RESUMO
Migration is essential for the laminar stratification and connectivity of neurons in the central nervous system. In the retina, photoreceptors (PRs) migrate to positions according to birthdate, with early-born cells localizing to the basal-most side of the outer nuclear layer. It was proposed that apical progenitor mitoses physically drive these basal translocations non-cell autonomously, but direct evidence is lacking, and whether other mechanisms participate is unknown. Here, combining loss- or gain-of-function assays to manipulate cell cycle regulators (Sonic hedgehog, Cdkn1a/p21) with an in vivo lentiviral labelling strategy, we demonstrate that progenitor division is one of two forces driving basal translocation of rod soma. Indeed, replacing Shh activity rescues abnormal rod translocation in retinal explants. Unexpectedly, we show that rod differentiation also promotes rod soma translocation. While outer segment function or formation is dispensable, Crx and SNARE-dependent synaptic function are essential. Thus, both non-cell and cell autonomous mechanisms underpin PR soma sublaminar positioning in the mammalian retina.
Assuntos
Neurossecreção , Células Fotorreceptoras Retinianas Bastonetes , Animais , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Proteínas Hedgehog/metabolismo , Retina/metabolismo , Diferenciação Celular , MamíferosRESUMO
Emerging evidence suggests that intracellular molecules and organelles transfer between cells during embryonic development, tissue homeostasis and disease. We and others recently showed that transplanted and host photoreceptors engage in bidirectional transfer of intracellular material in the recipient retina, a process termed material transfer (MT). We used cell transplantation, advanced tissue imaging approaches, genetic and pharmacologic interventions and primary cell culture to characterize and elucidate the mechanism of MT. We show that MT correlates with donor cell persistence and the accumulation of donor-derived proteins, mitochondria and transcripts in acceptor cells in vivo. MT requires cell contact in vitro and is associated with the formation of stable microtubule-containing protrusions, termed photoreceptor nanotubes (Ph NTs), that connect donor and host cells in vivo and in vitro. Ph NTs mediate GFP transfer between connected cells in vitro. Furthermore, interfering with Ph NT outgrowth by targeting Rho GTPase-dependent actin remodelling inhibits MT in vivo. Collectively, our observations provide evidence for horizontal exchange of intracellular material via nanotube-like connections between neurons in vivo.
Assuntos
Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/ultraestrutura , Retina/citologia , Actinas/metabolismo , Animais , Transporte Biológico , Sobrevivência Celular , Vesículas Extracelulares , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/metabolismo , Retina/fisiologia , Retinoblastoma/metabolismo , Retinoblastoma/patologia , Transducina/metabolismo , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
The goal of photoreceptor transplantation is to establish functional synaptic connectivity between donor cells and second-order neurons in the host retina. There is, however, limited evidence of donor-host photoreceptor connectivity post-transplant. In this report, we investigated the effect of the host retinal environment on donor photoreceptor neurite outgrowth in vivo and identified a neurite outgrowth-promoting effect of host Crx(-/-) retinas following transplantation of purified photoreceptors expressing green fluorescent protein (GFP). To investigate the noncell autonomous factors that influence donor cell neurite outgrowth in vitro, we established a donor-host coculture system using postnatal retinal aggregates. Retinal cell aggregation is sensitive to several factors, including plate coating substrate, cell density, and the presence of Müller glia. Donor photoreceptors exhibit motility in aggregate cultures and can engraft into established aggregate structures. The neurite outgrowth-promoting phenotype observed in Crx(-/-) recipients in vivo is recapitulated in donor-host aggregate cocultures, demonstrating the utility of this surrogate in vitro approach. The removal of Müller glia from host aggregates reduced donor cell neurite outgrowth, identifying a role for this cell type in donor-host signaling. Although disruption of chondroitin sulfate proteoglycans in aggregates had no effect on the neurite outgrowth of donor photoreceptors, disruption of Rho/ROCK signaling enhanced outgrowth. Collectively, these data show a novel role of Crx, Müller glia, and Rho/ROCK signaling in controlling neurite outgrowth and provide an accessible in vitro model that can be used to screen for factors that regulate donor-host connectivity. Stem Cells 2019;37:529-541.
