Jarid2 promotes temporal progression of retinal progenitors via repression of Foxp1.

Transitions in competence underlie the ability of CNS progenitors to generate a diversity of neurons and glia. Retinal progenitor cells in mouse generate early-born cell types embryonically and late-born cell types largely postnatally. We find that the transition from early to late progenitor competence is regulated by Jarid2. Loss of Jarid2 results in extended production of early cell types and extended expression of early progenitor genes. Jarid2 can regulate histone modifications, and we find reduction of repressive mark H3K27me3 on a subset of early progenitor genes with loss of Jarid2, most notably Foxp1. We show that Foxp1 regulates the competence to generate early-born retinal cell types, promotes early and represses late progenitor gene expression, and is required for extending early retinal cell production after loss of Jarid2. We conclude that Jarid2 facilitates progression of retinal progenitor temporal identity by repressing Foxp1, which is a primary regulator of early temporal patterning.

Neuronal apoptosis drives remodeling states of microglia and shifts in survival pathway dependence.

Microglia serve critical remodeling roles that shape the developing nervous system, responding to the changing neural environment with phagocytosis or soluble factor secretion. Recent single-cell sequencing (scRNAseq) studies have revealed the context-dependent diversity in microglial properties and gene expression, but the cues promoting this diversity are not well defined. Here, we ask how interactions with apoptotic neurons shape microglial state, including lysosomal and lipid metabolism gene expression and dependence on Colony-stimulating factor 1 receptor (CSF1R) for survival. Using early postnatal mouse retina, a CNS region undergoing significant developmental remodeling, we performed scRNAseq on microglia from mice that are wild-type, lack neuronal apoptosis (Bax KO), or are treated with CSF1R inhibitor (PLX3397). We find that interactions with apoptotic neurons drive multiple microglial remodeling states, subsets of which are resistant to CSF1R inhibition. We find that TAM receptor Mer and complement receptor 3 are required for clearance of apoptotic neurons, but that Mer does not drive expression of remodeling genes. We show TAM receptor Axl is negligible for phagocytosis or remodeling gene expression but is consequential for microglial survival in the absence of CSF1R signaling. Thus, interactions with apoptotic neurons shift microglia toward distinct remodeling states and through Axl, alter microglial dependence on survival pathway, CSF1R.

On the Generation and Regeneration of Retinal Ganglion Cells.

Retinal development follows a conserved neurogenic program in vertebrates to orchestrate the generation of specific cell types from multipotent progenitors in sequential but overlapping waves. In this program, retinal ganglion cells (RGCs) are the first cell type generated. RGCs are the final output neurons of the retina and are essential for vision and circadian rhythm. Key molecular steps have been defined in multiple vertebrate species to regulate competence, specification, and terminal differentiation of this cell type. This involves neuronal-specific transcription factor networks, regulators of chromatin dynamics and miRNAs. In mammals, RGCs and their optic nerve axons undergo neurodegeneration and loss in glaucoma and other optic neuropathies, resulting in irreversible vision loss. The incapacity of RGCs and axons to regenerate reinforces the need for the design of efficient RGC replacement strategies. Here we describe the essential molecular pathways for the differentiation of RGCs in vertebrates, as well as experimental manipulations that extend the competence window for generation of this early cell type from late progenitors. We discuss recent advances in regeneration of retinal neurons in vivo in both mouse and zebrafish and discuss possible strategies and barriers to achieving RGC regeneration as a therapeutic approach for vision restoration in blinding diseases such as glaucoma.

Developmental Apoptosis Promotes a Disease-Related Gene Signature and Independence from CSF1R Signaling in Retinal Microglia.

