Splotch (Sp) and splotch-delayed (Spd) are allelic mutations on chromosome 1 of the mouse. Embryos homozygous for either allele have neural tube defects (NTDs) and deficiencies in neural crest cell (NCC) derived structures. The fact that Spd mouse mutants sometimes have deficiencies in NCC derivatives in the absence of an NTD led to the hypothesis that neurulation and the release of NCCs may depend on a regulatory event that is common to both processes. Therefore, it may be possible to understand the cause of NTDs in these mutants by examining the basis of aberrant NCC derivatives. Caudal neural tubes were excised from day 9 Sp and Spd embryos and placed into gelatin-coated tissue culture dishes, or 3-dimensional basement membrane matrigel, and cultured for 72 hours. A cytogenetic marker was used to genotype the embryos. In planar cultures, no morphological differences were observed between NCCs from neural tube explants of Spd mutants compared to those from heterozygous or wild-type embryos. However, there appeared to be a delay in the release of NCCs from the neural tube in both Sp and Spd mutants, which was particularly evident in Sp. After 24 hours in culture, the extent of NCC outgrowth, as well as the number of NCCs emigrating from explanted neural tubes, was significantly lower in Sp and Spd mutant cultures than in controls. No differences were observed in the mitotic indices among cells which had emigrated. By 72 hours, mutant cultures and their non-mutant counterparts were similar in terms of outgrowth, cell number, and migratory capability. After 24 hours in 3-dimensional basement membrane matrigel, cell outgrowth from Sp explants was also significantly less than controls. The pattern of NCC outgrowth in both types of culture conditions indicates a 24 hour delay in mutant cultures compared to controls. This stems from a delay in the release of NCCs from the neural tube, suggesting that the defect lies within the neuroepithelium with respect to the release of NCCs.

Splotch and splotch-delayed mutants have anomalies in certain neural crest cell derivatives as well as neural tube defects. A genetic marker was used to identify mutant, heterozygote, and wild-type embryos within a litter, which enabled us to make intergenotypic comparisons. Histological studies of the lumbosacral region of day 15 and day 16 embryos indicated that the splotch-delayed mutant had similar but less severe defects in spinal ganglion development than those reported for splotch (Auerbach: Journal of Experimental Zoology 127:305-329, 1954). The ganglia were extensively reduced in size, residual, or missing in the splotch-delayed mutant, whereas in the splotch mutant, they were virtually nonexistent. Paired comparison analyses showed that all mutant embryos had a significant reduction in their volume of lumbosacral spinal ganglia when compared to their heterozygous and/or wild-type littermates. Also, some heterozygotes were found to have spinal ganglia volumes that were significantly reduced when compared to wild-type embryos. The volume of spinal ganglia was not related to the severity of the neural tube defect. In fact, three mutant embryos, which did not exhibit a neural tube defect, had spinal ganglia volumes comparable to or less than those mutants with open neural tube lesions or curly tails. This shows that the formation of abnormal neural crest cell derivatives is not a result of the neural tube closure defect. We hypothesize that the two anomalies observed in these mutants have a common etiological basis.

The majority of data that contribute to our understanding of mammalian embryonic development This article reviews the results of investigations that have examined splotch locus mutants. These studies include histological analyses at the structural and ultrastructural levels, gene-teratogen interactions, as well as outcomes from immunohistochemical, biochemical, cellular, and molecular approaches. Information gathered from the analyses of these mutants has contributed to our understanding of neurulation and neural crest cell emigration, and has provided clues as to how these two fundamental processes may be developmentally related. Early linkage analysis showed Sp belonged to linkage group XIII and was positioned between the coat colour mutation leaden (In) and the hair follicle mutation fuzzy (fz).910 Recombination frequencies between Spd and either In orfz were found to be similar to those of Sp,7 supporting the idea that Spd was indeed allelic to Sp. Since then, the splotch locus has been mapped more specifically to band C4 of chromosome 111 and investigations to define this locus further using DNA probes are currently in progress. HOMOZYGOUS FEATURESMutations at the Sp locus are classified as semidominant lethal,7 with differences in homozygous phenotype being the factor that distinguishes between splotch and splotch delayed. Most Sp homozygotes develop spina bifida (lumbosacral rachischisis) and over half exhibit exencephaly (cranioschisis) as well, owing to lack of closure in the hindbrain region.12 On occasion, these mutants may develop only a tail flexion defect that results in a curly tail.'3 However, all mutants die in utero at approximately 13 or 14 days of gestation.'2 This is in contrast to the Spd mutant which develops only spina bifida and survives until birth, hence undergoing delayed death.7Unlike Sp and Spd, splotch retarded homozygotes are presumed to die before implantation.8 This mutation is more severe than Sp or Spd as it involves a cytogenetically detectable deletion of the 1C4 band, which constitutes approximately 2% of the total physical length of chromosome 1.11

The allelic loci splotch (Sp) and splotch-delayed (Spd) cause neural tube defects (NTDs) in mice homozygous for either of these genes. The polymorphic enzyme isocitrate dehydrogenase (Idh-1) in conjunction with a recombination suppressor was used as a genetic marker to identify embryos homozygous for these alleles. A split dose of all-trans retinoic acid (RA) totalling 5.0 mg/kg administered on gestation day 9/15 and 9/18 (days/h) significantly reduced the frequencies of NTD and of mutant genotypes in marked Spd embryos examined on day 16 without significantly increasing the resorption frequency. There was a nonsignificant decrease in the frequencies of NTD and mutant genotypes in embryos examined on day 11 of gestation. Thus, retinoic acid treatment was associated with selective mortality of the homozygous Spd mutants. No evidence of selective mortality was observed in RA-treated Sp embryos.

Single‐step purification of boar sperm P68/62 that is cross‐reactive with a polyclonal antibody against sulfolipidimmobilizing protein 1 (SLIP1) was achieved by chromatofocusing. This method is useful for obtaining P68/62 in quantity. The two proteins, P68 and P62, were antigenically related, since the antibody generated specifically against the 68‐kDa band reacted with both the 68‐ and 62‐kDa bands. Like rat testis SLIP1, purified boar sperm P68/62 bound to sulfogalactosylglycerolipid (SGG) and inhibited sperm‐egg binding in a dose‐dependent manner when added exogenously to sperm‐egg coincubates. This inhibitory effect occurred at the level of the zona pellucida (ZP), and further studies showed that biotinylated boar sperm P68/62 bound to the ZP of unfertilized mouse eggs. Furthermore, biotinylated boar sperm P68/62 bound to isolated ZP of unfertilized eggs from other species, including pig, rat, cat, dog, and human, as well as to ZP of intact fertilized mouse eggs and preimplantation embryos of various developmental stages, although the degree of its binding to the ZP of intact eight‐cell embryos, morulae, and blastocysts was much lower than that of fertilized eggs and two‐cell embryos. These results suggest that P68/62 of capacitated sperm must act together with other sperm surface proteins/molecules that regulate zona binding specificity within homologous species and in unfertilized eggs. Together with our previous findings, we suggest that rather than being a true ZP receptor, sperm P68/62 may be involved in the initial step of sperm‐ZP binding that is adhesive in nature. Mol. Reprod. Dev. 49:203–216, 1998 © 1998 Wiley‐Liss, Inc.