Assuntos
Neuroglia/metabolismo , Crescimento Neuronal/genética , Células Fotorreceptoras/metabolismo , Quinases Associadas a rho/metabolismo , Animais , Técnicas de Genotipagem , Humanos , Camundongos , Transdução de SinaisRESUMO
ATRX is a chromatin remodeling protein that is mutated in several intellectual disability disorders including alpha-thalassemia/mental retardation, X-linked (ATR-X) syndrome. We previously reported the prevalence of ophthalmological defects in ATR-X syndrome patients, and accordingly we find morphological and functional visual abnormalities in a mouse model harboring a mutation occurring in ATR-X patients. The visual system abnormalities observed in these mice parallels the Atrx-null retinal phenotype characterized by interneuron defects and selective loss of amacrine and horizontal cells. The mechanisms that underlie selective neuronal vulnerability and neurodegeneration in the central nervous system upon Atrx mutation or deletion are unknown. To interrogate the cellular specificity of Atrx for its retinal neuroprotective functions, we employed a combination of temporal and lineage-restricted conditional ablation strategies to generate five different conditional knockout mouse models, and subsequently identified a non-cell-autonomous requirement for Atrx in bipolar cells for inhibitory interneuron survival in the retina. Atrx-deficient retinal bipolar cells exhibit functional, structural and molecular alterations consistent with impairments in neuronal activity and connectivity. Gene expression changes in the Atrx-null retina indicate defective synaptic structure and neuronal circuitry, suggest excitotoxic mechanisms of neurodegeneration, and demonstrate that common targets of ATRX in the forebrain and retina may contribute to similar neuropathological processes underlying cognitive impairment and visual dysfunction in ATR-X syndrome.
Assuntos
Deficiência Intelectual Ligada ao Cromossomo X/genética , Proteína Nuclear Ligada ao X/genética , Talassemia alfa/genética , Animais , Cromatina , Modelos Animais de Doenças , Interneurônios/metabolismo , Masculino , Deficiência Intelectual Ligada ao Cromossomo X/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mutação , Neurônios/metabolismo , Proteínas Nucleares/genética , Retina/metabolismo , Células Bipolares da Retina/metabolismo , Proteína Nuclear Ligada ao X/metabolismo , Talassemia alfa/metabolismoRESUMO
Sonic Hedgehog (Shh) is a secreted morphogen that is an essential regulator of patterning and growth. The Shh full-length protein undergoes autocleavage in the endoplasmic reticulum to generate the biologically active N-terminal fragment (ShhN), which is destined for secretion. We identified sortilin (Sort1), a member of the VPS10P-domain receptor family, as a new Shh trafficking receptor. We demonstrate that Sort-Shh interact by performing coimmunoprecipitation and proximity ligation assays in transfected cells and that they colocalize at the Golgi. Sort1 overexpression causes re-distribution of ShhN and, to a lesser extent, of full-length Shh to the Golgi and reduces Shh secretion. We show loss of Sort1 can partially rescue Hedgehog-associated patterning defects in a mouse model that is deficient in Shh processing, and we show that Sort1 levels negatively regulate anterograde Shh transport in axons in vitro and Hedgehog-dependent axon-glial interactions in vivo Taken together, we conclude that Shh and Sort1 can interact at the level of the Golgi and that Sort1 directs Shh away from the pathways that promote its secretion.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas Hedgehog/metabolismo , Animais , Astrócitos/citologia , Axônios/metabolismo , Células CHO , Células COS , Proliferação de Células , Chlorocebus aethiops , Cricetinae , Cricetulus , Técnicas de Inativação de Genes , Complexo de Golgi/metabolismo , Mutação/genética , Nervo Óptico/metabolismo , Células PC12 , Ligação Proteica , Processamento de Proteína Pós-Traducional , Transporte Proteico , Ratos , Células Ganglionares da Retina/metabolismo , Via SecretóriaRESUMO
The utilization of fluorescent reporter transgenes to discriminate donor versus host cells has been a mainstay of photoreceptor transplantation research, the assumption being that the presence of reporter+ cells in outer nuclear layer (ONL) of transplant recipients represents the integration of donor photoreceptors. We previously reported that GFP+ cells in the ONL of cone-GFP transplanted retinas exhibited rod-like characteristics, raising the possibility that GFP signal in recipient tissue may not be a consequence of donor cell integration. To investigate the basis for this mismatch, we performed a series of transplantations using multiple transgenic donor and recipient models, and assessed cell identity using nuclear architecture, immunocytochemistry, and DNA prelabeling. Our results indicate that GFP+ cells in the ONL fail to exhibit hallmark elements of donor cells, including nuclear hetero/euchromatin architecture. Furthermore, GFP signal does not appear to be a consequence of classic donor/host cell fusion or transfating post-transplant, but is most likely due to material exchange between donor and host photoreceptors. This transfer can be mediated by rods and cones, is bidirectional between donor and host cells, requires viable photoreceptors, occurs preferentially at sites of outer limiting membrane disruption and can be detected in second-order retinal neurons and Müller glia. Collectively, these data warrant re-evaluation of the use of lineage tracing fluorescent reporters in transplantation studies involving the retina and other CNS tissues. Furthermore, the reinterpretation of previous functional rescue data, based on material exchange, rather than cell integration, may offer a novel approach to vision rescue. Stem Cells 2017;35:932-939.
