RESUMEN
Coatomer complexes function in the sorting and trafficking of proteins between subcellular organelles. Pathogenic variants in coatomer subunits or associated factors have been reported in multi-systemic disorders, i.e., coatopathies, that can affect the skeletal and central nervous systems. We have identified loss-of-function variants in COPB2, a component of the coatomer complex I (COPI), in individuals presenting with osteoporosis, fractures, and developmental delay of variable severity. Electron microscopy of COPB2-deficient subjects' fibroblasts showed dilated endoplasmic reticulum (ER) with granular material, prominent rough ER, and vacuoles, consistent with an intracellular trafficking defect. We studied the effect of COPB2 deficiency on collagen trafficking because of the critical role of collagen secretion in bone biology. COPB2 siRNA-treated fibroblasts showed delayed collagen secretion with retention of type I collagen in the ER and Golgi and altered distribution of Golgi markers. copb2-null zebrafish embryos showed retention of type II collagen, disorganization of the ER and Golgi, and early larval lethality. Copb2+/- mice exhibited low bone mass, and consistent with the findings in human cells and zebrafish, studies in Copb2+/- mouse fibroblasts suggest ER stress and a Golgi defect. Interestingly, ascorbic acid treatment partially rescued the zebrafish developmental phenotype and the cellular phenotype in Copb2+/- mouse fibroblasts. This work identifies a form of coatopathy due to COPB2 haploinsufficiency, explores a potential therapeutic approach for this disorder, and highlights the role of the COPI complex as a regulator of skeletal homeostasis.
Asunto(s)
Huesos/metabolismo , Proteína Coat de Complejo I/genética , Proteína Coatómero/genética , Discapacidades del Desarrollo/genética , Discapacidad Intelectual/genética , Osteoporosis/genética , Animales , Ácido Ascórbico/farmacología , Huesos/efectos de los fármacos , Huesos/patología , Encéfalo/diagnóstico por imagen , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Niño , Preescolar , Proteína Coat de Complejo I/deficiencia , Proteína Coatómero/química , Proteína Coatómero/deficiencia , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Discapacidades del Desarrollo/diagnóstico por imagen , Discapacidades del Desarrollo/metabolismo , Discapacidades del Desarrollo/patología , Embrión no Mamífero , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/patología , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Regulación del Desarrollo de la Expresión Génica , Aparato de Golgi , Haploinsuficiencia , Humanos , Discapacidad Intelectual/diagnóstico por imagen , Discapacidad Intelectual/metabolismo , Discapacidad Intelectual/patología , Masculino , Ratones , Osteoporosis/tratamiento farmacológico , Osteoporosis/metabolismo , Osteoporosis/patología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Índice de Severidad de la Enfermedad , Pez CebraRESUMEN
SWI/SNF-related intellectual disability disorders (SSRIDDs) are rare neurodevelopmental disorders characterized by developmental disability, coarse facial features, and fifth digit/nail hypoplasia that are caused by pathogenic variants in genes that encode for members of the SWI/SNF (or BAF) family of chromatin remodeling complexes. We have identified 12 individuals with rare variants (10 loss-of-function, 2 missense) in the BICRA (BRD4 interacting chromatin remodeling complex-associated protein) gene, also known as GLTSCR1, which encodes a subunit of the non-canonical BAF (ncBAF) complex. These individuals exhibited neurodevelopmental phenotypes that include developmental delay, intellectual disability, autism spectrum disorder, and behavioral abnormalities as well as dysmorphic features. Notably, the majority of individuals lack the fifth digit/nail hypoplasia phenotype, a hallmark of most SSRIDDs. To confirm the role of BICRA in the development of these phenotypes, we performed functional characterization of the zebrafish and Drosophila orthologs of BICRA. In zebrafish, a mutation of bicra that mimics one of the loss-of-function variants leads to craniofacial defects possibly akin to the dysmorphic facial features seen in individuals harboring putatively pathogenic BICRA variants. We further show that Bicra physically binds to other non-canonical ncBAF complex members, including the BRD9/7 ortholog, CG7154, and is the defining member of the ncBAF complex in flies. Like other SWI/SNF complex members, loss of Bicra function in flies acts as a dominant enhancer of position effect variegation but in a more context-specific manner. We conclude that haploinsufficiency of BICRA leads to a unique SSRIDD in humans whose phenotypes overlap with those previously reported.
