Gene Loci Related To Spatial Patterning Of Central Nervous System And Gene Expression Pattern Analysis In Goldfish

The developmental phenotype system of gynogenetic haploid goldfish embryo is a suitable analytic system for investigations on genomic regulatory mechanisms and general principles of genes selective expression pattern at the whole genome level. Since haploid embryos have only one set of genome, the alleles with different developmental effect could be separated and identified by analyzing the phenotype of the haploid. Once the alleles identified, the function and regulation of the genes, especially the effects of epigenetic modification on gene expression during development and related epigenetic modification mechanism, can be readily investigated. By means of observation on living embryos and paraffin section, statistic analysis of different haploid phenotype, whole-mount in situ hybrization and frozen section of different haploid phenotype, we analyzed the gene loci related to spatial patterning of goldfish eye and brain ventricle and the expression pattern of one related gene. These results would be the basic for further identification of gene related to the spatial patterning of eye and brain, and provide some valuable cues for further research on the genetic and epigeneticmechanisms controlling the regulation of gene expression during development of eye and brain.The main contents, results and conclusions of this research are as follows:1. Observations on living embryonic brains and their microstructure of brain ventricle revealed that there are two brain ventricle phenotypes in gynogenetic haploid embryos. One phenotype is as normal as that of the control inbreeding diploid embryos, which has normal differentiated forebrain, midbrain and hindbrain. Another phenotype is obviously abnormal, the brain patterning is irregular and no distinct brain ventricle was observed.2. The ratio of haploid embryos with normal brain pattern to that with abnormal brain pattern is 1:3. This ratio indicated that there are at lest two different gene loci involved in the spatial patterning of the brain ventricle. Since the possibility that deleterious recessive mutant alleles exist on both of the two gene loci had been excluded in this experiment, the phenotype represented the expressional state rather than the genotype of these two genes. Therefore, the ratio of 1 : 3 suggested that the expressing probability for each copy of the two genes is 50%,and the regulatory mechanism of the expression is based on two set of chromosomes, controlled by the rule of diploid-dependent regulatory mechanism.3. Analyzing the temporal and spatial expression pattern of vsxl, a gene that plays a rule in development of central nervous system and eye, by whole-mount in situ hybridization and frozen section. The results showed that at the 16-somite stage, expression of vsxl was detected in all the inbreeding goldfish diploid embryos in spinal cord. Slightly later in development, vsxl was expressed in the developing hindbrain and the level of expression was increasing. At the 7 day after fertilization, the expression of vsxl was restricted to the inner nuclear layer of retina. The temporal and spatial expression pattern of vsxl in the haploid embryos was just the same as in diploid embryos, but vsxl was only expressed in part of the haploid embryos. As to the different phenotypes of eye, it was found that vsxl was expressed in all the examined normal eye haploid phenotype and none of the examined cup-less haploid phenotype was detected the expression of vsxl. But in the bubble-eye haploid phenotype, vsxl was expressed in some embryos but not in other embryos. This observation correlated well with the expectantdistribution in the three phenotypes. Since haploid embryos has only one set of genome, this result also revealed that the expression probability of maternal vsxl was 50% in haploids.4. The 50% expression probability of maternal vsxl in haploid embryos suggested that allele-specific methylation might occur during oogenesis in goldfish. Both the methylated and unmethylated allele of vsxl was distributed into eggs by chance during meiosis, which resulted in only 50% of the gynogenetic haploid embryos expressed vsxl. 100% expression probability of vsxlin inbreeding diploid embryos suggested that fertilization might promote the reprogramming process of gene expression and was essential for the normal development in vertebrate.