ABSTRACT
Ongoing cell therapy trials have demonstrated the need for precision control of donor cell behavior within the recipient tissue. We present a methodology to guide stem cell-derived and endogenously regenerated neurons by engineering the microenvironment. Being an "approachable part of the brain," the eye provides a unique opportunity to study neuron fate and function within the central nervous system. Here, we focused on retinal ganglion cells (RGCs)-the neurons in the retina are irreversibly lost in glaucoma and other optic neuropathies but can potentially be replaced through transplantation or reprogramming. One of the significant barriers to successful RGC integration into the existing mature retinal circuitry is cell migration toward their natural position in the retina. Our in silico analysis of the single-cell transcriptome of the developing human retina identified six receptor-ligand candidates, which were tested in functional in vitro assays for their ability to guide human stem cell-derived RGCs. We used our lead molecule, SDF1, to engineer an artificial gradient in the retina, which led to a 2.7-fold increase in donor RGC migration into the ganglion cell layer (GCL) and a 3.3-fold increase in the displacement of newborn RGCs out of the inner nuclear layer. Only donor RGCs that migrated into the GCL were found to express mature RGC markers, indicating the importance of proper structure integration. Together, these results describe an "in silico-in vitro-in vivo" framework for identifying, selecting, and applying soluble ligands to control donor cell function after transplantation.
Subject(s)
Retina , Retinal Ganglion Cells , Infant, Newborn , Humans , Stem Cells , Neurogenesis , Cell MovementABSTRACT
Many retinal diseases lead to the loss of retinal neurons and cause visual impairment. The adult mammalian retina has little capacity for regeneration. By contrast, teleost fish functionally regenerate their retina following injury, and Müller glia (MG) are the source of regenerated neurons. The proneural transcription factor Ascl1 is upregulated in MG after retinal damage in zebrafish and is necessary for regeneration. Although Ascl1 is not expressed in mammalian MG after injury, forced expression of Ascl1 in mouse MG induces a neurogenic state in vitro and in vivo after NMDA (N-methyl-d-aspartate) damage in young mice. However, by postnatal day 16, mouse MG lose neurogenic capacity, despite Ascl1 overexpression. Loss of neurogenic capacity in mature MG is accompanied by reduced chromatin accessibility, suggesting that epigenetic factors limit regeneration. Here we show that MG-specific overexpression of Ascl1, together with a histone deacetylase inhibitor, enables adult mice to generate neurons from MG after retinal injury. The MG-derived neurons express markers of inner retinal neurons, synapse with host retinal neurons, and respond to light. Using an assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), we show that the histone deacetylase inhibitor promotes accessibility at key gene loci in the MG, and allows more effective reprogramming. Our results thus provide a new approach for the treatment of blinding retinal diseases.
Subject(s)
Nerve Regeneration , Neurogenesis , Neuroglia/cytology , Neurons/cytology , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Epistasis, Genetic/drug effects , Female , Histone Deacetylase Inhibitors/pharmacology , Hydroxamic Acids/pharmacology , Male , Mice , Nerve Regeneration/drug effects , Neural Pathways/drug effects , Neurogenesis/drug effects , Neuroglia/metabolism , Neurons/drug effects , Neurons/metabolism , Retina/cytology , Retina/metabolism , Synapses/drug effects , Synapses/metabolismABSTRACT
Damage to neuronal tissues in mammals leads to permanent loss of tissue function that can have major health consequences. While mammals have no inherent regenerative capacity to functionally repair neuronal tissue, other species such as amphibians and teleost fish readily replace damaged tissue. The exploration of development and native regeneration can thus inform the process of inducing regeneration in non-regenerative systems, which can be used to develop new therapeutics. Increasing evidence points to an epigenetic component in the regulation of the changes in cellular gene expression necessary for regeneration. In this review, we compare evidence of epigenetic roles in development and regeneration of neuronal tissue. We have focused on three key systems of important clinical significance: the neural retina, the inner ear, and the spinal cord in regenerative and non-regenerative species. While evidence for epigenetic regulation of regeneration is still limited, changes in DNA accessibility, histone acetylation and DNA methylation have all emerged as key elements in this process. To date, most studies have used broadly acting experimental manipulations to establish a role for epigenetics in regeneration, but the advent of more targeted approaches to modify the epigenome will be critical to dissecting the relative contributions of these regulatory factors in this process and the development of methods to stimulate the regeneration in those organisms like ourselves where only limited regeneration occurs in these neural systems.