Microglia have important remodeling functions in neurodevelopment, aging, and disease, with evidence for molecular diversity. However, the signaling pathways and environmental cues that drive diverse states of microglia are incompletely understood. We profiled microglia of a discrete developing CNS region, the murine retina. We found distinct transcriptional signatures for retinal microglia across development and peak postnatal density of a population that resembles aging and disease-associated microglia (DAM) and CD11c+ microglia of developing white matter. While TREM2 signaling modulates the expression of select genes, the DAM-related signature is significantly reduced in retinas lacking Bax, a proapoptotic factor required for neuronal death. Furthermore, we found postnatal retinal microglia highly expressing CD11c are resistant to loss or inhibition of colony stimulating factor 1 receptor (CSF1R), while most microglia can be eliminated in Bax knockout retina. Thus, developmental apoptosis promotes a microglia gene signature linked to CSF1R independence that shares features with microglia in developing white matter and in disease.

Complement Targets Newborn Retinal Ganglion Cells for Phagocytic Elimination by Microglia.

Microglia play important roles in shaping the developing CNS, and at early stages they have been proposed to regulate progenitor proliferation, differentiation, and neuronal survival. However, these studies reveal contradictory outcomes, highlighting the complexity of these cell-cell interactions. Here, we investigate microglia function during embryonic mouse retina development, where only microglia, progenitors, and neurons are present. In both sexes, we determine that microglia primarily interact with retinal neurons and find that depletion of microglia via conditional KO of the Csf1 receptor results in increased density of retinal ganglion cells (RGCs). Pharmacological inhibition of microglia also results in an increase in RGCs, with no effect on retinal progenitor proliferation, RGC genesis, or apoptosis. We show that microglia in the embryonic retina are enriched for phagocytic markers and observe engulfment of nonapoptotic Brn3-labeled RGCs. We investigate the molecular pathways that can mediate cell engulfment by microglia and find selective downregulation of complement pathway components with microglia inhibition, and further show that C1q protein marks a subset of RGCs in the embryonic retina. KO of complement receptor 3 (CR3; Itgam), which is only expressed by microglia, results in increased RGC density, similar to what we observed after depletion or inhibition of microglia. Thus, our data suggest that microglia regulate neuron elimination in the embryonic mouse retina by complement-mediated phagocytosis of non-apoptotic newborn RGCs.SIGNIFICANCE STATEMENT Microglia are emerging as active and important participants in regulating neuron number in development, during adult neurogenesis, and following stem cell therapies. However, their role in these contexts and the mechanisms involved are not fully defined. Using a well-characterized in vivo system, we provide evidence that microglia regulate neuronal elimination by complement-mediated engulfment of nonapoptotic neurons. This work provides a significant advancement of the field by defining in vivo molecular mechanisms for microglia-mediated cell elimination. Our data add to a growing body of evidence that microglia are essential for proper nervous system development. In addition, we elucidate microglia function in the developing retina, which may shed light on microglia involvement in the context of retinal injury and disease.

Developmental roles of microglia: A window into mechanisms of disease.

Microglia are engineers of the central nervous system (CNS) both in health and disease. In addition to the canonical immunological roles of clearing damaging entities and limiting the spread of toxicity and death, microglia remodel the CNS throughout life. While they have been extensively studied in disease and injury, due to their highly variable functions, their precise role in these contexts still remains uncertain. Over the past decade, we have greatly expanded our understanding of microglial function, including their essential homeostatic roles during development. Here, we review these developmental roles, identify parallels in disease, and speculate whether developmental mechanisms re-emerge in disease and injury. Developmental Dynamics 248:98-117, 2019. © 2018 Wiley Periodicals, Inc.

Complement C3-Targeted Gene Therapy Restricts Onset and Progression of Neurodegeneration in Chronic Mouse Glaucoma.