Asunto(s)
Proteínas Cromosómicas no Histona/genética , Discapacidades del Desarrollo/genética , Mutación Missense , Fenotipo , Proteínas Supresoras de Tumor/genética , Adolescente , Animales , Niño , Preescolar , Proteínas de Drosophila/genética , Drosophila melanogaster , Femenino , Genes Dominantes , Variación Genética , Haploinsuficiencia , Humanos , Lactante , Masculino , Microscopía Confocal , Neuroglía/metabolismo , Neuronas/metabolismo , Unión Proteica , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
[This corrects the article DOI: 10.1371/journal.pgen.1008841.].
RESUMEN
Hypomyelination, a neurological condition characterized by decreased production of myelin sheets by glial cells, often has no known etiology. Elucidating the genetic causes of hypomyelination provides a better understanding of myelination, as well as means to diagnose, council, and treat patients. Here, we present evidence that YIPPEE LIKE 3 (YPEL3), a gene whose developmental role was previously unknown, is required for central and peripheral glial cell development. We identified a child with a constellation of clinical features including cerebral hypomyelination, abnormal peripheral nerve conduction, hypotonia, areflexia, and hypertrophic peripheral nerves. Exome and genome sequencing revealed a de novo mutation that creates a frameshift in the open reading frame of YPEL3, leading to an early stop codon. We used zebrafish as a model system to validate that YPEL3 mutations are causative of neuropathy. We found that ypel3 is expressed in the zebrafish central and peripheral nervous system. Using CRISPR/Cas9 technology, we created zebrafish mutants carrying a genomic lesion similar to that of the patient. Our analysis revealed that Ypel3 is required for development of oligodendrocyte precursor cells, timely exit of the perineurial glial precursors from the central nervous system (CNS), formation of the perineurium, and Schwann cell maturation. Consistent with these observations, zebrafish ypel3 mutants have metabolomic signatures characteristic of oligodendrocyte and Schwann cell differentiation defects, show decreased levels of Myelin basic protein in the central and peripheral nervous system, and develop defasciculated peripheral nerves. Locomotion defects were observed in adult zebrafish ypel3 mutants. These studies demonstrate that Ypel3 is a novel gene required for perineurial cell development and glial myelination.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/genética , Vaina de Mielina/patología , Neurogénesis/genética , Proteínas Supresoras de Tumor/genética , Animales , Plexo Braquial/diagnóstico por imagen , Niño , Análisis Mutacional de ADN , Modelos Animales de Enfermedad , Embrión no Mamífero , Femenino , Mutación del Sistema de Lectura , Sustancia Gris/diagnóstico por imagen , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/diagnóstico por imagen , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/patología , Humanos , Imagen por Resonancia Magnética , Neuroglía/patología , Oligodendroglía , Nervio Ciático/diagnóstico por imagen , Sustancia Blanca/diagnóstico por imagen , Secuenciación del Exoma , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
SPONASTRIME dysplasia is an autosomal-recessive spondyloepimetaphyseal dysplasia characterized by spine (spondylar) abnormalities, midface hypoplasia with a depressed nasal bridge, metaphyseal striations, and disproportionate short stature. Scoliosis, coxa vara, childhood cataracts, short dental roots, and hypogammaglobulinemia have also been reported in this disorder. Although an autosomal-recessive inheritance pattern has been hypothesized, pathogenic variants in a specific gene have not been discovered in individuals with SPONASTRIME dysplasia. Here, we identified bi-allelic variants in TONSL, which encodes the Tonsoku-like DNA repair protein, in nine subjects (from eight families) with SPONASTRIME dysplasia, and four subjects (from three families) with short stature of varied severity and spondylometaphyseal dysplasia with or without immunologic and hematologic abnormalities, but no definitive metaphyseal striations at diagnosis. The finding of early embryonic lethality in a Tonsl-/- murine model and the discovery of reduced length, spinal abnormalities, reduced numbers of neutrophils, and early lethality in a tonsl-/- zebrafish model both support the hypomorphic nature of the identified TONSL variants. Moreover, functional studies revealed increased amounts of spontaneous replication fork stalling and chromosomal aberrations, as well as fewer camptothecin (CPT)-induced RAD51 foci in subject-derived cell lines. Importantly, these cellular defects were rescued upon re-expression of wild-type (WT) TONSL; this rescue is consistent with the hypothesis that hypomorphic TONSL variants are pathogenic. Overall, our studies in humans, mice, zebrafish, and subject-derived cell lines confirm that pathogenic variants in TONSL impair DNA replication and homologous recombination-dependent repair processes, and they lead to a spectrum of skeletal dysplasia phenotypes with numerous extra-skeletal manifestations.
