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1.
Development ; 147(24)2020 12 28.
Artigo em Inglês | MEDLINE | ID: mdl-33158926

RESUMO

Ocular coloboma is a congenital eye malformation, resulting from a failure in optic fissure closure (OFC) and causing visual impairment. There has been little study of the epithelial fusion process underlying closure in the human embryo and coloboma aetiology remains poorly understood. We performed RNAseq of cell populations isolated using laser capture microdissection to identify novel human OFC signature genes and probe the expression profile of known coloboma genes, along with a comparative murine analysis. Gene set enrichment patterns showed conservation between species. Expression of genes involved in epithelial-to-mesenchymal transition was transiently enriched in the human fissure margins during OFC at days 41-44. Electron microscopy and histological analyses showed that cells transiently delaminate at the point of closure, and produce cytoplasmic protrusions, before rearranging to form two continuous epithelial layers. Apoptosis was not observed in the human fissure margins. These analyses support a model of human OFC in which epithelial cells at the fissure margins undergo a transient epithelial-to-mesenchymal-like transition, facilitating cell rearrangement to form a complete optic cup.


Assuntos
Coloboma/genética , Anormalidades do Olho/genética , Olho/ultraestrutura , Disco Óptico/ultraestrutura , Animais , Apoptose/genética , Sequência de Bases/genética , Coloboma/patologia , Transição Epitelial-Mesenquimal/genética , Olho/patologia , Anormalidades do Olho/patologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Microdissecção e Captura a Laser , Camundongos , Microscopia Eletrônica
2.
Semin Cell Dev Biol ; 91: 55-65, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-29198497

RESUMO

Embryonic development of the vertebrate eye begins with the formation of an optic vesicle which folds inwards to form a double-layered optic cup with a fissure on the ventral surface, known as the optic fissure. Closure of the optic fissure is essential for subsequent growth and development of the eye. A defect in this process can leave a gap in the iris, retina or optic nerve, known as a coloboma, which can lead to severe visual impairment. This review brings together current information about genes and pathways regulating fissure closure from human coloboma patients and animal models. It focuses especially on current understanding of the morphological changes and processes of epithelial remodelling occurring at the fissure margins.


Assuntos
Coloboma/embriologia , Olho/embriologia , Disco Óptico/embriologia , Transtornos da Visão/embriologia , Animais , Coloboma/genética , Olho/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Morfogênese/genética , Disco Óptico/metabolismo , Transdução de Sinais/genética , Transtornos da Visão/genética
3.
Ophthalmology ; 126(6): 888-907, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30653986

RESUMO

PURPOSE: To develop a comprehensive next-generation sequencing panel assay that screens genes known to cause developmental eye disorders and inherited eye disease and to evaluate its diagnostic yield in a pediatric cohort with malformations of the globe, anterior segment anomalies, childhood glaucoma, or a combination thereof. DESIGN: Evaluation of diagnostic test. PARTICIPANTS: Two hundred seventy-seven children, 0 to 16 years of age, diagnosed with nonsyndromic or syndromic developmental eye defects without a genetic diagnosis. METHODS: We developed a new oculome panel using a custom-designed Agilent SureSelect QXT target capture method (Agilent Technologies, Santa Clara, CA) to capture and perform parallel high-throughput sequencing analysis of 429 genes associated with eye disorders. Bidirectional Sanger sequencing confirmed suspected pathogenic variants. MAIN OUTCOME MEASURES: Collated clinical details and oculome molecular genetic results. RESULTS: The oculome design covers 429 known eye disease genes; these are subdivided into 5 overlapping virtual subpanels for anterior segment developmental anomalies including glaucoma (ASDA; 59 genes), microphthalmia-anophthalmia-coloboma (MAC; 86 genes), congenital cataracts and lens-associated conditions (70 genes), retinal dystrophies (RET; 235 genes), and albinism (15 genes), as well as additional genes implicated in optic atrophy and complex strabismus (10 genes). Panel development and testing included analyzing 277 clinical samples and 3 positive control samples using Illumina sequencing platforms; more than 30× read depth was achieved for 99.5% of the targeted 1.77-Mb region. Bioinformatics analysis performed using a pipeline based on Freebayes and ExomeDepth to identify coding sequence and copy number variants, respectively, resulted in a definitive diagnosis in 68 of 277 samples, with variability in diagnostic yield between phenotypic subgroups: MAC, 8.2% (8 of 98 cases solved); ASDA, 24.8% (28 of 113 cases solved); other or syndromic, 37.5% (3 of 8 cases solved); RET, 42.8% (21 of 49 cases solved); and congenital cataracts and lens-associated conditions, 88.9% (8 of 9 cases solved). CONCLUSIONS: The oculome test diagnoses a comprehensive range of genetic conditions affecting the development of the eye, potentially replacing protracted and costly multidisciplinary assessments and allowing for faster targeted management. The oculome enabled molecular diagnosis of a significant number of cases in our sample cohort of varied ocular birth defects.


