RESUMEN
The Drosophila corneal lens is entirely composed of chitin and other apical extracellular matrix components, and it is not known how it acquires the biconvex shape that enables it to focus light onto the retina. We show here that the zona pellucida domain-containing protein Dusky-like is essential for normal corneal lens morphogenesis. Dusky-like transiently localizes to the expanded apical surfaces of the corneal lens-secreting cells and prevents them from undergoing apical constriction and apicobasal contraction. Dusky-like also controls the arrangement of two other zona pellucida domain proteins, Dumpy and Piopio, external to the developing corneal lens. Loss of either dusky-like or dumpy delays chitin accumulation and disrupts the outer surface of the corneal lens. We find that artificially inducing apical constriction by activating myosin contraction is sufficient to similarly alter chitin deposition and corneal lens morphology. These results demonstrate the importance of cell shape in controlling the morphogenesis of overlying apical extracellular matrix structures such as the corneal lens.
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Proteínas de Drosophila , Cristalino , Morfogénesis , Animales , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Cristalino/citología , Cristalino/metabolismo , Cristalino/crecimiento & desarrollo , Quitina/metabolismo , Matriz Extracelular/metabolismo , Córnea/metabolismo , Córnea/citología , Córnea/crecimiento & desarrollo , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Forma de la CélulaRESUMEN
The role extracellular matrix (ECM) in multiple events of morphogenesis has been well described, little is known about its specific role in early eye development. One of the first morphogenic events in lens development is placodal thickening, which converts the presumptive lens ectoderm from cuboidal to pseudostratified epithelium. This process occurs in the anterior pre-placodal ectoderm when the optic vesicle approaches the cephalic ectoderm and is regulated by transcription factor Pax6 and secreted BMP4. Since cells and ECM have a dynamic relationship of interdependence and modulation, we hypothesized that the ECM evolves with cell shape changes during lens placode formation. This study investigates changes in optic ECM including both protein distribution deposition, extracellular gelatinase activity and gene expression patterns during early optic development using chicken and mouse models. In particular, the expression of Timp2, a metalloprotease inhibitor, corresponds with a decrease in gelatinase activity within the optic ECM. Furthermore, we demonstrate that optic ECM remodeling depends on BMP signaling in the placode. Together, our findings suggest that the lens placode plays an active role in remodeling the optic ECM during early eye development.
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Matriz Extracelular , Regulación del Desarrollo de la Expresión Génica , Cristalino , Factor de Transcripción PAX6 , Animales , Matriz Extracelular/metabolismo , Ratones , Cristalino/metabolismo , Cristalino/crecimiento & desarrollo , Cristalino/citología , Factor de Transcripción PAX6/metabolismo , Factor de Transcripción PAX6/genética , Proteínas del Ojo/metabolismo , Proteínas del Ojo/genética , Proteína Morfogenética Ósea 4/metabolismo , Proteína Morfogenética Ósea 4/genética , Embrión de Pollo , Proteínas de Homeodominio/metabolismo , Proteínas de Homeodominio/genética , Inhibidor Tisular de Metaloproteinasa-2/metabolismo , Inhibidor Tisular de Metaloproteinasa-2/genética , Factores de Transcripción Paired Box/metabolismo , Factores de Transcripción Paired Box/genética , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , Transducción de Señal , Pollos/genética , Ojo/metabolismo , Ojo/crecimiento & desarrollo , Ojo/embriologíaRESUMEN
Estuarine mangroves are often considered nurseries for the Atlantic Goliath grouper juveniles. Yet, the contributions of different estuarine primary producers and habitats as sources of organic matter during early ontogenetic development remain unclear. Given the species' critically endangered status and protection in Brazil, obtaining biological samples from recently settled recruits in estuaries is challenging. In this study, we leveraged a local partnership with fishers and used stable isotope (C and N) profiles from the eye lenses of stranded individuals or incidentally caught by fishery to reconstruct the trophic and habitat changes of small juveniles. The eye lens grows by the apposition of protein-rich layers. Once these layers are formed, they become inert, allowing to make inferences on the trophic ecology and habitat use along the development of the individual until its capture. We used correlations between fish size and the entire eye lens size, along with estuarine baselines, to reconstruct the fish size and trophic positions for each of the lens layers obtained. We then used dominant primary producers and basal sources from mangrove sheltered, exposed estuarine and marine habitats to construct an ontogenetic model of trophic and habitat support changes since maternal origins. Our model revealed marine support before the juveniles reached 25 mm (standard length), followed by a rapid increase in reliance on mangrove sheltered sources, coinciding with the expected size at settlement. After reaching 60 mm, individuals began to show variability. Some remained primarily supported by the mangrove sheltered area, while others shifted to rely more on the exposed estuarine area around 150 mm. Our findings indicate that while mangroves are critical for settlement, as Goliath grouper juveniles grow, they can utilize organic matter produced throughout the estuary. This underscores the need for conservation strategies that focus on seascape connectivity, as protecting just one discrete habitat may not be sufficient to preserve this endangered species and safeguard its ecosystem functions.
