Abstract
Purpose: :
The zinc-finger nuclear factor CTCF is critical for normal development. However its role in eye formation is still mostly uncharacterized. Previous studies have suggested that CTCF regulates the expression of the transcription factor Pax6, and that its overexpression in transgenic mice results in ocular defects. In this work we have tested the hypothesis that CTCF is involved in the control of eye development, using an in vivo loss-of-function approach.
Methods: :
Fluorescein labeled control morpholinos (MO) or MO that block CTCF translation were injected in the neural tube of stage 9-11 chick embryos, followed by electroporation into the optic vesicles. The embryos were collected 7, 24 and 72 hours later and processed for Western blot analysis, or fixed and prepared for histological and immunohistochemical examination. Apoptotic cell death and cell proliferation were analyzed by TUNEL labeling and immunohistochemistry for phosphorylated histone 3 (pH3) respectively. Dorso-ventral patterning of the retina was analyzed by expression of specific markers. Results were examined by fluorescence microscopy and image analysis was performed using ImageJ software.
Results: :
Macroscopic and histological examination showed that MOs were efficiently delivered into the retinal neuroepithelium. Western blot analysis confirmed CTCF downregulation in CTCF-MO treated embryos compared to controls. Inhibition of CTCF in vivo during early development resulted in microphthalmia, chorio-retinal coloboma, and an expansion in the expression domain of the transcription factor Pax2. At the cellular level, CTCF inhibition produced an increase in apoptosis in the developing retina, whereas the proliferation rate remained unaffected.
Conclusions: :
Our results show that inhibition of CTCF causes microphthalmia, possibly due to an increase in apoptosis that is not accompanied by alterations in cell proliferation rates. This phenotype is accompanied by chorio-retinal coloboma and dorso-ventral patterning abnormalities of the retina. Such observations are consistent with the hypothesis that CTCF plays an important role in eye development. Studies are underway to characterize the molecular mechanisms involved in CTCF action in the eye.
Keywords: development • gene/expression • transcription factors