Purpose: In adult mammals, retinal stem cells (RCS) found in the ciliary margin exhibit a limited proliferation and differentiation ability comparing with that in lower vertebrates, while this mechanism remains unknown. Recently, ephrinA3 has been identified as a negative regulator of adult neurogenesis in the mammalian central nervous system. This project was aimed to explore the roles of ephrinA3 in the regulation of RCS proliferation and differentiation.

Methods: Expression of ephrinA3 on mouse eye was analyzed by immunohistochemistry and western blot. To determine the functional significance of ephrinA3 in RSC regulation, mice deficient for ephrinA3 were examined. RSC proliferation was compared in vivo in adult wild-type and ephrinA3^-/- mice using 5-bromo-2-deoxyuridine (BrdU) pulse labeling. In vitro, quantification of RSC proliferation and differentiation were carried out using neurosphere cultures.

Results: Expression of ephrinA3 is increased in the retina and ciliary epithelium along maturation in mouse, and its expression reached the peak in the adult. Deletion of ephrinA3 greatly enhanced the proliferation of RSCs in the adult in vivo and in vitro. Results of RT-PCR show that the neurospheres derived from ephrinA3^-/- mouse express higher levels of neural progenitor cell markers, such as Sox2, neurogenin, and photoreceptor progenitor cell markers, including Crx, Nrl and Nr2e3, as compared to RSCs of wild-type mice. RSCs isolated from ephrinA3^-/- mice exhibit higher potential to differentiate into retinal neurons and photoreceptor cells as compared to wild-type RSCs

Conclusion: EphrinA3 is an endogenous inhibitor regulating the proliferation and neurogenic potential of RSCs in adult mice.