The Axd (axial defects) mutation, which causes open neural tube defects (NTD) in 25-30% of d 14 mouse embryos of heterozygous (Axd/+) matings and curly tails (CT) in 31%, can be useful in studying neurovertebral morphogenesis. Current interest focuses on the role of methionine in neurulation, because supplementation of dams with this essential amino acid (70 mg/kg body wt) has been shown to reduce by 41% the incidence of NTD among embryos of Axd/+ x Axd/+ crosses when administered on d 8 and 9 of pregnancy. The present experiments were undertaken to determine whether supplementation of dams with a higher dose of methionine could effect a greater level of remediation. At a dose of 180 mg/kg body wt, the amino acid produced a reduction in NTD of 47%, similar to that produced at 70 mg/kg body wt. Supplementation of dams of reciprocal matings of Axd/+ x BALB/cByJ or CF-1 mice allowed assessment of the effects of methionine in heterozygous embryos, which exhibit CT. The amino acid had no effect on Axd/+ embryos from the BALB crosses, but the frequency of CT declined significantly among embryos of the CF-1 outcrosses. Maternal supplements of folinic acid (33 mg/kg) or vitamin B-12 (330 mg/kg) did not alter the incidence of NTD or CT among Axd embryos. No difference in methionine concentration was detected in the serum of Axd/+ and +/+ dams.

The Axd (axial defects) mouse model system (Essien et al., Teratology 42:183-194, '90) is characterized by a dominant mutation which causes posterior open neural tube defects (NTD) and a variety of tail anomalies (curly tails, or CT). Repeated backcrosses to BALB/cByJ mice have resulted in a 50% increase in Axd penetrance among neonates of heterozygous matings and loss of a correlation with maternal tail phenotype. Analysis of D12-D18 embryos from Axd/+ x Axd/+ matings indicates that soft tissues can superficially heal over some lesions from open NTD and that some curly tails can straighten (macroscopically) as gestation proceeds. Similarly, in embryos of Axd/+ x BALB crosses, there is remediation of approximately 33% of the tail flexion defects by birth. Numerous studies show that maternal nutritional status can affect the development of the neural tube and related axial structures. One nutrient of special interest is methionine, which is required for neurulation in cultured rat embryos (Coelho et al., J. Nutr. 119:1716, '89). Thus, the major question addressed by this study was whether supplemental methionine administered to Axd/+ dams crossed to Axd/+ males would alter the prenatal expression of the gene. When given IP (70 mg/kg) on D8 and D9, methionine resulted in a 41% reduction (from 29% to 17%) in the incidence of NTD in D 14 embryos (P less than 0.01).

A cell preparation method by which large numbers of embryonic chick skeletal muscle cells may be obtained is described. The procedure requires fewer manipulations and much less time than standard trypsinization. By the criteria used, both methods are comparable with respect to percent viable cells and survival of plated cells. However, in addition to the ease of preparation, the mechanical dissociation method offers the significant advantage that the cell suspension is greatly enriched for myoblasts without the necessity of an additional preplating step.

A new autosomal mutation, Axd (axial defects), is described. Axd segregates in a simple Mendelian fashion, and it is dominant with incomplete penetrance and variable expressivity. The phenotype of Axd heterozygotes ranges from a variety of tail anomalies to visibly normal tails. Approximately 12% of neonates from curly-tail (CT) F1 (Axd/+) x F1 (Axd/+) matings exhibit open neural tube defects (NTD) in the lumbosacral region and 16% have curly tails. Mean litter sizes and resorption rates comparable to wild type indicate that homozygosity for Axd is not obligately lethal. Genetic background plays a major role in Axd expression. Strains such as BALB/cByJ allow the highest penetrance of the mutation in single dose (46%), whereas, in CF-1 mice Axd is recessive. The tail phenotype of heterozygous Axd/+ dams, in part reflective of their genetic background, correlates with the incidence of NTD in F2 offspring: CT mothers produce significantly more neonates with frank NTD than normal tail mothers. At the one embryonic period examined for this study (D13/D14 post-coitus), an 85% higher incidence of total axial defects is observed than among the F2 at birth. Unchanging litter size and the relative increase in phenotypically normal offspring by birth suggest that Axd acts by delaying posterior neural tube closure. One of the most significant findings in this study is that maternal age influences the survival of Axd embryos in utero. Axd/+ dams older than 8 months yield fewer mean implants, higher resorption rates, and fewer viable embryos with axial defects than do Axd/+ dams younger than 8 months. Axd is not allelic to nor linked to the Sp (splotch) gene which also affects neurulation.

The Axd mutation in the mouse acts by an unknown mechanism to cause lumbosacral open neural tube defects and a variety of tail anomalies. Retinoic acid (RA) plays a number of different physiological and developmental roles and has been shown to affect neurulation in mice and other species. Indeed, reports have shown that this biologically active compound (or its metabolites) at low dose can alter the incidence of neural tube defects (NTD) in curly-tail (ct), splotch (Sp), and delayed splotch (Spd) mice, strains that are genetically predisposed to such abnormalities. The aim of the present study was to determine if RA administered under similar conditions would affect the penetrance or expression of the Axd mutation or survival of Axd homozygotes. Axd/+ and +/+ dams were exposed to RA intraperitoneally (5 mg/kg) on D9 postcoitus. No difference in incidence or extent of neural tube defects or other axial anomalies was detected among embryos of Axd/+ dams given RA compared with those administered vehicle only. This finding is consistent with the diversity of gene-controlled steps required for neurulation and the differing sensitivities of specific mutants to rescue by extrinsic agents.