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Andrea Sophia Viczian, Zahra Motahari, Kelley Ledford, Matthew Theisen, Michael Ezra Zuber; Roles for Noggin and Tbx3 in retinal progenitor cell specification and determination.. Invest. Ophthalmol. Vis. Sci. 201657(12):.
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© ARVO (1962-2015); The Authors (2016-present)
Identifying the signaling systems required for the conversion of pluripotent cells to a retinal progenitor cell fate will streamline the production of cultures for cell replacement therapy. While extrinsic and intrinsic factors have been independently identified, how they work together remains unknown. We tested the hypothesis that the eye field transcription factor, Tbx3, functions downstream of the BMP and Activin inhibitor Noggin, to regulate retinal progenitor formation.
Immunohistochemistry was used to determine the fate of pluripotent cells expressing Noggin, Tbx3, or Noggin with Tbx3 morpholinos, when transplanted to host Xenopus laevis embryos. To determine if retinal formation required Tbx3, retinal progenitors from embryos injected with Tbx3 morpholinos were also transplanted to host embryos. To monitor changes in live embryos, we generated transgenic reporter lines expressing eGFP under the control of the six6 enhancer. To identify regulators of eye field formation, mRNA for candidate genes were injected into the six6 reporter line and the effect on GFP expression was determined.
Tbx3-expressing donor cells generated retina when transplanted to the eye field, but not when transplanted to the posterior neural plate or the lateral endoderm. Knockdown of Tbx3 in Noggin-expressing pluripotent cells blocked the ability of Noggin to induce retina. Retinal progenitor cells, isolated from Tbx3 morpholino-injected embryos failed to form retina, even when transplanted directly to the host eye field. We identified two regulatory regions of the six6 promoter, a 3’ region that drives expression at eye field and early optic vesicle stages, and a 5’ region sufficient for expression at optic cup and later developmental stages. Using the six6 reporter animals, we found that Noggin induces, while constitutively active BMP signaling represses six6 expression.
We found that Tbx3 functions downstream of, and is required for, the retinal inducing activity of Noggin. Furthermore, we found that Tbx3 is sufficient to determine a neural, but not a retinal fate. We have also identified regions of the six6 promoter, that drive early and late retinal expression. Finally, we demonstrate the six6 reporter line can serve as a tool to test candidate molecules for their ability to regulate retinal progenitor formation.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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