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 cardiovascular abnormalities are associated with low heritability, hindering investigations into the underlying genetic causes of CHD. Heterotaxy, the most highly heritable cardiovascular abnormality, is frequently shown to arise from mutation of the ciliome. Mutation of the X-linked transcription factor ZIC3, a member of the Zic family of transcriptional regulators, is associated with both isolated CHDs and Heterotaxy but 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 truncation, with the latter two phenotypes redolent of elevated canonical WNT signalling. Previous work has shown that ZIC proteins can 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 found 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. Detailed phenotyping 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 cardiac situs 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.