Subject(s)
Epigenesis, Genetic/genetics , Nerve Regeneration/genetics , Animals , HumansABSTRACT
The retina is a complex neuronal structure that converts light energy into visual perception. Many specialized aspects of the primate retina, including a cone rich macula for high acuity vision, ocular size, and cell type diversity are not found in other animal models. In addition, the unique morphologies and distinct laminar positions of cell types found in the retina make this model system ideal for the study of neuronal cell fate specification. Many key early events of human retinal development are inaccessible to investigation as they occur during gestation. For these reasons, it has been necessary to develop retinal model systems to gain insight into human-specific retinal development and disease. Recent advances in culturing retinal tissue have generated new systems for retinal research and have moved us closer to generating effective regenerative therapies for vision loss. Here, we describe the strengths, weaknesses, and future directions for different human retinal model systems including dissociated primary tissue, explanted primary tissue, retinospheres, and stem cell-derived retinal organoids.
Subject(s)
Cell Culture Techniques/trends , Retina/metabolism , Retina/physiology , Cell Culture Techniques/methods , Cell Differentiation/physiology , Humans , Induced Pluripotent Stem Cells , Models, Biological , Organoids/metabolism , Primary Cell Culture/methods , Retinal Cone Photoreceptor Cells/metabolism , Retinal Degeneration/metabolismABSTRACT
Müller glia (MG) in mammalian retinas are incapable of regenerating neurons after damage, whereas the MG in lower vertebrates regenerate functional neurons. Identification of cell signaling pathways and gene regulatory networks that regulate MG-mediated regeneration is key to harnessing the regenerative potential of MG. Here, we study how NFkB-signaling influences glial responses to damage and reprogramming of MG into neurons in the rodent retina. We find activation of NFkB and dynamic expression of NFkB-associated genes in MG after damage, however damage-induced NFkB activation is inhibited by microglia ablation. Knockout of NFkB in MG suppressed the accumulation of immune cells after damage. Inhibition of NFkB following NMDA-damage significantly enhanced the reprogramming of Ascl1-overexpressing MG into neuron-like cells. scRNA-seq of retinal glia following inhibition of NFkB reveals coordination with signaling via TGFß2 and suppression of NFI and Id transcription factors. Inhibition of Smad3 signal transducer or Id transcription factors increased numbers of neuron-like cells produced by Ascl1-overexpressing MG. We conclude that NFkB is a key signaling hub that is activated in MG after damage, mediates the accumulation of immune cells, and suppresses the neurogenic potential of MG.
Subject(s)
Ependymoglial Cells , Neuroglia , Animals , Cell Proliferation/physiology , Ependymoglial Cells/metabolism , Mammals/metabolism , NF-kappa B/metabolism , Neuroglia/metabolism , Neurons/metabolism , Regeneration , Retina , Signal Transduction , Transcription Factors/metabolismABSTRACT
Ischemic preconditioning (IPC) is a phenomenon whereby a brief, non-injurious ischemic exposure enhances tolerance to a subsequent ischemic challenge. The mechanism of IPC has mainly been studied in rodent stroke models where gray matter (GM) constitutes about 85% of the cerebrum. In humans, white matter (WM) is 50% of cerebral volume and is a critical component of stroke damage. We developed a novel CNS WM IPC model using the mouse optic nerve (MON) and identified the involved immune signaling pathways. Here we tested the hypothesis that microglia are necessary for WM IPC. Microglia were depleted by treatment with the colony stimulating factor 1 receptor (CSF1R) inhibitor PLX5622. MONs were exposed to transient ischemia in vivo, acutely isolated 72 h later, and subjected to oxygen-glucose deprivation (OGD) to simulate a severe ischemic injury (i.e., stroke). Functional and structural axonal recovery was assessed by recording compound action potentials (CAPs) and by microscopy using quantitative stereology. Microglia depletion eliminated IPC-mediated protection. In control mice, CAP recovery was improved in preconditioned MONs compared with non-preconditioned MONs, however, in PLX5622-treated mice, we observed no difference in CAP recovery between preconditioned and non-preconditioned MONs. Microgliadepletion also abolished IPC protective effects on axonal integrity and survival of mature (APC+ ) oligodendrocytes after OGD. IPC-mediated protection was independent of retinal injury suggesting it results from mechanistic processes intrinsic to ischemia-exposed WM. We conclude that preconditioned microglia are critical for IPC in WM. The "preconditioned microglia" phenotype might protect against other CNS pathologies and is a neurotherapeutic horizon worth exploring.
