Towards Mammalian Retinal Regeneration: Cell Signaling in Re-Differentiating Mouse Müller Glia and Transcriptional Regulation of the Axonal Guidance Receptor EPHA5

Damage to the neural retina or optic nerve results in irreversible blindness because the mammalian retina cannot regenerate. For stem cell therapies to regenerate a functional retina, multiple challenges must be overcome. These include identification of stem cell sources, targeted differentiation into particular retinal neuronal cell types, delivery to the damaged area, survival and integration into the remaining retinal tissue, and establishment of appropriate connections between cells. Müller glia have been proposed as potential retinal stem cells, but attempts to re-differentiate Müller glia into retinal neurons have shown poor yield and incomplete differentiation. In cultured retinal progenitor cells, differential activation of MAPK versus STAT3 cell signaling pathways by the cytokine ciliary neurotrophic factor (CNTF) can promote neuronal versus glial differentiation, respectively. Specific Aim 1 measured MAPK and STAT3 signaling and neuronal versus glial gene expression in a mouse Müller cell line during re-differentiation and in response to treatment with CNTF. At different stages of in vitro re-differentiation, ImM10 Müller glia activated neurogenic MAPK and did not change gliogenic STAT3 signaling pathways. Exogenous CNTF did not alter MAPK or STAT3 signaling or the final expression levels of neuronal and glial genes. Overall, ImM10 cells do not respond to CNTF as previously reported for primary Müller glia, and their neurogenic potential is not altered by CNTF. Another challenge facing regenerative therapies will be re-establishment of appropriate connections between the retina and the visual areas of the brain. The axonal guidance receptors EphA5 and EphA6 are expressed in gradients across the retina, and changes in their expression alter the location of retinal ganglion cell (RGC) synaptic termination zones. Therefore, new RGCs will need to express EphA5 and EphA6 at levels appropriate for their retinal location. Specific Aim 2 addressed the regulatory hierarchy controlling EphA5 and EphA6 expression in the mouse retina. POU4F2 is a RGC-specific transcription factor that when deleted results in aberrant RGC projections, suggesting potential defects in expression of axonal guidance genes. KLF16 is a recently-identified regulator of EphA5 that is expressed after POU4F2 in newly-differentiated RGCs, but KLF16’s regulation and spatial expression patterns in the retina have not been characterized. In wild-type mice, KLF16 expression was equal in nasal retina comparted to temporal retina. In Pou4f2 knockout mice, expression of EphA5, EphA6, and Klf16 was unchanged compared to wild-type mice. Unexpectedly, there was increased expression of a commonly used reference gene, β-2 microglobulin (B2m), which has been implicated in retinogeniculate synaptic remodeling. Thus POU4F2 may repress B2m expression, but does not regulate EphA5, EphA6, or Klf16 expression. In conclusion, in vitro differentiation of ImM10 Müller glia results in conflicting activation of MAPK and STAT3 signaling that may, in part, explain the limited neurogenic potential of this cell line. This emphasizes the necessity of finding alternative stem cell sources for regenerative therapies. In the Pou4f2-/- mouse, axonal misrouting is not a result of dysregulation of EphA5, EphA6, or Klf16, but may involve dysregulation of B2m. Further studies are needed to identify the transcriptional regulators of axonal guidance genes that could be exploited to promote axon outgrowth, guidance, and connectivity of stem cell-generated RGCs for development of regenerative therapies.