RESUMO
Ocular diseases resulting in death of the retinal pigment epithelium (RPE) lead to vision loss and blindness. There are currently no FDA-approved strategies to restore damaged RPE cells. Stimulating intrinsic regenerative responses within damaged tissues has gained traction as a possible mechanism for tissue repair. Zebrafish possess remarkable regenerative abilities, including within the RPE; however, our understanding of the underlying mechanisms remains limited. Here, we conducted an F0 in vivo CRISPR-Cas9-mediated screen of 27 candidate RPE regeneration genes. The screen involved injection of a ribonucleoprotein complex containing three highly mutagenic guide RNAs per target gene followed by PCR-based genotyping to identify large intragenic deletions and MATLAB-based automated quantification of RPE regeneration. Through this F0 screening pipeline, eight positive and seven negative regulators of RPE regeneration were identified. Further characterization of one candidate, cldn7b, revealed novel roles in regulating macrophage/microglia infiltration after RPE injury and in clearing RPE/pigment debris during late-phase RPE regeneration. Taken together, these data support the utility of targeted F0 screens for validating pro-regenerative factors and reveal novel factors that could regulate regenerative responses within the zebrafish RPE.
Assuntos
Epitélio Pigmentado da Retina , Peixe-Zebra , Animais , Epitélio Pigmentado da Retina/fisiologia , Peixe-Zebra/genética , Sistemas CRISPR-Cas/genéticaRESUMO
The retinal pigment epithelium (RPE) plays numerous critical roles in maintaining vision and this is underscored by the prevalence of degenerative blinding diseases like age-related macular degeneration (AMD), in which visual impairment is caused by progressive loss of RPE cells. In contrast to mammals, zebrafish possess the ability to intrinsically regenerate a functional RPE layer after severe injury. The molecular underpinnings of this regenerative process remain largely unknown yet hold tremendous potential for developing treatment strategies to stimulate endogenous regeneration in the human eye. In this study, we demonstrate that the mTOR pathway is activated in RPE cells post-genetic ablation. Pharmacological and genetic inhibition of mTOR activity impaired RPE regeneration, while mTOR activation enhanced RPE recovery post-injury, demonstrating that mTOR activity is essential for RPE regeneration in zebrafish. RNA-seq of RPE isolated from mTOR-inhibited larvae identified a number of genes and pathways dependent on mTOR activity at early and late stages of regeneration; amongst these were components of the immune system, which is emerging as a key regulator of regenerative responses across various tissue and model systems. Our results identify crosstalk between macrophages/microglia and the RPE, wherein mTOR activity is required for recruitment of macrophages/microglia to the RPE injury site. Macrophages/microglia then reinforce mTOR activity in regenerating RPE cells. Interestingly, the function of macrophages/microglia in maintaining mTOR activity in the RPE appeared to be inflammation-independent. Taken together, these data identify mTOR activity as a key regulator of RPE regeneration and link the mTOR pathway to immune responses in facilitating RPE regeneration.
Assuntos
Degeneração Macular , Epitélio Pigmentado da Retina , Animais , Degeneração Macular/genética , Degeneração Macular/metabolismo , Mamíferos/metabolismo , Regeneração/genética , Epitélio Pigmentado da Retina/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
The retinal pigment epithelium (RPE) resides at the back of the eye and performs functions essential for maintaining the health and integrity of adjacent retinal and vascular tissues. At present, the limited reparative capacity of mammalian RPE, which is restricted to small injuries, has hindered progress to understanding in vivo RPE regenerative processes. Here, a detailed methodology is provided to facilitate the study of in vivo RPE repair utilizing the zebrafish, a vertebrate model capable of robust tissue regeneration. This protocol describes a transgenic nitroreductase/metronidazole (NTR/MTZ)-mediated injury paradigm (rpe65a:nfsB-eGFP), which results in ablation of the central two-thirds of the RPE after 24 h treatment with MTZ, with subsequent tissue recovery. Focus is placed on RPE ablations in larval zebrafish and methods for testing the effects of pharmacological compounds on RPE regeneration are also outlined. Generation and validation of RpEGEN, a MATLAB script created to automate quantification of RPE regeneration based on pigmentation, is also discussed. Beyond active RPE repair mechanisms, this protocol can be expanded to studies of RPE degeneration and injury responses as well as the effects of RPE damage on adjacent retinal and vascular tissues, among other cellular and molecular processes. This zebrafish system holds significant promise in identifying genes, networks, and processes that drive RPE regeneration and RPE disease-related mechanisms, with the long-term goal of applying this knowledge to mammalian systems and, ultimately, toward therapeutic development.
