Retinal Ganglion Cell Differentiation Research Articles

Systematic identification of intergenic long-noncoding RNAs in mouse retinas using full-length isoform sequencing

BackgroundA great mass of long noncoding RNAs (lncRNAs) have been identified in mouse genome and increasing evidences in the last decades have revealed their crucial roles in diverse biological processes. Nevertheless, the biological roles of lncRNAs in the mouse retina remains largely unknown due to the lack of a comprehensive annotation of lncRNAs expressed in the retina.ResultsIn this study, we applied the long-reads sequencing strategy to unravel the transcriptomes of developing mouse retinas and identified a total of 940 intergenic lncRNAs (lincRNAs) in embryonic and neonatal retinas, including about 13% of them were transcribed from unannotated gene loci. Subsequent analysis revealed that function of lincRNAs expressed in mouse retinas were closely related to the physiological roles of this tissue, including 90 lincRNAs that were differentially expressed after the functional loss of key regulators of retinal ganglion cell (RGC) differentiation. In situ hybridization results demonstrated the enrichment of three class IV POU-homeobox genes adjacent lincRNAs (linc-3a, linc-3b and linc-3c) in ganglion cell layer and indicated they were potentially RGC-specific.ConclusionsIn summary, this study systematically annotated the lincRNAs expressed in embryonic and neonatal mouse retinas and implied their crucial regulatory roles in retinal development such as RGC differentiation.

Human retinal ganglion cell axon regeneration by recapitulating developmental mechanisms: effects of recruitment of the mTOR pathway.

The poor axon regeneration in the central nervous system (CNS) often leads to permanent functional deficit following disease or injury. For example, degeneration of retinal ganglion cell (RGC) axons in glaucoma leads to irreversible loss of vision. Here, we have tested the hypothesis that the mTOR pathway regulates the development of human RGCs and that its recruitment after injury facilitates axon regeneration. We observed that the mTOR pathway is active during RGC differentiation, and using the induced pluripotent stem cell model of neurogenesis show that it facilitates the differentiation, function and neuritogenesis of human RGCs. Using a microfluidic model, we demonstrate that recruitment of the mTOR pathway facilitates human RGC axon regeneration after axotomy, providing evidence that the recapitulation of developmental mechanism(s) might be a viable approach for facilitating axon regeneration in the diseased or injured human CNS, thus helping to reduce and/or recover loss of function.

Opposing Effects of Growth and Differentiation Factors in Cell-Fate Specification

Following ocular trauma or in diseases such as glaucoma, irreversible vision loss is due to the death of retinal ganglion cell (RGC) neurons. Although strategies to replace these lost cells include stem cell replacement therapy, few differentiated stem cells turn into RGC-like neurons. Understanding the regulatory mechanisms of RGC differentiation invivo may improve outcomes of cell transplantation by directing the fate of undifferentiated cells toward mature RGCs. Here, we report a new mechanism by which growth and differentiation factor-15 (GDF-15), a ligand in the transforming growth factor-beta (TGF-β) superfamily, strongly promotes RGC differentiation in the developing retina invivo in rodent retinal progenitor cells (RPCs) and in human embryonic stem cells (hESCs). This effect is in direct contrast to the closely related ligand GDF-11, which suppresses RGC-fate specification. We find these opposing effects are due in part to GDF-15's ability to specifically suppress Smad-2, but not Smad-1, signaling induced by GDF-11, which can be recapitulated by pharmacologic or genetic blockade of Smad-2 invivo to increase RGC specification. No other retinal cell types were affected by GDF-11 knockout, but a slight reduction in photoreceptor cells was observed by GDF-15 knockout in the developing retina invivo. These data define a novel regulatory mechanism of GDFs' opposing effects and their relevance in RGC differentiation and suggest a potential approach for advancing ESC-to-RGC cell-based replacement therapies.

A Chrnb3-Cre BAC transgenic mouse line for manipulation of gene expression in retinal ganglion cells.