Subject(s)
Ischemic Preconditioning , Stroke , White Matter , Animals , Cerebral Cortex/metabolism , Ischemic Preconditioning/methods , Mice , Microglia/metabolism , Stroke/metabolism , White Matter/metabolismABSTRACT
Müller glial cells (MG) generate retinal progenitor (RPC)-like cells after injury in non-mammalian species, although this does not occur in the mammalian retina. Studies have profiled gene expression in these cells to define genes that may be relevant to their differences in neurogenic potential. However, less is known about differences in micro-RNA (miRNA) expression. In this study, we compared miRNAs from RPCs and MG to identify miRNAs more highly expressed in RPCs, and others more highly expressed in MG. To determine whether these miRNAs are relevant to the difference in neurogenic potential between these two cell types, we tested them in dissociated cultures of MG using either mimics or antagomiRs to increase or reduce expression, respectively. Among the miRNAs tested, miR-25 and miR-124 overexpression, or let-7 antagonism, induced Ascl1 expression and conversion of â¼40% of mature MG into a neuronal/RPC phenotype. Our results suggest that the differences in miRNA expression between MG and RPCs contribute to their difference in neurogenic potential, and that manipulations in miRNAs provide a new tool with which to reprogram MG for retinal regeneration.
Subject(s)
Ependymoglial Cells/metabolism , MicroRNAs/metabolism , Neurogenesis/genetics , Animals , Antagomirs/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Differentiation/genetics , Cells, Cultured , Female , Male , Mice , Mice, Transgenic , MicroRNAs/genetics , Nerve Regeneration/physiology , Retina/cytology , Retina/metabolism , Retinal Neurons/metabolism , TransfectionABSTRACT
The basic body plan and major physiological axes have been highly conserved during mammalian evolution, yet only a small fraction of the human genome sequence appears to be subject to evolutionary constraint. To quantify cis- versus trans-acting contributions to mammalian regulatory evolution, we performed genomic DNase I footprinting of the mouse genome across 25 cell and tissue types, collectively defining â¼8.6 million transcription factor (TF) occupancy sites at nucleotide resolution. Here we show that mouse TF footprints conjointly encode a regulatory lexicon that is â¼95% similar with that derived from human TF footprints. However, only â¼20% of mouse TF footprints have human orthologues. Despite substantial turnover of the cis-regulatory landscape, nearly half of all pairwise regulatory interactions connecting mouse TF genes have been maintained in orthologous human cell types through evolutionary innovation of TF recognition sequences. Furthermore, the higher-level organization of mouse TF-to-TF connections into cellular network architectures is nearly identical with human. Our results indicate that evolutionary selection on mammalian gene regulation is targeted chiefly at the level of trans-regulatory circuitry, enabling and potentiating cis-regulatory plasticity.
Subject(s)
Conserved Sequence/genetics , Evolution, Molecular , Mammals/genetics , Regulatory Sequences, Nucleic Acid/genetics , Transcription Factors/genetics , Transcription Factors/metabolism , Animals , DNA Footprinting , Gene Expression Regulation, Developmental/genetics , Gene Regulatory Networks/genetics , Humans , MiceABSTRACT
Photoreceptors--the light-sensitive cells in the vertebrate retina--have been extremely well-characterized with regards to their biochemistry, cell biology and physiology. They therefore provide an excellent model for exploring the factors and mechanisms that drive neural progenitors into a differentiated cell fate in the nervous system. As a result, great progress in understanding the transcriptional network that controls photoreceptor specification and differentiation has been made over the last 20 years. This progress has also enabled the production of photoreceptors from pluripotent stem cells, thereby aiding the development of regenerative medical approaches to eye disease. In this Review, we outline the signaling and transcription factors that drive vertebrate photoreceptor development and discuss how these function together in gene regulatory networks to control photoreceptor cell fate specification.