Assuntos
Técnicas de Transferência de Genes , Proteínas de Fluorescência Verde/metabolismo , Células Fotorreceptoras de Vertebrados/citologia , Células Fotorreceptoras de Vertebrados/transplante , Animais , Membrana Celular/metabolismo , Camundongos , Células Fotorreceptoras de Vertebrados/metabolismo , Coloração e RotulagemRESUMO
Neurogenesis is regulated by the dynamic and coordinated activity of several extracellular signalling pathways, but the basis for crosstalk between these pathways remains poorly understood. Here we investigated regulatory interactions between two pathways that are each required for neural progenitor cell maintenance in the postnatal retina; Hedgehog (Hh) and Notch signalling. Both pathways are activated in progenitor cells in the postnatal retina based on the co-expression of fluorescent pathway reporter transgenes at the single cell level. Disrupting Notch signalling, genetically or pharmacologically, induces a rapid downregulation of all three Gli proteins and inhibits Hh-induced proliferation. Ectopic Notch activation, while not sufficient to promote Hh signalling or proliferation, increases Gli2 protein. We show that Notch regulation of Gli2 in Müller glia renders these cells competent to proliferate in response to Hh. These data suggest that Notch signalling converges on Gli2 to prime postnatal retinal progenitor cells and Müller glia to proliferate in response to Hh.
Assuntos
Células Ependimogliais/citologia , Proteínas Hedgehog/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Células-Tronco Neurais/citologia , Neurogênese/fisiologia , Receptores Notch/metabolismo , Animais , Proliferação de Células/fisiologia , Feminino , Fatores de Transcrição Kruppel-Like/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células-Tronco Neurais/metabolismo , Proteínas Nucleares/genética , RNA Mensageiro/genética , Receptores Notch/genética , Retina/citologia , Retina/embriologia , Transdução de Sinais , Fatores de Transcrição/genética , Proteína GLI1 em Dedos de Zinco , Proteína Gli2 com Dedos de ZincoRESUMO
Norrie disease (ND) is a congenital disorder characterized by retinal hypovascularization and cognitive delay. ND has been linked to mutations in 'Norrie Disease Protein' (Ndp), which encodes the secreted protein Norrin. Norrin functions as a secreted angiogenic factor, although its role in neural development has not been assessed. Here, we show that Ndp expression is initiated in retinal progenitors in response to Hedgehog (Hh) signaling, which induces Gli2 binding to the Ndp promoter. Using a combination of genetic epistasis and acute RNAi-knockdown approaches, we show that Ndp is required downstream of Hh activation to induce retinal progenitor proliferation in the retina. Strikingly, Ndp regulates the rate of cell-cycle re-entry and not cell-cycle kinetics, thereby uncoupling the self-renewal and cell-cycle progression functions of Hh. Taken together, we have uncovered a cell autonomous function for Ndp in retinal progenitor proliferation that is independent of its function in the retinal vasculature, which could explain the neural defects associated with ND.
