RESUMEN
Mutations in the genes encoding the Wnt receptor Frizzled-4 (FZD4), coreceptor LRP5, or the ligand Norrin disrupt retinal vascular development and cause ophthalmic diseases. Although Norrin is structurally unrelated to Wnts, it binds FZD4 and activates the canonical Wnt pathway. Here we show that the tetraspanin Tspan12 is expressed in the retinal vasculature, and loss of Tspan12 phenocopies defects seen in Fzd4, Lrp5, and Norrin mutant mice. In addition, Tspan12 genetically interacts with Norrin or Lrp5. Overexpressed TSPAN12 associates with the Norrin-receptor complex and significantly increases Norrin/beta-catenin but not Wnt/beta-catenin signaling, whereas Tspan12 siRNA abolishes transcriptional responses to Norrin but not Wnt3A in retinal endothelial cells. Signaling defects caused by Norrin or FZD4 mutations that are predicted to impair receptor multimerization are rescued by overexpression of TSPAN12. Our data indicate that Norrin multimers and TSPAN12 cooperatively promote multimerization of FZD4 and its associated proteins to elicit physiological levels of signaling.
Asunto(s)
Receptores Frizzled/metabolismo , Proteínas de la Membrana/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Retina/embriología , Transducción de Señal , beta Catenina/metabolismo , Animales , Diterpenos , Células Endoteliales/metabolismo , Receptores Frizzled/genética , Humanos , Ratones , Receptores Acoplados a Proteínas G/genética , Tetraspaninas , beta Catenina/genéticaRESUMEN
BACKGROUND: Macular corneal dystrophy (MCD) is a rare autosomal recessive disorder that is characterized by progressive corneal opacity that starts in early childhood and ultimately progresses to blindness in early adulthood. The aim of this study was to identify the cause of MCD in a black South African family with two affected sisters. METHODS: A multigenerational South African Sotho-speaking family with type I MCD was studied using whole exome sequencing. Variant filtering to identify the MCD-causal mutation included the disease inheritance pattern, variant minor allele frequency and potential functional impact. RESULTS: Ophthalmologic evaluation of the cases revealed a typical MCD phenotype and none of the other family members were affected. An average of 127 713 variants per individual was identified following exome sequencing and approximately 1.2 % were not present in any of the investigated public databases. Variant filtering identified a homozygous E71Q mutation in CHST6, a known MCD-causing gene encoding corneal N-acetyl glucosamine-6-O-sulfotransferase. This E71Q mutation results in a non-conservative amino acid change in a highly conserved functional domain of the human CHST6 that is essential for enzyme activity. CONCLUSION: We identified a novel E71Q mutation in CHST6 as the MCD-causal mutation in a black South African family with type I MCD. This is the first description of MCD in a black Sub-Saharan African family and therefore contributes valuable insights into the genetic aetiology of this disease, while improving genetic counselling for this and potentially other MCD families.
Asunto(s)
Distrofias Hereditarias de la Córnea/genética , Mutación , Sulfotransferasas/genética , Adulto , Córnea/patología , Distrofias Hereditarias de la Córnea/patología , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Homocigoto , Humanos , Masculino , Linaje , Fenotipo , Polimorfismo de Nucleótido Simple , Sudáfrica , Carbohidrato SulfotransferasasRESUMEN
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.
Asunto(s)
Ceguera/congénito , Proteínas del Ojo/genética , Factores de Transcripción de Tipo Kruppel/genética , Proteínas del Tejido Nervioso/genética , Enfermedades del Sistema Nervioso/genética , Retina/metabolismo , Espasmos Infantiles/genética , Vía de Señalización Wnt , Animales , Ceguera/genética , Ceguera/metabolismo , Ciclo Celular/genética , Proliferación Celular , Epistasis Genética , Proteínas del Ojo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Enfermedades Genéticas Ligadas al Cromosoma X , Proteínas Hedgehog/metabolismo , Humanos , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones , Ratones Transgénicos , Mutación , Neovascularización Fisiológica , Proteínas del Tejido Nervioso/metabolismo , Enfermedades del Sistema Nervioso/metabolismo , Unión Proteica , Retina/crecimiento & desarrollo , Degeneración Retiniana , Vasos Retinianos/crecimiento & desarrollo , Vasos Retinianos/metabolismo , Espasmos Infantiles/metabolismo , Vía de Señalización Wnt/genética , Proteína Gli2 con Dedos de ZincRESUMEN
The tumor microenvironment is a critical modulator of carcinogenesis; however, in many tumor types, the influence of the stroma during preneoplastic stages is unknown. Here we explored the relationship between pre-tumor cells and their surrounding stroma in malignant progression of the cerebellar tumor medulloblastoma (MB). We show that activation of the vascular regulatory signalling axis mediated by Norrin (an atypical Wnt)/Frizzled4 (Fzd4) inhibits MB initiation in the Ptch+/- mouse model. Loss of Norrin/Fzd4-mediated signalling in endothelial cells, either genetically or by short-term blockade, increases the frequency of pre-tumor lesions and creates a tumor-permissive microenvironment at the earliest, preneoplastic stages of MB. This pro-tumor stroma, characterized by angiogenic remodelling, is associated with an accelerated transition from preneoplasia to malignancy. These data expose a stromal component that regulates the earliest stages of tumorigenesis in the cerebellum, and a novel role for the Norrin/Fzd4 axis as an endogenous anti-tumor signal in the preneoplastic niche.
