Folate receptor alpha is necessary for neural plate cell apical constriction during Xenopus neural tube formation

Folate supplementation prevents up to 70% of neural tube defects (NTDs), which result from a failure of neural tube closure during embryogenesis. The elucidation of the mechanisms underlying folate action has been challenging. This study introduces Xenopus laevis as a model to determine the cellular and molecular mechanisms involved in folate action during neural tube formation. We show that knockdown of folate receptor-α (FRα) impairs neural tube formation and leads to NTDs. FRα knockdown in neural plate cells only is necessary and sufficient to induce NTDs. FRα-deficient neural plate cells fail to constrict, resulting in widening of the neural plate midline and defective neural tube closure. Pharmacological inhibition of folate action by methotrexate during neurulation induces NTDs by inhibiting folate interaction with its uptake systems. Our findings support a model for folate receptor interacting with cell adhesion molecules, thus regulating apical cell membrane remodeling and cytoskeletal dynamics necessary for neural plate folding. Further studies in this organism may unveil novel cellular and molecular events mediated by folate and lead to new means for preventing NTDs. D ev el o pm en t • A dv an ce a rt ic le Introduction Neural tube defects (NTDs) are among the most common serious morphological defects diagnosed in human fetuses and newborns with combined incidence of ~1/1,000. They result from the failure of the neural tube to close leading to exencephaly or spina bifida (Wallingford et al., 2013). Currently, there is no effective treatment for NTDs once the neural tube has failed to close and, since closure is completed by day 28 of gestation in humans, preventive therapy must be targeted to early pregnancy. A vast number of clinical studies have established that folate is a prominent environmental factor needed for appropriate neural tube closure (Detrait et al., 2005; MRC, 1991; Pitkin, 2007). Low blood folate levels in pregnant women are correlated with higher risk of NTDs in their offspring (Detrait et al., 2005; Smithells et al., 1976) and periconceptional supplementation of folate decreases the recurrence and occurrence of NTDs (MRC, 1991). Folate belongs to the vitamin B family. Folate-derived metabolites are required for DNA, protein and lipid methylation. In particular, folate participates in thymidine and purine synthesis, thus is specially needed for DNA replication and cell division, processes characteristic of rapid growth. Uptake of folate by mammalian cells is mediated by three membrane proteins, reduced-folate carrier, folate receptor (FR, also called FOLR or folate binding protein (folbp)), and the proton-coupled folate transporter (Antony, 1992; Antony, 1996; Sirotnak and Tolner, 1999). The mechanisms by which folate promotes neural tube formation are unclear. The importance of folate uptake systems for the formation of the neural tube becomes apparent from studies in humans and mice. Significant associations between single-nucleotide polymorphisms (SNPs) in different human folate transporters and myelomeningocele compared to the ethnically-paired healthy population have been found, including one SNP in folr1, 2 in folr2, 5 in folr3 and 2 in the reduced folate carrier (O'Byrne et al., 2010). In mice, knockout of folr1, Folbp1-/-, results in open neural tube by E9.5, when heterozygotes or wildtype littermate embryos show a closed neural tube. This phenotype leads to death of homozygous embryos in utero (Finnell et al., 2002; Piedrahita et al., 1999; Spiegelstein et al., 2004; Wallingford et al., 2013). The specific cellular mechanisms dependent on FRα (FOLR1) during mouse neural tube formation are unclear. Whether folate participation in methylation reactions to promote rapid growth is the aspect of folate action necessary for neural tube formation is not completely understood. D ev el o pm en t • A dv an ce a rt ic le Certain mutant variant of the methylenetetrahydrofolate reductase, one of the enzymes involved in folate metabolism, has been shown to increase the risk of incidence of NTDs in homozygous infants as well as in homozygous mothers’ offspring (Blom et al., 2006; van der Put et al., 1995; van der Put et al., 1996), although not every ethnic group studied exhibit this association (Koch et al., 1998; Mornet et al., 1997; Papapetrou et al., 1996; Speer et al., 1997; Wilcken and Wang, 1996). In addition, deficiencies in methylation disturb chick embryo neurulation (Afman et al., 2005; Afman et al., 2003). However, mice in which several enzymes involved in folate metabolism are disrupted do not show NTDs (Chen et al., 2001; Swanson et al., 2001; Watanabe et al., 1995). Moreover, screenings made in humans showed that there are cases with innate errors in folate metabolism but in whom NTDs are not overrepresented (Blom et al., 2006). Altogether, these studies suggest that the identification of relevant aspects of folate action that influence neural tube formation demands further investigation. Additional progress in the prevention of NTDs remains on hold due to the lack of understanding of the basic cellular and molecular mechanisms underlying folate action. Xenopus laevis animal model is especially suited for investigating events occurring during embryogenesis, because of the ready access to observation and manipulation of early developmental stages. Indeed, it has been the pioneer model system from which principles of the process of neurulation have been established (Davidson and Keller, 1999; Haigo et al., 2003; Kee et al., 2008; Keller et al., 1992). Here we use the Xenopus laevis model system to demonstrate that folate promotes neural tube formation by facilitating changes in neural plate cell shape required during neurulation, through a folate receptor-dependent action. The use of this animal model may lead to the understanding of the mechanisms of folate action during neural tube formation and hence, may help devising more effective therapies for the prevention of NTDs. D ev el o pm en t • A dv an ce a rt ic le