Early identification of mutant embryos prior to the manifestation of a defect facilitates the study of dysmorphogenesis. The In(l)lRk inversion was used as a cytogenetic marker to distinguish embryonic day 9 (D9) splotch (Sp) and splotch-delayed $(Sp sp{d})$ mouse mutants from heterozygous and wild-type littermates, and cellular aspects of abnormal neurulation and NCC migration were examined before inherent neural tube defects (NTDs) and deficiencies in neural crest cell (NCC) derivatives developed. In vitro analysis of NCC emigration from D9 neural tube explants revealed a delay in the release of NCCs from mutant neural tubes compared to controls, suggesting that the primary effect of the mutation was intrinsic to the neuroepithelium. Immunofluorescent localization of the neural cell adhesion molecule (N-CAM) antibody in situ demonstrated an increased intensity of antibody fluorescence in mutant tissue compared to controls, and further characterization by immunoblot analysis showed an altered embryonic N-CAM profile in both Sp and $Sp sp{d}$ mutants at D9 of gestation. The importance of N-CAMs in mediating cellular organization and communication has been well documented, supporting the idea that an alteration in this adhesion mechanism could result in the types of defects seen in splotch locus mouse mutants.

Cell adhesion molecules are known to play crucial roles in a variety of developmental processes. The neural cell adhesion molecule N-CAM is strongly implicated in neurulation and neural crest cell (NCC) migration and was thus studied in splotch (Sp) neural tube defect mutant embryos. At the 20 somite-stage of gestation day 9, Sp N-CAM was found to contain polysialic acid (PSA) side chains which are normally only present beginning at gestational day 11. Younger embryos at 12 and 14 somites also showed the presence of PSA on N-CAM, which was absent in controls. Enzymatic removal of PSA from N-CAM resulted in isoforms which migrated identically to PSA-free N-CAM isoforms in SDS-polyacrylamide gels. The post-translational modification of N-CAM appears to be the primary target of the Sp gene. In view of N-CAM's importance during development, an alteration at a critical stage is likely to result in the cascade of abnormalities seen in Sp mutants. / A new genotyping assay was also implemented for examination of N-CAM in Sp and other related wildtype strains.

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The splotch (sp) mutation has been identified as a mutation in the paired box gene, Pax-3. Heterozygous mice carrying this mutation are phenotypically normal, with the exception of a white spot on their bellies. Homozygous embryos do not live to birth, and suffer from a wide range of developmental defects, all of which result from delayed neural tube closure, or inadequate neural crest cell migration. Most notably, homozygotes have an increased rate of spina bifida with or without exencephaly. Retinoic acid (RA), which has been shown to be very important in the development of a number of systems, was shown to cause a selective mortality of the homozygous splotch embryos when administered maternally at day 9 p.c. (Moase and Trasler, 1987). Since cellular retinoic acid binding protein (CRABP) is localized to the tissues which are affected by both the splotch gene, and retinoic acid teratogenesis, its expression patterns in the developing splotch embryo were examined. It was found that the distribution of CRABP mRNA is normal, but its expression levels are excessive in splotch homozygous day 9 mouse embryos.

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