Asunto(s)
Carcinogénesis , Proteínas del Ojo/metabolismo , Receptores Frizzled/metabolismo , Meduloblastoma/fisiopatología , Proteínas del Tejido Nervioso/metabolismo , Transducción de Señal , Animales , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , RatonesRESUMEN
PURPOSE. Mice deficient in the secreted protein Norrin or its receptor Frizzled-4 (FZD4) exhibit incomplete vascularization of the neural retina. However, because of early retinal vascular defects in the knockout models, it has not been possible to study FZD4 contribution in ocular neovascular disease. To further understand the role of this signaling pathway in physiological and pathologic angiogenesis, the authors generated a monoclonal antibody that neutralizes FZD4 function in vivo. METHODS. Antibodies were generated by immunizing Fzd4 knockout mice with the cysteine-rich domain of FZD4. A monoclonal antibody (1.99.25) was discovered that antagonizes Norrin- and WNT3A-induced ß-catenin accumulation in vitro. 1.99.25 and an isotype-matched negative control antibody were evaluated in models of developmental retinal angiogenesis, oxygen-induced retinopathy, and retinal angiomatous proliferation. The authors also investigated the role of FZD4 in maintaining the blood-retina barrier in normal adult mice. RESULTS. Administration of 1.99.25 inhibited physiological and pathologic sprouting angiogenesis within the retina. Inhibition of FZD4 in developing retinal vascular networks caused the upregulation of PLVAP, a protein normally associated with fenestrated, immature endothelium in the CNS. In the adult neural retina, the administration of 1.99.25 induced PLVAP expression in the deep capillary bed and enabled extravasation of small and large molecules through the blood-retina barrier. CONCLUSIONS. These results demonstrate that FZD4 is required for physiological and pathologic angiogenesis in the retina and for regulation of retinal endothelial cell differentiation. The authors also show that FZD4 is critical for maintaining the integrity of the mature blood-retina barrier.
Asunto(s)
Barrera Hematorretinal/fisiología , Modelos Animales de Enfermedad , Receptores Frizzled/fisiología , Neovascularización Fisiológica/fisiología , Receptores Acoplados a Proteínas G/fisiología , Neovascularización Retiniana/metabolismo , Animales , Animales Recién Nacidos , Anticuerpos Neutralizantes/farmacología , Permeabilidad Capilar/fisiología , Proteínas del Ojo/antagonistas & inhibidores , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Receptores Frizzled/antagonistas & inhibidores , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neovascularización Fisiológica/efectos de los fármacos , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Oxígeno/toxicidad , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores de LDL/metabolismo , Neovascularización Retiniana/inducido químicamente , Neovascularización Retiniana/prevención & control , Vasos Retinianos/efectos de los fármacos , Vasos Retinianos/fisiologíaRESUMEN
During sprouting angiogenesis, groups of endothelial cells (ECs) migrate together in units called sprouts. In this study, we demonstrate that the vascular-specific secreted factor EGFL7 regulates the proper spatial organization of ECs within each sprout and influences their collective movement. In the homozygous Egfl7-knockout mice, vascular development is delayed in many organs despite normal EC proliferation, and 50% of the knockout embryos die in utero. ECs in the mutant vasculatures form abnormal aggregates and the vascular basement membrane marker collagen IV is mislocalized, suggesting that ECs fail to recognize the proper spatial position of their neighbors. Although the migratory ability of individual ECs in isolation is not affected by the loss of EGFL7, the aberrant spatial organization of ECs in the mutant tissues decreases their collective movement. Using in vitro and in vivo analyses, we showed that EGFL7 is a component of the interstitial extracellular matrix deposited on the basal sides of sprouts, a location suitable for conveying positional information to neighboring ECs. Taken together, we propose that EGFL7 defines the optimal path of EC movement by assuring the correct positioning of each EC in a nascent sprout.