The mechanisms by which retinal ganglion cells (RGCs) make specific connections during development is an intense area of research and have served as a model for understanding the general principles of circuit wiring. As such, genetic tools allowing for specific recombination in RGCs are critical to further our understanding of the cell-specific roles of different genes during these processes. However, many RGC-specific Cre lines have drawbacks, due to their broad expression in other cell types and/or retinorecipient regions or lack of expression in broad swaths of the retina. Here, we characterize a Cre BAC transgenic line driven by elements of the cholinergic receptor nicotinic beta 3 subunit (Chrnb3). We show that Cre expression is restricted to RGCs in the retina and sparsely expressed in the brain, importantly excluding retinorecipient regions. Furthermore, Chrnb3-Cre mice label a wide variety of RGCs distributed throughout the retina and Cre activity is detected embryonically, shortly following RGC differentiation. Finally, we find that Chrnb3-Cre-labeled RGCs innervate multiple retinorecipient areas that serve both image-forming and nonimage forming functions. Thus, this genetic tool will be of broad use to investigators studying the RGC-specific contributions of genes to visual circuit development.

Imaging and Differentiation of Retinal Ganglion Cells in Ex Vivo Experimental Optic Nerve Degeneration by Differential Interference Contrast Microscopy

ABSTRACTPurpose: Apoptotic loss of retinal ganglion cells (RGCs) is involved in various optic neuropathies, and its extent is closely related to visual impairment. Direct imaging and counting of RGCs is beneficial to the evaluation of RGC loss, but these processes are challenging with the conventional techniques, due to the transparency and hypo-reflectivity of RGCs as light-transmitting structures of the retina. Differential interference contrast (DIC) microscopy, which can provide real-time images of transparent specimens, is utilized to image neuronal cells including RGCs in the ganglion cell layer (GCL).Methods: Herein, we show that the neuronal cells within each GCL in an explanted rat retina, including the inner nuclear layer and the outer nuclear layer, can be imaged selectively by transmission-type DIC microscopy. RGCs were also differentiated from non-RGCs by the objective method.Results: RGCs were differentiated from non-RGCs in the GCL by their morphological features on DIC images with the aid of retrograde fluorescence labeling. Loss of RGCs was detected in optic-nerve-transection and retinal-ischemia-reperfusion models by DIC imaging. The images obtained from the reflection-type DIC microscopy were comparable to those from the transmission-type DIC microscopy.Conclusions: This method enables direct optical visualization of RGCs in experimental optic-nerve degeneration, thus providing the opportunity for more accurate evaluation of optic neuropathies as well as more effective investigation of diseases.

Loss of foxc1 in zebrafish reduces optic nerve size and cell number in the retinal ganglion cell layer

Mutation of FOXC1 causes Axenfeld-Rieger Syndrome (ARS) with early onset or congenital glaucoma. We assessed retinal ganglion cell (RGC) number in zebrafish due to CRISPR-mediated mutation and antisense inhibition of two-forkhead box transcription factors, foxc1a and foxc1b. These genes represent duplicated homologues of human FOXC1. Using a CRISPR induced null mutation in foxc1b, in combination with antisense inhibition of foxc1a, we demonstrate reduced cell number in the retinal ganglion cell layer of developing zebrafish eyes. As early as 5 days post fertilization (dpf), fewer RGCs are found in foxc1b homozygous mutants injected with foxc1a morpholinos, and a thinner optic nerve results. Our data illustrates that foxc1 is required for the expression of atonal homolog 7 (atoh7), a gene that is necessary for RGC differentiation. As markers of differentiated RGCs (pou4f2) are downregulated in foxc1b−/− mutants injected with foxc1a morpholinos and no cell death is observed, our results are consistent with defects in the differentiation of RGCs leading to reduced cell number, as opposed to increased cell death of RGCs or off targets effects of morpholino injection. Our zebrafish model demonstrates that aberrant regulation of RGC number could act in concert with other known glaucoma risk factors to influence the development of congenital and early onset glaucoma due to FOXC1 mutation.

