Zinc finger protein of the cerebellum 2 (Zic2) protects against teratogenesis during early mammalian development

Abstract

A key role of the master regulatory gene, Zinc finger protein of the cerebellum 2 (ZIC2/Zic2), is to transduce NODAL signalling in the mid-gastrula node to direct correct axial midline formation. Extensive studies confirm that significant ZIC2/Zic2 loss-of-function mutations induce a highly prevalent and severe congenital defect called holoprosencephaly (HPE), in which impaired midline formation disrupts dorso-ventral forebrain patterning and causes forebrain hemispheric separation to fail. Importantly, the inability of large-scale genome sequencing studies to genetically solve ~75% of human HPE cases suggests that environmental teratogens may frequently precipitate HPE pathogenesis by exacerbating the effects of 'mild' variants in HPE-associated genes. Therefore, the present study used the Kumba (Ku) mouse to investigate whether exposure to either hyperglycaemia or ethanol (EtOH) synergistically induces HPE or HPE-like defects in Zic2 haploinsufficient murine embryos. Contrary to previous reports that heterozygous Ku embryos maintain normal anterior development, the morphological, microCT and gene-expression analyses conducted herein showed that Zic2 haploinsufficiency significantly and frequently impairs midline formation during gastrulation and neurulation. As a result, ~50% of heterozygous Ku embryos exhibit significantly altered forebrain morphology by mid-gestation. Moreover, low/moderate-level EtOH exposure exacerbated these impacts both in whole embryo culture and in vivo, disrupting NODAL- and SHH-associated midline gene-expression patterns in the same manner and during the same developmental window as Ku allele homozygosity. By mid-gestation, EtOH-exposed heterozygous Ku embryos frequently exhibit dysmorphic forebrain lateral ventricles and a significantly reduced lateral ventricular volume. Likewise, neurulation-stage hyperglycaemia was found to selectively induce HPE-like gene-expression defects in axial midline structures in heterozygous Ku embryos, but not stage-matched controls. Thus, the present study demonstrates that Zic2 haploinsufficiency predisposes embryos to develop HPE-like defects in response to even low/moderate-level teratogen exposure, consistent with the notion that synergistic interactions between Zic2 variants and EtOH or hyperglycaemia frequently underlie HPE pathogenesis. These findings support current recommendations that women practice EtOH abstinence and maintain good glycaemic control in early pregnancy. Moreover, they identify membrane-based disruptions to gastrulation-stage NODAL signalling as a promising target for future research into the mechanisms that underlie EtOH-induced HPE. Show more