RESUMEN
AIMS: The adult mammalian heart has poor regenerative capacity. In contrast, the zebrafish heart retains a robust capacity for regeneration into adulthood. These distinct responses are consequences of a differential utilization of evolutionary-conserved gene regulatory networks in the damaged heart. To systematically identify miRNA-dependent networks controlling cardiac repair following injury, we performed comparative gene and miRNA profiling of the cardiac transcriptome in adult mice and zebrafish. METHODS AND RESULTS: Using an integrated approach, we show that 45 miRNA-dependent networks, involved in critical biological pathways, are differentially modulated in the injured zebrafish vs. mouse hearts. We study, more particularly, the miR-26a-dependent response. Therefore, miR-26a is down-regulated in the fish heart after injury, whereas its expression remains constant in the mouse heart. Targets of miR-26a involve activators of the cell cycle and Ezh2, a component of the polycomb repressive complex 2 (PRC2). Importantly, PRC2 exerts repressive functions on negative regulators of the cell cycle. In cultured neonatal cardiomyocytes, inhibition of miR-26a stimulates, therefore, cardiomyocyte proliferation. Accordingly, miR-26a knockdown prolongs the proliferative window of cardiomyocytes in the post-natal mouse heart. CONCLUSIONS: This novel strategy identifies a series of miRNAs and associated pathways, in particular miR-26a, which represent attractive therapeutic targets for inducing repair in the injured heart.
Asunto(s)
Proliferación Celular/genética , Redes Reguladoras de Genes/genética , MicroARNs/metabolismo , Cicatrización de Heridas/genética , Animales , Ciclo Celular , Perfilación de la Expresión Génica/métodos , Ratones Endogámicos C57BL , MicroARNs/genética , Miocitos Cardíacos/fisiología , Regeneración , Pez CebraRESUMEN
HMX1 is a homeobox-containing transcription factor implicated in eye development and responsible for the oculo-auricular syndrome of Schorderet-Munier-Franceschetti. HMX1 is composed of two exons with three conserved domains in exon 2, a homeobox and two domains called SD1 and SD2. The function of the latter two domains remains unknown. During retinal development, HMX1 is expressed in a polarized manner and thus seems to play a role in the establishment of retinal polarity although its exact role and mode of action in eye development are unknown. Here, we demonstrated that HMX1 dimerized and that the SD1 and homeodomains are required for this function. In addition, we showed that proper nuclear localization requires the presence of the homeodomain. We also identified that EPHA6, a gene implicated in retinal axon guidance, is one of its targets in eye development and showed that a dimerized HMX1 is needed to inhibit EPHA6 expression.
Asunto(s)
Proteínas de Homeodominio/metabolismo , Multimerización de Proteína , Receptor EphA4/metabolismo , Receptor EphA6/metabolismo , Retina/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Animales , Núcleo Celular/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Proteínas de Homeodominio/química , Humanos , Ratones Endogámicos C57BL , Proteínas Mutantes/metabolismo , Regiones Promotoras Genéticas/genética , Estructura Terciaria de Proteína , Transporte de Proteínas , Factores de Transcripción/química , Proteínas de Pez Cebra/químicaRESUMEN
Ocular development is controlled by a complex network of transcription factors, cell cycle regulators, and diffusible signaling molecules. Together, these molecules regulate cell proliferation, apoptosis and specify retinal fate. In the zebrafish (Danio rerio), hmx1 is a homeobox transcription factor implicated in eye and brain development. Hmx1 transcripts were detected in the nasal retina and lens as well as otic vesicles and pharyngeal arches by 24-32 hpf. Before this stage, transcripts were more uniformly expressed in the optic vesicle. Knockdown of hmx1 led to microphthalmia. Delayed withdrawal of retinal progenitors from the cell cycle resulting in retarded retinal differentiation was observed in morphant. The retina and brain also showed an increased cell death at 24 hpf. The polarized expression of hmx1 to the nasal part in the zebrafish retina strongly suggested an involvement in the nasal-temporal patterning. However, the key patterning genes tested so far were not regulated by hmx1. Altogether, these results suggest an important role for hmx1 in retinogenesis.