Assuntos
Epitélio Pigmentado da Retina , Peixe-Zebra , Animais , Animais Geneticamente Modificados , Mamíferos , Metronidazol/farmacologia , Nitrorredutases/genética , Peixe-Zebra/genéticaRESUMO
Loss of the retinal pigment epithelium (RPE) because of dysfunction or disease can lead to blindness in humans. Harnessing the intrinsic ability of the RPE to self-repair is an attractive therapeutic strategy; however, mammalian RPE is limited in its regenerative capacity. Zebrafish possess tremendous intrinsic regenerative potential in ocular tissues, including the RPE, but little is known about the mechanisms driving RPE regeneration. Here, utilizing transgenic and mutant zebrafish lines, pharmacological manipulations, transcriptomics, and imaging analyses, we identified elements of the immune response as critical mediators of intrinsic RPE regeneration. After genetic ablation, the RPE express immune-related genes, including leukocyte recruitment factors such as interleukin 34 We demonstrate that macrophage/microglia cells are responsive to RPE damage and that their function is required for the timely progression of the regenerative response. These data identify the molecular and cellular underpinnings of RPE regeneration and hold significant potential for translational approaches aimed toward promoting a pro-regenerative environment in mammalian RPE.
Assuntos
Cegueira/genética , Imunidade/genética , Interleucinas/genética , Regeneração/genética , Proteínas de Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/crescimento & desenvolvimento , Cegueira/parasitologia , Cegueira/terapia , Modelos Animais de Doenças , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Microglia/metabolismo , Microglia/patologia , Mutação/genética , Epitélio Pigmentado da Retina/crescimento & desenvolvimento , Epitélio Pigmentado da Retina/patologia , Transcriptoma/genética , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
Diseases that result in retinal pigment epithelium (RPE) degeneration, such as age-related macular degeneration (AMD), are among the leading causes of blindness worldwide. Atrophic (dry) AMD is the most prevalent form of AMD and there are currently no effective therapies to prevent RPE cell death or restore RPE cells lost from AMD. An intriguing approach to treat AMD and other RPE degenerative diseases is to develop therapies focused on stimulating endogenous RPE regeneration. For this to become feasible, a deeper understanding of the mechanisms underlying RPE development, injury responses and regenerative potential is needed. In mammals, RPE regeneration is extremely limited; small lesions can be repaired by the expansion of adjacent RPE cells, but large lesions cannot be repaired as remaining RPE cells are unable to functionally replace lost RPE tissue. In some injury paradigms, RPE cells proliferate but do not regenerate a morphologically normal monolayer, while in others, proliferation is pathogenic and results in further disruption to the retina. This is in contrast to non-mammalian vertebrates, which possess tremendous RPE regenerative potential. Here, we discuss what is known about RPE formation during development in mammalian and non-mammalian vertebrates, we detail the processes by which RPE cells respond to injury, and we describe examples of RPE-to-retina and RPE-to-RPE regeneration in non-mammalian vertebrates. Finally, we outline barriers to RPE-dependent regeneration in mammals that could potentially be overcome to stimulate a regenerative response from the RPE.
Assuntos
Degeneração Macular , Epitélio Pigmentado da Retina , Animais , Cegueira , Mamíferos , RetinaRESUMO
The retinal pigment epithelium (RPE) is a specialized monolayer of pigmented cells within the eye that is critical for maintaining visual system function. Diseases affecting the RPE have dire consequences for vision, and the most prevalent of these is atrophic (dry) age-related macular degeneration (AMD), which is thought to result from RPE dysfunction and degeneration. An intriguing possibility for treating RPE degenerative diseases like atrophic AMD is the stimulation of endogenous RPE regeneration; however, very little is known about the mechanisms driving successful RPE regeneration in vivo. Here, we developed a zebrafish transgenic model (rpe65a:nfsB-eGFP) that enabled ablation of large swathes of mature RPE. RPE ablation resulted in rapid RPE degeneration, as well as degeneration of Bruch's membrane and underlying photoreceptors. Using this model, we demonstrate for the first time that zebrafish are capable of regenerating a functional RPE monolayer after RPE ablation. Regenerated RPE cells first appear at the periphery of the RPE, and regeneration proceeds in a peripheral-to-central fashion. RPE ablation elicits a robust proliferative response in the remaining RPE. Subsequently, proliferative cells move into the injury site and differentiate into RPE. BrdU incorporation assays demonstrate that the regenerated RPE is likely derived from remaining peripheral RPE cells. Pharmacological disruption using IWR-1, a Wnt signaling antagonist, significantly reduces cell proliferation in the RPE and impairs overall RPE recovery. These data demonstrate that the zebrafish RPE possesses a robust capacity for regeneration and highlight a potential mechanism through which endogenous RPE regenerate in vivo.
