During the development of an embryo,the initiation of the coilinear expression of Hox genes is essential for the proper formation of the anteroposterior body axis.Retinoic acid (RA),a natural derivative of vitamin A,plays a role in vertebrate development by regulating Hox gene expression.CCCTC-binding factor (CTCF),an insulator protein that controls gene transcription,also regulates the expression of Hox genes by binding to the CTCF-binding sites (CBSs).It has been reported that upon RA signaling,retinoic acid response elements (RAREs) located in the Hox clusters become occupied.Interestingly,RAREs exist in close proximity with CBSs,and therefore when RA is bound,CTCF cannot bind.Without CTCF and its insulator activities,the repressive domain in the chromatin becomes open for gene transcription.Here,we examine the relationship between RA and CTCF during the RA-induced expression of the Hoxa cluster genes,using F9 murine embryonic teratocarcinoma cells as a model system.We treated F9 cells with RA for different time,confirmed the collinear expression of Hoxa genes,and validated CTCF-binding in F9 cells as well as in CTCF-overexpressing F9 cells,in the presence of RA.The present study suggests that RA and CTCF pose antagonistic effects on each other during vertebrate development to attain Hoxgene collinearity.