Assuntos
Variações do Número de Cópias de DNA/genética , Anormalidades do Olho/diagnóstico , Anormalidades do Olho/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Técnicas de Diagnóstico Molecular , Mutação/genética , Proteoma/genética , Adolescente , Criança , Pré-Escolar , Feminino , Genoma Humano , Humanos , Lactente , Recém-Nascido , Masculino , Linhagem
4.
Hum Mol Genet ; 23(10): 2511-26, 2014 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-24412933

RESUMO

Ocular coloboma is a congenital defect resulting from failure of normal closure of the optic fissure during embryonic eye development. This birth defect causes childhood blindness worldwide, yet the genetic etiology is poorly understood. Here, we identified a novel homozygous mutation in the SALL2 gene in members of a consanguineous family affected with non-syndromic ocular coloboma variably affecting the iris and retina. This mutation, c.85G>T, introduces a premature termination codon (p.Glu29*) predicted to truncate the SALL2 protein so that it lacks three clusters of zinc-finger motifs that are essential for DNA-binding activity. This discovery identifies SALL2 as the third member of the Drosophila homeotic Spalt-like family of developmental transcription factor genes implicated in human disease. SALL2 is expressed in the developing human retina at the time of, and subsequent to, optic fissure closure. Analysis of Sall2-deficient mouse embryos revealed delayed apposition of the optic fissure margins and the persistence of an anterior retinal coloboma phenotype after birth. Sall2-deficient embryos displayed correct posterior closure toward the optic nerve head, and upon contact of the fissure margins, dissolution of the basal lamina occurred and PAX2, known to be critical for this process, was expressed normally. Anterior closure was disrupted with the fissure margins failing to meet, or in some cases misaligning leading to a retinal lesion. These observations demonstrate, for the first time, a role for SALL2 in eye morphogenesis and that loss of function of the gene causes ocular coloboma in humans and mice.


Assuntos
Códon sem Sentido , Coloboma/genética , Fatores de Transcrição/genética , Adolescente , Animais , Criança , Consanguinidade , Análise Mutacional de DNA , Proteínas de Ligação a DNA , Olho/embriologia , Olho/patologia , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Feminino , Expressão Gênica , Genes Recessivos , Estudos de Associação Genética , Predisposição Genética para Doença , Células HEK293 , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Homozigoto , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator de Transcrição PAX2/genética , Fator de Transcrição PAX2/metabolismo , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Linhagem , Polimorfismo de Nucleotídeo Único , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo
6.
Nat Commun ; 15(1): 3567, 2024 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-38670973

RESUMO

The emergence of retinal progenitor cells and differentiation to various retinal cell types represent fundamental processes during retinal development. Herein, we provide a comprehensive single cell characterisation of transcriptional and chromatin accessibility changes that underline retinal progenitor cell specification and differentiation over the course of human retinal development up to midgestation. Our lineage trajectory data demonstrate the presence of early retinal progenitors, which transit to late, and further to transient neurogenic progenitors, that give rise to all the retinal neurons. Combining single cell RNA-Seq with spatial transcriptomics of early eye samples, we demonstrate the transient presence of early retinal progenitors in the ciliary margin zone with decreasing occurrence from 8 post-conception week of human development. In retinal progenitor cells, we identified a significant enrichment for transcriptional enhanced associate domain transcription factor binding motifs, which when inhibited led to loss of cycling progenitors and retinal identity in pluripotent stem cell derived organoids.