Asunto(s)
Retina/embriología , Factores de Transcripción/fisiología , Proteínas de Pez Cebra/fisiología , Animales , Región Branquial/embriología , Recuento de Células , Ciclo Celular , Proteínas de Unión al ADN/metabolismo , Oído/anomalías , Oído/embriología , Oído/patología , Embrión no Mamífero/metabolismo , Anomalías del Ojo/embriología , Anomalías del Ojo/genética , Anomalías del Ojo/patología , Técnica del Anticuerpo Fluorescente Indirecta , Silenciador del Gen/fisiología , Proteínas de Homeodominio/metabolismo , Hibridación in Situ , Microftalmía/embriología , Microftalmía/genética , Microftalmía/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transactivadores/metabolismo , Pez Cebra , Proteínas de Pez Cebra/metabolismoRESUMEN
Waardenburg anophthalmia syndrome, also known as microphthalmia with limb anomalies, ophthalmoacromelic syndrome, and anophthalmia-syndactyly, is a rare autosomal-recessive developmental disorder that has been mapped to 10p11.23. Here we show that this disease is heterogeneous by reporting on a consanguineous family, not linked to the 10p11.23 locus, whose two affected children have a homozygous mutation in SMOC1. Knockdown experiments of the zebrafish smoc1 revealed that smoc1 is important in eye development and that it is expressed in many organs, including brain and somites.
Asunto(s)
Mutación , Osteonectina/genética , Adulto , Secuencia de Bases , Niño , Consanguinidad , Ojo/crecimiento & desarrollo , Femenino , Dedos/diagnóstico por imagen , Dedos/crecimiento & desarrollo , Genes Recesivos , Humanos , Masculino , Datos de Secuencia Molecular , Linaje , Radiografía , Síndrome de Waardenburg/genéticaRESUMEN
PIKfyve is a kinase encoded by pip5k3 involved in phosphatidylinositols (PdtIns) pathways. These lipids building cell membranes have structural functions and are involved in complex intracellular regulations. Mutations in human PIP5K3 are associated with François-Neetens mouchetée fleck corneal dystrophy [Li, S., Tiab, L., Jiao, X., Munier, F.L., Zografos, L., Frueh, B.E., Sergeev, Y., Smith, J., Rubin, B., Meallet, M.A., Forster, R.K., Hejtmancik, J.F., Schorderet, D.F., 2005. Mutations in PIP5K3 are associated with François-Neetens mouchetee fleck corneal dystrophy. Am. J. Hum. Genet. 77, 54-63]. We cloned the zebrafish pip5k3 and report its molecular characterization and expression pattern in adult fish as well as during development. The zebrafish PIKfyve was 70% similar to the human homologue. The gene encompassed 42 exons and presented four alternatively spliced variants. It had a widespread expression in the adult organs and was localized in specific cell types in the eye as the cornea, lens, ganglion cell layer, inner nuclear layer and outer limiting membrane. Pip5k3 transcripts were detected in early cleavage stage embryos. Then it was uniformly expressed at 10 somites, 18 somites and 24 hpf. Its expression was then restricted to the head region at 48 hpf, 72 hpf and 5 dpf and partial expression was found in somites at 72 hpf and 5 dpf. In situ on eye sections at 3 dpf showed a staining mainly in lens, outer limiting membrane, inner nuclear layer and ganglion cell layer. A similar expression pattern was found in the eye at 5 dpf. A temporal regulation of the spliced variants was observed at 1, 3 and 5 dpf and they were also found in the adult eye.
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Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Proteínas de Pez Cebra/genética , Empalme Alternativo , Animales , Clonación Molecular , Embrión no Mamífero/enzimología , Ojo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , ARN Mensajero/metabolismo , Distribución Tisular , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismoRESUMEN
Several dysmorphic syndromes affect the development of both the eye and the ear, but only a few are restricted to the eye and the external ear. We describe a developmental defect affecting the eye and the external ear in three members of a consanguineous family. This syndrome is characterized by ophthalmic anomalies (microcornea, microphthalmia, anterior-segment dysgenesis, cataract, coloboma of various parts of the eye, abnormalities of the retinal pigment epithelium, and rod-cone dystrophy) and a particular cleft ear lobule. Linkage analysis and mutation screening revealed in the first exon of the NKX5-3 gene a homozygous 26 nucleotide deletion, generating a truncating protein that lacked the complete homeodomain. Morpholino knockdown expression of the zebrafish nkx5-3 induced microphthalmia and disorganization of the developing retina, thus confirming that this gene represents an additional member implicated in axial patterning of the retina.
