SUMMARY In response to retinal damage, the Müller glial cells (MGs) of the zebrafish retina have the ability to undergo a cellular reprogramming event in which they enter the cell cycle and divide asymmetrically, thereby producing multipotent retinal progenitors capable of regenerating lost retinal neurons. However, mammalian MGs do not exhibit such a proliferative and regenerative ability. Here, we identify Hippo pathway-mediated repression of the transcription cofactor YAP as a core regulatory mechanism that normally blocks mammalian MG proliferation and cellular reprogramming. MG-specific deletion of Hippo pathway components Lats1 and Lats2 , as well as transgenic expression of a Hippo non-responsive form of YAP (YAP5SA), resulted in dramatic Cyclin D1 upregulation, loss of adult MG identity, and attainment of a highly proliferative, progenitor-like cellular state. Our results reveal that mammalian MGs may have latent regenerative capacity that can be stimulated by repressing Hippo signaling.

SUMMARY A fundamental principle governing organ size and function is the fine balance between cell proliferation and differentiation. Here, we identify Ronin (Thap11) as a key transcriptional regulator of retinal progenitor cell (RPC) proliferation. RPC-specific loss of Ronin results in a phenotype strikingly similar to the G1- to S-phase arrest and photoreceptor degeneration observed in the Cyclin D1 null mutants. However, we determined that, rather than regulating canonical cell cycle genes, Ronin regulates a cohort of mitochondrial genes including components of the electron transport chain (ETC), which have been recently implicated as direct regulators of the cell cycle. Coincident with premature cell cycle exit, Ronin mutants exhibit deficient ETC activity, reduced ATP levels and increased oxidative stress that we ascribe to specific loss of subunits within complexes I, III and IV. These data implicate Ronin as a positive regulator of mitochondrial gene expression that coordinates mitochondrial activity and cell cycle progression.

Significance: The retina is critical for vision, and several diseases may alter its biomechanical properties. However, assessing the biomechanical properties of the retina nondestructively is a challenge due to its fragile nature and location within the eye globe. Advancements in Brillouin spectroscopy have provided the means for nondestructive investigations of retina biomechanical properties. Aim: We assessed the biomechanical properties of mouse retinas using Brillouin microscopy noninvasively and showed the potential of Brillouin microscopy to differentiate the type and layers of retinas based on stiffness. Approach: We used Brillouin microscopy to quantify stiffness of fresh and paraformaldehyde (PFA)-fixed retinas. As further proof-of-concept, we demonstrated a change in the stiffness of a retina with N-methyl-D-aspartate (NMDA)-induced damage, compared to an undamaged sample. Results: We found that the retina layers with higher cell body density had higher Brillouin modulus compared to less cell-dense layers. We have also demonstrated that PFA-fixed retina samples were stiffer compared with fresh samples. Further, NMDA-induced neurotoxicity leads to retinal ganglion cell (RGC) death and reactive gliosis, increasing the stiffness of the RGC layer. Conclusion: Brillouin microscopy can be used to characterize the stiffness distribution of the layers of the retina and can be used to differentiate tissue at different conditions based on biomechanical properties.

No abstract

In the retinas of non‐mammalian vertebrates, such as zebrafish, neural regeneration is mediated by the molecular reprogramming of MGs into progenitor‐like cells. While mammalian MGs do not exhibit the regenerative potential of the zebrafish, it has been shown that MGs of mammals display limited‐ and transient‐entry into the cell cycle in response to damage. Therefore, an intrinsic proliferative and/or regenerative block likely prevents mammalian MGs from undergoing sustained cell cycle re‐entry and acquiring a multipotential retinal progenitor‐like state. Using a gain‐of‐function strategy we bypassed the Hippo signaling pathway and induced expression of a mutant version of YAP (YAP5SA) in MGs. Lineage tracing and histological analyses of these YAP5SA‐mutant retinas showed that MGs that expressed YAP5SA entered the cell cycle and formed radial clusters of proliferative cells strikingly similar to clonally expanding MG‐derived progenitors of the regenerating zebrafish retina. This finding indicated that YAP5SA is capable of spontaneously driving MGs into the cell cycle. Whether this YAP‐mediated reprogramming of MGs is sufficient to induce differentiation of MGs into fully functional neurons is undetermined. Subsequent single cell RNA sequencing analysis of the YAP5SA‐mutant retinas revealed that the proliferative event of YAP5SA‐positive cells occurred coincident with a dramatic downregulation of genes required for mitochondrial metabolic processes indicating that a metabolic shift is part of the MG reprogramming event. In a variety of cellular contexts, mitochondrial function and the electron transport chain (ETC) activity have emerged as integral regulators of the cell cycle and cell fate. The factors regulating a mitochondrial metabolic reprogramming and cell cycle are coupled but unknown. We generated a mouse line to conditionally knock out (CKO) the mitochondrial transcription factor A (Tfam) to target the ETC. In the CKO developing retinas, loss of Tfam results in loss of ETC activity. Preliminary histological analysis of CKO developing retinas show that loss of Tfam delays progenitor cell cycle exit and reduces neurogenesis resulting in a hypoplastic adult retina. Our findings so far indicate that unless retinal progenitor cells have a functional ETC‐driven oxidative activity, they remain within the cell cycle compromising the normal neural developmental program. The overall goal of this studies will be to determine how the RPC cell cycle machinery interfaces with ETC to drive cell cycle exit and neural differentiation. A better understanding of these requirements will have broad implications for the entire field of neural regeneration.