Asunto(s)
Inestabilidad Cromosómica , Daño del ADN , Variación Genética , Anomalías Musculoesqueléticas/patología , FN-kappa B/genética , Osteocondrodisplasias/patología , Adolescente , Adulto , Alelos , Animales , Células Cultivadas , Niño , Preescolar , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Estudios de Asociación Genética , Humanos , Ratones , Ratones Noqueados , Anomalías Musculoesqueléticas/genética , Osteocondrodisplasias/genética , Secuenciación del Exoma , Adulto Joven , Pez CebraRESUMEN
The conserved oligomeric Golgi (COG) complex is involved in intracellular vesicular transport, and is composed of eight subunits distributed in two lobes, lobe A (COG1-4) and lobe B (COG5-8). We describe fourteen individuals with Saul-Wilson syndrome, a rare form of primordial dwarfism with characteristic facial and radiographic features. All affected subjects harbored heterozygous de novo variants in COG4, giving rise to the same recurrent amino acid substitution (p.Gly516Arg). Affected individuals' fibroblasts, whose COG4 mRNA and protein were not decreased, exhibited delayed anterograde vesicular trafficking from the ER to the Golgi and accelerated retrograde vesicular recycling from the Golgi to the ER. This altered steady-state equilibrium led to a decrease in Golgi volume, as well as morphologic abnormalities with collapse of the Golgi stacks. Despite these abnormalities of the Golgi apparatus, protein glycosylation in sera and fibroblasts from affected subjects was not notably altered, but decorin, a proteoglycan secreted into the extracellular matrix, showed altered Golgi-dependent glycosylation. In summary, we define a specific heterozygous COG4 substitution as the molecular basis of Saul-Wilson syndrome, a rare skeletal dysplasia distinct from biallelic COG4-CDG.
Asunto(s)
Síndrome del Cromosoma X Frágil/genética , Transporte de Proteínas/genética , Proteoglicanos/genética , Proteínas de Transporte Vesicular/genética , Adulto , Sustitución de Aminoácidos/genética , Animales , Animales Modificados Genéticamente/genética , Línea Celular , Niño , Preescolar , Retículo Endoplásmico/genética , Matriz Extracelular/genética , Femenino , Fibroblastos/patología , Glicosilación , Aparato de Golgi/genética , Heterocigoto , Humanos , Lactante , Masculino , Pez CebraRESUMEN
PURPOSE: Growth differentiation factor 11 (GDF11) is a key signaling protein required for proper development of many organ systems. Only one prior study has associated an inherited GDF11 variant with a dominant human disease in a family with variable craniofacial and vertebral abnormalities. Here, we expand the phenotypic spectrum associated with GDF11 variants and document the nature of the variants. METHODS: We present a cohort of six probands with de novo and inherited nonsense/frameshift (4/6 patients) and missense (2/6) variants in GDF11. We generated gdf11 mutant zebrafish to model loss of gdf11 phenotypes and used an overexpression screen in Drosophila to test variant functionality. RESULTS: Patients with variants in GDF11 presented with craniofacial (5/6), vertebral (5/6), neurological (6/6), visual (4/6), cardiac (3/6), auditory (3/6), and connective tissue abnormalities (3/6). gdf11 mutant zebrafish show craniofacial abnormalities and body segmentation defects that match some patient phenotypes. Expression of the patients' variants in the fly showed that one nonsense variant in GDF11 is a severe loss-of-function (LOF) allele whereas the missense variants in our cohort are partial LOF variants. CONCLUSION: GDF11 is needed for human development, particularly neuronal development, and LOF GDF11 alleles can affect the development of numerous organs and tissues.
