Abstract
ZIC2 mutation is known to cause holoprosencephaly (HPE). A subset of ZIC2 HPE probands harbour cardiovascular and visceral anomalies suggestive of laterality defects. 3D-imaging of novel mouse Zic2 mutants uncovers, in addition to HPE, laterality defects in lungs, heart, vasculature and viscera. A strong bias towards right isomerism indicates a failure to establish left identity in the lateral plate mesoderm (LPM), a phenotype that cannot be explained simply by the defective ciliogenesis previously noted in Zic2 mutants. Gene expression analysis showed that the left-determining NODAL-dependent signalling cascade fails to be activated in the LPM, and that the expression of Nodal at the node, which normally triggers this event, is itself defective in these embryos. Analysis of ChiP-seq data, in vitro transcriptional assays and mutagenesis reveals a requirement for a low-affinity ZIC2 binding site for the activation of the Nodal enhancer HBE, which is normally active in node precursor cells. These data show that ZIC2 is required for correct Nodal expression at the node and suggest a model in which ZIC2 acts at different levels to establish LR asymmetry, promoting both the production of the signal that induces left side identity and the morphogenesis of the cilia that bias its distribution.
Highlights
Laterality of the heart and viscera is determined by the NODAL pathway
The mouse phenotype reveals a laterality defect which shows a strong bias towards right-isomerism, indicative of a lack of left-sided identity in the lateral plate mesoderm during morphogenesis of these organ systems
This is supported by molecular data indicating that the left-determining NODAL signalling cascade fails to be activated in the lateral plate mesoderm (LPM)
Summary
Laterality of the heart and viscera is determined by the NODAL pathway. Bilateral symmetry is broken through generation of a leftward fluid flow by cilia within the node[9,10]. Ectopic activation of Pitx2c in the right lateral mesoderm only results in situs inversus whereas bilateral expression of Pitx2c results in two morphologically left sides, known as left-isomerism. Expression of Nodal at the node and of downstream genes in the LPM is reduced in the Zic2ku mutant[24], but ectopic right-sided or bilateral expression, which is present in iv embryos with nodal cilia defects[12,25], is not observed. Analysis of the phenotype reveals a strong bias towards right isomerism, indicative of defective left-sided identity specification during development This is supported by gene expression data revealing weak or absent Nodal node expression and loss of downstream gene expression in the LPM. Together with a previous study that identified a role for ZIC2 in ciliogenesis, our results suggest a model in which ZIC2 acts at multiple levels during the establishment of laterality, upstream of genes and events that are critical for the process to take place
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