Congenital heart disease (CHD) can occur in isolation or as part of a syndrome such as Heterotaxy, in which the laterality of internal organs is disrupted. Many CHDs are associated with low heritability, hindering investigations into the underlying genetic causes. Mutation of the X-linked ZIC3, a member of the Zic family of transcriptional regulators, is associated with both isolated CHDs and Heterotaxy yet the cellular and molecular causes underlying ZIC3-associated Heterotaxy remain unknown.
A genetic screen for mutations that affect murine embryogenesis identified a novel null allele of Zic3, called katun (Ka). Ka mutant embryos exhibit Heterotaxy and also incompletely penetrant, partial (posterior) axis duplications and anterior truncations, with the latter two phenotypes redolent of elevated canonical WNT signalling. Previous work has shown that ZIC proteins interact with TCF proteins to inhibit WNT/β-catenin-mediated transcription in model systems. This raises the possibility that dysregulated WNT signalling may underlie some cases of Heterotaxy and CHD.
Using mouse genetics we have shown that (i) ZIC3 loss-of-function leads to elevated WNT signalling and (ii) elevated WNT signalling is consistently associated with L-R axis and cardiac situs abnormalities in the absence of pronounced cilia defects (a common CHD cause). Detailed phenotyping and preliminary RNA-seq analysis of Ka mutant embryos showed that, during gastrulation, prospective definitive endoderm (DE) cells egress from the nascent mesoderm but fail to complete the mesoderm-to-epithelial transition and do not assemble basal basement membrane. Subsequently, DE cells do not completely clear from the emerging node, disturbing node morphogenesis and, presumably impairing nodal flow, resulting in L-R axis and cardiovascular abnormalities.
This work reveals that a specific dose of WNT activity is required for correct DE formation and is a pre-requisite for L-R axis establishment. Furthermore, it implicates genes involved in WNT signalling and DE formation as novel candidates for human CHD variants.