Genetic determinants of teratogen-induced abnormal development in mouse and rat embryos in vitro.

The response of an embryo to a teratogenic treatment is often critically dependent on its genetic makeup. However, in conventional in vivo studies of gene-teratogen interactions it may be difficult to distinguish between the effects of genes that are carried by the embryo and those that are carried by the mother. It is likewise not easy to determine whether an observed interaction is between a particular gene and the parent compound administered, or whether it is with a metabolite that has been generated by the maternal system. The use of whole rodent embryo culture offers certain advantages in the study of gene-teratogen interactions. Not only can the effects of metabolism and the maternal genotype be more carefully controlled, but the stage of development at which embryos of different genotypes are exposed can be matched. Rodent whole embryo culture has been used to a limited extent to study interactions between single gene mutations and teratogenic treatments, variations in responses of different strains to teratogens, as well as species differences in response to teratogens. These studies point to the need to precisely control the stage of development at the time of treatment in order to be able to make valid comparisons. But, even more important, they highlight the versatility of the whole embryo culture technique, and underscore the need for its wider use in evaluating the relative contribution of genes and environment to abnormal embryonic development.

DNA methyltransferase in normal and Dnmtn/Dnmtn mouse embryos.

The mouse genome experiences a large decrease in net 5-methylcytosine between fertilization and implantation; de novo methylation brings 5-methylcytosine to adult somatic cell levels between implantation and gastrulation. Very little is known of the regulation of demethylation or de novo methylation. Levels of the one known form of DNA methyltransferase are very high in early embryos, but the enzyme is localized to the cytoplasm during most of preimplantation development. We show here that DNA methyltransferase is found exclusively in nuclei of the conceptus after implantation, and that nuclei of proximal decidual cells are free of detectable DNA methyltransferase. High levels of DNA methyltransferase were seen in all tissues, including the developing nervous system, of 9.5- to 12.5-day embryos. The large maternal stores of DNA methyltransferase become limiting prior to embryonic day 9.5, as shown by barely detectable immunostaining in 9.5-day embryos homozygous for a loss-of-function mutation (Dnmtn) in the DNA methyltransferase gene. These mutant embryos failed to develop past the 25-somite stage and showed evidence of developmental delay and some developmental asynchrony. Normal embryonic and extraembryonic tissues contained similar levels of DNA methyltransferase, even though severely reduced methylation levels and a loss of imprinting have previously been observed in extraembryonic tissues. These findings suggest that methylation patterns are not a simple function of the concentration of DNA methyltransferase, and that unidentified factors must be involved in the regulation of de novo methylation during early development of the mouse.

Early sialylation on N-CAM in splotch neural tube defect mouse embryos.

The splotch (Sp) mutant mouse is a model for neural tube defects and Waardenburg syndrome type I. The neural tube defects that arise in Sp, which include spina bifida and exencephaly, are thought to be caused by a change in the timing of the cellular events which are taking place during neurulation. Cell adhesion molecules are strongly implicated in a variety of cell-cell interactions throughout development, thus the neural cell adhesion molecule (N-CAM) may play a role in neural tube formation and closure. The N-CAM in day 9 Sp embryos is altered showing a heavy 200 kD species rather than the 180 and 140 kD isoforms which are normally present at that developmental stage [Moase and Trasler (1991) Development 113:1049-1058]. These N-CAM isoforms normally become modified post-translationally by the addition of alpha-2,8 linked polysialosyl (PSA) units beginning at gestational day 11. Sp/Sp, Sp/+, and +/+ embryos were examined by Western blot analysis with an antibody (mAb 5A5) which specifically recognizes PSA residues on N-CAM. Mutant and heterozygote embryos display a sialylated N-CAM form at 20, 14, and 12 somite-stages which is absent in controls. Enzymatic removal of PSA on N-CAM resulted in a reduction of the 200 kD PSA-free N-CAM isoforms. These results in the observed 200 kD species, and suggest that the Sp gene is involved in the regulation of expression or the post-translational modification of N-CAM.

The splotch-delayed (Spd) mouse mutant carries a point mutation within the paired box of the Pax-3 gene.

