Nkx2-5 regulates cardiac growth through modulation of Wnt signaling by R-spondin3

Abstract

A complex regulatory network of morphogens and transcription factors is essential for normal cardiac development. Nkx2-5 is among the earliest known markers of cardiac mesoderm that is central to the regulatory pathways mediating second heart field (SHF) development. Here, we have examined the specific requirements for Nkx2-5 in the SHF progenitors. We show that Nkx2-5 potentiates Wnt signaling by regulating the expression of the R-spondin3 (Rspo3) gene during cardiogenesis. R-spondins are secreted factors and potent Wnt agonists that in part regulate stem cell proliferation. Our data show that Rspo3 is markedly downregulated in Nkx2-5 mutants and that Rspo3 expression is regulated by Nkx2-5. Conditional inactivation of Rspo3 in the Isl1 lineage resulted in embryonic lethality secondary to impaired development of SHF. More importantly, we find that Wnt signaling is significantly attenuated in Nkx2-5 mutants and that enhancing Wnt/ β-catenin signaling by pharmacological treatment or by transgenic expression of Rspo3 rescues the SHF defects in the conditional Nkx2-5 +/− mutants. We have identified a previously unrecognized genetic link between Nkx2-5 and Wnt signaling that supports continued cardiac growth and proliferation during development. Identification of Rspo3 in cardiac development provides a new paradigm in temporal regulation of Wnt signaling by cardiac-specific transcription factors.

Wnt/β-catenin signaling enables developmental transitions during valvulogenesis

“…2C,D,G,H) and previous Wnt reporter studies (Alfieri et al, 2010;Cai et al, 2013;Cambier et al, 2014;Gillers et al, 2015) indicate that Wnt signaling persists as cushions grow and elongate into mature valves. To explore Wnt signaling dynamics during the cushion/valve expansion phase, we assayed Wnt activity by Lef1 immunostaining.…”

“…Wnt reporter transgenic mice suggest that canonical Wnt signaling is active in AVC and OFT cushion mesenchyme during this growth phase (Gitler et al, 2003;Alfieri et al, 2010;Klaus et al, 2012;Gillers et al, 2015;Cai et al, 2013;Cambier et al, 2014). Known mitogenic roles of Wnt/β-catenin signaling in other developmental contexts (reviewed by Niehrs and Acebron, 2012) further support a role for Wnt in cushion expansion.…”

“…Previous surveys of Wnt/β-catenin activity in heart development have primarily used the TOPGAL and BATGAL transgenic reporters (Alfieri et al, 2010;Cai et al, 2013;Cambier et al, 2014;Gitler et al, 2003;Maretto et al, 2003) that are non-specific in some contexts (Al Alam et al, 2011;Barolo, 2006). Therefore, we examined patterns of Wnt signaling activity using a lacZ knock-in allele of Axin2, a well-established universal Wnt target gene (Jho et al, 2002;Lustig et al, 2002).…”

Maturing human pluripotent stem cell-derived cardiomyocytes in human engineered cardiac tissues

“…Additionally, anatomical blocks were occasionally observed that prevented continuous AP propagation, but which could be synchronized by means of electrical stimulation. Genetically, the hECT expression of MYH6 (α-MHC), TNNT2 (cardiac troponin-T), GJA1 (connexin-43) and NKX2-5 , a transcription factor for cardiac mesoderm specification [245], was demonstrated to be upregulated relative to age-matched aggregates, moreover the expression levels in the hECTs were in the range expressed in fetal and adult tissue. MYH7 (β-MHC) expression was significantly upregulated in hECTs relative to age-matched aggregates but also significantly less than found in either fetal or adult human tissue.…”

A common Shox2-Nkx2-5 antagonistic mechanism primes the pacemaking cell fate in the pulmonary vein myocardium and sinoatrial node

“…The evidence that Shox2 interacts with Nkx2-5 directly and their substantial genome-wide co-occupancy provide a potential mechanistic explanation for the antagonistic function of Shox2. However, although the inhibitory effect of Nkx2-5 over Isl1, Tbx3 and Hcn4 has been documented (Cambier et al, 2014;Mommersteeg et al, 2007b;Prall et al, 2007), the cis-regulatory elements associated with these genes that mediate such effects remain to be characterized. It is unclear whether Nkx2-5 inhibits the expression of these pacemaker genes directly, although it inhibits cardiac progenitor genes directly (Watanabe et al, 2012).…”