Assuntos
Degeneração Macular/genética , Regeneração/genética , Epitélio Pigmentado da Retina/crescimento & desenvolvimento , cis-trans-Isomerases/genética , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/crescimento & desenvolvimento , Apoptose/genética , Lâmina Basilar da Corioide/crescimento & desenvolvimento , Lâmina Basilar da Corioide/metabolismo , Diferenciação Celular/genética , Modelos Animais de Doenças , Proteínas de Fluorescência Verde/genética , Humanos , Imidas/administração & dosagem , Larva/genética , Larva/crescimento & desenvolvimento , Degeneração Macular/patologia , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/patologia , Quinolinas/administração & dosagem , Retina/crescimento & desenvolvimento , Retina/patologia , Epitélio Pigmentado da Retina/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
PURPOSE: The application of induced pluripotent stem cell-derived retinal pigmented epithelium (iPSC-RPE) in patients with retinal degenerative disease is making headway toward the clinic, with clinical trials already underway. Multiple groups have developed methods for RPE differentiation from pluripotent cells, but previous studies have shown variability in iPSC propensity to differentiate into RPE. METHODS: This study provides a comparison between 2 different methods for RPE differentiation: (1) a commonly used spontaneous continuously adherent culture (SCAC) protocol and (2) a more rapid, directed differentiation using growth factors. Integration-free iPSC lines were differentiated to RPE, which were characterized with respect to global gene expression, expression of RPE markers, and cellular function. RESULTS: We found that all 5 iPSC lines (iPSC-1, iPSC-2, iPSC-3, iPSC-4, and iPSC-12) generated RPE using the directed differentiation protocol; however, 2 of the 5 iPSC lines (iPSC-4 and iPSC-12) did not yield RPE using the SCAC method. Both methods can yield bona fide RPE that expresses signature RPE genes and carry out RPE functions, and are similar, but not identical to fetal RPE. No differences between methods were detected in transcript levels, protein localization, or functional analyses between iPSC-1-RPE, iPSC-2-RPE, and iPSC-3-RPE. Directed iPSC-3-RPE showed enhanced transcript levels of RPE65 compared to directed iPSC-2-RPE and increased BEST1 expression and pigment epithelium-derived factor (PEDF) secretion compared to directed iPSC-1-RPE. In addition, SCAC iPSC-3-RPE secreted more PEDF than SCAC iPSC-1-RPE. CONCLUSIONS: The directed protocol is a more reliable method for differentiating RPE from various pluripotent sources and some iPSC lines are more amenable to RPE differentiation.
Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Epitélio Pigmentado da Retina/citologia , Diferenciação Celular , Linhagem Celular , HumanosRESUMO
Stem cells provide a potentially unlimited source of cells for treating a plethora of human diseases. Regenerative therapies for retinal degenerative diseases are at the forefront of translation to the clinic, with stem cell-derived retinal pigment epithelium (RPE)-based treatments for age-related macular degeneration (AMD) already showing promise in human patients. Despite our expanding knowledge of stem cell biology, methods for deriving cells, including RPE have remained inefficient. Thus, there has been a push in recent years to develop more directed approaches to deriving cells for therapy. In this concise review, we summarize recent efforts that have been successful in improving RPE derivation efficiency by directing differentiation from human pluripotent stem cells using developmental cues important for normal RPE specification and maturation in vivo. In addition, potential obstacles for clinical translation are discussed. Finally, we review how derivation of RPE from human induced pluripotent stem cells (hiPSCs) provides in vitro models for studying mechanisms of retinal disease and discovering new avenues for treatment.
Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , Degeneração Macular/terapia , Epitélio Pigmentado da Retina/metabolismo , Transplante de Células-Tronco , Animais , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Células-Tronco Pluripotentes Induzidas/transplante , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Epitélio Pigmentado da Retina/patologiaRESUMO
PURPOSE: The purpose of this study was to better understand the role canonical/ß-catenin Wnt signaling plays in the differentiation of human embryonic stem cells (hESCs) into retinal pigmented epithelium (RPE), with the goal of improving methods for derivation. METHODS: Fluorescent reporters were generated to monitor RPE differentiating from hESCs by using a previously described 14-day derivation protocol. Reporters were used to test the effects of the canonical/ß-catenin Wnt pathway agonist CHIR99021 on differentiating RPE. Cells derived from differentiation studies were characterized by lineage-specific transcription factor expression, morphology, pigmentation, and function. The RPE derivation efficiency was determined from percentage positive PMEL17 expression. RESULTS: Fluorescent reporters mimicked expression of endogenous genes during 14-day differentiation to RPE. Analysis of Wnt pathway gene expression showed that the pathway components are expressed in differentiating RPE cells. Addition of CHIR99021 improved RPE derivation based on morphology, expression of RPE-specific lineage markers, and genes involved in melanogenesis. Additionally, expression of the neural retina marker CHX10 was suppressed during differentiation with CHIR99021. Addition of soluble WNT3A, but not WNT5A, had the same result. The CHIR99021-modified protocol yielded cell populations that were 97.77% ± 0.1% positive for the RPE marker PMEL17 at day 14. After cells were expanded to passage 3, they were shown to express RPE markers, carry out phagocytosis of rod outer segments, and secrete pigment epithelium-derived factor apically and vascular endothelial growth factor basally. CONCLUSIONS: Our findings demonstrated the importance of canonical/ß-catenin Wnt signaling in RPE differentiation and showed that manipulating the pathway significantly improves RPE derivation from hESC.
