Wnt/β-catenin signaling enables developmental transitions during valvulogenesis

Abstract

Heart valve development proceeds through coordinated steps by which endocardial cushions (ECs) form thin, elongated and stratified valves. Wnt signaling and its canonical effector β-catenin are proposed to contribute to endocardial-to-mesenchymal transformation (EMT) through postnatal steps of valvulogenesis. However, genetic redundancy and lethality have made it challenging to define specific roles of the canonical Wnt pathway at different stages of valve formation. We developed a transgenic mouse system that provides spatiotemporal inhibition of Wnt/β-catenin signaling by chemically inducible overexpression of Dkk1. Unexpectedly, this approach indicates canonical Wnt signaling is required for EMT in the proximal outflow tract (pOFT) but not atrioventricular canal (AVC) cushions. Furthermore, Wnt indirectly promotes pOFT EMT through its earlier activity in neighboring myocardial cells or their progenitors. Subsequently, Wnt/β-catenin signaling is activated in cushion mesenchymal cells where it supports FGF-driven expansion of ECs and then AVC valve extracellular matrix patterning. Mice lacking Axin2, a negative Wnt regulator, have larger valves, suggesting that accumulating Axin2 in maturing valves represents negative feedback that restrains tissue overgrowth rather than simply reporting Wnt activity. Disruption of these Wnt/β-catenin signaling roles that enable developmental transitions during valvulogenesis could account for common congenital valve defects.

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

“…While the effects of Wnt9b on sub-endocardial mesenchymal cell condensation are likely to be direct, we also observe changes in mesenchymal cell proliferation deep within the cushion mesenchyme following loss of endocardial Klf2 or Wnt9b that most likely reflect disruption of a still undefined chain of signals initiated by endocardial-mesenchymal cell WNT9B signaling. These studies confirm the proposed complexity of WNT signaling during valve development (Bosada et al, 2016; Gessert and Kuhl, 2010), and highlight the need to study this process in a highly specific spatio-temporal manner.…”

“…Analysis of cushion gene expression revealed a nearly complete loss of Wnt9b and markedly reduced levels of the known canonical WNT target genes Axin2 and Lef1 , as well as the WNT inhibitory proteins Notum and Apcdd1 (Figure 5A-C and Figure S5). A complex and dynamic pattern of expression of WNT ligands has been reported in developing heart valves (Alfieri et al, 2010), and reporters have shown abundant canonical WNT activity in the remodeling cushion (Bosada et al, 2016; Gitler et al, 2003) as well as in abluminal cells adjacent to the endothelial cells lining the site of valve development in the zebrafish heart (Pestel et al, 2016), suggesting that WNT signaling may play an important role in this process. However, the precise role played by WNTs in the remodeling of cushions to mature valves and the mechanisms that regulate WNT expression in the remodeling cushion have not been defined.…”

“…during EMT and later during cushion remodeling), and that multiple cell types (i.e. myocardial, mesenchymal, endocardial) may secrete and/or respond to WNT ligands (Bosada et al, 2016; Hurlstone et al, 2003; Liebner et al, 2004). Approaches that broadly inhibit canonical WNT signaling (e.g.…”

“…Approaches that broadly inhibit canonical WNT signaling (e.g. deletion of ß-catenin or over-expression of the canonical pathway inhibitor DKK1 (Bosada et al, 2016)) have failed to clearly define the roles of WNT signaling in cushion remodeling because early blockade of WNT signaling inhibits EMT and prevents cushion formation altogether (Hurlstone et al, 2003; Liebner et al, 2004). Our studies reveal that endocardial Wnt9b is required to slow mesenchymal cell proliferation and drive the sub-endocardial mesenchymal cell condensation by which a bulky cushion is remodeled into a mature valve cusp.…”

Increased canonical WNT/β-catenin signalling and myxomatous valve disease

“…In fact, it has been demonstrated that valvulogenesis continues after birth to adjust to the postnatal maturation of the heart. WNT/b-catenin signalling is active during heart valve development 6 and has been reported in human MVD. 2,7 There are 19 WNT family members which signal through the canonical (via b-catenin) pathway but can also signal through non-canonical pathways.…”