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Ecosistema , Especies en Peligro de Extinción , Cristalino , Animales , Cristalino/crecimiento & desarrollo , Brasil , Estuarios , Isótopos de Carbono/análisis , Isótopos de Nitrógeno/análisis , Lubina/fisiología , Lubina/crecimiento & desarrollo , Cadena Alimentaria , Monitoreo del AmbienteRESUMEN
BACKGROUND: Cellular differentiation is marked by temporally and spatially coordinated gene expression regulated at multiple levels. DNA methylation represents a universal mechanism to control chromatin organization and its accessibility. Cytosine methylation of CpG dinucleotides regulates binding of methylation-sensitive DNA-binding transcription factors within regulatory regions of transcription, including promoters and distal enhancers. Ocular lens differentiation represents an advantageous model system to examine these processes as lens comprises only two cell types, the proliferating lens epithelium and postmitotic lens fiber cells all originating from the epithelium. RESULTS: Using whole genome bisulfite sequencing (WGBS) and microdissected lenses, we investigated dynamics of DNA methylation and chromatin changes during mouse lens fiber and epithelium differentiation between embryos (E14.5) and newborns (P0.5). Histone H3.3 variant chromatin landscapes were also generated for both P0.5 lens epithelium and fibers by chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq). Tissue-specific features of DNA methylation patterns are demonstrated via comparative studies with embryonic stem (ES) cells and neural progenitor cells (NPCs) at Nanog, Pou5f1, Sox2, Pax6 and Six3 loci. Comparisons with ATAC-seq and RNA-seq data demonstrate that reduced methylation is associated with increased expression of fiber cell abundant genes, including crystallins, intermediate filament (Bfsp1 and Bfsp2) and gap junction proteins (Gja3 and Gja8), marked by high levels of histone H3.3 within their transcribed regions. Interestingly, Pax6-binding sites exhibited predominantly DNA hypomethylation in lens chromatin. In vitro binding of Pax6 proteins showed Pax6's ability to interact with sites containing one or two methylated CpG dinucleotides. CONCLUSIONS: Our study has generated the first data on methylation changes between two different stages of mammalian lens development and linked these data with chromatin accessibility maps, presence of histone H3.3 and gene expression. Reduced DNA methylation correlates with expression of important genes involved in lens morphogenesis and lens fiber cell differentiation.
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Cromatina , Histonas , Cristalino , Animales , Ratones , Diferenciación Celular/genética , ADN/metabolismo , Metilación de ADN , Expresión Génica , Histonas/metabolismo , Cristalino/crecimiento & desarrolloRESUMEN
We have previously shown that the conditional deletion of either the p110α catalytic subunit of phosphatidylinositol 3-kinase (PI3K), or its opposing phosphatase, phosphatase and tensin homolog (PTEN), had distinct effects on lens growth and homeostasis. The deletion of p110α reduced the levels of phosphorylated Akt and equatorial epithelial cell proliferation, and resulted in smaller transparent lenses in adult mice. The deletion of PTEN increased levels of phosphorylated Akt, altered lens sodium transport, and caused lens rupture and cataract. Here, we have generated conditional p110α/PTEN double-knockout mice, and evaluated epithelial cell proliferation and lens homeostasis. The double deletion of p110α and PTEN rescued the defect in lens size seen after the single knockout of p110α, but accelerated the lens rupture phenotype seen in PTEN single-knockout mice. Levels of phosphorylated Akt in double-knockout lenses were significantly higher than in wild-type lenses, but not as elevated as those reported for PTEN single-knockout lenses. These results showed that the double deletion of the p110α catalytic subunit of PI3K and its opposing phosphatase, PTEN, exacerbated the rupture defect seen in the single PTEN knockout and alleviated the growth defect observed in the single p110α knockout. Thus, the integrity of the PI3K signaling pathway was absolutely essential for proper lens homeostasis, but not for lens growth.
