ADAM10 mediates cleavage of N-cadherin ectodomain during retinal ganglion cell differentiation in primary cultured developing chick retinal cells

Abstract

Purpose: The purpose of this study is to investigate the role of ADAM10 mediated N-cadherin shedding in retinal ganglion cell differentiation in primary cultured embryonic chick retinal cells. Our research includes the effects of ADAM10 inhibition in ganglion and photoreceptor cells differentiation as well as in neurite outgrowths of ganglion cells.

Methods: We performed immunohistochemistry to examine the expression patterns of ADAM10 in developing retina using ED 5, 7 and 9 chick retinas. We cultured developing chick retinal cells from the embryonic day 6 (ED6) and treated specific ADAM10 inhibitor GI254023X or ADAM10-siRNA to inhibit the ADAM10 activity. Effect of ADAM10 inhibition in ganglion and photoreceptor cell differentiation was determined by immunofluorescence analysis and western blot analysis using specific cell type markers and antibodies.

Results: Immunohistochemistry showed that ADAM10 is ubiquitously expressed neuroblastic layer at ED5, byt highly expressed in the ganglion cell layer at ED7 and Ed9. Phase-contrast microscopy showed that long neurite extensions were present in untreated cultures at 24 h after plating, whereas GI254023X treatment decreased neurite extension. Immunostaining revealed that there were far fewer differentiated ganglion cells in GI254023X- or ADAM10-siRNA treated cultures compared to controls, whereas the photoreceptor cells were unaltered. The Pax6 protein was more strongly detected in the differentiated ganglion cells in control cultures compared to GI254023X- or ADAM10-siRNA treated cultures. By immunofluorescence analysis, N-cadherin (NTF) staining was strongly detected in photoreceptor cells but absent in ganglion cells.

Conclusions: Our results indicate that the inhibition of ADAM10 can inhibit Pax6 expression and N-cadherin ectodomian shedding in retinal cells, possibly affecting neurite outgrowth and ganglion cell differentiation but had no effect in photoreceptor cell differentiation.

Keywords: 688 retina • 500 differentiation • 531 ganglion cells