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Kimberly A Wong, Michael Trembley, Syafiq Abd Wahab, Andrea Sophia Viczian; Efficient retina formation requires suppression of both Activin and BMP signaling pathways in pluripotent cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):439.
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© ARVO (1962-2015); The Authors (2016-present)
Retina formation requires the correct spatiotemporal patterning of key regulatory factors. While it is known that repression of several signaling pathways lead to specification of retinal fates in vivo, we have found that treatment of pluripotent cells with only Noggin, a known BMP antagonist, can direct cells to become functional retinal cells. The aim of this study is to determine if Noggin affects intracellular signaling pathways other than BMP to efficiently direct this conversion.
We treated pluripotent Xenopus laevis animal caps with chemical inhibitors and dominant negative components of the BMP and Activin signaling pathways. Their effect on retina formation was determined using the Animal Cap Transplant (ACT) assay, in which treated pluripotent cells were transplanted into the eye field of sibling embryos at neural plate stage, after endogenous neural induction has already occurred. Signaling activity at the point of transplantation was determined by Western blot and semi-quantitative PCR (RT-PCR) to measure downstream protein and gene target expression.
Overexpressing Noggin in pluripotent cells resulted in a concentration-dependent suppression of both Smad1 and Smad2 phosphorylation, which act downstream of BMP and Activin receptors, respectively. This caused a decrease in downstream transcriptional ability, reflected by the reduced expression of both the endothelial marker, xk81, and the mesodermal marker, xbra. Expression of dominant negative BMP and Activin receptors or R-Smads revealed that retinal specification was increased when both pathways were inhibited simultaneously. Similar results were observed when the chemical inhibitors dorsomorphin and SB431542 were used to inhibit Smad1 and Smad2 phosphorylation, respectively.
Thus, the dual inhibition of BMP and Activin pathways promotes retinal specification in Xenopus tissue. This suggests that Noggin extrinsically modulates intercellular BMP and Activin signaling in order to efficiently specify retinal cell fate. Future studies could translate these findings to a mammalian culture assay, in order to efficiently produce retinal cells in culture.
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