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Cristalino , Fosfohidrolasa PTEN , Fosfatidilinositol 3-Quinasa , Animales , Homeostasis , Cristalino/crecimiento & desarrollo , Cristalino/metabolismo , Ratones , Ratones Noqueados , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Fosfatidilinositol 3-Quinasa/genética , Fosfatidilinositol 3-Quinasa/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
Cytokinetic membrane abscission is a spatially and temporally regulated process that requires ESCRT (endosomal sorting complexes required for transport)dependent control of membrane remodeling at the midbody, a subcellular organelle that defines the cleavage site. Alteration of ESCRT function can lead to cataract, but the underlying mechanism and its relation to cytokinesis are unclear. We found a lens-specific cytokinetic process that required PI3K-C2α (phosphatidylinositol-4-phosphate 3-kinase catalytic subunit type 2α), its lipid product PI(3,4)P2 (phosphatidylinositol 3,4-bisphosphate), and the PI(3,4)P2binding ESCRT-II subunit VPS36 (vacuolar protein-sorting-associated protein 36). Loss of each of these components led to impaired cytokinesis, triggering premature senescence in the lens of fish, mice, and humans. Thus, an evolutionarily conserved pathway underlies the cell typespecific control of cytokinesis that helps to prevent early onset cataract by protecting from senescence.
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Catarata/patología , Senescencia Celular , Citocinesis , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Cristalino/citología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositoles/metabolismo , Envejecimiento Prematuro , Animales , Evolución Biológica , Proteínas de Unión al Calcio/metabolismo , Catarata/metabolismo , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Humanos , Cristalino/crecimiento & desarrollo , Cristalino/metabolismo , Ratones , Mutación , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 4,5-Difosfato/metabolismo , Tubulina (Proteína)/metabolismo , Pez Cebra , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Genetic mutations in heat shock factor 4 (Hsf4) is associated with both congenital and age-related cataracts. Hsf4 regulates lens development through its ability to both activate and inhibit transcription. Previous studies suggested Hsf4 is involved in modulating cellular senescence depending on p21cip1 and p27 kip1 expression in MEF cells. Here, we found that Hsf4 acts as a suppressor of p21cip1 expression and plays an anti-senescence role during lens development. Knocking out Hsf4 facilitated UVB-induced cellular senescence in mouse lens epithelial cells (mLECs). p21cip1 was upregulated at both the mRNA and protein levels in HSF4-/- mLECs under control and UVB-treated conditions, and knockdown of p21cip1 by siRNA alleviated UVB-induced cellular senescence. HSF4 directly bound to the p21cip1 promoter and increased H3K27m3 levels at the p21cip1 proximal promoter region by recruiting the methyltransferase EZH2. In animal models, p21cip1 was gradually upregulated in wild-type mouse lenses with increasing age, while Hsf4 levels decreased. We generated a Hsf4 mutant mice line (Hsf4del-42) which displayed obvious congenital cataract phenotype. The expression of p21cip1 and senescence-associated cytokines were induced in the cataractous lenses of Hsf4del-42 mice. H3K27m3 and EZH2 levels decreased in p21cip1 promoters in the lenses of Hsf4del-42 mice. The SA-ß-Gal activities were positive in lens epithelia of aged Hsf4null zebrafish compared to wild-type lenses. p21cip1 and senescence-associated cytokines levels were also upregulated in lenses of Hsf4null zebrafish. Accordingly, we propose that HSF4 plays a protective role in lens epithelial cells against cellular senescence during lens development and aging, partly by fine-tuning p21cip1 expression.