Dysregulation of the complement system is implicated in neurodegeneration, including human and animal glaucoma. Optic nerve and retinal damage in glaucoma is preceded by local complement upregulation and activation, but whether targeting this early innate immune response could have therapeutic benefit remains undefined. Because complement signals through three pathways that intersect at complement C3 activation, here we targeted this step to restore complement balance in the glaucomatous retina and to determine its contribution to degeneration onset and/or progression. To achieve this, we combined adeno-associated virus retinal gene therapy with the targeted C3 inhibitor CR2-Crry. We show that intravitreal injection of AAV2.CR2-Crry produced sustained Crry overexpression in the retina and reduced deposition of the activation product complement C3d on retinal ganglion cells and the inner retina of DBA/2J mice. This resulted in neuroprotection of retinal ganglion cell axons and somata despite continued intraocular pressure elevation, suggesting a direct restriction of neurodegeneration onset and progression and significant delay to terminal disease stages. Our study uncovers a damaging effect of complement C3 or downstream complement activation in glaucoma, and it establishes AAV2.CR2-Crry as a viable therapeutic strategy to target pathogenic C3-mediated complement activation in the glaucomatous retina.

From Retina to Stem Cell and Back Again: Memories of a Chromatin Journey.

The use of retinal organoids requires efficient differentiation from induced pluripotent stem cells (iPSCs). In this issue of Cell Reports, Wang et al. (2018) examine how the chromatin landscape after iPSC programming predicts their ability to differentiate into retinal tissue.

C8orf46 homolog encodes a novel protein Vexin that is required for neurogenesis in Xenopus laevis.

Neural basic helix-loop helix (bHLH) transcription factors promote progenitor cell differentiation by activation of downstream target genes that coordinate neuronal differentiation. Here we characterize a neural bHLH target gene in Xenopus laevis, vexin (vxn; previously sbt1), that is homologous to human c8orf46 and is conserved across vertebrate species. C8orf46 has been implicated in cancer progression, but its function is unknown. Vxn is transiently expressed in differentiating progenitors in the developing central nervous system (CNS), and is required for neurogenesis in the neural plate and retina. Its function is conserved, since overexpression of either Xenopus or mouse vxn expands primary neurogenesis and promotes early retinal cell differentiation in cooperation with neural bHLH factors. Vxn protein is localized to the cell membrane and the nucleus, but functions in the nucleus to promote neural differentiation. Vxn inhibits cell proliferation, and works with the cyclin-dependent kinase inhibitor p27Xic1 (cdkn1b) to enhance neurogenesis and increase levels of the proneural protein Neurog2. We propose that vxn provides a key link between neural bHLH activity and execution of the neurogenic program.

Report on the National Eye Institute Audacious Goals Initiative: Replacement of Retinal Ganglion Cells from Endogenous Cell Sources.

This report emerges from a workshop convened by the National Eye Institute (NEI) as part of the "Audacious Goals Initiative" (AGI). The workshop addressed the replacement of retinal ganglion cells (RGCs) from exogenous and endogenous sources, and sought to identify the gaps in our knowledge and barriers to progress in devising cellular replacement therapies for diseases where RGCs die. Here, we briefly review relevant literature regarding common diseases associated with RGC death, the genesis of RGCs in vivo, strategies for generating transplantable RGCs in vitro, and potential endogenous cellular sources to regenerate these cells. These topics provided the clinical and scientific context for the discussion among the workshop participants and are relevant to efforts that may lead to therapeutic approaches for replacing RGCs. This report also summarizes the content of the workshop discussion, which focused on: (1) cell sources for RGC replacement and regeneration, (2) optimizing integration, survival, and synaptogenesis of new RGCs, and (3) approaches for assessing the outcomes of RGC replacement therapies. We conclude this report with a summary of recommendations, based on the workshop discussions, which may guide vision scientists seeking to develop therapies for replacing RGCs in humans.

Loss of Fractalkine Signaling Exacerbates Axon Transport Dysfunction in a Chronic Model of Glaucoma.

