Analysis Of Gene Expression Patterns And Isolation Of Specific Genes In The Avian Somite Development

The chicken embryo is an important model to understand the early vertebrate embryogenesis. As the organizer of chicken embryos, Hensen抯 node (H) possesses many unique properties, such as self-differentiation into notochord and somite (S). Somite is the source of axial bone, skeletal muscle and hypodermis. Segmental plate is the transition structure from H to S. Our research group first discovered that presomite cells (H and SP) occurred two commitments before somite forming by using the transplantation technique of dissociated cells. However, it is not clear about the molecular mechanisms of the two commitments. Isolation the differentially expressed genes between H and SP will help to understand the two commitments in the level of molecular biology, and to analyze the mechanisms for maintain H unique characteristics and the maturation of SP. For this goal, Differential Display of mRNA technique and Representational Difference Analysis of cDNA were used to compare the gene expression patterns between H and SP. The crucial factors that restrict the two methods were analyzed in detail. An improved method, which is simpler, more sensitive, fewer amounts of RNA and relatively lower false positives, was established based on the two techniques. First, it conquered the disadvantage of the information loss caused by multiple digestion with enzymes, ligation and hybridization-PCR of Representational Difference Analysis by decreasing the cycles of hybridization. Second, it improved the sensitivity to detect the differentially expressed genes by relaxing the hybridization system. Third, it avoided the burdensome labor in screening the differential fragments through repetition with different combinations of primers and high false positives of Differential Display of mRNA technique by reducing the complexity of PCR templates. Using this improved method, we analyzed the gene expression patterns of H and SP, achieving 13 differentially expressed cDNA fragments. One of them is expressed uniquely in SP, and two are expressed uniformly in H and SP, five are expressed equally in H and SP, while decreased by 2 or I fold in S. These isolated uniquely and differentially expressed genes may play roles in the commitments of presomite cells or maintain the special properties of H or maturation of SP, which gives new insights to further research their molecular mechanisms.