Altered signaling pathway in the dysmorphogenesis of telencephalon in the Gli3 depressed mouse embryo, Pdn/Pdn.

The responsible gene of genetic polydactyly/arhinencephaly mouse (Pdn/Pdn) is Gli3. Pdn/Pdn exhibits absence of the olfactory bulb, suggesting telencephalic dysmorphogenesis. It has been cleared that a transposon was inserted into intron 3 of the Gli3 gene in the Pdn mouse. Adequate PCR primers in the intron 3 and transposon allowed us to discriminate +/+, Pdn/+ and Pdn/Pdn embryos. After genotyping of the Pdn embryos using genomic DNA from the yolk sac membrane, gene expressions in the embryo proper were analyzed by DNA microarray, real-time PCR and whole-mount in situ hybridization (WISH) methods. DNA microarray detected 368 depressed and 425 over-expressed genes in the Pdn/Pdn mouse embryos on day 9 of gestation. In these genes, six signaling pathway and 20 transcription factor genes were included. From these genes, we further investigated Gli3, Emx2, Wnt8b and Wnt7b gene expressions using real-time PCR and WISH, and depression of these gene expression amounts and altered expression patterns were confirmed. Although alterations of Shh and Fgf8 gene expressions were not detected in the DNA microarray, as these genes have been closed up in the telencephalic morphogenesis, we investigated these gene expressions by real-time PCR and WISH. Shh gene expression amount and pattern were not changed. Alteration of Fgf8 gene expression amount was not detected also in the real-time PCR, but altered expression pattern was detected in the Pdn/Pdn embryos by WISH. From the present data, we suggested that Emx2, Wnt8b, Wnt7b and Fgf8 are the important Gli3 signaling pathway in the morphogenesis of telencephalon.

Gender-dependent differences in the incidence of ochratoxin A-induced neural tube defects in the Pdn/Pdn mouse.

Genetic polydactyly/arhinencephaly mouse embryo, Pdn/Pdn, exhibits suppression of Gli3 gene expression. Ochratoxin A (OTA) is a teratogen that causes neural tube defects (NTD) in mice. We investigated gender-dependent differences in the incidence of NTD induced by OTA in the Pdn/Pdn mouse. After administering 2 mg/kg OTA to Pdn/+ female mice, mated with Pdn/+ males, on day 7.5 of gestation, we examined the genotypes, sex and NTD of fetuses on day 18. Non-treated Pdn/Pdn had a 15.8% risk of NTD, and all NTD fetuses were female. When Pdn/Pdn embryos were exposed to OTA, the incidence of NTD increased to 16 (51.6%) of 31 Pdn/Pdn fetuses, and 10 (71.4%) of 14 male Pdn/Pdn fetuses exhibited NTD. From these results, it was speculated that NTD in OTA-treated male Pdn/Pdn were due to the synergistic effect between depressed Gli3 and altered sex-correlated gene expression from OTA treatment. After treatment with OTA, the embryos were recovered on day 9 and gene expressions, which were correlated with Gli3, telencephalic morphogenesis, formation of gonadal anlage, and gender-dependent differentiation were investigated. From real-time polymerase chain reaction analysis results, it was suggested that the manifestation of NTD in the male OTA-treated Pdn/Pdn might be due to the complicated altered gene expressions among Gli3, Wnt7b, Wnt8b, Fez1, Barx1, Lim1, Dmrt1, Igf1, Fog2, Dax1 and Sox9, and in particular, upregulation and gender-dependent difference in Barx1 and gender-dependent difference in Sox9 gene expressions might be noteworthy findings.

Comparative structural biology of the genome: nano-scale imaging of single nucleus from different kingdoms reveals the common physicochemical property of chromatin with a 40 nm structural unit.

Genome function is closely linked to the higher-order chromatin structures. To reveal a structural basis for the interphase chromatin organization, the 'on-substrate' lysis procedure was applied to nuclei isolated from human HeLa cells, chicken erythrocyte cells and yeast Schizosaccharomyces pombe, which possessed different intrinsic properties of the genomes such as histone composition and inter-nucleosomal distance. The isolated nuclei on a coverslip were successively treated with a detergent and a high-salt solution to extract the nuclear membrane and the nucleoplasm, and therefore, atomic force microscopy (AFM) visualized the structural changes in response to the lysis procedure. After the nucleoplasm was extracted, AFM clarified that chromatin fibers, approximately 40 nm in width, were partially released out of the nuclei and that the other chromatin still remaining in the nuclei was composed of granular structures with diameter of 80-100 nm. Thus, these results suggest that the approximately 40 nm fiber would be a stable structural unit and fold the 80-100 nm granules into a one-step higher unit. A common mechanism could be implied regardless of the intrinsic properties of the eukaryotic genomes.