Neurodegeneration in glaucoma results in decline and loss of retinal ganglion cells (RGCs), and is associated with activation of myeloid cells such as microglia and macrophages. The chemokine fractalkine (FKN or Cx3cl1) mediates communication from neurons to myeloid cells. Signaling through its receptor Cx3cr1 has been implicated in multiple neurodegenerative diseases, but the effects on neuronal pathology are variable. Since it is unknown how FKN-mediated crosstalk influences RGC degeneration in glaucoma, we assessed this in a chronic mouse model, DBA/2J. We analyzed a DBA/2J substrain deficient in Cx3cr1, and compared compartmentalized RGC degeneration and myeloid cell responses to those in standard DBA/2J mice. We found that loss of FKN signaling exacerbates axon transport dysfunction, an early event in neurodegeneration, with a significant increase in RGCs with somal accumulation of the axonal protein phosphorylated neurofilament, and reduced retinal expression of genes involved in axon transport, Kif1b, and Atp8a2. There was no change in the loss of Brn3-positive RGCs, and no difference in the extent of damage to the proximal optic nerve, suggesting that the loss of fractalkine signaling primarily affects axon transport. Since Cx3cr1 is specifically expressed in myeloid cells, we assessed changes in retinal microglial number and activation, changes in gene expression, and the extent of macrophage infiltration. We found that loss of fractalkine signaling led to innate immune changes within the retina, including increased infiltration of peripheral macrophages and upregulated nitric oxide synthase-2 (Nos-2) expression in myeloid cells, which contributes to the production of NO and can promote axon transport deficits. In contrast, resident retinal microglia appeared unchanged either in number, morphology, or expression of the myeloid activation marker ionized calcium binding adaptor molecule 1 (Iba1). There was also no significant increase in the proinflammatory gene interleukin 1 beta (Il1ß). We conclude that loss of fractalkine signaling causes a selective worsening of axon transport dysfunction in RGCs, which is linked to enhanced Nos-2 expression in myeloid cells. Our findings suggest that distinct mechanisms may contribute to different aspects of RGC decline in glaucoma, with axonal transport selectively altered after loss of Cx3cr1 in microglia and/or macrophages.

Glial coverage in the optic nerve expands in proportion to optic axon loss in chronic mouse glaucoma.

Within the white matter, axonal loss by neurodegeneration is coupled to glial cell changes in gene expression, structure and function commonly termed gliosis. Recently, we described the highly variable expansion of gliosis alebosco@neuro.utah.edu in degenerative optic nerves from the DBA/2J mouse model of chronic, age-related glaucoma. Here, to estimate and compare the levels of axonal loss with the expansion of glial coverage and axonal degeneration in DBA/2J nerves, we combined semiautomatic axon counts with threshold-based segmentation of total glial/scar areas and degenerative axonal profiles in plastic cross-sections. In nerves ranging from mild to severe degeneration, we found that the progression of axonal dropout is coupled to an increase of gliotic area. We detected a strong correlation between axon loss and the aggregate coverage by glial cells and scar, whereas axon loss did not correlate with the small fraction of degenerating profiles. Nerves with low to medium levels of axon loss displayed moderate glial reactivity, consisting of hypertrophic astrocytes, activated microglia and normal distribution of oligodendrocytes, with minimal reorganization of the tissue architecture. In contrast, nerves with extensive axonal loss showed prevalent rearrangement of the nerve, with loss of axon fascicle territories and enlarged or almost continuous gliotic and scar domains, containing reactive astrocytes, oligodendrocytes and activated microglia. These findings support the value of optic nerve gliotic expansion as a quantitative estimate of optic neuropathy that correlates with axon loss, applicable to grade the severity of optic nerve damage in mouse chronic glaucoma.

Ezh2 maintains retinal progenitor proliferation, transcriptional integrity, and the timing of late differentiation.