The splotch (Sp) mouse mutant displays defects in neural crest cell migration and neural tube closure and serves as a model for the study of spina bifida, exencephaly, and Waardenburg syndrome type I in humans. Recently, we have described alterations in the Pax-3 gene for the radiation-induced Spr and Sp2H alleles and for the original, spontaneously arising Sp allele. Another allele that arose spontaneously at the Sp locus, termed splotch-delayed (Spd), shows a less severe phenotype than the other Sp alleles, including the delayed death of homozygous embryos. To determine the molecular basis underlying this unique phenotype, we have analyzed the integrity of the Pax-3 gene in Spd mutant embryos. Nucleotide sequence analysis of cDNA and genomic clones revealed a G to C transversion at nucleotide 421 of the Pax-3 mRNA transcript, which results in a Gly to Arg substitution at position 42 of the Pax-3 protein (position 9 of the paired domain). The location of the mutated residue, its conservation in all other paired domain-containing proteins described to date, and the nonconservative nature of the substitution suggest that this mutation is responsible for the phenotype observed in the Spd mouse mutant.

A mutation within intron 3 of the Pax-3 gene produces aberrantly spliced mRNA transcripts in the splotch (Sp) mouse mutant.

The splotch (Sp) mouse mutant displays defects in neural tube closure in the form of exencephaly and spina bifida. Recently, mutations in the Pax-3 gene have been described in the radiation-induced Spr and Sp2H alleles. This led us to examine the integrity of the Pax-3 gene and its cellular mRNA transcript in the original, spontaneously arising Sp allele. A complex mutation in the Pax-3 gene including an A-->T transversion at the invariant 3' AG splice acceptor of intron 3 was identified in the Sp/Sp mutant. This genomic mutation abrogates the normal splicing of intron 3, resulting in the generation of four aberrantly spliced mRNA transcripts. Two of these Pax-3 transcripts make use of cryptic 3' splice sites within the downstream exon, generating small deletions which disrupt the reading frame of the transcripts. A third aberrant splicing event results in the deletion of exon 4, while a fourth retains intron 3. These aberrantly spliced mRNA transcripts are not expected to result in functional Pax-3 proteins and are thus responsible for the phenotype observed in the Sp mouse mutant.

Morphometric analysis of heterozygote dancer mice predisposed to 6-aminonicotinamide-induced cleft lip.

Mid-facial development is an extremely complex process involving coordinated events and precise timing. Cleft lip (CL) may result from the failed fusion of the lateral and medial nasal processes in the developing embryo. It has been postulated that spontaneous CL in the A/J strain of mice may be due to a predisposing face shape (Trasler, '68). This hypothesis was examined in mutant mice susceptible to teratogen-induced CL. Mice carrying the dancer (Dc) mutation in the heterozygous state rarely develop CL, whereas 90% of homozygotes (Dc/Dc) develop CL. Outcrossed heterozygotes show elevated susceptibility to 6-aminonicotinamide (6AN)-induced CL (Trasler et al., '84) and these were used to investigate face shape as a predisposing factor. Dc/+ and +/+ males were mated to R stock females, and embryos were collected on day 10/21 hr, when the nasal placodes are approximately at the oblong or crescent stage. Total nasal process areas and volumes, medial and lateral process areas and volumes, and medial jut lengths were measured from histological sections, and comparisons made between the two populations. The results indicate that compared to +/+ control, heads of embryos from the Dc/+ cross have significantly smaller mean total process areas and volumes (P less than 0.005), mean lateral process areas and volumes (P less than 0.005), mean medial process area and volumes (P less than 0.01), mean maximum head diameter (P less than 0.02), but similarly sized medial juts and crown rump lengths. Correlations between maximum head diameter and process size indicate that the Dc mutation may hinder normal development of the nasal processes. These reduced nasal processes may explain the underlying predisposition to 6AN-induced CL.

Segregation analysis reveals tight genetic linkage between the spontaneously arising neural tube defect gene splotch (Sp) and Pax-3 in an intraspecific mouse backcross.

