Abstract
ABSTRACTFolic acid supplementation can prevent neural tube defects, but the specific molecular mechanisms by which it does have not been elucidated. During neural plate morphogenesis, epithelial cell apical constriction cooperates with other events to drive tissue-bending, and when defective, can result in neural tube defects. A Rho-kinase deficient binding mutant of the apical constriction regulating protein, Shroom3 (Shroom3R1838C), is one of only a handful of mouse mutant lines with neural tube defects that can be rescued by folic acid supplementation. This provided a unique opportunity to probe the functional rescue of a protein linked to neural tube development by folic acid. Utilizing an epithelial cell culture model of apical constriction, it was observed that treatment with exogenous folic acid, as well as co-expression of the folic acid receptor Folr1, can rescue the function of the Rho-kinase binding deficient mutant of Shroom3 in vitro. It was also determined that the rescuing ability of folic acid is RhoA and Rho-kinase independent but myosin light chain kinase (MLCK) and Src-kinase dependent. Inhibition of Rho-kinase-dependent apical constriction in chick embryo neural epithelium was also observed to be rescued by exogenous folic acid and that treatment with folic acid is accompanied by elevated activated myosin light chain and MLCK. Furthermore, doubly heterozygous mouse embryos lacking one copy each of Shroom3 and Folr1 exhibit a low rate of neural tube defects and also have lower levels of activated myosin light chain and MLCK. These studies suggest a novel mechanism by which folic acid modifies epithelial cell shape during morphogenesis, shedding light onto how folic acid may prevent neural tube defects.
Highlights
Neural tube defects (NTDs) are a group of structural birth defects characterized by a failure of the early neural plate to undergo its normal morphogenetic program leading to spina bifida, anencephaly, and craniorachischisis
While previous studies have demonstrated that the folic acid receptor, Folr1, is required for apical constriction in the neural plate of Xenopus embryos (Balashova et al, 2017), Folr1 expressed in MDCK cells is not sufficient to induce AC in the presence or absence of exogenously added folic acid (Fig. 1A–B, Table 1) as determined by calculating the mean ratio of the apical and basal areas of transgenic cells
Because folic acid is capable of rescuing the neural tube defect phenotype in a percentage of Rho-kinase-binding deficient Shroom3 mouse mutant (Shroom3R1838C/R1838C) embryos (Marean et al, 2011; Das et al, 2014), MDCK cells were again used to test the role of Folr1 and folic acid on Shroom3-dependent AC
Summary
Neural tube defects (NTDs) are a group of structural birth defects characterized by a failure of the early neural plate to undergo its normal morphogenetic program leading to spina bifida, anencephaly, and craniorachischisis. Maternal dietary folic acid supplementation reduces the risk of NTDs in offspring of human populations throughout the world The predominant folate receptor that is required for normal neural tube closure appears to be Folr as neural tube defects are observed in homozygous mouse embryos lacking this gene but not in the Folr homozygous embryos (Piedrahita et al, 1999). Because the cellular mechanisms that regulate apical constriction are thought to be key in the morphogenesis of the neural tube (Nikolopoulou et al, 2017), an intriguing possible mechanism for the action of folic acid could be through the regulation of apical constriction
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