Michael Zuber - US grants

DESCRIPTION (provided by applicant): 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, tll 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 processes with 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 over express 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, integrate and survive in the mature retina (Aim 3). Together, these experiments will 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.

Project Summary Establishing and maintaining a postmitotic state is essential for normal development and organ function. Cells that fail to exit, or reenter, the cell cycle commonly die or continue proliferating unchecked. Despite the importance of these processes the mechanisms that drive cells into, and maintain them in, a non-proliferative state are incompletely understood. The long-term goal of this research is to identify the mechanisms that control these processes in the mammalian retina. The objectives of this application are to characterize a new molecule we have identified that is implicated in maintaining postmitotic retinal cells in a non-proliferative state. Our preliminary data support a central hypothesis that Maturin, regulates postnatal retinogenesis, and Maturin loss is sufficient for differentiated retinal cells to reenter the cell cycle and generate additional retina. The rationale for the proposed experiments is that that they will distinguish between two distinct models. Maturin may be required 1) to prevent the production of excess retinal cells, or 2) for normal retina formation, independent of proliferation. In Aim 1, morphometric analysis, flow cytometry and EdU labelling will determine where defects are first detected, if and when retinal cell number is altered, and if cycling RPCs are observed with retinal expansion. In Aim 2, we precisely define the temporal and spacial expression pattern to determine which cell types express Maturin, and if that expression is restricted to postmitotic cells. In Aim 3, mice with tamoxifen-inducible null alleles of Maturin will be used to determine precisely when Maturin loss triggers retinal expansion. This work is significant, as Maturin may be required, independent of proliferation, for normal retina development or maintenance. Alternatively, Maturin may control retina size by inhibiting reentry of retinal cells into the cell cycle. This would be an exciting possibility as it would suggest inhibiting Maturin could potentially reactivate retinogenesis in the mammalian eye. Therefore, this project perfectly fits within the NEI's audacious goal to ?Regenerate neurons and neural connections in the eye and visual system.?