Subject(s)
Cell Differentiation/physiology , Cell Lineage/physiology , Gene Regulatory Networks/physiology , Neural Stem Cells/physiology , Photoreceptor Cells, Vertebrate/cytology , Signal Transduction/physiology , Vertebrates/embryology , Animals , HumansABSTRACT
The primary glial cells in the retina, the Müller glia, differentiate from retinal progenitors in the first postnatal week. CNTF/LIF/STAT3 signaling has been shown to promote their differentiation; however, another key glial differentiation signal, BMP, has not been examined during this period of Müller glial differentiation. In the course of our analysis of the BMP signaling pathway, we observed a transient wave of Smad1/5/8 signaling in the inner nuclear layer at the end of the first postnatal week, from postnatal day (P) 5 to P9, after the end of neurogenesis. To determine the function of this transient wave, we blocked BMP signaling during this period in vitro or in vivo, using either a BMP receptor antagonist or noggin (Nog). Either treatment leads to a reduction in expression of the Müller glia-specific genes Rlbp1 and Glul, and the failure of many of the Müller glia to repress the bipolar/photoreceptor gene Otx2. These changes in normal Müller glial differentiation result in permanent disruption of the retina, including defects in the outer limiting membrane, rosette formation and a reduction in functional acuity. Our results thus show that Müller glia require a transient BMP signal at the end of neurogenesis to fully repress the neural gene expression program and to promote glial gene expression.
Subject(s)
Bone Morphogenetic Proteins/metabolism , Cell Differentiation/physiology , Ependymoglial Cells/physiology , Neurogenesis/physiology , Retina/growth & development , Signal Transduction/physiology , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Blotting, Western , Chromatin Immunoprecipitation , DNA Primers/genetics , Gene Knock-In Techniques , Immunohistochemistry , In Situ Hybridization , Mice , Mice, Inbred C57BL , Real-Time Polymerase Chain ReactionABSTRACT
Müller glial cells are the source of retinal regeneration in fish and birds; although this process is efficient in fish, it is less so in birds and very limited in mammals. It has been proposed that factors necessary for providing neurogenic competence to Müller glia in fish and birds after retinal injury are not expressed in mammals. One such factor, the proneural transcription factor Ascl1, is necessary for retinal regeneration in fish but is not expressed after retinal damage in mice. We previously reported that forced expression of Ascl1 in vitro reprograms Müller glia to a neurogenic state. We now test whether forced expression of Ascl1 in mouse Müller glia in vivo stimulates their capacity for retinal regeneration. We find that transgenic expression of Ascl1 in adult Müller glia in undamaged retina does not overtly affect their phenotype; however, when the retina is damaged, the Ascl1-expressing glia initiate a response that resembles the early stages of retinal regeneration in zebrafish. The reaction to injury is even more pronounced in Müller glia in young mice, where the Ascl1-expressing Müller glia give rise to amacrine and bipolar cells and photoreceptors. DNaseI-seq analysis of the retina and Müller glia shows progressive reduction in accessibility of progenitor gene cis-regulatory regions consistent with the reduction in their reprogramming. These results show that at least one of the differences between mammal and fish Müller glia that bears on their difference in regenerative potential is the proneural transcription factor Ascl1.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Ependymoglial Cells/metabolism , Regeneration , Retina/physiology , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Mice , Mice, TransgenicABSTRACT
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.