Differentiation of retinal ganglion cells from induced pluripotent stem cells: a review.

Glaucoma is a common optic neuropathy that is characterized by the progressive degeneration of axons and the loss of retinal ganglion cells (RGCs). Glaucoma is one of the leading causes of irreversible blindness worldwide. Current glaucoma treatments only slow the progression of RGCs loss. Induced pluripotent stem cells (iPSCs) are capable of differentiating into all three germ layer cell lineages. iPSCs can be patient-specific, making iPSC-derived RGCs a promising candidate for cell replacement. In this review, we focus on discussing the detailed approaches used to differentiate iPSCs into RGCs.

Polybenzyl Glutamate Biocompatible Scaffold Promotes the Efficiency of Retinal Differentiation toward Retinal Ganglion Cell Lineage from Human-Induced Pluripotent Stem Cells.

Optic neuropathy is one of the leading causes of irreversible blindness caused by retinal ganglion cell (RGC) degeneration. The development of induced pluripotent stem cell (iPSC)-based therapy opens a therapeutic window for RGC degeneration, and tissue engineering may further promote the efficiency of differentiation process of iPSCs. The present study was designed to evaluate the effects of a novel biomimetic polybenzyl glutamate (PBG) scaffold on culturing iPSC-derived RGC progenitors. The iPSC-derived neural spheres cultured on PBG scaffold increased the differentiated retinal neurons and promoted the neurite outgrowth in the RGC progenitor layer. Additionally, iPSCs cultured on PBG scaffold formed the organoid-like structures compared to that of iPSCs cultured on cover glass within the same culture period. With RNA-seq, we found that cells of the PBG group were differentiated toward retinal lineage and may be related to the glutamate signaling pathway. Further ontological analysis and the gene network analysis showed that the differentially expressed genes between cells of the PBG group and the control group were mainly associated with neuronal differentiation, neuronal maturation, and more specifically, retinal differentiation and maturation. The novel electrospinning PBG scaffold is beneficial for culturing iPSC-derived RGC progenitors as well as retinal organoids. Cells cultured on PBG scaffold differentiate effectively and shorten the process of RGC differentiation compared to that of cells cultured on coverslip. The new culture system may be helpful in future disease modeling, pharmacological screening, autologous transplantation, as well as narrowing the gap to clinical application.

Advances in the Differentiation of Retinal Ganglion Cells from Human Pluripotent Stem Cells.

Human pluripotent stem cell (hPSC) technology has revolutionized the field of biology through the unprecedented ability to study the differentiation of human cells in vitro. In the past decade, hPSCs have been applied to study development, model disease, develop drugs, and devise cell replacement therapies for numerous biological systems. Of particular interest is the application of this technology to study and treat optic neuropathies such as glaucoma. Retinal ganglion cells (RGCs) are the primary cell type affected in these diseases, and once lost, they are unable to regenerate in adulthood. This necessitates the development of strategies to study the mechanisms of degeneration as well as develop translational therapeutic approaches to treat early- and late-stage disease progression. Numerous protocols have been established to derive RGCs from hPSCs, with the ability to generate large populations of human RGCs for translational applications. In this review, the key applications of hPSCs within the retinal field are described, including the use of these cells as developmental models, disease models, drug development, and finally, cell replacement therapies. In greater detail, the current report focuses on the differentiation of hPSC-derived RGCs and the many unique characteristics associated with these cells in vitro including their genetic identifiers, their electrophysiological activity, and their morphological maturation. Also described is the current progress in the use of patient-specific hPSCs to study optic neuropathies affecting RGCs, with emphasis on the use of these RGCs for studying disease mechanisms and pathogenesis, drug screening, and cell replacement therapies in future studies.

