Neural tube defects (NTDs) are severe congenital malformations caused by failure of the neural tube to close during neurulation. Their etiology is complex involving both environmental and genetic factors. We have recently reported three mutations in the planar cell polarity gene VANGL1 associated with NTDs. The aim of the present study was to define the role of VANGL1 genetic variants in the development of NTDs in a large cohort of various ethnic origins. We identified five novel missense variants in VANGL1, p.Ser83Leu, p.Phe153Ser, p.Arg181Gln, p.Leu202Phe and p.Ala404Ser, occurring in sporadic and familial cases of spinal dysraphisms. All five variants affect evolutionary conserved residues and are absent from all controls analyzed. This study provides further evidence supporting the role of VANGL1 as a risk factor in the development of spinal NTDs.

Endogenous folate stores are required in preimplantation embryos of several species, but how folates are accumulated and whether they can be replenished has not been determined. Folates are generally taken up into cells by specific transporters, mainly the reduced folate carrier RFC1 (SLC19A1 protein) and the high-affinity folate receptors FOLR1 and FOLR2. Quantitative RT-PCR showed that Slc19a1 mRNA was expressed in mouse cumulus-oocyte complexes (COCs) and oocytes, whereas Folr1 showed expression only in preimplantation embryos, increasing from the 2-cell stage onward. The mRNAs encoding Folr2 and the intestinal folate transporter Slc46a1 were not detected. Methotrexate (MTX), an antifolate often used as a model substrate for folate transport, exhibited saturable transport in COCs and in preimplantation embryos starting at the 2-cell stage. However, folate transport characteristics differed between COCs and embryos. In COCs, transport of MTX and the reduced folate leucovorin was inhibited by the anion transport inhibitor SITS that blocks RFC1 but was insensitive to dynasore, a specific dynamin inhibitor that instead inhibits folate receptor-receptor mediated endocytosis, whereas the opposite was found in 2-cell embryos and blastocysts. The inhibitor profile and transport properties of MTX and leucovorin in COCs correspond to established transport characteristics of RFC1 (SLC19A1), whereas those in 2-cell embryos and blastocysts correspond with those of FOLR1, consistent with the mRNA expression patterns. Considerable folate was accumulated in COCs via RFC1, but the presence of cumulus cells did not enhance folate accumulation in the enclosed oocyte, indicating a lack of transfer from cumulus to oocyte.

As both Alx1/Cart1 and Dlx5 mouse mutations result in exencephaly, we hypothesize that changes in expression of these mesenchymal/ectodermal transcription factors may contribute to NTDs associated with Cecr2.

Background: Blastocyst stage embryos require a large pool of methyl groups, but the source is unknown. Results: Betaine-homocysteine methyltransferase (BHMT), which takes methyl groups from betaine, is highly active in mouse blastocysts and promotes development of cells that become the fetus. Conclusion: BHMT contributes to the methyl pool in the blastocyst. Significance: Betaine and BHMT promote embryo development.

The loss of Cecr2, which encodes a chromatin remodeling protein, has been associated with the neural tube defect (NTD) exencephaly and open eyelids in mice. Here, we show that two independent mutations of Cecr2 are also associated with specific inner ear defects. Homozygous mutant 18.5 days post coitus (dpc) fetuses exhibited smaller cochleae as well as rotational defects of sensory cells and extra cell rows in the inner ear reminiscent of planar cell polarity (PCP) mutants. Cecr2 was expressed in the neuroepithelium, head mesenchyme, and the cochlear floor. Although limited genetic interaction for NTDs was seen with Vangl2, a microarray analysis of PCP genes did not reveal a direct connection to this pathway. The mechanism of Cecr2 action in neurogenesis and inner ear development is likely complex.

Preimplantation mouse embryos of many strains become arrested at the 2-cell stage if the osmolarity of culture medium that normally supports development to blastocysts is raised to approximately that of their normal physiological environment in the oviduct. Arrest can be prevented if molecules that serve as "organic osmolytes" are present in the medium, because organic osmolytes, principally glycine, are accumulated by embryos to provide intracellular osmotic support and regulate cell volume. Medium with an osmolarity of 310 mOsM induced arrest of approximately 80% of CF1 mouse embryos at the 2-cell stage, in contrast to the approximately 100% that progressed beyond the 2-cell stage at 250 or 301 mOsM with glycine. The nature of this arrest induced by physiological levels of osmolarity is unknown. Arrest was reversible by transfer to lower-osmolarity medium at any point during the 2-cell stage, but not after embryos would normally have progressed to the 4-cell stage. Cessation of development likely was not due to apoptosis, as shown by lack of external annexin V binding, detectable cytochrome c release from mitochondria, or nuclear DNA fragmentation. Two-cell embryos cultured at 310 mOsM progressed through the S phase, and zygotic genome activation markers were expressed. However, most embryos failed to initiate the M phase, as evidenced by intact nuclei with decondensed chromosomes, low M-phase promoting factor activity, and an inactive form of CDK1, although a few blastomeres were arrested in metaphase. Thus, embryos become arrested late in the G(2) stage of the second embryonic cell cycle when stressed by physiological osmolarity in the absence of organic osmolytes.

Although neural tube defects (NTDs) are common in humans, little is known about their multifactorial genetic causes. While most mouse models involve NTDs caused by a single mutated gene, we have previously described a multigenic system involving susceptibility to NTDs. In mice with a mutation in Cecr2, the cranial NTD exencephaly shows strain-specific differences in penetrance, with 74% penetrance in BALB/cCrl and 0% penetrance in FVB/N. Whole genome linkage analysis showed that a region of chromosome 19 was partially responsible for this difference in penetrance. We now reveal by genetic analysis of three subinterval congenic lines that the chromosome 19 region contains more than one modifier gene. Analysis of embryos showed that although a Cecr2 mutation causes wider neural tubes in both strains, FVB/N embryos overcome this abnormality and close. A microarray analysis comparing neurulating female embryos from both strains identified differentially expressed genes within the chromosome 19 region, including Arhgap19, which is expressed at a lower level in BALB/cCrl due to a stop codon specific to that substrain. Modifier genes in this region are of particular interest because a large portion of this region is syntenic to human chromosome 10q25, the site of a human susceptibility locus.