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Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Factores de Transcripción del Choque Térmico/deficiencia , Cristalino/patología , Proteínas de Pez Cebra/deficiencia , Proteínas de Pez Cebra/genética , Envejecimiento/genética , Animales , Animales Modificados Genéticamente , Catarata/genética , Catarata/patología , Línea Celular , Senescencia Celular/genética , Senescencia Celular/efectos de la radiación , Metilación de ADN , Modelos Animales de Enfermedad , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Células Epiteliales/patología , Células Epiteliales/efectos de la radiación , Regulación del Desarrollo de la Expresión Génica , Técnicas de Inactivación de Genes , Factores de Transcripción del Choque Térmico/genética , Histonas/genética , Histonas/metabolismo , Humanos , Cristalino/citología , Cristalino/crecimiento & desarrollo , Cristalino/efectos de la radiación , Ratones , Regiones Promotoras Genéticas , Rayos Ultravioleta/efectos adversos , Pez Cebra , Proteínas de Pez Cebra/metabolismoRESUMEN
A hallmark feature of lens development and differentiation is the complete elimination of organelles from the center of the eye lens. A long unanswered question in lens biology is what are the mechanisms that control the elimination of organelles during the terminal remodeling program to form mature lens fiber cells? Recent advances have expanded our understanding of these mechanisms including newly discovered signaling pathways, proteasomal regulators, autophagy proteins, transcription factors and the hypoxic environment of the lens itself. These recent discoveries suggest that distinct mechanisms coordinate the elimination of the nucleus, mitochondria, endoplasmic reticulum and Golgi apparatus during lens fiber cell differentiation. Since regulation of organelle number and distribution is also a feature of the terminal remodeling programs of more complex cell-types and tissues, these advances are likely to impact a wide-variety of fields.
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Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Cristalino/crecimiento & desarrollo , Animales , Autofagia , Diferenciación Celular , Humanos , Cristalino/citología , Mitocondrias/metabolismo , Modelos AnimalesRESUMEN
In the process of exploring new methods for cataract treatment, lens regeneration is an ideal strategy for effectively restoring accommodative vision and avoiding postoperative complications and has great clinical potential. Lens regeneration, which is not a simple repetition of lens development, depends on the complex regulatory network comprising the FGF, BMP/TGF-ß, Notch, and Wnt signaling pathways. Current research mainly focuses on in situ and in vitro lens regeneration. On the one hand, the possibility of the autologous stem cell in situ regeneration of functional lenses has been confirmed; on the other hand, both embryonic stem cells and induced pluripotent stem cells have been induced into lentoid bodies in vitro which are similar to the natural lens to a certain extent. This article will briefly summarize the regulatory mechanisms of lens development, describe the recent progress of lens regeneration, explore the key molecular signaling pathways, and, more importantly, discuss the prospects and challenges of their clinical applications to provide reference for clinical transformations.
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Catarata , Cristalino , Regeneración/fisiología , Animales , Catarata/metabolismo , Catarata/patología , Catarata/terapia , Diferenciación Celular , Células Madre Embrionarias/trasplante , Humanos , Técnicas In Vitro , Células Madre Pluripotentes Inducidas/trasplante , Cristalino/crecimiento & desarrollo , Cristalino/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Vía de Señalización Wnt/fisiologíaRESUMEN
High myopia is a leading cause of blindness worldwide. Myopia progression may lead to pathological changes of lens and affect the outcome of lens surgery, but the underlying mechanism remains unclear. Here, we find an increased lens size in highly myopic eyes associated with up-regulation of ß/γ-crystallin expressions. Similar findings are replicated in two independent mouse models of high myopia. Mechanistic studies show that the transcription factor MAF plays an essential role in up-regulating ß/γ-crystallins in high myopia, by direct activation of the crystallin gene promoters and by activation of TGF-ß1-Smad signaling. Our results establish lens morphological and molecular changes as a characteristic feature of high myopia, and point to the dysregulation of the MAF-TGF-ß1-crystallin axis as an underlying mechanism, providing an insight for therapeutic interventions.
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Cristalino/patología , Factores de Transcripción Maf/metabolismo , Miopía Degenerativa/patología , Factor de Crecimiento Transformador beta1/metabolismo , beta-Cristalinas/biosíntesis , gamma-Cristalinas/biosíntesis , Animales , Femenino , Humanos , Cristalino/crecimiento & desarrollo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Regiones Promotoras Genéticas/genética , Proteínas Smad/metabolismo , Regulación hacia Arriba/genéticaRESUMEN
Accurate methods for age determination are critical to the knowledge of wildlife populations' age structure and, therefore, to their successful management. The reliability of age estimation may have profound economic and ecological consequences on the management of the European wild rabbits, Oryctolagus cuniculus, in its native and introduced range, where it is a keystone species and a major pest, respectively. As in other mammal species, European rabbits' age is often estimated using the Gompertz relationship between age and lens' weight. The growth rate formula has been developed based on data collected from European rabbits introduced in Australia, where a single subspecies (O. cuniculus cuniculus, Occ) is present. However, this curve has never been validated in the species native range, the Iberian Peninsula, where two subspecies (Occ, and O. c. algirus, Oca) coexist naturally. In this study, we tested the relationship between age and lens' weight using 173 Occ and 112 Oca wild rabbits that were surveyed in two experimental facilities in Spain. Our findings show that, in the native range, the published growth curve formula fits well Occ but not Oca data. Therefore, we recommend using the formula reported in this study to estimate the age of Oca (Lens dry weight = 240 × 10(-64.9/(Age+32))). This study supports Oca rabbits' distinctiveness revealed by previous studies, which suggests that management interventions should be applied to protect this subspecies whose distribution range is very narrow and whose populations seem to be declining. More broadly, our findings point to the importance of testing the suitability of growth curves defined for other species with different genetic forms as occurs in the European wild rabbit case.
