Abstract
Abstract: :
Purpose: Pax6 is a paired domain, homeobox transcription factor well known to play a primary role in eye development in organisms ranging from Drosophila to humans. Mutations in Pax6 lead to defects in eye formation including eyeless in Drosophila and aniridia in humans. Methods: We studied the role of Pax6 in chick eye development, which allowed us to easily perform localized transgenesis in ovo. In order to study the effects of Pax6 overactivation and repression we used a Pax6 dominant positive construct containing the VP16 transcriptional activation domain (Pax6–Pos) and a dominant negative construct linked to the Engrailed repressor domain (Pax6–Neg) (kind gifts of Dr. Nakamura). These were used in electroporation experiments where the developing left eye of a day 3 embryo was electroporated with one of the constructs and recovered from day 7 to 14 afterwards. Results: During normal development antibody staining shows that the Pax6 protein is first expressed at day 2 in the optic vesicle and cephalic ectoderm and then at day 4 in the future corneal epithelium, lens vesicle and developing retina. Electroporation with Pax6–Neg (EnR) showed a range of effects ranging from no left eye formation, in 40–55 % of the cases, to small or abnormally formed eyes. On the other hand, electroporation with the Pax6–Pos (VP16) construct leads to disruption of the normal dorsoventral patterning of the eye or to enlarged eyes. The choroid fissure, which is normally localized in the ventral region of the retina is mislocalized to the dorsal region and/or undergoes duplication one to three times. Conclusions: These results emphasize the necessity for a tight control of Pax6 activity during development as both increased and lowered Pax6 activity has deleterious effects on eye development.
Keywords: immunohistochemistry • gene modifiers