Assuntos
Cegueira/congênito , Proteínas do Olho/genética , Fatores de Transcrição Kruppel-Like/genética , Proteínas do Tecido Nervoso/genética , Doenças do Sistema Nervoso/genética , Retina/metabolismo , Espasmos Infantis/genética , Via de Sinalização Wnt , Animais , Cegueira/genética , Cegueira/metabolismo , Ciclo Celular/genética , Proliferação de Células , Epistasia Genética , Proteínas do Olho/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Doenças Genéticas Ligadas ao Cromossomo X , Proteínas Hedgehog/metabolismo , Humanos , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos Transgênicos , Mutação , Neovascularização Fisiológica , Proteínas do Tecido Nervoso/metabolismo , Doenças do Sistema Nervoso/metabolismo , Ligação Proteica , Retina/crescimento & desenvolvimento , Degeneração Retiniana , Vasos Retinianos/crescimento & desenvolvimento , Vasos Retinianos/metabolismo , Espasmos Infantis/metabolismo , Via de Sinalização Wnt/genética , Proteína Gli2 com Dedos de ZincoRESUMO
The inhibition of MyoD expression is important for obtaining muscle progenitors that can replenish the satellite cell niche during muscle repair. Progenitors could be derived from either embryonic stem cells or satellite cells. Hedgehog (Hh) signaling is important for MyoD expression during embryogenesis and adult muscle regeneration. To date, the mechanistic understanding of MyoD regulation by Hh signaling is unclear. Here, we demonstrate that the Hh effector, Gli2, regulates MyoD expression and associates with MyoD gene elements. Gain- and loss-of-function experiments in pluripotent P19 cells show that Gli2 activity is sufficient and required for efficient MyoD expression during skeletal myogenesis. Inhibition of Hh signaling reduces MyoD expression during satellite cell activation in vitro. In addition to regulating MyoD expression, Hh signaling regulates MyoD transcriptional activity, and MyoD activates Hh signaling in myogenic conversion assays. Finally, Gli2, MyoD, and MEF2C form a protein complex, which enhances MyoD activity on skeletal muscle-related promoters. We therefore link Hh signaling to the function and expression of MyoD protein during myogenesis in stem cells.
Assuntos
Regulação da Expressão Gênica/fisiologia , Proteínas Hedgehog/metabolismo , Proteína MyoD/biossíntese , Células-Tronco Pluripotentes/metabolismo , Células Satélites de Músculo Esquelético/metabolismo , Transdução de Sinais/fisiologia , Animais , Linhagem Celular , Proteínas Hedgehog/genética , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Fatores de Transcrição MEF2 , Camundongos , Camundongos Transgênicos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Desenvolvimento Muscular/fisiologia , Fatores de Regulação Miogênica/genética , Fatores de Regulação Miogênica/metabolismo , Células-Tronco Pluripotentes/citologia , Células Satélites de Músculo Esquelético/citologia , Proteína Gli2 com Dedos de ZincoRESUMO
Human stem cell-derived organoids enable both disease modeling and serve as a source of cells for transplantation. Human retinal organoids are particularly important as a source of human photoreceptors; however, the long differentiation period required and lack of vascularization in the organoid often results in a necrotic core and death of inner retinal cells before photoreceptors are fully mature. Manipulating the in vitro environment of differentiating retinal organoids through the incorporation of extracellular matrix components could influence retinal development. We investigated the addition of hyaluronan (HA), a component of the interphotoreceptor matrix, as an additive to promote long-term organoid survival and enhance retinal maturation. HA treatment had a significant reduction in the proportion of proliferating (Ki67+) cells and increase in the proportion of photoreceptors (CRX+), suggesting that HA accelerated photoreceptor commitment in vitro. HA significantly upregulated genes specific to photoreceptor maturation and outer segment development. Interestingly, prolonged HA-treatment significantly decreased the length of the brush border layer compared to those in control retinal organoids, where the photoreceptor outer segments reside; however, HA-treated organoids also had more mature outer segments with organized discs structures, as revealed by transmission electron microscopy. The brush border layer length was inversely proportional to the molar mass and viscosity of the hyaluronan added. This is the first study to investigate the role of exogenous HA, viscosity, and polymer molar mass on photoreceptor maturation, emphasizing the importance of material properties on organoid culture. STATEMENT OF SIGNIFICANCE: Retinal organoids are a powerful tool to study retinal development in vitro, though like many other organoid systems, can be highly variable. In this work, Shoichet and colleagues investigated the use of hyaluronan (HA), a native component of the interphotoreceptor matrix, to improve photoreceptor maturation in developing human retinal organoids. HA promoted human photoreceptor differentiation leading to mature outer segments with disc formation and more uniform and healthy retinal organoids. These findings highlight the importance of adding components native to the developing retina to generate more physiologically relevant photoreceptors for cell therapy and in vitro models to drive drug discovery and uncover novel disease mechanisms.
