Embryonic retinal stem cells give rise to the diverse assortment of neuronal and glial cells, which form the mature eye. In retinal injury and blinding diseases, such as glaucoma and retinitis pigmentosa, retinal cells are lost and never regenerate. Replacement of damaged retinal cells with multipotent retinal stem (RS) cells has been hampered by an inability to isolate and culture sufficient quantities of these rare cells. Once the intrinsic and extrinsic cues regulating their generation and differentiation are identified, retinal stem cells will become a precious resource for cell replacement therapies. Molecular and genetic evidence shows that embryonic RS cells are specified in the anterior neural plate by the combined action of eye field transcription factors (ET, Rx, Pax6, Six3, Lhx2, til and Optx2). Following RS cell specification, additional extrinsic and intrinsic cues direct their differentiation into mature cell types in the eye. We recently discovered that co-expression of the eye field transcription factors is sufficient to induce RS cells and fully functional eyes at high frequency in the frog embryo. In the frog Xenopus laevis, RS cells develop at room temperature in a simple salt solution in just hours. Modern molecular and genetic techniques make it possible to manipulate and monitor gene expression in living embryos at virtually any developmental stage. The frog eye shares common molecular, developmental, structural, and functional processeswith other vertebrate species, including humans. These strengths make Xenopus a uniquely suited system to analyze RS cells and eye development. RNA blastomere injections will be used to overexpress cocktails of wild-type with function blocking forms of eye field transcription factors (EFTFs) in developing embryos. These experiments will determine the minimum number of EFTFs necessary and sufficient to generate multipotent RS cells and ectopic eyes in vivo.
(Aim 1). Next, cultured RS cells will be used to determine the combination of extrinsic and intrinsic factors regulating their proliferation and differentiation (Aim 2). Finally, we will transplant in vitro generated RS cells into the embryo to determine if they differentiate, integrateand survive in the mature retina (Aim 3). Together, these experimentswill identify the effectors regulating RS cell specification, proliferation and differentiation and test the ability of in vitro generated RS cells to repopulate the mature retina. These studies will advance our basic understanding of the underlying mechanisms regulating retinal development, and provide the fundamental work necessary for the ultimate goal of using RS cells to treat blinding diseases.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY015748-04
Application #
7496924
Study Section
Neurogenesis and Cell Fate Study Section (NCF)
Program Officer
Greenwell, Thomas
Project Start
2005-09-30
Project End
2010-08-31
Budget Start
2008-09-01
Budget End
2010-08-31
Support Year
4
Fiscal Year
2008
Total Cost
$253,127
Indirect Cost
Name
Upstate Medical University
Department
Ophthalmology
Type
Schools of Medicine
DUNS #
058889106
City
Syracuse
State
NY
Country
United States
Zip Code
13210
Ledford, Kelley L; Martinez-De Luna, Reyna I; Theisen, Matthew A et al. (2017) Distinct cis-acting regions control six6 expression during eye field and optic cup stages of eye formation. Dev Biol 426:418-428
Martinez-De Luna, Reyna I; Ku, Ray Y; Aruck, Alexandria M et al. (2017) Müller glia reactivity follows retinal injury despite the absence of the glial fibrillary acidic protein gene in Xenopus. Dev Biol 426:219-235
Motahari, Zahra; Martinez-De Luna, Reyna I; Viczian, Andrea S et al. (2016) Tbx3 represses bmp4 expression and, with Pax6, is required and sufficient for retina formation. Development 143:3560-3572
Viczian, Andrea S; Zuber, Michael E (2014) A simple behavioral assay for testing visual function in Xenopus laevis. J Vis Exp :
Martinez-De Luna, Reyna I; Ku, Ray Yueh; Lyou, Yung et al. (2013) Maturin is a novel protein required for differentiation during primary neurogenesis. Dev Biol 384:26-40
Zuber, Michael E; Nihart, Heather S; Zhuo, Xinming et al. (2012) Site-specific transgenesis in Xenopus. Genesis 50:325-32
Choi, Rene Y; Engbretson, Gustav A; Solessio, Eduardo C et al. (2011) Cone degeneration following rod ablation in a reversible model of retinal degeneration. Invest Ophthalmol Vis Sci 52:364-73
Viczian, Andrea S; Zuber, Michael E (2010) Tissue determination using the animal cap transplant (ACT) assay in Xenopus laevis. J Vis Exp :
Zuber, Michael E (2010) Eye field specification in Xenopus laevis. Curr Top Dev Biol 93:29-60
Viczian, Andrea S; Solessio, Eduardo C; Lyou, Yung et al. (2009) Generation of functional eyes from pluripotent cells. PLoS Biol 7:e1000174