Asunto(s)
Oído/anomalías , Anomalías del Ojo/genética , Proteínas de Homeodominio/genética , Factores de Transcripción/genética , Anciano , Animales , Consanguinidad , Embrión de Mamíferos/metabolismo , Embrión no Mamífero/metabolismo , Anomalías del Ojo/embriología , Femenino , Feto/metabolismo , Proteínas de Homeodominio/biosíntesis , Humanos , Masculino , Ratones , Persona de Mediana Edad , Datos de Secuencia Molecular , Especificidad de Órganos , Linaje , Síndrome , Factores de Transcripción/biosíntesis , Pez Cebra/embriología , Pez Cebra/metabolismoRESUMEN
RPE65 is the retinal isomerase essential for conversion of all-trans-retinyl ester to 11-cis-retinol in the visual cycle. Leber's congenital amaurosis (LCA), an autosomal recessive form of RP resulting in blindness, is commonly caused by mutations in the Rpe65 gene. Whereas the molecular mechanisms by which these mutations contribute to retinal disease remain largely unresolved, affected patients show marked RPE damage and photoreceptor degeneration. We evaluated gene expression in Rpe65-/- mouse model of LCA before and at the onset of photoreceptor cell death in 2, 4, and 6 month old animals. Microarray analysis demonstrates altered expression of genes involved in phototransduction, apoptosis regulation, cytoskeleton organization, and extracellular matrix (ECM) constituents. Cone-specific phototransduction genes are strongly decreased, reflecting early loss of cones. In addition, remaining rods show modified expression of genes encoding components of the cytoskeleton and ECM. This may affect rod physiology and interaction with the adjacent RPE and lead to loss of survival signals, as reflected by the alteration of apoptosis-related genes Together, these results suggest that RPE65 defect triggers an overall remodeling of the neurosensitive retina that may, in turn, disrupt photoreceptor homeostasis and induce apoptosis signaling cascade toward retinal cell death.
Asunto(s)
Ceguera/genética , Proteínas del Ojo/genética , Regulación de la Expresión Génica , Animales , Apoptosis/genética , Ceguera/etiología , Ceguera/patología , Proteínas Portadoras , Proteínas del Citoesqueleto/genética , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Proteínas de la Matriz Extracelular/genética , Perfilación de la Expresión Génica , Ratones , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , Células Fotorreceptoras/patología , Visión Ocular/genética , cis-trans-Isomerasas/genéticaRESUMEN
To identify candidate genes that are responsible for motoneurone degeneration, we combined laser capture microdissection with microarray technology. We analysed gene expression in pure motoneurones from two mouse mutants that develop motoneurone degeneration, progressive motor neuronopathy and wobbler. At a presymptomatic age, there was a significant differential expression of a restricted number of genes (25 and 72 in progressive motor neuronopathy and wobbler respectively, of 22 600 transcripts screened). We compared these results to our previous analyses in the copper-zinc superoxide dismutase mutant mouse (SOD1(G93A)) in which we observed a de-regulation of 27 genes. Some of these genes were de-regulated uniquely in one mouse mutant and some have already been identified in cell death pathways implicated in amyotrophic lateral sclerosis and animal models of motoneurone degeneration (i.e. de-regulation of intermediate filaments, axonal transport, the ubiquitin-proteasome system and excitotoxicity). One gene, vimentin, was differentially up-regulated in all mouse mutants; this main candidate gene has been confirmed by in situ hybridization and immunohistochemistry to be expressed in motoneurones in all mouse mutants. Furthermore, vimentin expression correlated with the state of motoneurone degeneration. These results identify early molecular changes that may be involved in the pathogenesis of motoneurones leading to cell death and favour a complex multipathway induction of the disease; surprisingly, there was no important modification in cell death-associated genes. This is the first study to show a clear difference in the genes that are de-regulated at an early stage in three different mouse models of motoneurone disease.
Asunto(s)
Ratones Mutantes Neurológicos , Enfermedad de la Neurona Motora/fisiopatología , Neuronas Motoras , Degeneración Nerviosa/fisiopatología , Animales , Animales Recién Nacidos , Muerte Celular , Separación Celular/métodos , Sistemas de Computación , Citoplasma/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Expresión Génica , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Cuerpos de Inclusión/patología , Rayos Láser , Ratones , Microdisección , Enfermedad de la Neurona Motora/genética , Enfermedad de la Neurona Motora/patología , Neuronas Motoras/metabolismo , Degeneración Nerviosa/genética , Degeneración Nerviosa/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , Médula Espinal/patología , Vimentina/genética , Vimentina/metabolismoRESUMEN
To identify candidate genes that may be involved in motoneuron degeneration, we combined laser capture microdissection with microarray technology. Gene expression in motoneurons was analyzed during the progression of the disease in transgenic SOD1(G93A) mice that develop motoneuron loss. Three major observations were made: first, there was only a small number of genes that were differentially expressed in motoneurons at a pre-symptomatic age (27 out of 34 000 transcripts). Secondly, there is an early specific up-regulation of the gene coding for the intermediate filament vimentin that is increased even further during disease progression. Using in situ hybridization and immunohistochemical analysis, we show that vimentin expression was not only elevated in motoneurons but that the protein formed inclusions in the motoneuron cytoplasm. Thirdly, a time-course analysis of the motoneurons at a symptomatic age (90 and 120 days) showed a modest de-regulation of only a few genes associated with cell death pathways; however, a massive up-regulation of genes involved in cell growth and/or maintenance was observed. This is the first description of the gene profile of SOD1(G93A) motoneurons during disease progression and unexpectedly, no widespread induction of cell death-associated genes was detected in motoneurons of SOD1(G93A) mice.