Abstract
Purpose::
Previous studies have demonstrated roles for Notch signaling in regulating cell fates and proliferation in the developing retina. Inhibition of Notch-1 expression during early development leads to premature neuronal differentiation. However, the specific mechanisms and down-stream molecules by which Notch mediates these effects in the chicken retina remain uncertain. In this study, we interfered with Notch signaling by using different methods and determined the outcomes on the differentiation and proliferation of embryonic chicken retinal cells.
Methods::
We used DAPT, a gamma-secretase inhibitor, to block cleavage and activation of the intracellular domain of the Notch receptor, and double stranded (ds) RNA to silence the expression of bHLH factors (Hes1 and Hes5) that can be downstream of Notch signaling. We cultured embryonic day 5 or day 6 retinal cells under control or treatment conditions for 1-4 days. We identified ganglion cells and/or amacrine cells, photoreceptors and proliferating cells by using antibodies to HuD and/or calretinin, visinin and BrdU respectively.
Results::
We found that blocking Notch signaling with DAPT resulted in increased numbers of cells that differentiated as amacrine cells (but not photoreceptors) with no effect on cell proliferation. Transfection of cells with dsRNA to Hes1 resulted in increased numbers of cells that differentiated as photoreceptors, but not as ganglion or amacrine cells. Transfection of cells with dsRNA to Hes5 resulted in reduced numbers of cells that differentiated as photoreceptors, ganglion cells and amacrine cells.
Conclusions::
We find that inhibition of different components of the Notch-signaling pathway had different outcomes on the fates of embryonic chicken retinal progenitors. We propose that Hes1 normally inhibits the photoreceptor fate of embryonic chicken retinal cells, whereas Hes5 may act in opposition to Hes1 with respect to photoreceptor differentiation.
Keywords: retinal development • transcription factors • photoreceptors