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Kimberly Wong, Michael A Trembley, Andrea Sophia Viczian; Retina formation requires suppression of BMP and Activin pathways in pluripotent cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):4034.
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© ARVO (1962-2015); The Authors (2016-present)
Retina formation requires the correct spatiotemporal expression of key regulatory proteins. Signaling through the bone morphogenetic protein (BMP) pathway represses the formation of neural and retinal fates. It has been shown that Noggin acts as a morphogen to specify neural cell types at low concentrations, and retinal cell types at higher concentrations. The aim of our study is to determine if the higher concentration of Noggin affects signaling pathways other than BMP.
We treated pluripotent Xenopus laevis tissue (animal caps) with chemical inhibitors and function-altering components of the BMP and Activin/Nodal signaling pathway. Animal caps were removed from the embryos at the blastula stage and cultured until neural plate stage. Their effect on retina formation was determined using the Animal Cap Transplant (ACT) assay, in which the animal caps were transplanted into the eye field of sibling embryos. Signaling activity was determined by Western blot and semi-quantitative PCR (RT-PCR) to measure downstream protein and gene target expression.
Overexpressing Noggin in animal caps resulted in a concentration-dependent suppression of both Smad1 and Smad2 phosphorylation, which act downstream of BMP and Activin/Nodal receptors, respectively. This caused a decrease in downstream transcriptional ability, reflected by the reduced expression of mesodermal marker, Xbra, and endothelial marker, Xk81. However, we also observed that Cerberus was less effective at blocking Smad1/5/8 phosphorylation, yet it can specify retina as efficiently as Noggin in ACT assays. Cerberus has been shown to block the Activin/Nodal pathway, as well as the BMP pathway, suggesting that there is a specific balance between the two pathways that is required to direct a retinal fate. The use of dominant negative BMP and activin receptors 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 Smad2/3 phosphorylation, respectively.
Thus, the dual inhibition of BMP and activin pathways promotes retinal specification in Xenopus tissue. Future studies will translate these findings to a mammalian culture assay, in order to efficiently produce a large percentage of retinal cells in vitro.
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