Assuntos
Diferenciação Celular , Ácido Hialurônico , Organoides , Retina , Ácido Hialurônico/farmacologia , Ácido Hialurônico/química , Humanos , Organoides/efeitos dos fármacos , Organoides/citologia , Organoides/metabolismo , Diferenciação Celular/efeitos dos fármacos , Retina/efeitos dos fármacos , Retina/citologia , Retina/crescimento & desenvolvimento , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Células Fotorreceptoras de Vertebrados/citologia , Células Fotorreceptoras de Vertebrados/metabolismoRESUMO
SNARE-mediated vesicular transport is thought to play roles in photoreceptor glutamate exocytosis and photopigment delivery. However, the functions of Synaptosomal-associated protein (SNAP) isoforms in photoreceptors are unknown. Here, we revisit the expression of SNAP-23 and SNAP-25 and generate photoreceptor-specific knockout mice to investigate their roles. Although we find that SNAP-23 shows weak mRNA expression in photoreceptors, SNAP-23 removal does not affect retinal morphology or vision. SNAP-25 mRNA is developmentally regulated and undergoes mRNA trafficking to photoreceptor inner segments at postnatal day 9 (P9). SNAP-25 knockout photoreceptors develop normally until P9 but degenerate by P14 resulting in severe retinal thinning. Photoreceptor loss in SNAP-25 knockout mice is associated with abolished electroretinograms and vision loss. We find mistrafficked photopigments, enlarged synaptic vesicles, and abnormal synaptic ribbons which potentially underlie photoreceptor degeneration. Our results conclude that SNAP-25, but not SNAP-23, mediates photopigment delivery and synaptic functioning required for photoreceptor development, survival, and function.
Assuntos
Células Fotorreceptoras de Vertebrados , Proteínas Qb-SNARE , Proteínas Qc-SNARE , Proteína 25 Associada a Sinaptossoma , Animais , Camundongos , Transporte Biológico , Citoesqueleto , Ácido Glutâmico , Camundongos Knockout , RNA Mensageiro , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/metabolismo , Proteína 25 Associada a Sinaptossoma/metabolismo , Células Fotorreceptoras de Vertebrados/citologia , Células Fotorreceptoras de Vertebrados/metabolismoRESUMO
BACKGROUND: The discovery of material transfer between transplanted and host mouse photoreceptors has expanded the possibilities for utilizing transplanted photoreceptors as potential vehicles for delivering therapeutic cargo. However, previous research has not directly explored the capacity for human photoreceptors to engage in material transfer, as human photoreceptor transplantation has primarily been investigated in rodent models of late-stage retinal disease, which lack host photoreceptors. METHODS: In this study, we transplanted human stem-cell derived photoreceptors purified from human retinal organoids at different ontological ages (weeks 10, 14, or 20) into mouse models with intact photoreceptors and assessed transfer of human proteins and organelles to mouse photoreceptors. RESULTS: Unexpectedly, regardless of donor age or mouse recipient background, human photoreceptors did not transfer material in the mouse retina, though a rare subset of donor cells (< 5%) integrated into the mouse photoreceptor cell layer. To investigate the possibility that a species barrier impeded transfer, we used a flow cytometric assay to examine material transfer in vitro. Interestingly, dissociated human photoreceptors transferred fluorescent protein with each other in vitro, yet no transfer was detected in co-cultures of human and mouse photoreceptors, suggesting that material transfer is species specific. CONCLUSIONS: While xenograft models are not a tractable system to study material transfer of human photoreceptors, these findings demonstrate that human retinal organoid-derived photoreceptors are competent donors for material transfer and thus may be useful to treat retinal degenerative disease.