Concurrent research has recently characterized Sp2H, a radiation induced mutation at the splotch (Sp) locus, and found alterations in the murine paired box gene, Pax-3, in homozygous Sp2H DNA. It was proposed that Sp and Pax-3 are the same gene. This report presents additional genetic evidence in support of this finding through linkage studies. Southern blot analysis of genomic DNAs from a panel of 125 intraspecific [(Sp/+ x CBA/J)F1-Sp x CBA/J] backcross mice reveals no crossover between Pax-3 and the spontaneously occurring splotch allele, Sp. This positions Pax-3 within 2.9 cM of the Sp locus (95% confidence interval) and suggests tight genetic linkage between the two marker genes.

Immunohistochemical localization of chondroitin and heparan sulfate proteoglycans in pre-spina bifida splotch mouse embryos.

The splotch (Sp) mutation on mouse chromosome I is a genetic model for the neural tube defects spina bifida and exencephaly. Embryos carrying Sp or its allele splotch-delayed (Spd), have been shown to have delays in neural tube closure, and neural crest cell emigration, as well as a reduction in extracellular space around the neural tube. Pre-spina bifida Sp and Spd embryos have abnormalities of notochord, mesoderm and neuroepithelial development. Chondroitin sulphate proteoglycans (CSPG) and heparan sulfate proteoglycans (HSPG) have been shown to play essential roles during neural tube closure and neural crest cell emigration and migration and thus might well be affected by the splotch mutation. Therefore, the effects of Sp and Spd on the temporal and spatial distributions of CSPG and HSPG were studied in pre-spina bifida embryos cytogenetically identified as Sp/Sp (Spd/Spd), Sp/ + (Spd/ +) or +/+. Immunohistochemical localization of CSPG by means of the CS-56 monoclonal antibody showed that in Sp/Sp head sections, the neuroepithelial basement membranes stained more intensely at 5-, 10-, and 15-somite stages, whereas similar staining was observed at 16- and 19-somite stages compared with matched +/+ sections. In caudal sections Sp/Sp again showed a more intense stain for CSPG in the neuroepithelial basement membranes in all sections (except one comparison, in which staining was similar) from embryos of 14-, 15-, 16-, and 19-somite stages, compared to matched +/+ sections. Heterozygotes did not differ consistently from the mutant or the normal (+/+) embryos in CS-56 stain intensity.(ABSTRACT TRUNCATED AT 250 WORDS)

N-CAM alterations in splotch neural tube defect mouse embryos.

The splotch (Sp) mouse is a model for both neurulation defects and defects in neural crest cell (NCC) derivatives. Since neurulation and NCC emigration from the neural tube occur at similar times in development, we suggest that these two events share a mechanism that, if disrupted, leads to malformations in both developmental pathways. Previous studies have shown that the underlying defect in these mutants may involve a mechanism that alters cellular organization and communication. Cell adhesion molecules (CAMs) have been linked with such interactions and because some, including N-CAM, are involved in neural development, we were interested in their pattern of expression in the splotch mutant. Immunolocalization studies showed similar temporospatial distributions of N-CAM antibody in embryonic day 9 mutants and controls. However, mutant embryos had a much higher intensity of anti-N-CAM fluorescence compared to controls. Further characterization using immunoblot analysis revealed that Sp mutants have an altered N-CAM polypeptide profile. Two N-CAM isoforms (Mr 140K and 180K, K = 10(3] are normally present at this time of development. However, extracts from Sp embryos display a heavier N-CAM species (Mr 200K), as well as an altered 140K isoform. Heterozygotes also exhibit a different N-CAM profile, displaying a band between 180K and 200K in addition to the normal 180K and 140K species. Microheterogeneity was also observed in mutant and heterozygous embryos carrying Spd, an allele of Sp. However, these differences were less dramatic than that of Sp. The Sp locus may be involved in post-translational modification of N-CAM. An aberration in N-CAM processing could be the primary target of the mutation that leads to the development abnormalities observed in this mouse mutant.

Abnormalities of neural tube formation in pre-spina bifida splotch-delayed mouse embryos.