Epigenetic regulation, including histone modification, is a critical component of gene regulation, although precisely how this contributes to the development of complex tissues such as the neural retina is still being explored. We show that during retinal development in mouse, there are dynamic patterns of expression of the polycomb repressive complex 2 (PRC2) catalytic subunit EZH2 in retinal progenitors and some differentiated cells, as well as dynamic changes in the histone modification H3K27me3. Using conditional knockout of Ezh2 using either Pax6-αCre or Six3-Cre, we find selective reduction in postnatal retinal progenitor proliferation, disruption of retinal lamination, and enhanced differentiation of several late born cell types in the early postnatal retina, including photoreceptors and Müller glia, which are ultimately increased in number and become reactive. RNA-seq identifies many non-retinal genes upregulated with loss of Ezh2, including multiple Hox genes and the cell cycle regulator Cdkn2a, which are established targets of EZH2-mediated repression. ChIP analysis confirms loss of the H3K27me3 modification at these loci. Similar gene upregulation is observed in retinal explants treated with an EZH2 chemical inhibitor. There is considerable overlap with EZH2-regulated genes reported in non-neural tissues, suggesting that EZH2 can regulate similar genes in multiple lineages. Our findings reveal a conserved role for EZH2 in constraining the expression of potent developmental regulators to maintain lineage integrity and retinal progenitor proliferation, as well as regulating the timing of late differentiation.

In vivo dynamics of retinal microglial activation during neurodegeneration: confocal ophthalmoscopic imaging and cell morphometry in mouse glaucoma.

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with distinct functions and gene expression profiles. The roles of microglial activation in health, injury and disease remain incompletely understood due to their dynamic and complex regulation in response to changes in their microenvironment. Thus, it is critical to non-invasively monitor and analyze changes in microglial activation over time in the intact organism. In vivo studies of microglial activation have been delayed by technical limitations to tracking microglial behavior without altering the CNS environment. This has been particularly challenging during chronic neurodegeneration, where long-term changes must be tracked. The retina, a CNS organ amenable to non-invasive live imaging, offers a powerful system to visualize and characterize the dynamics of microglia activation during chronic disorders. This protocol outlines methods for long-term, in vivo imaging of retinal microglia, using confocal ophthalmoscopy (cSLO) and CX3CR1(GFP/+) reporter mice, to visualize microglia with cellular resolution. Also, we describe methods to quantify monthly changes in cell activation and density in large cell subsets (200-300 cells per retina). We confirm the use of somal area as a useful metric for live tracking of microglial activation in the retina by applying automated threshold-based morphometric analysis of in vivo images. We use these live image acquisition and analyses strategies to monitor the dynamic changes in microglial activation and microgliosis during early stages of retinal neurodegeneration in a mouse model of chronic glaucoma. This approach should be useful to investigate the contributions of microglia to neuronal and axonal decline in chronic CNS disorders that affect the retina and optic nerve.

Neurodegeneration severity can be predicted from early microglia alterations monitored in vivo in a mouse model of chronic glaucoma.

Microglia serve key homeostatic roles, and respond to neuronal perturbation and decline with a high spatiotemporal resolution. The course of all chronic CNS pathologies is thus paralleled by local microgliosis and microglia activation, which begin at early stages of the disease. However, the possibility of using live monitoring of microglia during early disease progression to predict the severity of neurodegeneration has not been explored. Because the retina allows live tracking of fluorescent microglia in their intact niche, here we investigated their early changes in relation to later optic nerve neurodegeneration. To achieve this, we used the DBA/2J mouse model of inherited glaucoma, which develops progressive retinal ganglion cell degeneration of variable severity during aging, and represents a useful model to study pathogenic mechanisms of retinal ganglion cell decline that are similar to those in human glaucoma. We imaged CX3CR1(+/GFP) microglial cells in vivo at ages ranging from 1 to 5 months by confocal scanning laser ophthalmoscopy (cSLO) and quantified cell density and morphological activation. We detected early microgliosis at the optic nerve head (ONH), where axonopathy first manifests, and could track attenuation of this microgliosis induced by minocycline. We also observed heterogeneous and dynamic patterns of early microglia activation in the retina. When the same animals were aged and analyzed for the severity of optic nerve pathology at 10 months of age, we found a strong correlation with the levels of ONH microgliosis at 3 to 4 months. Our findings indicate that live imaging and monitoring the time course and levels of early retinal microgliosis and microglia activation in glaucoma could serve as indicators of future neurodegeneration severity.

