Efficient retina formation requires suppression of both Activin and BMP signaling pathways in pluripotent cells.
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, addition of only Noggin, a known BMP antagonist, can convert pluripotent Xenopus laevis animal cap cells to functional retinal cells. The aim of this study is to determine the intracellular molecular events that occur during this conversion. Surprisingly, blocking BMP signaling alone failed to mimic Noggin treatment. Overexpressing Noggin in pluripotent cells resulted in a concentration-dependent suppression of both Smad1 and Smad2 phosphorylation, which act downstream of BMP and Activin signaling, respectively. This caused a decrease in downstream targets: endothelial marker, xk81, and mesodermal marker, xbra. We treated pluripotent cells with dominant-negative receptors or the chemical inhibitors, dorsomorphin and SB431542, which each target either the BMP or Activin signaling pathway. We determined the effect of these treatments on retina formation using the Animal Cap Transplant (ACT) assay; in which treated pluripotent cells were transplanted into the eye field of host embryos. We found that inhibition of Activin signaling, in the presence of BMP signaling inhibition, promotes efficient retinal specification in Xenopus tissue, mimicking the affect of adding Noggin alone. In whole embryos, we found that the eye field marker, rax, expanded when adding both dominant-negative Smad1 and Smad2, as did treating the cells with both dorsomorphin and SB431542. Future studies could translate these findings to a mammalian culture assay, in order to more efficiently produce retinal cells in culture.
Department of Neuroscience and Physiology, SUNY Upstate Medical University, Syracuse, NY 13210, USA The Center for Vision Research, SUNY Eye Institute, Upstate Medical University, Syracuse, NY 13210, USA.
Kimberly A
KA
Wong
Department of Pharmacology and Physiology, Aab Cardiovascular Research Institute, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.
Michael
M
Trembley
Department of Molecular Biology, Weill Cornell Graduate School of Medical Sciences, New York, NY 10021, USA.
Syafiq
S
Abd Wahab
Department of Ophthalmology, SUNY Upstate Medical University, Syracuse, NY 13210, USA Department of Neuroscience and Physiology, SUNY Upstate Medical University, Syracuse, NY 13210, USA The Center for Vision Research, SUNY Eye Institute, Upstate Medical University, Syracuse, NY 13210, USA ViczianA@upstate.edu.
Andrea S
AS
Viczian
Activin; Animal cap; BMP; Dorsomorphin; Noggin; R-SMAD; Retina; SB431542; SMAD1; SMAD2; Xenopus