Three-Dimensional Retinal Organoids Facilitate the Investigation of Retinal Ganglion Cell Development, Organization and Neurite Outgrowth from Human Pluripotent Stem Cells

Retinal organoids are three-dimensional structures derived from human pluripotent stem cells (hPSCs) which recapitulate the spatial and temporal differentiation of the retina, serving as effective in vitro models of retinal development. However, a lack of emphasis has been placed upon the development and organization of retinal ganglion cells (RGCs) within retinal organoids. Thus, initial efforts were made to characterize RGC differentiation throughout early stages of organoid development, with a clearly defined RGC layer developing in a temporally-appropriate manner expressing a complement of RGC-associated markers. Beyond studies of RGC development, retinal organoids may also prove useful for cellular replacement in which extensive axonal outgrowth is necessary to reach post-synaptic targets. Organoid-derived RGCs could help to elucidate factors promoting axonal outgrowth, thereby identifying approaches to circumvent a formidable obstacle to RGC replacement. As such, additional efforts demonstrated significant enhancement of neurite outgrowth through modulation of both substrate composition and growth factor signaling. Additionally, organoid-derived RGCs exhibited diverse phenotypes, extending elaborate growth cones and expressing numerous guidance receptors. Collectively, these results establish retinal organoids as a valuable tool for studies of RGC development, and demonstrate the utility of organoid-derived RGCs as an effective platform to study factors influencing neurite outgrowth from organoid-derived RGCs.

Defined Conditions for Differentiation of Functional Retinal Ganglion Cells From Human Pluripotent Stem Cells.

We aimed to establish an efficient method for retinal ganglion cell (RGC) differentiation from human pluripotent stem cells (hPSCs) using defined factors. To define the contribution of specific signal pathways to RGC development and optimize the differentiation of hPSCs toward RGCs, we examined RGC differentiation in three stages: (1) eye field progenitors expressing the eye field transcription factors (EFTFs), (2) RGC progenitors expressing MATH5, and (3) RGCs expressing BRN3B and ISLET1. By monitoring the condition that elicited the highest yield of cells expressing stage-specific markers, we determined the optimal concentrations and combinations of signaling pathways required for efficient generation of RGCs from hPSCs. Precise modulation of signaling pathways, including Wnt, insulin growth factor-1, and fibroblast growth factor, in combination with mechanical isolation of neural rosette cell clusters significantly enriched RX and PAX6 double-positive eye field progenitors from hPSCs by day 12. Furthermore, Notch signal inhibition facilitated differentiation into MATH5-positive progenitors at 90% efficiency by day 20, and these cells further differentiated to BRN3B and ISLET1 double-positive RGCs at 45% efficiency by day 40. RGCs differentiated via this method were functional as exemplified by their ability to generate action potentials, express microfilament components on neuronal processes, and exhibit axonal transportation of mitochondria. This protocol offers highly defined culture conditions for RGC differentiation from hPSCs and in vitro disease model and cell source for transplantation for diseases related to RGCs.

Integral bHLH factor regulation of cell cycle exit and RGC differentiation.

In the developing mouse embryo, the bHLH transcription factor Neurog2 is transiently expressed by retinal progenitor cells and required for the initial wave of neurogenesis. Remarkably, another bHLH factor, Ascl1, normally not present in the embryonic Neurog2 retinal lineage, can rescue the temporal phenotypes of Neurog2 mutants. Here we show that Neurog2 simultaneously promotes terminal cell cycle exit and retinal ganglion cell differentiation, using mitotic window labeling and integrating these results with retinal marker quantifications. We also analyzed the transcriptomes of E12.5 GFP-expressing cells from Neurog2GFP/+ , Neurog2GFP/GFP , and Neurog2Ascl1KI/GFP eyes, and validated the most significantly affected genes using qPCR assays. Our data support the hypothesis that Neurog2 acts at the top of a retinal bHLH transcription factor hierarchy. The combined expression levels of these downstream factors are sufficiently induced by ectopic Ascl1 to restore RGC genesis, highlighting the robustness of this gene network during retinal ganglion cell neurogenesis. Developmental Dynamics 247:965-975, 2018. © 2018 Wiley Periodicals, Inc.