MicroRNA maintenance of cone outer segments.

The function of cone photoreceptors depends upon the formation and maintenance of outer segments, which are lost in degenerative diseases. reveal a critical role for microRNAs, specifically miR-182 and miR-183, in the maintenance of these specialized structures.

The ETS transcription factor Etv1 mediates FGF signaling to initiate proneural gene expression during Xenopus laevis retinal development.

Fibroblast growth factor signaling plays a significant role in the developing eye, regulating both patterning and neurogenesis. Members of the Pea3/Etv4-subfamily of ETS-domain transcription factors (Etv1, Etv4, and Etv5) are transcriptional activators that are downstream targets of FGF/MAPK signaling, but whether they are required for eye development is unknown. We show that in the developing Xenopus laevis retina, etv1 is transiently expressed at the onset of retinal neurogenesis. We found that etv1 is not required for eye specification, but is required for the expression of atonal-related proneural bHLH transcription factors, and is also required for retinal neuron differentiation. Using transgenic reporters we show that the distal atoh7 enhancer, which is required for the initiation of atoh7 expression in the Xenopus retina, is responsive to both FGF signaling and etv1 expression. Thus, we conclude that Etv1 acts downstream of FGF signaling to regulate the initiation of neurogenesis in the Xenopus retina.

Polycomb repressive complex PRC2 regulates Xenopus retina development downstream of Wnt/ß-catenin signaling.

The histone methyltransferase complex PRC2 controls key steps in developmental transitions and cell fate choices; however, its roles in vertebrate eye development remain unknown. Here, we report that in Xenopus, PRC2 regulates the progression of retinal progenitors from proliferation to differentiation. We show that the PRC2 core components are enriched in retinal progenitors and downregulated in differentiated cells. Knockdown of the PRC2 core component Ezh2 leads to reduced retinal progenitor proliferation, in part due to upregulation of the Cdk inhibitor p15(Ink4b). In addition, although PRC2 knockdown does not alter eye patterning, retinal progenitor gene expression or expression of the neural competence factor Sox2, it does cause suppression of proneural bHLH gene expression, indicating that PRC2 is crucial for the initiation of neural differentiation in the retina. Consistent with this, knocking down or blocking PRC2 function constrains the generation of most retinal neural cell types and promotes a Müller glial cell fate decision. We also show that Wnt/ß-catenin signaling acting through the receptor Frizzled 5, but independent of Sox2, regulates expression of key PRC2 subunits in the developing retina. This is consistent with a role for this pathway in coordinating proliferation and the transition to neurogenesis in the Xenopus retina. Our data establish PRC2 as a regulator of proliferation and differentiation during eye development.

Early reduction of microglia activation by irradiation in a model of chronic glaucoma.

Glaucoma is a neurodegenerative disease that results in the progressive decline and ultimate death of retinal ganglion cells (RGCs). While multiple risk factors are associated with glaucoma, the mechanisms leading to onset and progression of the disease remain unknown. Molecular analysis in various glaucoma models has revealed involvement of non-neuronal cell populations, including astrocytes, Mueller glia and microglia, at early stages of glaucoma. High-dose irradiation was reported to have a significant long-term protective effect in the DBA/2J (D2) mouse model of glaucoma, although the cellular and molecular basis for this effect remains unclear. In particular, the acute effects of irradiation on specific cell populations, including non-neuronal cells, in the D2 retina and nerve have not been assessed. Here we report that irradiation induces transient reduction in proliferating microglia within the optic nerve head and glial lamina within the first week post-irradiation. This was accompanied by reduced microglial activation, with no effect on astrocyte gliosis in those regions. At later stages we confirm that early high-dose irradiation of the mouse head results in improvement of axonal structural integrity and anterograde transport function, without reduction of intraocular pressure. Thus reduced microglial activation induced by irradiation at early stages is associated with reduced optic nerve and retinal neurodegeneration in the D2 mouse model of glaucoma.