Mensuration And Bioinformatics Analysis Of Gene Expression During The Process Of Chondrogenesis

Objective: The aim of the present study was to investigate gene expression during the critical period of mice embryonal limb bud chondrogenesis in order to better understand the complexities mechanisms that control this process and its relationship to the cell cycle changes that occur in the critical period.Method: Mice embryonal anterior limb buds was cut from mice embryo during 10.5～14.5 days of the prenatal development by micromanipulation, poly(A+) RNA was isolated from the five anterior limb buds cell populations and subjected to a gene expression analysis using a microarray containing～34,000 known mice genes and～39,000 expressed sequence tags (ESTs). The critical period of mice embryonal limb bud development was investigated by biochemistry, histology, immunohistochemistry and microarray analysis.Results: A≥2-fold difference in the expression of 2998 known genes or ESTs was observed in the five days. The differences in expression of several of the genes detected by the microarray hybridization were confirmed by Real-time PCR analyses. There were obvious increases in mRNAs for TIFs (transcription initiation factors) and RNA polymerases during the critical period. Such as TFⅡA(Gtf2a2), TFⅡB(Gtf2b),TFDⅡD(Taf5, -6, -7, -9, -10, -12, -13), TFⅡE(Gtf2e1, -2e2), TFⅡF(Gtf2f2), TFⅡH(cdk7, Gtf2h1, -2, -3, -4, Ercc3, Ccnh, Mant1); RNA polymerasesⅠ(Rpo1-1, -1-2, -1-3, -1-4), RNA polymerasesⅡ(Pol2a, -b, -c, -e, -g, -k, -I, -j)and RNA polymerasesⅢ(Sin, Rpc8, Polr3b, -d, -k). Ccnh, Cdk7 and Mnat1 were also observed in cell cycle gens changes, this finding revealed a complex pattern of gene expression during the critical period of mice embryonal limb bud development, including many genes not yet reported to be expressed by mice embryonal limb bud chondrogenesis.Conclusion: The 10.5～14.5 days of the prenatal development is the critical period of mice embryonal limb bud development. Changes in gene expression associated with the critical period were observed in a wide variety of genes , including genes encoding extracellular matrix proteins , transcription initiation factors, and growth factors. At least 99 of the genes that were regulated in response to chondrogenesis were also found to be expressed in several pathways. Our results indicate that the simplicity of the system makes it possible to explain the complexities mechanisms partially during the critical period of mice embryonal limb bud chondrogenesis.