Summary
The mammalian retina does not regenerate following retinal damage. In contrast, regeneration occurs
in teleost fish through the proliferation and reprogramming of Müller glia into neurogenic progenitor cells
that replace the retinal cells in deficit. Thus, a goal of retinal regenerative medicine is to efficiently direct
mammalian Müller glia into a neurogenic progenitor state. Several barriers prevent this and they include
inefficient cell cycle reentry and proliferation, the lack of dedifferentiation (loss of glial properties), and
inability to transition into a neurogenic state. However, recent studies show that this type of
reprogramming is possible with intervention, and continued research is needed to push Müller glia-
based reprogramming into the realm of structural and functional regeneration. For this R21, we will
determine if p27Kip1 inactivation in mouse Müller glia drives these cells toward a neurogenic progenitor
state. p27Kip1 is a cyclin-dependent kinase inhibitor protein that has multiple functions in the cell cycle,
differentiation, cell migration, metabolism, and gene expression. Prior work from our lab showed that
p27Kip1 inactivation in the absence of injury caused Müller glia proliferation and migration into the outer
nuclear layer. We now have preliminary data showing that aged retinas exhibit robust proliferation after
p27Kip1 inactivation, and that a subset of p27Kip1-inactivated Müller glia express a transcription factor
associated with retinal neurogenesis. In this project, we will determine the phenotypic changes of
p27Kip1-inactivated Müller glia in aged retinas in the presence or absence of two types of induced
retinal damage; N-methyl-D-aspartate excitotoxicity to target inner retinal neurons, and thermal laser
lesions to target photoreceptors and RPE. While initial studies will incorporate noninvasive ocular
imaging in live animals and immunohistology of fixed tissue, the primary method of data collection will
be single cell RNA sequencing. Multiple conditions and timepoints will be studied, and to leverage the
capabilities of existing technology and reduce costs, we will optimize methods for retinal cell
cryopreservation and sample multiplexing. Through analysis of the datasets generated here
accompanied by integration of existing datasets, we will be able to identify new cell states in p27Kip1-
inactivated Müller glia, determine if they dedifferentiate and acquire neurogenic properties, and identify
selective responses to different types of injury. These studies will provide the needed resolution to
understand how blocking p27Kip1 function impacts the ability of Müller glia to enter into a neurogenic
progenitor state. Additionally, the successful development of retinal cell cryopreservation and sample
multiplexing protocols will enhance the ability of vision research labs to expand upon more complex
experimental designs in a cost effective manner.