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Envejecimiento/fisiología , Animales Salvajes , Cristalino/crecimiento & desarrollo , Tamaño de los Órganos , Animales , Ecosistema , Europa (Continente) , ConejosRESUMEN
The vertebrate lens is a valuable model system for investigating the gene expression changes that coordinate tissue differentiation due to its inclusion of two spatially separated cell types, the outer epithelial cells and the deeper denucleated fiber cells that they support. Zebrafish are a useful model system for studying lens development given the organ's rapid development in the first several days of life in an accessible, transparent embryo. While we have strong foundational knowledge of the diverse lens crystallin proteins and the basic gene regulatory networks controlling lens development, no study has detailed gene expression in a vertebrate lens at single cell resolution. Here we report an atlas of lens gene expression in zebrafish embryos and larvae at single cell resolution through five days of development, identifying a number of novel putative regulators of lens development. Our data address open questions about the temperospatial expression of α-crystallins during lens development that will support future studies of their function and provide the first detailed view of ß- and γ-crystallin expression in and outside the lens. We describe divergent expression in transcription factor genes that occur as paralog pairs in the zebrafish. Finally, we examine the expression dynamics of cytoskeletal, membrane associated, RNA-binding, and transcription factor genes, identifying a number of novel patterns. Overall these data provide a foundation for identifying and characterizing lens developmental regulatory mechanisms and revealing targets for future functional studies with potential therapeutic impact.
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Células Epiteliales/metabolismo , Cristalino/metabolismo , Transcriptoma/genética , alfa-Cristalinas/genética , gamma-Cristalinas/genética , Animales , Células Epiteliales/citología , Cristalino/crecimiento & desarrollo , Pez Cebra , alfa-Cristalinas/metabolismo , gamma-Cristalinas/metabolismoRESUMEN
In humans, TDRD7 (tudor domain containing 7) mutations lead to a syndrome combining congenital cataracts (CCs) and non-obstructive azoospermia (NOA), characterized by abnormal lens development and spermiogenesis. However, the molecular mechanism underlying TDRD7's functions in eye and testicular development are still largely unknown. Here, we show that the depletion of this gene in mice and humans resulted in the accumulation of autophagosomes and the disruption of macroautophagic/autophagic flux. The disrupted autophagic flux in tdrd7-deficient mouse embryonic fibroblasts (MEFs) was caused by a failure of autophagosome fusion with lysosomes. Furthermore, transcriptome analysis and biochemical assays showed that TDRD7 might directly bind to Tbc1d20 mRNAs and downregulate its expression, which is a key regulator of autophagosome maturation, resulting in the disruption of autophagosome maturation. In addition, we provide evidence to show that TDRD7-mediated autophagosome maturation maintains lens transparency by facilitating the removal of damaged proteins and organelles from lens fiber cells and the biogenesis of acrosome. Altogether, our results showed that TDRD7 plays an essential role in the maturation of autophagosomes and that tdrd7 deletion results in eye defects and testicular abnormalities in mice, implicating disrupted autophagy might be the mechanism that contributes to lens development and spermiogenesis defects in human.Abbreviations: CB: chromatoid bodies; CC: congenital cataract; CTSD: cathepsin D; DMSO: dimethyl sulfoxide; LAMP1: lysosomal-associated membrane protein 1; LECs: lens epithelial cells; MAP1LC3/LC3/Atg8: microtubule-associated protein 1 light chain 3; MEFs: mouse embryonic fibroblasts; NOA: non-obstructive azoospermia; OFZ: organelle-free zone; RG: RNA granules; SQSTM1/p62: sequestosome 1; TBC1D20: TBC1 domain family member 20; TDRD7: tudor domain containing 7; TEM: transmission electron microscopy; WT: wild type.
