Retinal ganglion cell (RGC) degeneration is a hallmark of glaucoma, the most prevalent cause of irreversible blindness. Thus, therapeutic strategies are needed to protect and replace these projection neurons. One innovative approach is to promote de novo genesis of RGCs via manipulation of endogenous cell sources. Here, we demonstrate that the pluripotency regulator gene Krüppel-like factor 4 (Klf4) is sufficient to change the potency of lineage-restricted retinal progenitor cells to generate RGCs in vivo. Transcriptome analysis disclosed that the overexpression of Klf4 induces crucial regulators of RGC competence and specification, including Atoh7 and Eya2. In contrast, loss-offunction studies in mice and zebrafish demonstrated that Klf4 is not essential for generation or differentiation of RGCs during retinogenesis. Nevertheless, induced RGCs (iRGCs) generated upon Klf4 overexpression migrate to the proper layer and project axons aligned with endogenous fascicles that reach the optic nerve head. Notably, iRGCs survive for up to 30 days after in vivo generation. We identified Klf4 as a promising candidate for reprogramming retinal cells and regenerating RGCs in the retina. This article has an associated 'The people behind the papers' interview.

Retinal ganglion cell (RGC) degeneration is a hallmark of glaucoma, the most prevalent cause of irreversible blindness. Thus, innovative therapeutic strategies are needed to protect and replace these projection neurons. It has been shown that endogenous glial cells of the retina, Müller cells, can be directly reprogrammed into late-born retinal interneurons. However, since RGCs are the first neurons born during development, the replacement of damaged RGCs requires the reprograming to an early neurogenic state.Here, we demonstrate that the pluripotency regulator Klf4 is sufficient to reprogram the potency of lineage-restricted retinal progenitor cells (RPCs) to generate RGCs in vivo.Transcriptome analysis disclosed that the overexpression of Klf4 induces crucial regulators of RGC competence and specification, including Atoh7 and Eya2. In contrast, loss-of-function studies in mice and zebrafish demonstrated that Klf4 is not essential for generation or differentiation of RGCs during retinogenesis. Nevertheless, induced RGCs (iRGCs) generated upon Klf4 overexpression migrate to the proper layer and project axons aligned with endogenous fascicles that reach the optic nerve head.Notably, iRGCs survive for up to 30 days after in vivo reprogramming. Finally, we demonstrate that Klf4 converts Müller cells into neurons that express markers of RGCs.Altogether, we identified Klf4 as a promising tool to reprogram retinal cells and regenerate RGCs in the mature retina. Significance StatementCell fate determination is a key process for development, regeneration and for the design of therapeutic strategies that involve cellular reprogramming. This work shows that the manipulation of a single pluripotency regulator (Klf4) is sufficient to reprogram restricted progenitor cells in vivo. These reprogrammed progenitors reacquire the potency to generate retinal ganglion cells. Ganglion cell degeneration is the leading cause of irreversible blindness; therefore, manipulation of ganglion cell competence is of relevance for human health. Our findings point to Klf4 as a promising tool to develop therapeutic strategies for the replacement of damaged ganglion cells.1 1 among the cells that underwent recombination (CRE+) when MG CTR and MG KLF4 groups were compared ( Figure 7E-H). These results raise the possibility that Klf4 may reprogram activated Müller glial cells to the RGC fate. DiscussionWe show here that although Klf4 is not essential for RGC generation during retinal development in either mouse or zebrafish retinas, it is sufficient to induce de novo genesis of RGCs in vivo outside their developmental window. Late retinal progenitors overexpressing Klf4 exit the cell cycle prematurely, reside mostly in the ganglion cell and inner plexiform layers, contain molecular signatures of RGCs, and project axons towards the initial segment of the optic nerve. Notably, cell cycle exit was accompanied by strong upregulation of Atoh7, a master regulator of the transcription network for RGC differentiation. Even though KLF4-induced RGCs (iRGCs) d...

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Temporal identity factors regulate competence of neural progenitors to generate specific cell types in a time-dependent manner, but how they operate remains poorly defined. In the developing mouse retina, the Ikaros zinc finger transcription factor Ikzf1 regulates production of early-born cell types, except cone photoreceptors. In this study we show that, during early stages of retinal development, another Ikaros family protein, Ikzf4, functions redundantly with Ikzf1 to regulate cone photoreceptor production. Using CUT&RUN and functional assays, we show that Ikzf4 binds and represses genes involved in late-born rod photoreceptor specification, hence favoring cone production. At late stages, when Ikzf1 is no longer expressed in progenitors, we show that Ikzf4 re-localizes to target genes involved in gliogenesis and is required for Müller glia production. We report that Ikzf4 regulates Notch signaling genes and is sufficient to activate the Hes1 promoter through two Ikzf GGAA binding motifs, suggesting a mechanism by which Ikzf4 may influence gliogenesis. These results uncover a combinatorial role for Ikaros family members during nervous system development and provide mechanistic insights on how they temporally regulate cell fate output.