Abstract
Purpose::
We have previously demonstrated that retinal stem cells/progenitors can be enriched prospectively as the side population (SP) cells, using the Hoechst dye efflux assay (Bhattacharya et al., 2003, IOVS, 44:2764). The molecular determinant of SP cell phenotype is ABCG2, a half transporter belonging to ATP-binding cassette (ABC) super family of transmembrane proteins. ABCG2 expression is developmentally regulated in retina and associated with lineage commitment. Like that of Notch1, a key regulator of retinal stem cells/progenitors, ABCG2 expression is preferentially associated with proliferating progenitors and decreases with the onset of differentiation. Here, we demonstrate that ABCG2 is one of the targets of Notch signaling for the maintenance of retinal stem cells/progenitors in an uncommitted state.
Methods::
Effects on retinal SP cells, progenitor properties and differentiation was examined in response to perturbation of ABCG2 expression by retrovirus-mediated transduction and siRNA mediated gene silencing. Effects on ABCG2 expression was examined in response to perturbation of Notch signaling by forced expression of NICD or culturing retinal stem cells/progenitors in the presence of DAPT. Reporter activities, driven by ABCG2 promoter, were analyzed in response to perturbation of Notch signaling to evaluate the direct effects of Notch signaling on ABCG2 expression.
Results::
There was a significant increase and decrease in the proportion of cells expressing differentiation markers and neural progenitor markers, respectively, in response to attenuation in ABCG2 expression. Converse was observed in response to forced expression of ABCG2. Accentuation of Notch signaling led to an increased expression of ABCG2, accompanied by increase in the proportion of cells with progenitor properties, and in co-transfection assays enhanced reporter activities via ABCG2 promoter. DAPT had inverse effects.
Conclusions::
Together, these observations suggest ABCG2 is one of the downstream targets of Notch signaling in stem cells/progenitors which contributes towards their maintenance. Supported by COBRE (Neurosensory Research) and Research to Prevent Blindness.
Keywords: retinal development • proliferation • cell-cell communication