial tissue movements. Gene profiling analyses and live imaging reveal that this reorganization is dependent on extracellular matrix proteins, in particular on the expression of fibronectin1b. We show that blood flow and klf2a, a major endocardial flow-responsive gene, control these cell behaviours and fibronectin1b synthesis. Our results uncover a unique multicellular layering process leading to leaflet formation and demonstrate that endocardial mechanotransduction and valve morphogenesis are coupled via cellular rearrangements mediated by fibronectin synthesis.

Hemodynamic Forces Sculpt Developing Heart Valves through a KLF2-WNT9B Paracrine Signaling Axis

“…A remarkable finding in our gene expression studies was the lack of any change in Nfatc1 , a gene expressed by endocardial cells that is known to play a critical role in cushion remodeling similar to that observed for KLF2 (Chang et al, 2004), and a lack of change in expression of Trpv4 , Pkd2 ( Trpp2 ) or Fn1 (Figure S5), genes previously associated with klf2a function in the developing zebrafish heart valve (Heckel et al, 2015; Steed et al, 2016). To further investigate potential regulation of NFATC1 by KLF2 we stained Nfatc1 Klf2 KO and control cushions with anti-NFATC1 antibodies.…”

“…Moreover, confocal analysis demonstrated co-expression of wnt9b and klf2a by individual endocardial cells at these sites (Figure 7B, C). Prior studies have demonstrated that klf2a expression in the endocardial cells overlying the developing AV valve is strictly dependent upon blood flow and hemodynamic shear forces (Heckel et al, 2015; Steed et al, 2016; Vermot et al, 2009). As previously shown for klf2a , wnt9b expression was extinguished or severely reduced in the hearts of silent heart ( tnnt2a ) mutant fish that lack blood flow (Sehnert et al, 2002) (Figure 7D, E).…”

“…Physical forces generated by flowing blood have long been proposed to instruct development of the heart, and of heart valves in particular (Hogers et al, 1999; Hove et al, 2003; Sedmera et al, 1999; Vermot et al, 2009; Yalcin et al, 2010, Steed, 2016 #7936). However, many of the cellular processes proposed to be regulated by hemodynamic forces during heart valve development take place away from the flowing blood, especially the sub-endocardial mesenchymal cell remodeling by which cardiac cushions are remodeled to mature valves.…”

“…Seminal studies in zebrafish embryos have established that heart valves fail to form when blood flow through the heart is mechanically blocked (Hove et al, 2003), and have identified endocardial klf2a as a critical effector of fluid shear forces in the developing valve, the expression of which changes rapidly and coordinately with maneuvers that alter hemodynamic forces (Heckel et al, 2015; Renz et al, 2015; Steed et al, 2016; Vermot et al, 2009). These findings are consistent with the identification of Klf2 as a gene regulated by fluid shear in studies of cultured human endothelial cells (Clark et al, 2011; Dekker et al, 2002; Huddleson et al, 2004; Parmar et al, 2006; Zhou et al, 2015), with the observation that endocardial KLF2 expression is similarly altered by changes in blood flow in the embryonic chicken heart (Groenendijk et al, 2005), with the fact that KLF2 expression in both the mouse and human cardiovascular systems follows predicted shear forces with remarkable cellular resolution (Clark et al, 2011; Dekker et al, 2002; Huddleson et al, 2004; Parmar et al, 2006; Zhou et al, 2015), and with the observation that endocardial KLF2 is expressed in a graded manner predicted by exposure to shear forces in developing valves (Figure 1A-E) (Lee et al, 2006).…”

“…Expression of the transcription factor KLF2 is regulated by fluid shear in cultured human endothelial cells ex vivo and in mouse and human vascular endothelium in vivo (Dekker et al, 2002; Dekker et al, 2005; Lee et al, 2006; Parmar et al, 2005; Zhou et al, 2015). In the developing zebrafish heart klf2a is strongly expressed in endothelial cells overlying the developing valve and strictly dependent upon hemodynamic forces, and loss of klf2a confers valve defects like those observed with loss of blood flow or shear (Heckel et al, 2015; Steed et al, 2016; Vermot et al, 2009). Whether KLF2 plays a similar requisite role in mammalian heart development and, more importantly, whether and how information from hemodynamic forces sensed by endocardial cells in contact with blood might be relayed through KLF2 to instruct underlying cells not exposed to blood during the development of heart valves is unknown.…”