Overexpression of Grainyhead-like 3 causes spina bifida and interacts genetically with mutant alleles of Grhl2 and Vangl2 in mice

Abstract

The genetic basis of human neural tube defects (NTDs), such as anencephaly and spina bifida (SB), is complex and heterogeneous. Grainyhead-like genes represent candidates for involvement in NTDs based on the presence of SB and exencephaly in mice carrying loss-of-function alleles of Grhl2 or Grhl3. We found that reinstatement of Grhl3 expression, by bacterial artificial chromosome (BAC)-mediated transgenesis, prevents SB in Grhl3-null embryos, as in the Grhl3 hypomorphic curly tail strain. Notably, however, further increase in expression of Grhl3 causes highly penetrant SB. Grhl3 overexpression recapitulates the spinal NTD phenotype of loss-of-function embryos, although the underlying mechanism differs. However, it does not phenocopy other defects of Grhl3-null embryos such as abnormal axial curvature, cranial NTDs (exencephaly) or skin barrier defects, the latter being rescued by the Grhl3-transgene. Grhl2 and Grhl3 can form homodimers and heterodimers, suggesting a possible model in which defects arising from overexpression of Grhl3 result from sequestration of Grhl2 in heterodimers, mimicking Grhl2 loss of function. This hypothesis predicts that increased abundance of Grhl2 would have an ameliorating effect in Grhl3 overexpressing embryo. Instead, we observed a striking additive genetic interaction between Grhl2 and Grhl3 gain-of-function alleles. Severe SB arose in embryos in which both genes were expressed at moderately elevated levels that individually do not cause NTDs. Furthermore, moderate Grhl3 overexpression also interacted with the Vangl2Lp allele to cause SB, demonstrating genetic interaction with the planar cell polarity signalling pathway that is implicated in mouse and human NTDs.

Meta-Analysis of Grainyhead-Like Dependent Transcriptional Networks: A Roadmap for Identifying Novel Conserved Genetic Pathways

“…In vertebrates, Grainyhead-like orthologues display a conserved functional homology of regulating processes, required for epithelial development. Studies in mice [2,5], Xenopus [6] and zebrafish [7] have determined remarkable conserved functional homology to Drosophila grh , apparent in the morphogenic development of the epidermal barrier [8,9,10,11], closure of the neural tube [12,13,14,15], maintenance of neural cell survival [16], and the growth and development of the craniofacial skeleton [17,18]. Together, these findings point to a critical role for Grhl function in the development of all organisms within the animal kingdom.…”

“…Studies of human Grainyhead-like ( Grhl ) orthologues have begun to uncover the role that loss of function or gene variants have in adult-onset diseases, such as hearing loss/deafness [22,23], defects of epidermal integrity and, particularly, numerous forms of cancer [9,24,25,26]. The Grhl -genes are also implicated in the aetiology of a number of human diseases, including cleft palate [27,28], spina bifida [5,14,29], cancer [30], ectodermal dysplasia [28] and asthma [31]. This incredible functional diversity is mediated, at least in part, through a grh / Grhl transcription factor binding to a promoter or enhancer element and regulating the subsequent transactivation or repression of target effector genes through recruitment of transcriptional co-factors.…”

Functional Validation of CLDN Variants Identified in a Neural Tube Defect Cohort Demonstrates Their Contribution to Neural Tube Defects

“…To investigate the potential pathogenic role of the rare and novel missense CLDN variants identified in NTD patients, we used an overexpression assay in the chick embryo model to examine the effect of these variants on neural tube closure. Previous studies have shown that knockdown or overexpression of NTD-related genes can lead to similar NTD phenotypes (Ting et al, 2003;Yu et al, 2006;Gustavsson et al, 2007;De Castro et al, 2018). The chick embryo is an excellent model to study neural tube closure for several reasons: (1) the tissue and cellular behaviors that regulate neurulation in the chick have been well-studied, (2) the morphogenetic movements regulating neural tube closure in chick and human embryos are predicted to be very similar, (3) neural tube closure occurs over a short period of time (∼28 h) making it easy to collect a large number of embryos (Schoenwolf and Franks, 1984;Smith and Schoenwolf, 1987;Schoenwolf et al, 1988;Schoenwolf and Alvarez, 1989;Schoenwolf, 1991;Smith et al, 1994;Moury and Schoenwolf, 1995;Sausedo et al, 1997;Lawson et al, 2001;Kinoshita et al, 2008;Nishimura et al, 2012;Chen et al, 2017), and (4) the neural tube is easily accessible for manipulation in ex ovo culture.…”

Genetic variants in GRHL3 and risk for neural tube defects: A case–control and case–parent triad/control study

“…Evidence from animal studies has suggested that Grhl3 is a component of the PCP pathway, which functions as a key transcriptional regulator (Caddy et al, ). It interacts genetically with Vangl2 in epidermal healing and neural tube closure (De Castro, Gustavsson, et al, ; Dworkin, Jane, & Darido, ). In an animal study, Ting et al () found that all Grhl3 knockout mice embryos displayed full penetrance with spina bifida and curled tail, caused by a primary failure of neural tube closure.…”