Laminin \u03b33 plays an important role in retinal lamination, photoreceptor organisation and ganglion cell differentiation

Laminins are heterotrimeric glycoproteins of the extracellular matrix. Eleven different laminin chains have been identified in vertebrates. They are ubiquitously expressed in the human body, with a distinct tissue distribution. Laminin expression in neural retina and their functional role during human retinogenesis is still unknown. This study investigated the laminin expression in human developing and adult retina, showing laminin α1, α5, β1, β2 and γ1 to be predominantly expressed in Bruch’s membrane and the inner limiting membrane. Laminin-332 and laminin γ3 expression were mainly observed in the neural retina during retinal histogenesis. These expression patterns were largely conserved in pluripotent stem cell-derived retinal organoids. Blocking of laminin γ3 function in retinal organoids resulted in the disruption of laminar organisation and synapse formation, the loss of photoreceptor organisation and retinal ganglion cells. Our data demonstrate a unique temporal and spatial expression for laminins and reveal a novel role for laminin γ3 during human retinogenesis.

Expression profiling of cell-intrinsic regulators in the process of differentiation of human iPSCs into retinal lineages

BackgroundDifferentiation of human induced pluripotent stem cells (hiPSCs) into retinal lineages offers great potential for medical application. Therefore, it is of crucial importance to know the key intrinsic regulators of differentiation and the specific biomarker signatures of cell lineages.MethodsIn this study, we used microarrays to analyze transcriptomes of terminally differentiated retinal ganglion cell (RGC) and retinal pigment epithelium (RPE) lineages, as well as intermediate retinal progenitor cells of optic vesicles (OVs) derived from hiPSCs. In our analysis, we specifically focused on the classes of transcripts that encode intrinsic regulators of gene expression: the transcription factors (TFs) and epigenetic chromatin state regulators. We applied two criteria for the selection of potentially important regulators and markers: firstly, the magnitude of fold-change of upregulation; secondly, the contrasted pattern of differential expression between OV, RGC and RPE lineages.ResultsWe found that among the most highly overexpressed TF-encoding genes in the OV/RGC lineage were three members of the Collier/Olfactory-1/Early B-cell family: EBF1, EBF2 and EBF3. Knockdown of EBF1 led to significant impairment of differentiation of hiPSCs into RGCs. EBF1 was shown to act upstream of ISL1 and BRN3A, the well-characterized regulators of RGC lineage specification. TF-encoding genes DLX1, DLX2 and INSM1 were the most highly overexpressed genes in the OVs, indicating their important role in the early stages of retinal differentiation. Along with MITF, the two paralogs, BHLHE41 and BHLHE40, were the most robust TF markers of RPE cells. The markedly contrasted expression of ACTL6B, encoding the component of chromatin remodeling complex SWI/SNF, discriminated hiPSC-derived OV/RGC and RPE lineages.ConclusionsWe identified novel, potentially important intrinsic regulators of RGC and RPE cell lineage specification in the process of differentiation from hiPSCs. We demonstrated the crucial role played by EBF1 in differentiation of RGCs. We identified intrinsic regulator biomarker signatures of these two retinal cell types that can be applied with high confidence to confirm the cell lineage identities.

Elevated expression of human bHLH factor ATOH7 accelerates cell cycle progression of progenitors and enhances production of avian retinal ganglion cells

The production of vertebrate retinal projection neurons, retinal ganglion cells (RGCs), is regulated by cell-intrinsic determinants and cell-to-cell signaling events. The basic-helix-loop-helix (bHLH) protein Atoh7 is a key neurogenic transcription factor required for RGC development. Here, we investigate whether manipulating human ATOH7 expression among uncommitted progenitors can promote RGC fate specification and thus be used as a strategy to enhance RGC genesis. Using the chicken retina as a model, we show that cell autonomous expression of ATOH7 is sufficient to induce precocious RGC formation and expansion of the neurogenic territory. ATOH7 overexpression among neurogenic progenitors significantly enhances RGC production at the expense of reducing the progenitor pool. Furthermore, forced expression of ATOH7 leads to a minor increase of cone photoreceptors. We provide evidence that elevating ATOH7 levels accelerates cell cycle progression from S to M phase and promotes cell cycle exit. We also show that ATOH7-induced ectopic RGCs often exhibit aberrant axonal projection patterns and are correlated with increased cell death during the period of retinotectal connections. These results demonstrate the high potency of human ATOH7 in promoting early retinogenesis and specifying the RGC differentiation program, thus providing insight for manipulating RGC production from stem cell-derived retinal organoids.