Assuntos
Diferenciação Celular , Retina , Análise de Célula Única , Células-Tronco , Humanos , Análise de Célula Única/métodos , Retina/citologia , Retina/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Organoides/metabolismo , Organoides/citologia , Regulação da Expressão Gênica no Desenvolvimento , Cromatina/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , RNA-Seq , Linhagem da Célula , Transcriptoma
7.
EMBO Mol Med ; 15(10): e17393, 2023 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-37642150

RESUMO

Deafness affects 5% of the world's population, yet there is a lack of treatments to prevent hearing loss due to genetic causes. Norrie disease is a recessive X-linked disorder, caused by NDP gene mutation. It manifests as blindness at birth and progressive sensorineural hearing loss, leading to debilitating dual sensory deprivation. To develop a gene therapy, we used a Norrie disease mouse model (Ndptm1Wbrg ), which recapitulates abnormal retinal vascularisation and progressive hearing loss. We delivered human NDP cDNA by intravenous injection of adeno-associated viral vector (AAV)9 at neonatal, juvenile and young adult pathological stages and investigated its therapeutic effects on the retina and cochlea. Neonatal treatment prevented the death of the sensory cochlear hair cells and rescued cochlear disease biomarkers as demonstrated by RNAseq and physiological measurements of auditory function. Retinal vascularisation and electroretinograms were restored to normal by neonatal treatment. Delivery of NDP gene therapy after the onset of the degenerative inner ear disease also ameliorated the cochlear pathology, supporting the feasibility of a clinical treatment for progressive hearing loss in people with Norrie disease.

8.
Front Genet ; 13: 884722, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35651932

RESUMO

Aims and Rationale: The inner retina is supplied by three intraretinal capillary plexi whereas the outer retina is supplied by the choroidal circulation: NDP is essential for normal intraretinal vascularisation. Pathogenic variants in NDP (Xp11.3) may result in either a severe retinal phenotype associated with hearing loss (Norrie Disease) or a moderate retinal phenotype (Familial Exudative Vitreoretinopathy, FEVR). However, little is known about whether the nature or location of the NDP variant is predictive of severity. In this systematic review we summarise all reported NDP variants and draw conclusions about whether the nature of the NDP variant is predictive of the severity of the resulting ocular pathology and associated hearing loss and intellectual disability. Findings: 201 different variants in the NDP gene have been reported as disease-causing. The pathological phenotype that may result from a disease-causing NDP variant is quite diverse but generally comprises a consistent cluster of features (retinal hypovascularisation, exudation, persistent foetal vasculature, tractional/exudative retinal detachment, intellectual disability and hearing loss) that vary predictably with severity. Previous reviews have found no clear pattern in the nature of NDP mutations that cause either FEVR or Norrie disease, with the exception that mutations affecting cysteine residues have been associated with Norrie Disease and that visual loss amongst patients with Norrie disease tends to be more severe if the NDP mutation results in an early termination of translation as opposed to a missense related amino acid change. A key limitation of previous reviews has been variability in the case definition of Norrie disease and FEVR amongst authors. We thus reclassified patients into two groups based only on the severity of their retinal disease. Of the reported pathogenic variants that have been described in more than one patient, we found that any given variant caused an equivalent severity of retinopathy each time it was reported with very few exceptions. We therefore conclude that specific NDP mutations generally result in a consistent retinal phenotype each time they arise. Reports by different authors of the same variant causing either FEVR or Norrie disease conflict primarily due to variability in the authors' respective case definitions rather than true differences in disease severity.