Subject(s)
Cell Differentiation , Polycomb Repressive Complex 2/metabolism , Retina/cytology , Retina/metabolism , Animals , Cell Proliferation , Chromatin Assembly and Disassembly , Enhancer of Zeste Homolog 2 Protein , Gene Expression Regulation , Mice , Stem Cells/cytology , Stem Cells/metabolism , Transcription, GeneticABSTRACT
Non-mammalian vertebrates have a robust ability to regenerate injured retinal neurons from Müller glia (MG) that activate the gene encoding the proneural factor Achaete-scute homolog 1 (Ascl1; also known as Mash1 in mammals) and de-differentiate into progenitor cells. By contrast, mammalian MG have a limited regenerative response and fail to upregulate Ascl1 after injury. To test whether ASCL1 could restore neurogenic potential to mammalian MG, we overexpressed ASCL1 in dissociated mouse MG cultures and intact retinal explants. ASCL1-infected MG upregulated retinal progenitor-specific genes and downregulated glial genes. Furthermore, ASCL1 remodeled the chromatin at its targets from a repressive to an active configuration. MG-derived progenitors differentiated into cells that exhibited neuronal morphologies, expressed retinal subtype-specific neuronal markers and displayed neuron-like physiological responses. These results indicate that a single transcription factor, ASCL1, can induce a neurogenic state in mature MG.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Neuroglia/metabolism , Regeneration , Retina/cytology , Retinal Neurons/cytology , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Biomarkers/metabolism , Cell Proliferation , Cells, Cultured , Cellular Reprogramming , Chromatin Assembly and Disassembly , Cloning, Molecular , Epidermal Growth Factor/pharmacology , Gene Expression Regulation , HEK293 Cells , Histones/metabolism , Humans , In Vitro Techniques , Lentivirus/genetics , Lentivirus/metabolism , Luminescent Proteins/metabolism , Mice , Mice, Inbred C57BL , Neurogenesis , Neuroglia/cytology , Patch-Clamp Techniques , Retina/metabolism , Retinal Neurons/drug effects , Retinal Neurons/metabolism , Red Fluorescent ProteinABSTRACT
During early patterning of the neural plate, a single region of the embryonic forebrain, the eye field, becomes competent for eye development. The hallmark of eye field specification is the expression of the eye field transcription factors (EFTFs). Experiments in fish, amphibians, birds, and mammals have demonstrated largely conserved roles for the EFTFs. Although some of the key signaling events that direct the synchronized expression of these factors to the eye field have been elucidated in fish and frogs, it has been more difficult to study these mechanisms in mammalian embryos. In this study, we have used two different methods for directed differentiation of mouse embryonic stem cells (mESCs) to generate eye field cells and retina in vitro to test for a role of the PDZ domain-containing protein GIPC1 in the specification of the mammalian eye primordia. We find that the overexpression of a dominant-negative form of GIPC1 (dnGIPC1), as well as the downregulation of endogenous GIPC1, is sufficient to inhibit the development of eye field cells from mESCs. GIPC1 interacts directly with IGFR and participates in Akt1 activation, and pharmacological inhibition of Akt1 phosphorylation mimics the dnGIPC1 phenotype. Our data, together with previous studies in Xenopus, support the hypothesis that the GIPC1-PI3K-Akt1 pathway plays a key role in eye field specification in vertebrates.
Subject(s)
Adaptor Proteins, Signal Transducing/biosynthesis , Eye Proteins/biosynthesis , Mouse Embryonic Stem Cells/metabolism , Proto-Oncogene Proteins c-akt/biosynthesis , Retina/metabolism , Signal Transduction/physiology , Animals , Cell Differentiation/physiology , HEK293 Cells , Humans , Mice , Retina/cytology , Xenopus laevisABSTRACT
Most regions of the vertebrate central nervous system develop by the sequential addition of different classes of neurons and glia. This phenomenon has been best characterized in laminated structures like the retina and the cerebral cortex, in which the progenitor cells in these structures are thought to change in their competence as development proceeds to generate different types of neurons in a stereotypic sequence that is conserved across vertebrates. We previously reported that conditional deletion of Dicer prevents the change in competence of progenitors to generate later-born cell types, suggesting that specific microRNAs (miRNAs) are required for this developmental transition. In this report, we now show that three miRNAs, let-7, miR-125, and miR-9, are key regulators of the early to late developmental transition in retinal progenitors: (i) members of these three miRNA families increase over the relevant developmental period in normal retinal progenitors; (ii) inhibiting the function of these miRNAs produces changes in retinal development similar to Dicer CKO; (iii) overexpression of members of these three miRNA families in Dicer-CKO retinas can rescue the phenotype, allowing their progression to late progenitors; (iv) overexpression of these miRNAs can accelerate normal retinal development; (v) microarray and computational analyses of Dicer-CKO retinal cells identified two potential targets of the late-progenitor miRNAs: Protogenin (Prtg) and Lin28b; and (vi) overexpression of either Lin28 or Prtg can maintain the early progenitor state. Together, these data demonstrate that a conserved miRNA pathway controls a key step in the progression of temporal identity in retinal progenitors.