Asunto(s)
Proteínas Morfogenéticas Óseas , Anomalías Craneofaciales/genética , Factores de Diferenciación de Crecimiento , Animales , Proteínas Morfogenéticas Óseas/genética , Factores de Diferenciación de Crecimiento/genética , Humanos , Mutación Missense , Fenotipo , Columna Vertebral , Pez Cebra/genéticaRESUMEN
Determination of variant pathogenicity represents a major challenge in the era of high-throughput sequencing. Erroneous categorization may result if variants affect genes that are in fact dispensable. We demonstrate that this also applies to rare, apparently unambiguous truncating mutations of an established disease gene. By whole-exome sequencing (WES) in a consanguineous family with congenital non-syndromic deafness, we unexpectedly identified a homozygous nonsense variant, p.Arg1066*, in AHI1, a gene associated with Joubert syndrome (JBTS), a severe recessive ciliopathy. None of four homozygotes expressed any signs of JBTS, and one of them had normal hearing, which also ruled out p.Arg1066* as the cause of deafness. Homozygosity mapping and WES in the only other reported JBTS family with a homozygous C-terminal truncation (p.Trp1088Leufs*16) confirmed AHI1 as disease gene, but based on a more N-terminal missense mutation impairing WD40-repeat formation. Morpholinos against N-terminal zebrafish Ahi1, orthologous to where human mutations cluster, produced a ciliopathy, but targeting near human p.Arg1066 and p.Trp1088 did not. Most AHI1 mutations in JBTS patients result in truncated protein lacking WD40-repeats and the SH3 domain; disease was hitherto attributed to loss of these protein interaction modules. Our findings indicate that normal development does not require the C-terminal SH3 domain. This has far-reaching implications, considering that variants like p.Glu984* identified by preconception screening ('Kingsmore panel') do not necessarily indicate JBTS carriership. Genomes of individuals with consanguineous background are enriched for homozygous variants that may unmask dispensable regions of disease genes and unrecognized false positives in diagnostic large-scale sequencing and preconception carrier screening.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Estudios de Asociación Genética , Mutación , Anomalías Múltiples/diagnóstico , Anomalías Múltiples/genética , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras del Transporte Vesicular , Animales , Encéfalo/patología , Cerebelo/anomalías , Mapeo Cromosómico , Consanguinidad , Análisis Mutacional de ADN , Modelos Animales de Enfermedad , Evolución Molecular , Exoma , Anomalías del Ojo/diagnóstico , Anomalías del Ojo/genética , Femenino , Orden Génico , Genes Recesivos , Sitios Genéticos , Heterocigoto , Secuenciación de Nucleótidos de Alto Rendimiento , Homocigoto , Humanos , Enfermedades Renales Quísticas/diagnóstico , Enfermedades Renales Quísticas/genética , Imagen por Resonancia Magnética , Masculino , Modelos Moleculares , Linaje , Conformación Proteica , Retina/anomalías , Pez Cebra/genéticaRESUMEN
We describe a consanguineous Iraqi family with Leber congenital amaurosis (LCA), Joubert syndrome (JBTS), and polycystic kidney disease (PKD). Targeted next-generation sequencing for excluding mutations in known LCA and JBTS genes, homozygosity mapping, and whole-exome sequencing identified a homozygous missense variant, c.317G>C (p.Arg106Pro), in POC1B, a gene essential for ciliogenesis, basal body, and centrosome integrity. In silico modeling suggested a requirement of p.Arg106 for the formation of the third WD40 repeat and a protein interaction interface. In human and mouse retina, POC1B localized to the basal body and centriole adjacent to the connecting cilium of photoreceptors and in synapses of the outer plexiform layer. Knockdown of Poc1b in zebrafish caused cystic kidneys and retinal degeneration with shortened and reduced photoreceptor connecting cilia, compatible with the human syndromic ciliopathy. A recent study describes homozygosity for p.Arg106ProPOC1B in a family with nonsyndromic cone-rod dystrophy. The phenotype associated with homozygous p.Arg106ProPOC1B may thus be highly variable, analogous to homozygous p.Leu710Ser in WDR19 causing either isolated retinitis pigmentosa or Jeune syndrome. Our study indicates that POC1B is required for retinal integrity, and we propose POC1B mutations as a probable cause for JBTS with severe PKD.