9.
Stem Cell Reports ; 17(11): 2421-2437, 2022 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-36240775

RESUMO

Usher syndrome-associated retinitis pigmentosa (RP) causes progressive retinal degeneration, which has no cure. The pathomechanism of Usher type 1B (USH1B)-RP caused by MYO7A mutation remains elusive because of the lack of faithful animal models and limited knowledge of MYO7A function. Here, we analyzed 3D retinal organoids generated from USH1B patient-derived induced pluripotent stem cells. Increased differential gene expression occurred over time without excessive photoreceptor cell death in USH1B organoids compared with controls. Dysregulated genes were enriched first for mitochondrial functions and then proteasomal ubiquitin-dependent protein catabolic processes and RNA splicing. Single-cell RNA sequencing revealed MYO7A expression in rod photoreceptor and Müller glial cells corresponding to upregulation of stress responses in NRL+ rods and apoptotic signaling pathways in VIM+ Müller cells, pointing to the defensive mechanisms that mitigate photoreceptor cell death. This first human model for USH1B-RP provides a representation of patient retina in vivo relevant for development of therapeutic strategies.


Assuntos
Organoides , Retinose Pigmentar , Animais , Humanos , Miosina VIIa , Organoides/patologia , Patologia Molecular , Miosinas/genética , Miosinas/metabolismo , Retina/metabolismo , Retinose Pigmentar/genética , Retinose Pigmentar/patologia , Células Fotorreceptoras Retinianas Bastonetes/patologia
10.
JCI Insight ; 7(3)2022 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-35132964

RESUMO

Norrie disease is caused by mutation of the NDP gene, presenting as congenital blindness followed by later onset of hearing loss. Protecting patients from hearing loss is critical for maintaining their quality of life. This study aimed to understand the onset of pathology in cochlear structure and function. By investigating patients and juvenile Ndp-mutant mice, we elucidated the sequence of onset of physiological changes (in auditory brainstem responses, distortion product otoacoustic emissions, endocochlear potential, blood-labyrinth barrier integrity) and determined the cellular, histological, and ultrastructural events leading to hearing loss. We found that cochlear vascular pathology occurs earlier than previously reported and precedes sensorineural hearing loss. The work defines a disease mechanism whereby early malformation of the cochlear microvasculature precedes loss of vessel integrity and decline of endocochlear potential, leading to hearing loss and hair cell death while sparing spiral ganglion cells. This provides essential information on events defining the optimal therapeutic window and indicates that early intervention is needed. In an era of advancing gene therapy and small-molecule technologies, this study establishes Ndp-mutant mice as a platform to test such interventions and has important implications for understanding the progression of hearing loss in Norrie disease.


Assuntos
Cegueira/congênito , Gerenciamento Clínico , Potenciais Evocados Auditivos do Tronco Encefálico/fisiologia , Previsões , Doenças Genéticas Ligadas ao Cromossomo X/fisiopatologia , Perda Auditiva Neurossensorial/fisiopatologia , Audição/fisiologia , Doenças do Sistema Nervoso/fisiopatologia , Degeneração Retiniana/fisiopatologia , Espasmos Infantis/fisiopatologia , Adolescente , Adulto , Animais , Cegueira/complicações , Cegueira/fisiopatologia , Cegueira/terapia , Criança , Pré-Escolar , Modelos Animais de Doenças , Feminino , Seguimentos , Doenças Genéticas Ligadas ao Cromossomo X/complicações , Doenças Genéticas Ligadas ao Cromossomo X/terapia , Perda Auditiva Neurossensorial/diagnóstico , Perda Auditiva Neurossensorial/etiologia , Humanos , Masculino , Camundongos , Camundongos Mutantes , Doenças do Sistema Nervoso/complicações , Doenças do Sistema Nervoso/terapia , Degeneração Retiniana/complicações , Degeneração Retiniana/terapia , Espasmos Infantis/complicações , Espasmos Infantis/terapia , Adulto Jovem
11.
Elife ; 82019 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-31162046

RESUMO

Epithelial fusion underlies many vital organogenic processes during embryogenesis. Disruptions to these cause a significant number of human birth defects, including ocular coloboma. We provide robust spatial-temporal staging and unique anatomical detail of optic fissure closure (OFC) in the embryonic chick, including evidence for roles of apoptosis and epithelial remodelling. We performed complementary transcriptomic profiling and show that Netrin-1 (NTN1) is precisely expressed in the chick fissure margin during fusion but is immediately downregulated after fusion. We further provide a combination of protein localisation and phenotypic evidence in chick, humans, mice and zebrafish that Netrin-1 has an evolutionarily conserved and essential requirement for OFC, and is likely to have an important role in palate fusion. Our data suggest that NTN1 is a strong candidate locus for human coloboma and other multi-system developmental fusion defects, and show that chick OFC is a powerful model for epithelial fusion research.