Assuntos
Células-Tronco Embrionárias/citologia , Proteínas do Olho/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Degeneração Macular/genética , Fatores de Crescimento Neural/metabolismo , RNA/genética , Epitélio Pigmentado da Retina/metabolismo , Serpinas/metabolismo , beta Catenina/genética , Diferenciação Celular , Células Cultivadas , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Reação em Cadeia da Polimerase em Tempo Real , Epitélio Pigmentado da Retina/embriologia , Via de Sinalização Wnt , beta Catenina/metabolismoRESUMO
Vascular endothelial growth factor (VEGF) plays a crucial role in developmental and pathological angiogenesis. Expression of VEGF in quiescent adult tissue suggests a potential role in the maintenance of mature blood vessels. We demonstrate, using a Vegf-lacZ reporter mouse model, that VEGF is expressed by arterial but not by venous or capillary endothelial cells (ECs) in vivo. Using an in vitro model, we show that arterial shear stress of human umbilical vein ECs (HUVECs) decreases apoptosis and increases VEGF expression, which is mediated by the induction of Krüppel-like factor 2 (KLF2). Additionally, shear stress stimulates the expression of VEGF receptor 2 (VEGFR2) and is associated with its activation. Knockdown of VEGF in shear stressed HUVECs blocks the protective effect of shear stress, resulting in EC apoptosis equivalent to that in control ECs cultured under static conditions. Similarly, treatment of ECs subjected to arterial shear stress with the VEGF receptor tyrosine kinase inhibitor SU1498, or VEGFR2 neutralizing antiserum, led to increased apoptosis, demonstrating that the mechanoprotection from increased shear is mediated by VEGFR2. Taken together, these studies suggest that arterial flow induces VEGF-VEGFR2 autocrine-juxtacrine signaling, which is a previously unidentified mechanism for vascular EC survival in adult arterial blood vessels.
Assuntos
Células Endoteliais/citologia , Células Endoteliais/metabolismo , Estresse Mecânico , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Artérias/citologia , Capilares/citologia , Sobrevivência Celular , Ativação Enzimática , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Camundongos , Receptores de Fatores de Crescimento do Endotélio Vascular/biossíntese , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/biossíntese , Veias/citologiaRESUMO
BACKGROUND: GATA4 and FOG2 proteins are required for normal cardiac development in mice. It has been proposed that GATA4/FOG2 transcription complex exercises its function through gene activation as well as repression; however, targets of GATA4/FOG2 action in the heart remain elusive. RESULTS: Here we report identification of the Lhx9 gene as a direct target of the GATA4/FOG2 complex. We demonstrate that the developing mouse heart normally expresses truncated isoforms of Lhx9 - Lhx9alpha and Lhx9beta, and not the Lhx9-HD isoform that encodes a protein with an intact homeodomain. At E9.5 Lhx9alpha/beta expression is prominent in the epicardial primordium, septum transversum while Lhx9-HD is absent from this tissue; in the E11.5 heart LHX9alpha/beta-positive cells are restricted to the epicardial mesothelium. Thereafter in the control hearts Lhx9alpha/beta epicardial expression is promptly down-regulated; in contrast, mouse mutants with Fog2 gene loss fail to repress Lhx9alpha/beta expression. Chromatin immunoprecipitation from the E11.5 hearts demonstrated that Lhx9 is a direct target for GATA4 and FOG2. In transient transfection studies the expression driven by the cis-regulatory regions of Lhx9 was repressed by FOG2 in the presence of intact GATA4, but not the GATA4ki mutant that is impaired in its ability to bind FOG2. CONCLUSION: In summary, the Lhx9 gene represents the first direct target of the GATA4/FOG2 repressor complex in cardiac development.