Mutation in the Zebrafish cct2 Gene Leads to Abnormalities of Cell Cycle and Cell Death in the Retina: A Model of CCT2-Related Leber Congenital Amaurosis.

PurposeThe compound heterozygous mutations in the β subunit of chaperonin containing TCP-1 (CCT), encoded by CCT2, lead to the Leber congenital amaurosis (LCA). In this study, a cct2 mutant line of zebrafish was established to investigate the role of CCT2 mutations in LCA in vertebrates.MethodsA cct2 mutant zebrafish line was produced using the CRISPR-Cas9 system. Changes in the eyes of developing wild-type and mutant larvae were monitored using microscopy, immunostaining, TUNEL, and EdU assays. Phenotypic rescue of mutant phenotype was investigated by injection of CCT2 RNA into zebrafish embryos.ResultsThe cct2 mutation (L394H-7del) led to the synthesis of a mutated cctβ protein with the L394H replacement and deletion of 7 amino acid residues (positions 395–401). The homozygous cct2-L394H-7del mutant exhibited a small eye phenotype at 2 days post fertilization (dpf) and was embryonically lethal after 5 dpf. In homozygous cct2-L394H-7del mutants, the retinal ganglion cell differentiation was attenuated, retinal cell cycle was affected, and the neural retinal cell death was significantly increased at 2 dpf compared with wild-type. Injection of RNA encoding wild-type human CCTβ rescued the small eye phenotype, reduced retinal cell death, and restored the levels of CCTβ protein and the major client protein Gβ1 that were significantly reduced in the homozygous cct2-L394H-7del mutant compared with wild-type. These results indicate that cct2 plays an essential role in retinal development by regulating the cell cycle.ConclusionsThe retinal pathology observed in the homozygous cct2-L394H-7del mutants resembles the retinal pathology of human LCA patients.

The dynamics of native Atoh7 protein expression during mouse retinal histogenesis, revealed with a new antibody

The Atoh7 transcription factor catalyzes the rate-limiting step in the specification of retinal ganglion cells (RGCs). As a tool to study vertebrate retinal development, we validate an antibody that recognizes human and mouse Atoh7 polypeptide, using informative knockout and transgenic mouse tissues and overexpression experiments. The transient features of Atoh7 protein expression during retinal neurogenesis match the expected pattern at the tissue and cellular level. Further, we compare endogenous Atoh7 to established RGC markers, reporter mouse lines and cell cycle markers, demonstrating the utility of the antibody to investigate molecular mechanisms of retinal histogenesis.

Tet-mediated DNA hydroxymethylation regulates retinal neurogenesis by modulating cell-extrinsic signaling pathways.