Assuntos
Coloboma/genética , Evolução Molecular , Olho/crescimento & desenvolvimento , Netrina-1/genética , Animais , Apoptose/genética , Embrião de Galinha , Galinhas , Coloboma/patologia , Sequência Conservada/genética , Células Epiteliais/metabolismo , Olho/patologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Camundongos , Palato/crescimento & desenvolvimento , Palato/patologia , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento
12.
Sci Rep ; 9(1): 2314, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30783126

RESUMO

Irreversible photoreceptor cell death is a major cause of blindness in many retinal dystrophies. A better understanding of the molecular mechanisms underlying the progressive loss of photoreceptor cells remains therefore crucial. Abnormal expression of microRNAs (miRNAs) has been linked with the aetiology of a number of retinal dystrophies. However, their role during the degenerative process remains poorly understood. Loss of cone photoreceptors in the human macula has the greatest impact on sight as these cells provide high acuity vision. Using a Chrnb4-cre; Dicerflox/flox conditional knockout mouse (Dicer CKO) to delete Dicer1 from cone cells, we show that cone photoreceptor cells degenerate and die in the Dicer-deleted retina. Embryonic eye morphogenesis appeared normal in Dicer CKO mice. Cone photoreceptor abnormalities were apparent by 3 weeks of age, displaying either very short or absent outer segments. By 4 months 50% of cones were lost and cone function was impaired as assessed by electroretinography (ERG). RNAseq analysis of the Dicer CKO retina revealed altered expression of genes involved in the visual perception pathway. These data show that loss of Dicer1 leads to early-onset cone cell degeneration and suggest that Dicer1 is essential for cone photoreceptor survival and homeostasis.


Assuntos
Morte Celular/fisiologia , Visão de Cores/fisiologia , RNA Helicases DEAD-box/metabolismo , Integrases/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores Nicotínicos/metabolismo , Células Fotorreceptoras Retinianas Cones/citologia , Células Fotorreceptoras Retinianas Cones/metabolismo , Ribonuclease III/metabolismo , Acuidade Visual/fisiologia , Animais , Morte Celular/genética , Visão de Cores/genética , RNA Helicases DEAD-box/genética , Eletrorretinografia , Feminino , Integrases/genética , Masculino , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Receptores Nicotínicos/genética , Ribonuclease III/genética , Acuidade Visual/genética
13.
Stem Cell Reports ; 9(6): 1898-1915, 2017 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-29153988

RESUMO

Loss of cone photoreceptors, crucial for daylight vision, has the greatest impact on sight in retinal degeneration. Transplantation of stem cell-derived L/M-opsin cones, which form 90% of the human cone population, could provide a feasible therapy to restore vision. However, transcriptomic similarities between fetal and stem cell-derived cones remain to be defined, in addition to development of cone cell purification strategies. Here, we report an analysis of the human L/M-opsin cone photoreceptor transcriptome using an AAV2/9.pR2.1:GFP reporter. This led to the identification of a cone-enriched gene signature, which we used to demonstrate similar gene expression between fetal and stem cell-derived cones. We then defined a cluster of differentiation marker combination that, when used for cell sorting, significantly enriches for cone photoreceptors from the fetal retina and stem cell-derived retinal organoids, respectively. These data may facilitate more efficient isolation of human stem cell-derived cones for use in clinical transplantation studies.


Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Degeneração Retiniana/genética , Opsinas de Bastonetes/genética , Transcriptoma/genética , Diferenciação Celular/genética , Feto/citologia , Feto/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Células-Tronco Pluripotentes Induzidas/transplante , Retina/crescimento & desenvolvimento , Retina/metabolismo , Retina/patologia , Células Fotorreceptoras Retinianas Cones/transplante , Degeneração Retiniana/patologia
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