Asunto(s)
Proteínas de Ciclo Celular/genética , Enfermedades Cerebelosas/genética , Anomalías del Ojo/genética , Enfermedades Renales Quísticas/genética , Mutación , Retina/anomalías , Anomalías Múltiples , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Proteínas de Ciclo Celular/metabolismo , Enfermedades Cerebelosas/metabolismo , Enfermedades Cerebelosas/patología , Cerebelo/anomalías , Niño , Cilios/metabolismo , Cilios/ultraestructura , Anomalías del Ojo/metabolismo , Anomalías del Ojo/patología , Técnicas de Silenciamiento del Gen , Humanos , Irak , Riñón/patología , Enfermedades Renales Quísticas/metabolismo , Enfermedades Renales Quísticas/patología , Amaurosis Congénita de Leber/genética , Amaurosis Congénita de Leber/metabolismo , Masculino , Ratones , Datos de Secuencia Molecular , Linaje , Retina/metabolismo , Retina/patología , Pez CebraRESUMEN
In adaptating to perpetual darkness, cave species gradually lose eyes and body pigmentation and evolve alternatives for exploring their environments. Although troglodyte features evolved independently many times in cavefish, we do not yet know whether independent evolution of these characters involves common genetic mechanisms. Surface-dwelling and many cave-dwelling species make the freshwater teleost genus Sinocyclocheilus an excellent model for studying the evolution of adaptations to life in constant darkness. We compared the mature retinal histology of surface and cave species in Sinocyclocheilus and found that adult cavefish showed a reduction in the number and length of photoreceptor cells. To identify genes and genetic pathways that evolved in constant darkness, we used RNA-seq to compare eyes of surface and cave species. De novo transcriptome assemblies were developed for both species, and contigs were annotated with gene ontology. Results from cave-dwelling Sinocyclocheilus revealed reduced transcription of phototransduction and other genes important for retinal function. In contrast to the blind Mexican tetra cavefish Astyanax mexicanus, our results on morphologies and gene expression suggest that evolved retinal reduction in cave-dwelling Sinocyclocheilus occurs in a lens-independent fashion by the reduced proliferation and downregulation of transcriptional factors shown to have direct roles in retinal development and maintenance, including cone-rod homeobox (crx) and Wnt pathway members. These results show that the independent evolution of retinal degeneration in cavefish can occur by different developmental genetic mechanisms.
Asunto(s)
Adaptación Biológica , Evolución Biológica , Ojo/patología , Degeneración Retiniana/genética , Animales , Secuencia de Bases , Cuevas , Characidae/genética , Cipriniformes/genética , Oscuridad , Ojo/metabolismo , Pigmentación/genética , Degeneración Retiniana/patología , Transcriptoma/genética , Vía de Señalización WntRESUMEN
People with NR5A1 mutations experience testicular dysgenesis, ovotestes, or adrenal insufficiency, but we do not completely understand the origin of this phenotypic diversity. NR5A1 is expressed in gonadal soma precursor cells before expression of the sex-determining gene SRY. Many fish have two co-orthologs of NR5A1 that likely partitioned ancestral gene subfunctions between them. To explore ancestral roles of NR5A1, we knocked out nr5a1a and nr5a1b in zebrafish. Single-cell RNA-seq identified nr5a1a-expressing cells that co-expressed genes for steroid biosynthesis and the chemokine receptor Cxcl12a in 1-day postfertilization (dpf) embryos, as does the mammalian adrenal-gonadal (interrenal-gonadal) primordium. In 2dpf embryos, nr5a1a was expressed stronger in the interrenal-gonadal primordium than in the early hypothalamus but nr5a1b showed the reverse. Adult Leydig cells expressed both ohnologs and granulosa cells expressed nr5a1a stronger than nr5a1b. Mutants for nr5a1a lacked the interrenal, formed incompletely differentiated testes, had no Leydig cells, and grew far larger than normal fish. Mutants for nr5a1b formed a disorganized interrenal and their gonads completely disappeared. All homozygous mutant genotypes lacked secondary sex characteristics, including male breeding tubercles and female sex papillae, and had exceedingly low levels of estradiol, 11-ketotestosterone, and cortisol. RNA-seq showed that at 21dpf, some animals were developing as females and others were not, independent of nr5a1 genotype. By 35dpf, all mutant genotypes greatly under-expressed ovary-biased genes. Because adult nr5a1a mutants form gonads but lack an interrenal and conversely, adult nr5a1b mutants lack a gonad but have an interrenal, the adrenal, and gonadal functions of the ancestral nr5a1 gene partitioned between ohnologs after the teleost genome duplication, likely owing to reciprocal loss of ancestral tissue-specific regulatory elements. Identifying such elements could provide hints to otherwise unexplained cases of Differences in Sex Development.
