Abstract
Abstract: :
Purpose: Control of cell differentiation in eye development is important for normal vision function. Homeobox Pax6 gene expression during early embryonic stages plays a dominant role in this process. The purpose of the present study is to explore the regulatory effect of CTCF on Pax6 expression affecting eye development. Methods: CTCF transgenic mice were established by microinjecting linealized CMV–CTCF construct into fertilized mouse oocytes. Injected oocytes were implanted in the uterus of foster mothers through canulation of the ovarian duct. Mouse embryos were collected at stage E11 and E14. PCR reactions with a pair primer specific for CMV–CTCF construct were performed to identify CTCF transgenic mice. Embryo sections were prepared for immunochemistry studies. Northern and western blots were performed to detect gene and protein expressions. CTCF was overexpressed by transient and stable transfection in eye–derived cells. Results: In the mouse embryo, overexpression of CTCF, a zinc finger (ZF) protein and transcription regulator, suppresses Pax6 gene expression. This effect in CTCF transgenic mice also caused defects in ocular development, which resulted in eyeless and small eye phenotypes. Further studies indicated in eye–derived cells that up–regulation of CTCF expression using a tetracycline turn–on system significantly suppressed Pax6 protein expression. In addition, overexpression of CTCF diminished Pax6 reporter activity, suggesting that interaction of CTCF and Pax6 gene is through transcription control in the 5’–flanking region upstream from the Pax6 P0 promoter. In contrast, down–regulation of CTCF expression by knockdown of CTCF mRNA markedly enhanced Pax6 expression in these cells. Conclusions: We have investigated the role of CTCF in regulating eye development through control of Pax6 gene transcription. This is the first example, to our knowledge, showing such interdependence between CTCF and PAX6 in the control of eye development.
Keywords: cornea: epithelium • gene/expression • genetics