Abstract
Abstract: :
Purpose: CNTF promotes glia cell differentiation in brain. This study has characterized functions of STAT3 and MAPK, the downstream targets of CNTF, in Müller cell differentiation. Methods: Eighty CD1 and C57Bl/6 mice were used in this study. Timed–pregnant CD–1 and C57Bl/6 mice were purchased from Charles River Laboratories (Boston, MA) and Jackson Laboratories (Bar Harbor, ME). All mouse protocols were in accordance with ARVO guidelines and were approved by the Yale IACUC. Most of the litters were born on E19.5, which was considered equivalent to postnatal day 0 (PN0). Embryonic day 17.5 (E17.5) embryos and postnatal day 1 (PN0) retinas were dissected in cooled phosphate–buffered saline (PBS). Retina explants were cultured in serum–free medium supplemented with L–glutamine and antibiotics. Müller cells were isolated from PN5 retinas for monolayer culture. PD98059 and/or CNTF were added in the culture medium 5 hours after isolation and kept for 7 to 10 days. Treated and control retina samples were collected for immunohistochemistry and western blot. Recombinant wild–type STAT3, constitutively active and dominant negative forms of STAT3 adenoviruses were used to infect Müller cells in vitro. Results: Both STAT3 and MAPK are activated by CNTF in Müller cells. Müller cell numbers increased after CNTF treatment of E17.5 cultured retina explants but not of explants cultured from PN1. Pretreatment of retinal explants with PD98059 inhibited the CNTF–induced increase in Müller cell numbers. Activation of STAT3 by CNTF treatment produced a mislocation of Müller cells, but only in E17.5 retina explants. Müller cells remained in the inner part of the outer retina layers and never spanned the whole retina. In vitro studies of Müller cells showed significant acceleration of migration after infection by dominant negative STAT3 adenovirus compared with controls. Conclusions: Our results indicate that MAPK may regulate Müller cell proliferation after CNTF treatment whereas STAT3 is more likely involved in Müller cell migration.
Keywords: signal transduction • Muller cells • retinal development