Subject(s)
MicroRNAs/genetics , MicroRNAs/metabolism , Neurogenesis/genetics , Neurogenesis/physiology , Retina/embryology , Animals , Cell Differentiation , DEAD-box RNA Helicases/deficiency , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mice, Knockout , Mice, Transgenic , Neural Stem Cells/cytology , Neural Stem Cells/metabolism , RNA-Binding Proteins , Retina/growth & development , Retina/metabolism , Ribonuclease III/deficiency , Ribonuclease III/genetics , Ribonuclease III/metabolismABSTRACT
During retinal development, photoreceptors and bipolar cells express the transcription factor Otx2. Blimp1 is transiently expressed in Otx2+ cells. Blimp1 deletion results in excess bipolar cell formation at the expense of photoreceptors. In principle, Blimp1 could be expressed only in Otx2+ cells that are committed to photoreceptor fate. Alternatively, Blimp1 could be expressed broadly in Otx2+ cells and silenced to allow bipolar cell development. To distinguish between these alternatives, we followed the fate of Blimp1 expressing cells using Blimp1-Cre mice and Lox-Stop-Lox reporter strains. We observed that Blimp1+ cells gave rise to all photoreceptors, but also to one third of bipolar cells, consistent with the latter alternative: that Blimp1 inhibits bipolar competence in Otx2+ cells and must be silenced to allow bipolar cell generation. To further test this hypothesis, we looked for transitioning rod photoreceptors in Blimp1 conditional knock-out (CKO) mice carrying the NRL-GFP transgene, which specifically labels rods. Control animals lacked NRL-GFP+ bipolar cells. In contrast, about half of the precociously generated bipolar cells in Blimp1 CKO mice co-expressed GFP, suggesting that rods become re-specified as bipolar cells. Birthdating analyses in control and Blimp1 CKO mice showed that bipolar cells were birthdated as early as E13.5 in Blimp1 CKO mice, five days before this cell type was generated in the wild-type retina. Taken together, our data suggest that early Otx2+ cells upregulate photoreceptor and bipolar genes, existing in a bistable state. Blimp1 likely forms a cross-repressive network with pro-bipolar factors such that the winner of this interaction stabilizes the photoreceptor or bipolar state, respectively.
Subject(s)
Cell Differentiation/physiology , Photoreceptor Cells, Vertebrate/cytology , Retina/cytology , Transcription Factors/physiology , Animals , Chromatin Immunoprecipitation , Mice , Mice, Inbred C57BL , Mice, Knockout , Positive Regulatory Domain I-Binding Factor 1 , Transcription Factors/geneticsABSTRACT
The mechanisms of cell fate diversification in the retina are not fully understood. The seven principal cell types of the neural retina derive from a population of multipotent progenitors during development. These progenitors give rise to multiple cell types concurrently, suggesting that progenitors are a heterogeneous population. It is thought that differences in progenitor gene expression are responsible for differences in progenitor competence (i.e. potential) and, subsequently, fate diversification. To elucidate further the mechanisms of fate diversification, we assayed the expression of three transcription factors made by retinal progenitors: Ascl1 (Mash1), Ngn2 (Neurog2) and Olig2. We observed that progenitors were heterogeneous, expressing every possible combination of these transcription factors. To determine whether this progenitor heterogeneity correlated with different cell fate outcomes, we conducted Ascl1- and Ngn2-inducible expression fate mapping using the CreER™/LoxP system. We found that these two factors gave rise to markedly different distributions of cells. The Ngn2 lineage comprised all cell types, but retinal ganglion cells (RGCs) were exceedingly rare in the Ascl1 lineage. We next determined whether Ascl1 prevented RGC development. Ascl1-null mice had normal numbers of RGCs and, interestingly, we observed that a subset of Ascl1+ cells could give rise to cells expressing Math5 (Atoh7), a transcription factor required for RGC competence. Our results link progenitor heterogeneity to different fate outcomes. We show that Ascl1 expression defines a competence-restricted progenitor lineage in the retina, providing a new mechanism to explain fate diversification.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Lineage , Gene Expression Regulation, Developmental , Retina/embryology , Retina/metabolism , Stem Cells/cytology , Stem Cells/metabolism , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Mice , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Retina/cytologyABSTRACT