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Autofagosomas/metabolismo , Cristalino/crecimiento & desarrollo , Ribonucleoproteínas/fisiología , Espermatogénesis , Animales , Autofagosomas/fisiología , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Humanos , Lisosomas/metabolismo , Ratones , Ribonucleoproteínas/metabolismoRESUMEN
Macroautophagy/autophagy is known to be important for intracellular quality control in the lens. GJA8 is a major gap junction protein in vertebrate lenses. Mutations in GJA8 cause cataracts in humans. The well-known cataractogenesis mechanism is that mutated GJA8 leads to abnormal assembly of gap junctions, resulting in defects in intercellular communication among lens cells. In this study, we observed that ablation of Gja8b (a homolog of mammalian GJA8) in zebrafish led to severe defects in organelle degradation, an important cause of cataractogenesis in developing lens. The role of autophagy in organelle degradation in lens remains disputable. Intriguingly, we also observed that ablation of Gja8b induced deficient autophagy in the lens. More importantly, in vivo treatment of zebrafish with rapamycin, an autophagy activator that inhibits MAPK/JNK and MTORC1 signaling, stimulated autophagy in the lens and relieved the defects in organelle degradation, resulting in the mitigation of cataracts in gja8b mutant zebrafish. Conversely, inhibition of autophagy by treatment with the chemical reagent 3-MA blocked these recovery effects, suggesting the important roles of autophagy in organelle degradation in the lens in gja8b mutant zebrafish. Further studies in HLE cells revealed that GJA8 interacted with ATG proteins. Overexpression of GJA8 stimulated autophagy in HLE cells. These data suggest an unrecognized cataractogenesis mechanism caused by ablation of Gja8b and a potential treatment for cataracts by stimulating autophagy in the lens.Abbreviations: 3-MA: 3-methyladenine; ATG: autophagy related; AV: autophagic vacuoles; Dpf: days post fertilization; GJA1: gap junction protein alpha 1; GJA3: gap junction protein alpha 3; GJA8: gap junction protein alpha 8; Hpf: hours post fertilization; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; PtdIns3K: class III phosphatidylinositol 3-kinase; WT: wild type.
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Autofagia/efectos de los fármacos , Autofagia/genética , Catarata/genética , Conexinas/antagonistas & inhibidores , Conexinas/genética , Sirolimus/farmacología , Proteínas de Pez Cebra/antagonistas & inhibidores , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Pez Cebra/fisiología , Adenina/análogos & derivados , Adenina/farmacología , Animales , Animales Modificados Genéticamente , Autofagia/fisiología , Catarata/patología , Catarata/fisiopatología , Conexinas/fisiología , Cristalino/anomalías , Cristalino/crecimiento & desarrollo , Cristalino/fisiología , Mutación , Proteínas de Pez Cebra/fisiologíaAsunto(s)
Cicatriz/diagnóstico por imagen , Lesiones Oculares/diagnóstico por imagen , Cristalino/diagnóstico por imagen , Cristalino/crecimiento & desarrollo , Cámara Anterior/diagnóstico por imagen , Cicatriz/complicaciones , Lesiones Oculares/complicaciones , Humanos , Masculino , Persona de Mediana Edad , Tomografía de Coherencia ÓpticaRESUMEN
Mutations in cis-regulatory elements play important roles for phenotypic changes during evolution. Eye degeneration in the blind mole rat (BMR; Nannospalax galili) and other subterranean mammals is significantly associated with widespread divergence of eye regulatory elements, but the effect of these regulatory mutations on eye development and function has not been explored. Here, we investigate the effect of mutations observed in the BMR sequence of a conserved noncoding element upstream of Tdrd7, a pleiotropic gene required for lens development and spermatogenesis. We first show that this conserved element is a transcriptional repressor in lens cells and that the BMR sequence partially lost repressor activity. Next, we recapitulated evolutionary changes in this element by precisely replacing the endogenous regulatory element in a mouse line by the orthologous BMR sequence with CRISPR-Cas9. Strikingly, this repressor replacement caused a more than 2-fold upregulation of Tdrd7 in the developing lens; however, increased mRNA level does not result in a corresponding increase in TDRD7 protein nor an obvious lens phenotype, possibly explained by buffering at the posttranscriptional level. Our results are consistent with eye degeneration in subterranean mammals having a polygenic basis where many small-effect mutations in different eye-regulatory elements collectively contribute to phenotypic differences.
