Folic acid modifies the shape of epithelial cells during morphogenesis via a Folr1 and MLCK dependent mechanism [RESEARCH ARTICLE]
INTRODUCTION
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. Notably, maternal dietary folic acid supplementation reduces the risk of NTDs in offspring of human populations throughout the world (Group, 1991; Berry et al., 1999; López-Camelo et al., 2005; De Wals et al., 2007). In some animal models, maternal folic acid supplementation similarly decreases the frequency of NTDs (Harris, 2009; Harris and Juriloff, 2010; Marean et al., 2011). Although folic acid appears to be a powerful aid in the global effort to prevent birth defects, the specific cellular mechanisms by which folic acid prevents them remain unidentified.
In mammals, folate is transported across cell membranes through three major folate transport protein types: the reduced folate carrier, the proton-coupled folate transporter, and the folate receptors. There are three folate receptors encoded by the genes FOLR1, FOLR2, and FOLR3 in humans and two in mice (Folr1/Folr2) that mediate folic acid intake through endocytosis (Zhao et al., 2011; Chen et al., 2013). The predominant folate receptor that is required for normal neural tube closure appears to be Folr1 as neural tube defects are observed in homozygous mouse embryos lacking this gene but not in the Folr2 homozygous embryos (Piedrahita et al., 1999). Folr1 expression is enriched in the neural epithelium of mouse embryos and Folr1 protein (Folate receptor alpha) is localized apically during neural tube closure (Barber et al., 1999; Saitsu et al., 2003; Kur et al., 2014). In Folr1 morpholino-treated Xenopus embryos neural tube closure defects occur due to the failure of neural epithelial cell apical constriction, or the lack of adopting a wedge-like shape (Balashova et al., 2017). 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.
A major component of the apical constriction machinery in numerous vertebrate tissues is the cytoskeletal protein Shroom3, an F-actin and Rho-kinase binding protein that facilitates non-muscle myosin activation and subsequent contraction of the apical cellular junctions (Haigo et al., 2003; Nishimura and Takeichi, 2008; Chung et al., 2010; Plageman et al., 2010; Plageman et al., 2011; Ernst et al., 2012; Das et al., 2014). Shroom3 functions by recruiting Rho-kinase to apical cell junctions, facilitating the activation of non-muscle myosin II and actomyosin contraction thereby reducing the apical area of epithelial cells. Loss of function mutations in the SHROOM3 gene of humans and mice result in NTDs that include exencephaly, anencephaly, and spina bifida (Hildebrand and Soriano, 1999; Lemay et al., 2015). The importance of Rho-kinase binding to Shroom3 function is highlighted by the finding that a missense mutation of Shroom3 that inhibits Rho-kinase binding (Shroom3R1838C) also causes NTDs that are similar to the mouse loss of function allele (Marean, et al., 2011; Das et al., 2014; Zalewski et al., 2016). Interestingly, the phenotype in Shroom3R1838C/R1838C homozygous embryos can be partially alleviated by folic acid supplementation (Marean et al., 2011). Given this result and the knowledge of how this mutation inhibits Shroom3 function, it provides a unique opportunity to probe the mechanism of folic acid rescue of NTDs.
In this study, the mechanism of folic acid rescue of Shroom3 function was analyzed using both a cell culture model of apical constriction and mouse and chicken embryos. It was determined that folic acid and the folic acid receptor, Folr1, can rescue the function of the Rho-kinase-binding deficient mutation of Shroom3. Chemical inhibition experiments support the role for myosin light chain kinase (MLCK) mediating the functional rescue in cell culture. Further investigation demonstrated that folic acid can also rescue non-muscle myosin activation and apical constriction in embryos treated with a Rho-kinase inhibitor. The effect was also coincident with an increase in junctional MLCK activation in response to folic acid. Finally, it was determined that both non-muscle myosin and MLCK activation are decreased in Shroom3/Folr1 doubly heterozygous embryos. These results provide details of a potential mechanism by which folic acid facilitates morphogenesis and/or prevents disruptions in this process in developmental defects.