The splotch-delayed homozygous mutant (Spd/Spd) develops spina bifida with or without exencephaly, has spinal ganglia abnormalities, and delays in posterior neuropore closure and neural crest cell emigration. The heterozygote (Spd/+) has a pigmentation defect, and occasionally neural tube defects. To investigate the underlying mechanisms, we compared the neuroepithelium in the posterior neuropore region of cytogenetically identified 15-18 somite pair Spd/Spd, Spd/+, and +/+ mouse embryos by transmission electron and light microscopy. The notochordal area and cell number in the non-fused neuroepithelium region of Spd/Spd and Spd/+ embryos were significantly reduced compared to those of normal (+/+) embryos, which suggests an abnormality in notochord elongation. In the mesoderm, the mean cell number and mean ratio of cell number to area in the non-fused region were significantly lower in the Spd/Spd compared with +/+ embryos. The distance of exposed neuroepithelium above the mesoderm in the just-fused region was significantly lower in the Spd/Spd versus +/+ embryos, which may indicate an insufficient force exerted by the mesoderm during neural tube closure. Within the neuroepithelium, significantly more intercellular space was found in Spd/Spd than in +/+ embryos indicating disorganization. The basal lamina was poorly organized and the formation delayed around the neural tube in Spd/Spd and Spd/+ embryos. All together, these results suggest an early abnormality in interactions among the neuroepithelium, mesoderm, and notochord, which may lead to the delay or inhibition of neural tube closure observed in Spd/Spd mutants.

Delayed neural crest cell emigration from Sp and Spd mouse neural tube explants.

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.

Spinal ganglia reduction in the splotch-delayed mouse neural tube defect mutant.

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.

Interaction between the splotch mutation and retinoic acid in mouse neural tube defects in vitro.

The interaction between the splotch gene (Sp) and all-trans retinoic acid (RA) was investigated using cytogenetically marked Sp/+ and +/+ mouse embryos cultured in the presence of RA. Retinoic acid retarded the development of and had a teratogenic effect on mouse embryos in culture. In particular, RA had seemingly opposite effects on the posterior neural tube, inducing abnormally early fusion in some embryos and causing a dose-dependent delay in others. When the effects of RA on identified Sp/+ and +/+ embryos were compared, the only observed difference in their responses was in the degree of the delay in posterior neuropore (PNP) closure. At the end of the culture period, among the untreated control embryos, the Sp heterozygotes showed retardation of PNP closure compared to +/+ embryos. In addition, the RA treatment was found to have induced a greater delay in posterior neural tube closure in Sp/+ than in +/+ embryos. The basis for this difference in response to RA is presumed to be the retardation of PNP closure that is caused by the Sp gene in heterozygous form. The effects of the gene and the teratogen are additive and the gene carriers thus have greater mean PNP lengths at the end of culture. Since the length of the PNP is an indication of an embryo's likelihood of developing spina bifida, this provides an explanation for the observation that Sp/+ embryos are more sensitive to the spina bifida-causing effects of RA than are +/+ embryos.

Histological comparison of the effects of the splotch gene and retinoic acid on the closure of the mouse neural tube.

The splotch gene (Sp) and all-trans retinoic acid (RA) interact to cause spina bifida in mouse embryos. To investigate the mechanisms of action of the two, the spinal regions of Sp homozygotes, RA-treated wild-type, and control wild-type embryos were examined histologically by light microscopy on day 9 of gestation. The mean numbers of cells per section in the neural tube, mesoderm, and notochord were determined, along with the percentages of mitotic and pyknotic nuclei and the numbers of migrating neural crest cells. As well, the effect of Sp and RA on the extracellular matrix was studied histochemically with Alcian blue staining for glycosaminoglycans. The main defect in Sp homozygotes was a marked reduction in the number of migrating neural crest cells and the amount of extracellular matrix around the neural tube. Retinoic acid, on the other hand, caused a number of disruptions in the embryo, including abnormalities in the position of the notochord and the shape of the neural tube. Sp and RA delay neural tube closure and thus cause neural tube defects, through different mechanisms. However, the combined effects of the gene and teratogen on the embryo lead to a greater inhibition of neural tube closure than when either is present separately.

