Elevated canonical Wnt signalling disrupts development of the embryonic midline and may underlie cases of ZIC3-associated Heterotaxy

Abstract

Heterotaxy is a congenital abnormality where thoraco-abdominal organs demonstrate abnormal arrangement across the left-right axis of the body, affecting their placement and orientation. The left-right axis is established early in embryogenesis when unidirectional signals emanate from the node at the midline to initiate distinct molecular pathways on the left and right sides of the developing embryo. The gene most commonly mutated in human cases of Heterotaxy is the X-linked ZIC3, but the mechanism by which the ZIC3 transcription factor prevents Heterotaxy remains unknown. A genetic screen for mutations that affect murine embryogenesis identified a novel null allele of Zic3, called katun (Ka). The mutant embryos exhibit Heterotaxy and also incompletely penetrant, partial (posterior) axis duplications and anterior truncation. These latter two phenotypes are redolent of elevated canonical Wnt signalling.

ZIC3 is a member of the Zic family of transcriptional regulators and previous work has shown that the ZICs can interact with TCF proteins to inhibit Wnt/β-catenin mediated transcription when overexpressed in cell lines. Additionally, analysis of Ka and Tcf7l1-/- embryos reveals similar ectopic expression of Foxa2, a direct target of Wnt/β-catenin mediated transcription. This raises the possibility that ZIC3 directly interacts with TCF7L1 to repress the expression of Wnt target genes during gastrulation and consequently, dysregulated Wnt signalling may contribute to Heterotaxy. Via analysis of the murine batface (Bfc) gain-of-function allele of β-catenin that results in elevated Wnt/β-catenin signalling, we have shown that Bfc recapitulates the ectopic expression of Foxa2 seen in Ka mutants. Furthermore, the node of both Ka and Bfc homozygous embryos is misshapen with patches of non-ciliated cells that express endoderm genes. Moreover we find that human ZIC3-Heterotaxy associated mutations have impaired ability to inhibit Wnt/β-catenin signalling. Together this provides strong evidence that Wnt dysregulation may underlie cases of ZIC3-associated Heterotaxy. Show more