Assuntos
Retina , Degeneração Retiniana , Humanos , Animais , Camundongos , Doadores de Tecidos , Células Fotorreceptoras de Vertebrados , Degeneração Retiniana/terapia , Bioensaio , Modelos Animais de DoençasRESUMO
The hedgehog (Hh) signaling pathway is involved in numerous developmental and adult processes with many links to cancer. In vertebrates, the activity of the Hh pathway is mediated primarily through three Gli transcription factors (Gli1, 2 and 3) that can serve as transcriptional activators or repressors. The identification of Gli target genes is essential for the understanding of the Hh-mediated processes. We used a comparative genomics approach using the mouse and human genomes to identify 390 genes that contained conserved Gli binding sites. RT-qPCR validation of 46 target genes in E14.5 and P0.5 retinal explants revealed that Hh pathway activation resulted in the modulation of 30 of these targets, 25 of which demonstrated a temporal regulation. Further validation revealed that the expression of Bok, FoxA1, Sox8 and Wnt7a was dependent upon Sonic Hh (Shh) signaling in the retina and their regulation is under positive and negative controls by Gli2 and Gli3, respectively. We also show using chromatin immunoprecipitation that Gli2 binds to the Sox8 promoter, suggesting that Sox8 is an Hh-dependent direct target of Gli2. Finally, we demonstrate that the Hh pathway also modulates the expression of Sox9 and Sox10, which together with Sox8 make up the SoxE group. Previously, it has been shown that Hh and SoxE group genes promote Müller glial cell development in the retina. Our data are consistent with the possibility for a role of SoxE group genes downstream of Hh signaling on Müller cell development.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Retina/metabolismo , Fatores de Transcrição SOXE/metabolismo , Animais , Humanos , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fatores de Transcrição SOXE/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína GLI1 em Dedos de ZincoRESUMO
BACKGROUND: We have previously shown that the transcription factor AP-2α (Tcfap2a) is expressed in postmitotic developing amacrine cells in the mouse retina. Although retina-specific deletion of Tcfap2a did not affect retinogenesis, two other family members, AP-2ß and AP-2γ, showed expression patterns similar to AP-2α. RESULTS: Here we show that, in addition to their highly overlapping expression patterns in amacrine cells, AP-2α and AP-2ß are also co-expressed in developing horizontal cells. AP-2γ expression is restricted to amacrine cells, in a subset that is partially distinct from the AP-2α/ß-immunopositive population. To address possible redundant roles for AP-2α and AP-2ß during retinogenesis, Tcfap2a/b-deficient retinas were examined. These double mutants showed a striking loss of horizontal cells and an altered staining pattern in amacrine cells that were not detected upon deletion of either family member alone. CONCLUSIONS: These studies have uncovered critical roles for AP-2 activity in retinogenesis, delineating the overlapping expression patterns of Tcfap2a, Tcfap2b, and Tcfap2c in the neural retina, and revealing a redundant requirement for Tcfap2a and Tcfap2b in horizontal and amacrine cell development.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Retina/embriologia , Fator de Transcrição AP-2/genética , Células Amácrinas/citologia , Células Amácrinas/fisiologia , Animais , Camundongos , Organogênese/fisiologia , Retina/fisiologiaRESUMO
The microenvironment profoundly influences tumor initiation across numerous tissues but remains understudied in brain tumors. In the cerebellum, canonical Wnt signaling controlled by Norrin/Frizzled4 (Fzd4) activation in meningeal endothelial cells is a potent inhibitor of preneoplasia and tumor progression in mouse models of Sonic hedgehog medulloblastoma (Shh-MB). Single-cell transcriptome profiling and phenotyping of the meninges indicate that Norrin/Frizzled4 sustains the activation of meningeal macrophages (mMΦs), characterized by Lyve1 and CXCL4 expression, during the critical preneoplastic period. Depleting mMΦs during this period enhances preneoplasia and tumorigenesis, phenocopying the effects of Norrin loss. The anti-tumorigenic function of mMΦs is derived from the expression of CXCL4, which counters CXCL12/CXCR4 signaling in pre-tumor cells, thereby inhibiting cell-cycle progression and promoting migration away from the pre-tumor niche. These findings identify a pivotal role for mMΦs as key mediators in chemokine-regulated anti-cancer crosstalk between the stroma and pre-tumor cells in the control of MB initiation.
