Purchase this article with an account.
K. M. Beach, J. Phillips, D. C. Otteson; Ciliary Neurotrophic Factor Regulation of Gfap Expression in Mouse Muller Glia-Derived Neurospheres. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2654.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Muller glia are a source of stem cells in the mammalian retina. In vivo, ciliary neurotrophic factor (CNTF) upregulates glial acidic fibrillary protein (Gfap) expression in Muller glia via phosphorylation of STAT3. We examined STAT3 phosphorylation and Gfap expression in mouse Muller-derived retinal stem cells following CNTF treatment or fibroblast growth factor 2 (FGF2)-induced differentiation.
Neurospheres were generated from conditionally immortalized mouse Muller cells (ImM10) by culturing in serum-free Neurobasal medium with epidermal growth factor (EGF) + FGF2. STAT3 phosphorylation was assayed by Western blots of total proteins from CNTF treated neurospheres or dissociated mouse retinal cells. Gfap expression was assayed by quantitative RT-PCR in total RNA isolated from neurospheres treated with CNTF (0, 2, 20, and 200 ng/ml, 24 hrs) or differentiated neurospheres (primed with FGF2, 5 days, followed by growth factor withdrawal, 5 days). RT-PCR data was analyzed using Relative Expression Software Tool (REST).
FGF2-induced differentiation of Muller glial-derived neurospheres resulted in a 9.3-fold increase in Gfap mRNA expression (p=0.03) vs. neurospheres. CNTF did not alter Gfap mRNA expression in neurospheres at any concentration tested (p=0.28, 0 vs. 200 ng/ml). Although CNTF increased phosphylation of STAT3 in dissociated retinal cells by 3.6-fold, only a 1.31 increase in p-STAT3 was detected in CNTF treated neurospheres.
The minimal changes in STAT3 phosphorylation and lack of significant increase in Gfap expression in Muller-derived neurospheres treated with CNTF was unexpected. These results suggest that Gfap upregulation during FGF2 induced differentation may result from activation of alternate signaling pathways. Understanding the mechanisms regulating glial activation in Muller-derived stem cells will be important in identifying strategies to block gliogenesis and promote neurogenesis.
This PDF is available to Subscribers Only