Adult stem cells reside in hypoxic niches, and embryonic stem cells (ESCs) are derived from a low oxygen environment. However, it is not clear whether hypoxia is critical for stem cell fate since for example human ESCs (hESCs) are able to self-renew in atmospheric oxygen concentrations as well. We now show that hypoxia can govern cell fate decisions since hypoxia alone can revert hESC- or iPSC-derived differentiated cells back to a stem cell-like state, as evidenced by re-activation of an Oct4-promoter reporter. Hypoxia-induced "de-differentiated" cells also mimic hESCs in their morphology, long-term self-renewal capacity, genome-wide mRNA and miRNA profiles, Oct4 promoter methylation state, cell surface markers TRA1-60 and SSEA4 expression, and capacity to form teratomas. These data demonstrate that hypoxia can influence cell fate decisions and could elucidate hypoxic niche function.
Subject(s)
Cell Lineage , Pluripotent Stem Cells/cytology , Adult , Animals , Basic Helix-Loop-Helix Transcription Factors/metabolism , Biomarkers/metabolism , Cell Dedifferentiation/drug effects , Cell Hypoxia/drug effects , Cell Line , Cell Lineage/drug effects , Embryonic Stem Cells/cytology , Embryonic Stem Cells/drug effects , Embryonic Stem Cells/metabolism , Green Fluorescent Proteins/metabolism , Histone Deacetylases/metabolism , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Mice , Models, Biological , Octamer Transcription Factor-3/metabolism , Oxygen/pharmacology , Pluripotent Stem Cells/drug effects , Pluripotent Stem Cells/metabolismABSTRACT
Regeneration of neurons has important implications for human health, and the retina provides an accessible system to study the potential of replacing neurons following injury. In previous work, we generated transgenic mice in which neurogenic transcription factors were expressed in Müller glia (MG) and showed that they stimulated neurogenesis following inner retinal damage. It was unknown, however, whether the timing or mode of injury mattered in this process. Here, we explored these parameters on induced neurogenesis from MG and show that MG expressing Ascl1 will generate new bipolar neurons with similar efficiency irrespective of injury mode or timing. However, MG that express Ascl1-Atoh1 produce a new type of retinal ganglion-like cell after outer retinal damage, which is absent with inner retinal damage. Our data suggest that although cell fate is primarily dictated by neurogenic transcription factors, the inflammatory state of MG relative to injury can influence the outcome of induced neurogenesis.
Subject(s)
Ependymoglial Cells , Retina , Mice , Animals , Humans , Ependymoglial Cells/metabolism , Retina/metabolism , Neurogenesis/physiology , Retinal Ganglion Cells , Mice, Transgenic , Transcription Factors/metabolism , Neuroglia/metabolism , Cell Proliferation/physiology , MammalsABSTRACT
Retinal degeneration in mammals causes permanent loss of vision, due to an inability to regenerate naturally. Some non-mammalian vertebrates show robust regeneration, via Muller glia (MG). We have recently made significant progress in stimulating adult mouse MG to regenerate functional neurons by transgenic expression of the proneural transcription factor Ascl1. While these results showed that MG can serve as an endogenous source of neuronal replacement, the efficacy of this process is limited. With the goal of improving this in mammals, we designed a small molecule screen using sci-Plex, a method to multiplex up to thousands of single nucleus RNA-seq conditions into a single experiment. We used this technology to screen a library of 92 compounds, identified, and validated two that promote neurogenesis in vivo. Our results demonstrate that high-throughput single-cell molecular profiling can substantially improve the discovery process for molecules and pathways that can stimulate neural regeneration and further demonstrate the potential for this approach to restore vision in patients with retinal disease.