Study On Function Genomics Of Early Human Embryo And Characterization Of Novel Genes During Embryogenesis

Elucidation of gene expression and regulation is the core questions in human developmental biology. After fertilization, embryonic development occurs in a fixed order and according to a precise schedule set by the genetic program contained in the chromosomes. And this program determines when and where lines of differentiated cells will emerge, when and where different proteins vary in time and space during development. The temporo-spatial patterns of genes in various species shape the morphology and functional abilities of individual species.Although the morphological changes that occur during human embryonic development have long been known, to date, little is known about the underlying molecular mechanisms. Elucidation of the global patterns of gene expression and regulation is of fundamental importance for decoding the biological programs that control human embryogenesis.With the advent of genomic sequencing and microarray analysis of gene expression, the major characteristics of the transcriptional programs that control the development of a number of model organisms have been determined. However, very few such studies have been performed in mammals, especially in humans. Especially on the critical developmental period when the embryo switches from mainly rapid cell proliferation to the development of organs, i.e., the period from about 20 to 60 days post-fertilization, or approximately the 4th-9th weeks (Carnegie stages 10-23) of human embryonic development.We have now filled this important knowledge gap by utilizing a variety of functional genomics techniques to carry out gene expressing profiling on human embryos during the 4th-9th weeks of development.(1) Constructing cDNAlibrary is a reliable method to study the cells, organs, tissues and even multi-cellular organism-specific gene expression. In this research, we successfully constructed full-length cDNA libraries from three individual early human embryos (4 week、5 week、6 week) by applying the latest gateway BP recombination technology. The qualify identification shows that the libraries with high qualities were suitable to screen novel rare expressed genes.In order to isolate and identify new genes expressed during early human development, we screen the low-abundance clones through the methods of colony in situ hybridization. Partial sequences were generated from 5’end of 3581 low-abundance cDNA clones, and by searching in the Batch BLAST of NCBI,350 cDNA fragments (10%) appeared to be unknown in genetic databases.(2) Human embryogenesis is believed to involve an integrated set of complex yet coordinated development of different organs and tissues mediated by the changes in the spatiotemporal expression of many genes. In this study, we analyse a genome-wide microarray of the gene expression profiles at the 4-9th week during human embryonic development, a period when most organs developed. About half of all human genes are expressed and 18.6% of the expressed genes were significantly regulated during this important period. We further identified over 5000 regulated genes, most of which were previously not known to be associated with animal development. Our study filled an important gap in mammalian developmental studies by identifying functional pathways involved in this critical but previously not studied period. Our study also revealed that the genes involved here are distinct from those during early embryogenesis, which include three groups of maternal genes. Furthermore, we discovered that genes in a given developmental process are coordinately regulated. This led us to develop an easily searchable database of this entire collection of gene expression profiles, allowing for identifying new genes important for a particular developmental process/pathway and deducing the potential function of a novel gene.(3) Screened in the low abundance of 350 new genes, for studying the fu nction of these new genes, firstly we investigated the expression patterns of ne w genes in human transcriptome microarray.The results showed that most of th e new genes does not express or sustained low expression in 7-9 weeks huma n embryo, but there are a small part of the gene expression increase. The resu It indicate that this part of the new gene may be involved in 7-9 weeks of hu man embryogenesis.Plac9 is one of the new genes and its 7-9 week expression increased sign ificantly (p<0.05). Then we further study plac9 function through the real-time f luorescence quantitative PCR, RT-PCR and immunofluorescence experiments tec hniques. The results showed that plac9 supposed to be secreted into the extrace llular region by cutting the signal peptide, thus to participate in the regulation of embryonic bone development.In all, by cDNA library construction, screening and expression microarray analysis, we characterized the major characteristics of the transcriptional programs that control the early human embryogenesis. Our analysis suggested that genes with similar expression profiles tend to function in the same biochemical and cellular processes during the 4th-9th week of human embryo development. This further prompted us to establish a searchable web-based database that can be used to deduce the potential function of a novel gene by virtue of its expression profiles. We then use this database to predict the possible function of a novel gene, plac9, and use experimental techniques to validate the hypothesis. The consistency between the experiment findings and prediction based on our database further suggested that this should serve as a highly valuable resource for studying vertebrate development in general and human embryogenesis in particular.