Retinoic acid-induced selective mortality of splotch-delayed mouse neural tube defect mutants.

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.

A chromosome marker for the early detection of mouse embryos carrying the neural tube defect mutation splotch.

A major problem in the study of neural tube defects caused by the splotch (Sp) gene in the mouse has been the identification of gene carriers or potentially affected embryos at an early stage of development, since the gene's effects become visible only late in gestation or after birth. To aid in the identification of Sp carriers, we have developed a technique using a Robertsonian translocation as a marker for this gene. The accuracy of identification is reduced by crossing-over between the Sp locus and the centromere but, because of crossover suppression in the particular cross used, there was only 23.2% recombination compared with the known map distance of 36%. Paternal age had no effect on the frequency of recombination, but individual males differed significantly in the degree of crossover suppression.

Reduction in the frequency of neural tube defects in splotch mice by retinoic acid.

In the homozygous state, the splotch (Sp) gene causes spina bifida and exencephaly. Close to 25% of the embryos from Sp/ + X Sp/+ litters are affected. The frequency of these defects is significantly reduced by maternal treatment with 5 mg/kg retinoic acid on day 9 of gestation. There is no significant increase in the resorption frequency with this treatment, indicating that the fall in the frequency of neural tube defects is not due to differential mortality of the affected fetuses. The effects of retinoic acid are time specific, with treatment at different times on day 9 having the greatest influence on either the anterior or posterior neuropore. Treatment on day 8 with the same dose of retinoic acid causes an increase in both resorptions and neural tube defects, although only the increase in the former was significant.

Left cleft lip predominance and genetic similarities of L line and CL/Fr strain mice.

Newborn litters of the L line and CL/Fr and A/JFr strains were examined, and sex, frequency and type of cleft lip (left, right or bilateral) were recorded. Embryos and fetuses from crosses between these strains and line were collected on days 13 to 16 of gestation, and frequency and type of cleft lip recorded. Overall cleft frequencies in L X CL/Fr, CL/Fr X L, and CL/Fr X A/JFr crosses (female stated first) were similar, while in A/JFr X L (10.3%) they were significantly lower than in L X A/JFr (23.3%). The data suggested that the same maternal effect genes were present in CL/Fr and the related L line and absent from A/JFr. In the L, CL/Fr, and A/JFr newborns, there was a tendency for males to have higher frequencies of cleft lip and bilateral cleft lip and the latter was significant for L. Left cleft lip frequency was significantly higher than right for L and CL/Fr newborns and in embryos of the CL/Fr X L and L X CL/Fr cross. No significant differences in laterality were found in the A/JFr strain, A/JFr X L, L X A/JFr, and CL/Fr X A/JFr crosses. It was concluded that (1) the embryonic and maternal effect genes for cleft lip are similar or identical in CL/Fr and L; and (2) using data from the literature, there are additional genetic factor(s) increasing left cleft lip occurrence acting in the embryo, which are present in CL/Fr, L, A/HeJ, A/He, and A/St and absent from A/JKt, A/J, A/JFr, and A/WySn.

Gene-teratogen interaction and its morphological basis in retinoic acid-induced mouse spina bifida.

Homozygotes for the splotch (Sp) mutation in the mouse have spina bifida, whereas the heterozygotes have a white belly spot but otherwise appear normal. Spina bifida can be induced by maternal treatment with retinoic acid. Female SWV strain mice were treated intraperitoneally with retinoic acid suspended in peanut oil 8 days/12 hours after they had been mated to either Sp/+ or +/+ males. Probit analysis of the dose-response data suggests that the presence of the Sp gene causes an increased susceptibility of the embryo to the spina bifida-causing effects of retinoic acid. To study the nature of this increase litters were obtained on gestation day 9 from untreated SWV females mated as above. The mean length of the posterior neuropore (the length of the posterior neural tube that has not yet closed) was determined for each somite number between 14 and 26 and was found to be significantly greater in embryos from the Sp/+ cross. This delay of closure of the neural tube in Sp/+ cross embryos could explain the observed increase in their susceptibility to retinoic acid.