Assuntos
Neoplasias Cerebelares , Meduloblastoma , Camundongos , Animais , Meduloblastoma/metabolismo , Proteínas Hedgehog/metabolismo , Células Endoteliais/metabolismo , Via de Sinalização Wnt , Neoplasias Cerebelares/metabolismo , Microambiente TumoralRESUMO
Cell therapy holds tremendous promise for vision restoration; yet donor cell survival and integration continue to limit efficacy of these strategies. Transplanted photoreceptors, which mediate light sensitivity in the retina, transfer cytoplasmic components to host photoreceptors instead of integrating into the tissue. Donor cell material transfer could, therefore, function as a protein augmentation strategy to restore photoreceptor function. Biomaterials, such as hyaluronan-based hydrogels, can support donor cell survival but have not been evaluated for effects on material transfer. With increased survival, we hypothesized that we would achieve greater material transfer; however, the opposite occurred. Photoreceptors delivered to the subretinal space in mice in a hyaluronan and methylcellulose (HAMC) hydrogel showed reduced material transfer. We examined mitochondria transfer in vitro and cytosolic protein transfer in vivo and demonstrate that HAMC significantly reduced transfer in both contexts, which we ascribe to reduced cell-cell contact. Nanotube-like donor cell protrusions were significantly reduced in the hydrogel-transplanted photoreceptors compared to the saline control group, which suggests that HAMC limits the contact required to the host retina for transfer. Thus, HAMC can be used to manipulate the behaviour of transplanted donor cells in cell therapy strategies.
Assuntos
Ácido Hialurônico , Hidrogéis , Camundongos , Animais , Retina , Materiais BiocompatíveisRESUMO
BACKGROUND: Retinitis pigmentosa (RP) is an inherited retinal disease that results in photoreceptor degeneration, leading to severe vision loss or blindness. Due to its genetic heterogeneity, developing a new gene therapy to correct every genetic mutation contributing to its progression is infeasible. Photoreceptor transplantation can be harnessed to restore vision; however, this approach is limited by poor cell survival and synaptic integration into the neural retina. Thus, we developed a combined cell and gene therapy that is expected to protect photoreceptors in most, if not all, cases of RP. METHODS: Human embryonic stem cells (hESCs) modified with our FailSafe™ system were genetically engineered to overexpress sCX3CL1, an inhibitor of microglia activation that has been shown to preserve photoreceptor survival and function in mouse models of RP, independent of the genetic cause. These cells were differentiated into human retinal pigment epithelium (hRPE) cells and used as therapeutic cells due to their longevity and safety, both of which have been demonstrated in preclinical and clinical studies. Transgenic hRPE were delivered into the subretinal space of immunodeficient mice and the rd10 mouse model of RP to evaluate donor cell survival and retention of transgene expression. The outer nuclear layer was quantified to assess photoreceptor protection. RESULTS: Transgenic FailSafe™ hRPE (FS-hRPE) cells can survive for at least four months in the retina of immunodeficient mice and retain transgene expression. However, these cells do not persist beyond two weeks post-injection in the retina of immunocompetent rd10 recipients, despite Cyclosporine A treatment. Nevertheless, sCX3CL1-expressing FailSafe™ hRPE cells prevented photoreceptor degeneration in a local acting manner during the duration of their presence in the subretinal space. CONCLUSIONS: Transgenic hESCs differentiate into hRPE cells and retain sCX3CL1 transgene expression both in vitro and in vivo. Moreover, hRPE cells delivered to the subretinal space of rd10 mice prevented photoreceptor degeneration in a local-acting manner, suggesting that this approach could have applications for preserving photoreceptors in specific subregions of the retina, such as the macula. Overall, our study not only reveals the potential of a combined cell and gene therapy for the treatment of RP, but also the possibility of using hRPE cells to deliver therapeutic biologics in situ to treat diseases over long-term.
