Abstract: Müller cells (MC) are considered dormant retinal progenitor cells in mammals. Previous studies demonstrated ephrin-As act as negative regulators of neural progenitor cells in the retina and brain. It remains unclear whether the lack of ephrin-A2/A3 is sufficient to promote the neurogenic potential of MC. Here we investigated whether the MC is the primary retinal cell type expressing ephrin-A2/A3 and their role on the neurogenic potential of Müller cells. In this study, we showed that ephrin-A2/A3 and their receptor EphA4 were expressed in retina and especially enriched in MC. The level of ephrinAs/EphA4 expression increased as the retina matured that is correlated with the reduced proliferative and progenitor cell potential of MC. Next, we investigated the proliferation in primary MC cultures isolated from wild-type and A2–/– A3–/– mice by 5-ethynyl-2′-deoxyuridine (EdU) incorporation. We detected a significant increase of EdU+ cells in MC derived from A2–/– A3–/– mice. Next, we investigated the role of ephrin-A2/A3 in mice undergoing photoreceptor degeneration such as Rhodopsin knockout (Rho–/–) mice. To further evaluate the role of ephrin-A2/A3 in MC proliferation in vivo, EdU was injected intraperitoneally to adult wild-type, A2–/– A3–/– , Rho–/– and Rho–/– A2–/– A3–/–  mice and the numbers of EdU+ cells distributed among different layers of the retina. EphrinAs/EphA4 expression was upregulated in the retina of Rho–/– mice compared to the wild-type mice. In addition, cultured MC derived from ephrin-A2–/– A3–/– mice also expressed higher levels of progenitor cell markers and exhibited higher proliferation potential than those from wild-type mice. Interestingly, we detected a significant increase of EdU+ cells in the retinas of adult ephrin-A2–/– A3–/– mice mainly in the inner nuclear layer; and these EdU+ cells were co-localized with MC marker, cellular retinaldehyde-binding protein, suggesting some proliferating cells are from MC. In Rhodopsin knockout mice (Rho–/– A2–/– A3–/– mice), a significantly greater amount of EdU+ cells were located in the ciliary body, retina and RPE than that of Rho–/–  mice. Comparing between 6 and 12 weeks old Rho–/– A2–/– A3–/– mice, we recorded more EdU+ cells in the outer nuclear layer in the 12-week-old mice undergoing severe retinal degeneration. Taken together, Ephrin-A2/A3 are negative regulators of the proliferative and neurogenic potentials of MC. Absence of ephrin-A2/A3 promotes the migration of proliferating cells into the outer nuclear layer and may lead to retinal cell regeneration. All experimental procedures were approved by the Animal Care and Use Committee at Schepens Eye Research Institute, USA (approval No. S-353-0715) on October 24, 2012.

Key words: endogenous stem cell, EphA4, ephrin-A2, ephrin-A3, ephrins, Müller cell, photoreceptor cell regeneration, retinal degeneration, retinal regeneration, retinal stem cell