DNA hydroxymethylation has recently been shown to play critical roles in regulating gene expression and terminal differentiation events in a variety of developmental contexts. However, little is known about its function during eye development. Methylcytosine dioxygenases of the Tet family convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), an epigenetic mark thought to serve as a precursor for DNA demethylation and as a stable mark in neurons. Here, we report a requirement for Tet activity during zebrafish retinal neurogenesis. In tet2-/-;tet3-/- mutants, retinal neurons are specified but most fail to terminally differentiate. While differentiation of the first born retinal neurons, the retinal ganglion cells (RGCs), is less affected in tet2-/-;tet3-/- mutants than other retinal cell types, the majority of RGCs do not undergo terminal morphogenesis and form axons. Moreover, the few photoreceptors that differentiate in tet2-/-;tet3-/- mutants fail to form outer segments, suggesting that Tet function is also required for terminal morphogenesis of differentiated retinal neurons. Mosaic analyses revealed a surprising cell non-autonomous requirement for tet2 and tet3 activity in facilitating retinal neurogenesis. Through a combination of candidate gene analysis, transcriptomics and pharmacological manipulations, we identified the Notch and Wnt pathways as cell-extrinsic pathways regulated by tet2 and tet3 activity during RGC differentiation and morphogenesis. Transcriptome analyses also revealed the ectopic expression of non-retinal genes in tet2-/-;tet3-/- mutant retinae, and this correlated with locus-specific reduction in 5hmC. These data provide the first evidence that Tet-dependent regulation of 5hmC formation is critical for retinal neurogenesis, and highlight an additional layer of complexity in the progression from retinal progenitor cell to differentiated retinal neuron during development of the vertebrate retina.

Modeling Glaucoma: Retinal Ganglion Cells Generated from Induced Pluripotent Stem Cells of Patients with SIX6 Risk Allele Show Developmental Abnormalities.

Glaucoma represents a group of multifactorial diseases with a unifying pathology of progressive retinal ganglion cell (RGC) degeneration, causing irreversible vision loss. To test the hypothesis that RGCs are intrinsically vulnerable in glaucoma, we have developed an in vitro model using the SIX6 risk allele carrying glaucoma patient-specific induced pluripotent stem cells (iPSCs) for generating functional RGCs. Here, we demonstrate that the efficiency of RGC generation by SIX6 risk allele iPSCs is significantly lower than iPSCs-derived from healthy, age- and sex-matched controls. The decrease in the number of RGC generation is accompanied by repressed developmental expression of RGC regulatory genes. The SIX6 risk allele RGCs display short and simple neurites, reduced expression of guidance molecules, and immature electrophysiological signature. In addition, these cells have higher expression of glaucoma-associated genes, CDKN2A and CDKN2B, suggesting an early onset of the disease phenotype. Consistent with the developmental abnormalities, the SIX6 risk allele RGCs display global dysregulation of genes which map on developmentally relevant biological processes for RGC differentiation and signaling pathways such as mammalian target of rapamycin that integrate diverse functions for differentiation, metabolism, and survival. The results suggest that SIX6 influences different stages of RGC differentiation and their survival; therefore, alteration in SIX6 function due to the risk allele may lead to cellular and molecular abnormalities. These abnormalities, if carried into adulthood, may make RGCs vulnerable in glaucoma. Stem Cells 2017;35:2239-2252.

Programmed mitophagy is essential for the glycolytic switch during cell differentiation.

Retinal ganglion cells (RGCs) are the sole projecting neurons of the retina and their axons form the optic nerve. Here, we show that embryogenesis-associated mouse RGC differentiation depends on mitophagy, the programmed autophagic clearance of mitochondria. The elimination of mitochondria during RGC differentiation was coupled to a metabolic shift with increased lactate production and elevated expression of glycolytic enzymes at the mRNA level. Pharmacological and genetic inhibition of either mitophagy or glycolysis consistently inhibited RGC differentiation. Local hypoxia triggered expression of the mitophagy regulator BCL2/adenovirus E1B 19-kDa-interacting protein 3-like (BNIP3L, best known as NIX) at peak RGC differentiation. Retinas from NIX-deficient mice displayed increased mitochondrial mass, reduced expression of glycolytic enzymes and decreased neuronal differentiation. Similarly, we provide evidence that NIX-dependent mitophagy contributes to mitochondrial elimination during macrophage polarization towards the proinflammatory and more glycolytic M1 phenotype, but not to M2 macrophage differentiation, which primarily relies on oxidative phosphorylation. In summary, developmentally controlled mitophagy promotes a metabolic switch towards glycolysis, which in turn contributes to cellular differentiation in several distinct developmental contexts.