Asunto(s)
Glándulas Suprarrenales/metabolismo , Proteínas de Unión al ADN/genética , Disgenesia Gonadal/genética , Gónadas/metabolismo , Factores de Transcripción/genética , Proteínas de Pez Cebra/genética , Glándulas Suprarrenales/embriología , Animales , Proteínas de Unión al ADN/metabolismo , Femenino , Gónadas/embriología , Masculino , Fenotipo , Procesos de Determinación del Sexo , Factores de Transcripción/metabolismo , Pez Cebra , Proteínas de Pez Cebra/metabolismoRESUMEN
Morphogenesis and mechanoelectrical transduction of the hair cell mechanoreceptor depend on the correct assembly of Usher syndrome (USH) proteins into highly organized macromolecular complexes. Defects in these proteins lead to deafness and vestibular areflexia in USH patients. Mutations in a non-USH protein, glutaredoxin domain-containing cysteine-rich 1 (GRXCR1), cause non-syndromic sensorineural deafness. To understand the deglutathionylating enzyme function of GRXCR1 in deafness, we generated two grxcr1 zebrafish mutant alleles. We found that hair bundles are thinner in homozygous grxcr1 mutants, similar to the USH1 mutants ush1c (Harmonin) and ush1ga (Sans). In vitro assays showed that glutathionylation promotes the interaction between Ush1c and Ush1ga and that Grxcr1 regulates mechanoreceptor development by preventing physical interaction between these proteins without affecting the assembly of another USH1 protein complex, the Ush1c-Cadherin23-Myosin7aa tripartite complex. By elucidating the molecular mechanism through which Grxcr1 functions, we also identify a mechanism that dynamically regulates the formation of Usher protein complexes.
Asunto(s)
Glutarredoxinas/metabolismo , Células Ciliadas Auditivas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Síndromes de Usher/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Animales , Perros , Glutatión/metabolismo , Aparato de Golgi/metabolismo , Células de Riñón Canino Madin Darby , Actividad Motora , Mutación/genética , Unión Proteica , Transporte de Proteínas , Estereocilios/metabolismo , Especificidad por SustratoRESUMEN
Advances in genomics and next-generation sequencing have provided clinical researchers with unprecedented opportunities to understand the molecular basis of human genetic disorders. This abundance of information places new requirements on traditional disease models, which have the potential to be used to confirm newly identified pathogenic mutations and test the efficacy of emerging therapies. The unique attributes of zebrafish are being increasingly leveraged to create functional disease models, facilitate drug discovery, and provide critical scientific bases for the development of new clinical tools for the diagnosis and treatment of human disease. In this short review and the accompanying poster, we highlight a few illustrative examples of the applications of the zebrafish model to the study of human health and disease.
Asunto(s)
Salud , Investigación Biomédica Traslacional , Pez Cebra/metabolismo , Animales , Modelos Animales de Enfermedad , Estudios de Asociación Genética , Humanos , Modelos GenéticosRESUMEN
Clarin-1 (CLRN1) is the causative gene in Usher syndrome type 3A, an autosomal recessive disorder characterized by progressive vision and hearing loss. CLRN1 encodes Clarin-1, a glycoprotein with homology to the tetraspanin family of proteins. Previous cell culture studies suggest that Clarin-1 localizes to the plasma membrane and interacts with the cytoskeleton. Mouse models demonstrate a role for the protein in mechanosensory hair bundle integrity, but the function of Clarin-1 in hearing remains unclear. Even less is known of its role in vision, because the Clrn1 knockout mouse does not exhibit a retinal phenotype and expression studies in murine retinas have provided conflicting results. Here, we describe cloning and expression analysis of the zebrafish clrn1 gene, and report protein localization of Clarin-1 in auditory and visual cells from embryonic through adult stages. We detect clrn1 transcripts as early as 24h post-fertilization, and expression is maintained through adulthood. In situ hybridization experiments show clrn1 transcripts enriched in mechanosensory hair cells and supporting cells of the inner ear and lateral line organ, photoreceptors, and cells of the inner retina. In mechanosensory hair cells, Clarin-1 is polarized to the apical cell body and the synapses. In the retina, Clarin-1 localizes to lateral cell contacts between photoreceptors and is associated with the outer limiting membrane and subapical processes emanating from Müller glial cells. We also find Clarin-1 protein in the outer plexiform, inner nuclear and ganglion cell layers of the retina. Given the importance of Clarin-1 function in the human retina, it is imperative to find an animal model with a comparable requirement. Our data provide a foundation for exploring the role of Clarin-1 in retinal cell function and survival in a diurnal, cone-dominant species.