Assuntos
Epitélio Pigmentado da Retina , Retinose Pigmentar , Humanos , Animais , Camundongos , Células Fotorreceptoras Retinianas Bastonetes , Retinose Pigmentar/genética , Retinose Pigmentar/terapia , Retina , Animais Geneticamente Modificados , Modelos Animais de Doenças , Quimiocina CX3CL1RESUMO
Neuronal repopulation achieved through transplantation or transdifferentiation from endogenous sources holds tremendous potential for restoring function in chronic neurodegenerative disease or acute injury. Key to the evaluation of neuronal engraftment is the definitive discrimination of new or donor neurons from preexisting cells within the host tissue. Recent work has identified mechanisms by which genetically encoded donor cell reporters can be transferred to host neurons through intercellular material transfer. In addition, labeling transplanted and endogenously transdifferentiated neurons through viral vector transduction can yield misexpression in host cells in some circumstances. These issues can confound the tracking and evaluation of repopulated neurons in regenerative experimental paradigms. Using the retina as an example, we discuss common reasons for artifactual labeling of endogenous host neurons with donor cell reporters and suggest strategies to prevent erroneous conclusions based on misidentification of cell origin.
RESUMO
The morphogen sonic hedgehog (Shh) plays a crucial role in development of the CNS, including the neural retina. Suppressor of fused (Sufu) has been recently identified as a critical regulator of Hh signaling in mammals. However, the precise roles that Sufu plays in the regulation of proliferation and cell-fate decisions in neural progenitors is unknown. Here, we have addressed these questions by conditionally deleting Sufu in mouse multipotent retinal progenitor cells (RPCs). Sufu deletion in RPCs results in transient increases in Hh activity and proliferation followed by developmentally premature cell-cycle exit. Importantly, we demonstrate a novel role for Sufu in the maintenance of multipotency in RPCs. Sufu-null RPCs downregulate transcription factors required to specify or maintain RPC identity (Rax, Vsx2) and multipotency (Pax6) but continue to express the neural progenitor marker Sox2. These cells fail to express retinal lineage-specific transcription factors, such as Math5, and adopt an amacrine or horizontal cell fate at the expense of all other classes of retinal neurons. Genetic elimination of Gli2 in Sufu-null RPCs attenuates Hh pathway activity and restores multipotency in neural progenitors. These data provide novel evidence that Sufu-mediated antagonism of Hh/Gli2 signaling is required to maintain RPC multipotency and identity.
Assuntos
Células-Tronco Multipotentes/fisiologia , Neurônios/fisiologia , Proteínas Repressoras/fisiologia , Retina/embriologia , Animais , Diferenciação Celular/genética , Proliferação de Células , Regulação para Baixo/genética , Feminino , Proteínas Hedgehog/antagonistas & inibidores , Proteínas Hedgehog/genética , Proteínas Hedgehog/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Gravidez , Distribuição Aleatória , Proteínas Repressoras/deficiência , Proteínas Repressoras/genética , Retina/citologia , Retina/fisiologia , Transdução de Sinais/genéticaRESUMO
PURPOSE: During mammalian eye development, the restriction of Wnt/ß-catenin signaling at the junction of the neural retina and the retinal pigment epithelium in the peripheral eyecup is required for the development of the ciliary margin, a non-neural region of the eyecup that is the precursor of the ciliary body and iris of the adult eye. METHODS: To identify genes that are modulated by ß-catenin activity in the embryonic retina, we performed gene expression profiling in Li(+)-treated retinal explants, a pharmacological model of ß-catenin activation. The Li(+)-modulated gene data set was searched for ß-catenin/T-cell specific transcription factor binding sites. RESULTS: Functional annotations of this data set revealed significant enrichments for genes involved in chromatin organization, neurogenesis, and cell motion/migration. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis confirmed the modulation of 12 genes in Li(+)-treated explants and retinas of mice with Cre-mediated induction of constitutively active ß-catenin (ß-cat(act)). In situ hybridization revealed ß-catenin-specific upregulation of cyclin-dependent kinase inhibitor 1A (P21) [Cdkn1a] and tumor necrosis factor receptor superfamily, member 19 (Tnfrsf19) in the developing retina consistent with the antineurogenic and proliferation changes associated with ectopic Wnt/ß-catenin signaling in the eyecup. CONCLUSIONS: This data set of Li(+)-modulated genes provides a valuable resource for characterizing the Wnt/ ß-catenin regulated gene network in eyecup patterning.