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Evolución Molecular , Cristalino/metabolismo , Ratas Topo/genética , Elementos Reguladores de la Transcripción/genética , Ribonucleoproteínas/genética , Animales , Femenino , Cristalino/crecimiento & desarrollo , Masculino , Ratones Transgénicos , Ribonucleoproteínas/metabolismoRESUMEN
Purpose: To reveal age-related changes of the human crystalline lens by using high-spatial resolution T1-weighted brain magnetic resonance imaging of patients under general anesthesia. Methods: We retrospectively identified 47 children (2-17 years) and 30 adults (18-70 years) without diabetes or eye disease, who required brain magnetic resonance imaging examinations under general anesthesia between 2012 and 2019. Normalized signal intensity of the crystalline lens and vitreous body, as well as equatorial diameter and axial thickness of the lens were assessed by using a three-dimensional T1-weighted magnetization prepared rapid acquisition gradient echo sequence of the brain with 0.9-mm spatial resolution. Patient dossiers were reviewed to record indication for magnetic resonance imaging examination and hypertension. Results: Advancing age was significantly correlated with increasing equatorial diameter of the infantile lens (r = 0. 74; 95% confidence interval, 0.58-0.85; P < .0001) and increasing crystalline lens signal intensity of the adult lens (r = 0.38; 95% confidence interval, 0.02-0.65; P = .0382), which remained significant after accounting for potential confounding variables. There was no significant correlation between age and axial thickness or vitreous body signal intensity in the children and adult cohort. Conclusions: The present study demonstrated that advancing age was significantly correlated with an increasing equatorial diameter of the infantile lens and with increasing crystalline lens signal intensity of the adult lens. These normative data can contribute to our understanding of age-related changes in eye health and function, especially in regard to the emmetropization process and should also be taken into account when investigating lens pathologies.
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Encéfalo/diagnóstico por imagen , Cristalino/anatomía & histología , Imagen por Resonancia Magnética , Neuroimagen , Adolescente , Adulto , Factores de Edad , Anciano , Niño , Preescolar , Femenino , Humanos , Imagenología Tridimensional , Cristalino/crecimiento & desarrollo , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Adulto JovenRESUMEN
The unique cellular organization and transparent function of the ocular lens depend on the continuous differentiation of immature epithelial cells on the lens anterior surface into mature elongated fiber cells within the lens core. A ubiquitous event during lens differentiation is the complete elimination of organelles required for mature lens fiber cell structure and transparency. Distinct pathways have been identified to mediate the elimination of non-nuclear organelles and nuclei. Recently, we reported the discovery of a unique structure in developing fiber cells of the chick embryo lens, called the Nuclear Excisosome, that is intractably associated with degrading nuclei during lens fiber cell differentiation. In the chick lens, the Nuclear Excisosome is derived from projections of adjacent cells contacting the nuclear envelope during nuclear elimination. Here, we demonstrate that, in contrast to the avian model, Nuclear Excisosomes in a primate model, Galago (bush baby) monkeys, are derived through the recruitment of mitochondria to form unique linear assemblies that define a novel primate Nuclear Excisosome. Four lenses from three monkeys aged 2-5 years were fixed in formalin, followed by paraformaldehyde, then processed for Airyscan confocal microscopy or transmission electron microscopy. For confocal imaging, fluorescent dyes labelled membranes, carbohydrate in the extracellular space, filamentous actin and nuclei. Fiber cells from Galago lenses typically displayed prominent linear structures within the cytoplasm with a distinctive cross-section of four membranes and lengths up to 30 µm. The outer membranes of these linear structures were observed to attach to the outer nuclear envelope membrane to initiate degradation near the organelle-free zone. The origin of these unique structures was mitochondria in the equatorial epithelium (not from plasma membranes of adjacent cells as in the chick embryo model). Early changes in mitochondria appeared to be the collapse of the cristae and modification of one side of the mitochondrial outer membrane to promote accumulation of protein in a dense cluster. As a mitochondrion surrounded the dense protein cluster, an outer mitochondrial membrane enclosed the protein to form a core and another outer mitochondrial membrane formed the outermost layer. The paired membranes of irregular texture between the inner core membrane and the outer limiting membrane appeared to be derived from modified mitochondrial cristae. Several mitochondria were involved in the formation and maturation of these unique complexes that apparently migrated around the fulcrum into the cytoplasm of nascent fiber cells where they were stabilized until the nuclear degradation was initiated. Thus, unlike in the chick embryo, the Galago lenses degraded nuclear envelopes with a Nuclear Excisosome derived from multiple mitochondria in the epithelium that formed novel linear assemblies in developing fiber cells. These findings suggest that recruitment of distinct structures is required for Nuclear Excisosome formation in different species.