Asunto(s)
Oído Interno/metabolismo , Proteínas de la Membrana/genética , Retina/metabolismo , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Animales , Línea Celular , Clonación Molecular , Cricetinae , Expresión Génica , Humanos , Mecanorreceptores/metabolismo , Proteínas de la Membrana/metabolismo , Especificidad de Órganos , Transporte de Proteínas , Retina/citología , Células Ganglionares de la Retina/metabolismo , Segmento Interno de las Células Fotorreceptoras Retinianas/metabolismo , Homología de Secuencia de Aminoácido , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismoRESUMEN
Usher syndrome is the most prevalent cause of hereditary deaf-blindness, characterized by congenital sensorineural hearing impairment and progressive photoreceptor degeneration beginning in childhood or adolescence. Diagnosis and management of this disease are complex, and the molecular changes underlying sensory cell impairment remain poorly understood. Here we characterize two zebrafish models for a severe form of Usher syndrome, Usher syndrome type 1C (USH1C): one model is a mutant with a newly identified ush1c nonsense mutation, and the other is a morpholino knockdown of ush1c. Both have defects in hearing, balance and visual function from the first week of life. Histological analyses reveal specific defects in sensory cell structure that are consistent with these behavioral phenotypes and could implicate Müller glia in the retinal pathology of Usher syndrome. This study shows that visual defects associated with loss of ush1c function in zebrafish can be detected from the onset of vision, and thus could be applicable to early diagnosis for USH1C patients.
Asunto(s)
Proteínas del Tejido Nervioso/metabolismo , Neuroglía/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Sinapsis/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/crecimiento & desarrollo , Animales , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Células Ciliadas Auditivas/efectos de los fármacos , Células Ciliadas Auditivas/metabolismo , Audición/efectos de los fármacos , Larva/efectos de los fármacos , Sistema de la Línea Lateral/efectos de los fármacos , Sistema de la Línea Lateral/metabolismo , Sistema de la Línea Lateral/fisiopatología , Estadios del Ciclo de Vida/efectos de los fármacos , Datos de Secuencia Molecular , Morfolinos/farmacología , Mutación/genética , Proteínas del Tejido Nervioso/genética , Células Fotorreceptoras de Vertebrados/efectos de los fármacos , Células Fotorreceptoras de Vertebrados/patología , Células Fotorreceptoras de Vertebrados/ultraestructura , Transporte de Proteínas/efectos de los fármacos , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Sinapsis/efectos de los fármacos , Sinapsis/patología , Sinapsis/ultraestructura , Visión Ocular/efectos de los fármacos , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
Usher syndrome is a genetically heterogeneous recessive disease characterized by hearing loss and retinitis pigmentosa (RP). It frequently presents with unexplained, often intrafamilial, variability of the visual phenotype. Although 9 genes have been linked with Usher syndrome, many patients do not have mutations in any of these genes, suggesting that there are still unidentified genes involved in the syndrome. Here, we have determined that mutations in PDZ domain-containing 7 (PDZD7), which encodes a homolog of proteins mutated in Usher syndrome subtype 1C (USH1C) and USH2D, contribute to Usher syndrome. Mutations in PDZD7 were identified only in patients with mutations in other known Usher genes. In a set of sisters, each with a homozygous mutation in USH2A, a frame-shift mutation in PDZD7 was present in the sister with more severe RP and earlier disease onset. Further, heterozygous PDZD7 mutations were present in patients with truncating mutations in USH2A, G protein-coupled receptor 98 (GPR98; also known as USH2C), and an unidentified locus. We validated the human genotypes using zebrafish, and our findings were consistent with digenic inheritance of PDZD7 and GPR98, and with PDZD7 as a retinal disease modifier in patients with USH2A. Pdzd7 knockdown produced an Usher-like phenotype in zebrafish, exacerbated retinal cell death in combination with ush2a or gpr98, and reduced Gpr98 localization in the region of the photoreceptor connecting cilium. Our data challenge the view of Usher syndrome as a traditional Mendelian disorder and support the reclassification of Usher syndrome as an oligogenic disease.
