De novogenesis of retinal ganglion cells by targeted expression of KLF4in vivo

Abstract

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...

Progenitor death drives retinal dysplasia and neuronal degeneration in a mouse model of ATRIP-Seckel syndrome

“…Measurements of visual acuity by optomotor response were performed using OptoMotry as previously described ( Cavalheiro et al, 2017 ; Rocha-Martins et al, 2019 ). Experimenters were blind in relation to mice genotypes, and the visual accuracy threshold was determined by systematic increments of the spatial frequency until the animal no longer responded.…”

RINT1 Loss Impairs Retinogenesis Through TRP53-Mediated Apoptosis

“…Measurements of visual acuity by optomotor response were performed using OptoMotry as previously described ( Cavalheiro et al, 2017 ; Rocha-Martins et al, 2019 ). Visual accuracy threshold was determined by systematic increments of the spatial frequency until the animal no longer responded.…”

On the Generation and Regeneration of Retinal Ganglion Cells

“…This may be due to redundancy with other Klf family members ( Jiang et al, 2008 ), since many are expressed in the developing retina ( Moore et al, 2009 ; Njaine et al, 2014 ). We recently showed that overexpression of Klf4 in late retinal progenitors generates induced RGCs outside of their developmental window ( Figure 1 ; Rocha-Martins et al, 2019 ). This study showed that Klf4 induced the reactivation of the early neurogenic program in late progenitors, changing their competence to generate RGCs that properly localized to the inner retina and projected axons into the optic nerve head ( Rocha-Martins et al, 2019 ).…”

“…The potential roles of other elements of this network, like fly Krüppel and Pdm , remain unknown. Recently, Klf4 , a member of the family of Krüppel -like factors was studied in the mouse retina, but no critical function in cell fate determination was described ( Moore et al, 2009 ; Fang et al, 2016 ; Rocha-Martins et al, 2019 ). This may be due to redundancy with other Klf family members ( Jiang et al, 2008 ), since many are expressed in the developing retina ( Moore et al, 2009 ; Njaine et al, 2014 ).…”

“…The critical factors are included above each developmental step at the top of the figure. Recently we showed that a RGC program may be reactivated in late RPCs upon Klf4 overexpression ( Rocha-Martins et al, 2019 ) to generate induced RGCs (green). Current RGC regenerative approaches apply strategies to induce or reactivate the embryonic molecular program on exogenous (induced pluripotent or embryonic stem cells) or endogenous (Müller glia) sources (left).…”