Asunto(s)
Núcleo Celular/ultraestructura , Cristalino/ultraestructura , Mitocondrias/metabolismo , Actinas/metabolismo , Animales , Diferenciación Celular , Núcleo Celular/metabolismo , Espacio Extracelular/metabolismo , Galago , Cristalino/crecimiento & desarrollo , Cristalino/metabolismoRESUMEN
The homeodomain transcription factors (TFs) Pax6 (OMIM: 607108) and Prox1 (OMIM: 601546) critically regulate gene expression in lens development. While PAX6 mutations in humans can cause cataract, aniridia, microphthalmia, and anophthalmia, among other defects, Prox1 deletion in mice causes severe lens abnormalities, in addition to other organ defects. Furthermore, the optimal dosage/spatiotemporal expression of these key TFs is essential for development. In lens development, Pax6 expression is elevated in cells of the anterior epithelium compared to fiber cells, while Prox1 exhibits the opposite pattern. Whether post-transcriptional regulatory mechanisms control these precise TF expression patterns is unknown. Here, we report the unprecedented finding that the cataract-linked RNA-binding protein (RBP), Celf1 (OMIM: 601074), post-transcriptionally regulates Pax6 and Prox1 protein expression in lens development. Immunostaining shows that Celf1 lens-specific conditional knockout (Celf1cKO) mice exhibit abnormal elevation of Pax6 protein in fiber cells and abnormal Prox1 protein levels in epithelial cells-directly opposite to their normal expression patterns in development. Furthermore, RT-qPCR shows no change in Pax6 and Prox1 transcript levels in Celf1cKO lenses, suggesting that Celf1 regulates these TFs on the translational level. Indeed, RNA-immunoprecipitation assays using Celf1 antibody indicate that Celf1 protein binds to Pax6 and Prox1 transcripts. Furthermore, reporter assays in Celf1 knockdown and Celf1-overexpression cells demonstrate that Celf1 negatively controls Pax6 and Prox1 translation via their 3' UTRs. These data define a new mechanism of RBP-based post-transcriptional regulation that enables precise control over spatiotemporal expression of Pax6 and Prox1 in lens development, thereby uncovering a new etiological mechanism for Celf1 deficiency-based cataract.
Asunto(s)
Proteínas CELF1/genética , Catarata/genética , Proteínas de Homeodominio/genética , Cristalino/metabolismo , Factor de Transcripción PAX6/genética , Proteínas Supresoras de Tumor/genética , Animales , Proteínas CELF1/antagonistas & inhibidores , Proteínas CELF1/deficiencia , Catarata/patología , Diferenciación Celular/genética , Células Epiteliales/metabolismo , Células Epiteliales/patología , Proteínas del Ojo/antagonistas & inhibidores , Proteínas del Ojo/genética , Regulación del Desarrollo de la Expresión Génica/genética , Humanos , Cristalino/crecimiento & desarrollo , Ratones , Ratones Noqueados , Proteínas de Unión al ARN/genéticaRESUMEN
Purpose: Studying the full shape crystalline lens geometry is important to understand the changes undergone by the crystalline lens leading to presbyopia, cataract, or failure of emmetropization, and to aid in the design and selection of intraocular lenses and new strategies for correction. We used custom-developed three-dimensional (3-D) quantitative optical coherence tomography (OCT) to study age-related changes in the full shape of the isolated human crystalline lens. Methods: A total of 103 ex vivo human isolated lenses from 87 subjects (age range, 0-56 years) were imaged using a 3-D spectral-domain OCT system. Lens models, constructed after segmentation of the surfaces and distortion correction, were used to automatically quantify central geometric parameters (lens thickness, radii of curvatures, and asphericities of anterior and posterior surfaces) and full shape parameters (lens volume, surface area, diameter, and equatorial plane position). Age-dependencies of these parameters were studied. Results: Most of the measured parameters showed a biphasic behavior, statistically significantly increasing (radii of curvature, lens volume, surface area, diameter) or decreasing (asphericities, lens thickness) very fast in the first two decades of life, followed by a slow but significant increase after age 20 years (for all the parameters except for the posterior surface asphericity and the equatorial plane position, that remained constant). Conclusions: Three-dimensional quantitative OCT allowed us to study the age-dependency of geometric parameters of the full isolated human crystalline lens. We found that most of the lens geometric parameters showed a biphasic behavior, changing rapidly before age 20 years and with a slower linear growth thereafter.