Abstract
Abstract: :
Purpose: : Evidence is emerging that stem cells may recruit multiple signaling pathways to maintain themselves in a proliferative and uncommitted state. We have demonstrated the involvement of Notch signaling in the regulation of ocular neural stem cells. Transcritpional profiling of ocular neural stem cells revealed the involvement of Wnt signaling in addition to Notch signaling in their regulation. Wnt signaling ensues when Wnt proteins interact with members of frizzled family of receptors. This interaction leads to inhibition of Gsk–3ß mediated destabilization of ß–catenin. The stabilized ß–catenin in association with Lef1/Tcf transcription factors regulates expression of multiple target genes includes cell cycle genes such as cyclinD1. Here we report the involvement of Wnt canonical pathway in the regulation of ocular stem cells. Methods: We examined the expression of the components of Wnt canonical pathway and its modulators in neural stem cells isolated from embryonic retina and adult ciliary epithelium (CE) by RT–PCR analysis. To study the involvement of Wnt signaling in the regulation of ocular neural stem cells, cell proliferation and differentiation were analyzed in the presence of Wnt3a/constitutively active ß–catenin/fzd8CRD. The activation of Wnt canonical pathway was determined by measuring levels of cytosolic ß–catenin and the activity of TOPFLASH/FOP FLASH reporter conctructs, transfected in the ocular neural stem cells. Results: Components belonging to the Wnt canonical pathway (i.e., Wnt proteins, Frizzled receptors and Lef–1) and their modulators (Sfrps and Dkks) are expressed in both retinal and CE stem cells. Their involvement in retinal development is reflected by their temporal pattern of expression during retinal histogenesis. Exposure of cells to Wnt3a led to an increase in cell proliferation, preceded by/or accompanied with an increase in levels of cytosolic ß–catenin and Lef–1 transcripts. The effects of Wnt3a were abrogated in the presence of Fzd8CRD, a negative regulator of Wnt–Frizzled interaction. Similar effects of cell proliferation as observed with Wnt3a were obtained when constitutively active ß–catenin was overexpressed in ocular neural stem cells. There was a significant increase in TOP FLASH reporter activities in the presence of Wnt3a, further confirming the activation of Wnt canonical pathway in the ocular neural stem cells. Conclusions: Taken together, our data suggest that the Wnt canonical pathway play an important role in the maintenance of ocular neural stem cells.
Keywords: retinal development • proliferation • cell–cell communication