Asunto(s)
Pérdida Auditiva/genética , Mutación , Receptores Acoplados a Proteínas G/genética , Retinitis Pigmentosa/genética , Síndromes de Usher/genética , Mutación del Sistema de Lectura , Genotipo , Homocigoto , Humanos , Masculino , Fenotipo , Hermanos , Síndrome , Síndromes de Usher/metabolismoRESUMEN
The early Caenorhabditis elegans embryo is well suited for investigating microtubule-dependent cell division processes. In the one-cell stage, the XMAP215 homologue ZYG-9, associated with the TACC protein TAC-1, promotes microtubule growth during interphase and mitosis, whereas the doublecortin domain protein ZYG-8 is required for anaphase spindle positioning. How ZYG-9, TAC-1 and ZYG-8 together ensure correct microtubule-dependent processes throughout the cell cycle is not fully understood. Here, we identify new temperature-sensitive alleles of zyg-9 and tac-1. Analysis of ZYG-9 and TAC-1 distribution in these mutants identifies amino acids important for centrosomal targeting and for stability of the two proteins. This analysis also reveals that TAC-1 is needed for correct ZYG-9 centrosomal enrichment. Moreover, we find that ZYG-9, but not TAC-1, is limiting for microtubule-dependent processes in one-cell-stage embryos. Using two of these alleles to rapidly inactivate ZYG-9-TAC-1 function, we establish that this complex is required for correct anaphase spindle positioning. Furthermore, we uncover that ZYG-9-TAC-1 and ZYG-8 function together during meiosis, interphase and mitosis. We also find that TAC-1 physically interacts with ZYG-8 through its doublecortin domain, and that in vivo TAC-1 and ZYG-8 are part of a complex that does not contain ZYG-9. Taken together, these findings indicate that ZYG-9-TAC-1 and ZYG-8 act in a partially redundant manner to ensure correct microtubule assembly throughout the cell cycle of early C. elegans embryos.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citología , Proteínas de Ciclo Celular/metabolismo , Ciclo Celular , Embrión no Mamífero/citología , Microtúbulos/metabolismo , Alelos , Anafase , Animales , Caenorhabditis elegans/embriología , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/química , Pérdida del Embrión , Embrión no Mamífero/metabolismo , Mutación/genética , Fenotipo , Unión Proteica , Estructura Terciaria de Proteína , Huso Acromático/metabolismoRESUMEN
The Caenorhabditis elegans genome encodes multiple isotypes of alpha-tubulin and beta-tubulin. Roles for a number of these tubulins in neuronal development have been described, but less is known about the isoforms that function during early embryonic development. Microtubules are required for multiple events after fertilization produces a one-cell zygote in C. elegans, including pronuclear migration, mitotic spindle assembly and function, and proper spindle positioning. Here we describe a conditional and dominant mis-sense mutation in the C. elegans alpha-tubulin gene tba-1 that disrupts pronuclear migration and positioning of the first mitotic spindle, and results in a highly penetrant embryonic lethality, at the restrictive temperature of 26 degrees C. Our analysis of the dominant tba-1 (or346ts) allele suggests that TBA-1 assembles into microtubules in early embryonic cells. However, we also show that reduction of tba-1 function using RNA interference results in defects much less severe than those caused by the dominant or346ts mutation, due to partial redundancy of TBA-1 and another alpha-tubulin called TBA-2. Reducing the function of both TBA-1 and TBA-2 results in severe defects in microtubule-dependent processes. We conclude that microtubules in the early C. elegans embryo are composed of both TBA-1 and TBA-2, and that the dominant tba-1(or346ts) mutation disrupts MT assembly or stability. Cell Motil.
Asunto(s)
Caenorhabditis elegans/embriología , Mitosis/fisiología , Tubulina (Proteína)/metabolismo , Animales , Caenorhabditis elegans/metabolismo , Centrosoma/metabolismo , Técnica del Anticuerpo Fluorescente , Genes Dominantes , Huso Acromático/metabolismo , Temperatura , Tubulina (Proteína)/genéticaRESUMEN
The mitotic spindle, which partitions replicated chromosomes to daughter cells during cell division, is composed of microtubule assemblies of alpha/beta-tubulin heterodimers. Positioning of the mitotic spindle influences the size and location of daughter cells, and can be important for the proper partitioning of developmental determinants. We describe two semi-dominant mis-sense mutations in tbb-2, one of two C. elegans beta-tubulin genes that are maternally expressed and together are required for microtubule-dependent processes in the early embryo. These mutations result in a posteriorly displaced and misoriented mitotic spindle during the first cell division. In contrast, a probable tbb-2 null allele is recessive, and when homozygous results in less severe spindle positioning defects and only partially penetrant embryonic lethality. Two of the tbb-2 mutations result in reduced levels of TBB-2 protein, and increased levels of a second maternally expressed beta-tubulin, TBB-1. However, levels of TBB-1 are not increased in a tbb-2 mutant with an allele that does not result in reduced levels of TBB-2 protein. We conclude that feedback regulation influences maternal beta-tubulin expression in C. elegans, but cannot fully restore normal microtubule function in the absence of one beta-tubulin isoform.