RESUMEN
Biocompatible fluorescent reporters with spectral properties spanning the entire visible spectrum are indispensable tools for imaging the biochemistry of living cells and organisms in real time. Here, we report the engineering of a fluorescent chemogenetic reporter with tunable optical and spectral properties. A collection of fluorogenic chromophores with various electronic properties enables to generate bimolecular fluorescent assemblies that cover the visible spectrum from blue to red using a single protein tag engineered and optimized by directed evolution and rational design. The ability to tune the fluorescence color and properties through simple molecular modulation provides a broad experimental versatility for imaging proteins in live cells, including neurons, and in multicellular organisms, and opens avenues for optimizing Förster resonance energy transfer (FRET) biosensors in live cells. The ability to tune the spectral properties and fluorescence performance enables furthermore to match the specifications and requirements of advanced super-resolution imaging techniques.
Asunto(s)
Diagnóstico por Imagen/métodos , Fluorescencia , Ingeniería de Proteínas/métodos , Animales , Materiales Biocompatibles , Técnicas Biosensibles , Color , Colorantes , Electrónica , Femenino , Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes , Proteínas Fluorescentes Verdes , Masculino , Neuronas , Ratas , Ratas Sprague-DawleyRESUMEN
The vertebrate central nervous system (CNS) is a fantastically complex organ composed of dozens of cell types within the neural and glial lineages. Its organization is laid down during development, through the localized and sequential production of subsets of neurons with specific identities. The principles and mechanisms that underlie the timely production of adequate classes of cells are only partially understood. Recent advances in molecular profiling describe the developmental trajectories leading to this amazing cellular diversity and provide us with cell atlases of an unprecedented level of precision. Yet, some long-standing questions pertaining to lineage relationships between neural progenitor cells and their differentiated progeny remain unanswered. Here, we discuss questions related to proliferation potential, timing of fate choices and restriction of neuronal output potential of individual CNS progenitors through the lens of lineage relationship. Unlocking methodological barriers will be essential to accurately describe CNS development at a cellular resolution.
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Células-Madre Neurales , Neuroglía , Diferenciación Celular , Linaje de la Célula , Sistema Nervioso Central , NeuronasRESUMEN
The architecture of renal glomeruli is acquired through intricate and still poorly understood developmental steps. In our study we identify a crucial glomerular morphogenetic event in nephrogenesis that drives the remodeling/separation of the prospective vascular pole (the future entrance of the glomerular arterioles) and the urinary pole (the tubular outflow). We demonstrate that this remodeling is genetically programmed. In fact, in mouse and human, the absence of HNF1B impairs the remodeling/separation of the two poles, leading to trapping and constriction of the tubular outflow inside the glomerulus. This aberration gives rise to obstructive glomerular dilations upon the initiation of primary urine production. In this context, we show that pharmacological decrease of glomerular filtration significantly contains cystic expansion. From a developmental point of view, our study discloses a crucial event on glomerular patterning affecting the "inside-outside" fate of the epithelia in the renal glomerulus.
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Enfermedades Renales/congénito , Glomérulos Renales/embriología , Humanos , Glomérulos Renales/patologíaRESUMEN
Following the discovery of (R)-roscovitine's beneficial effects in three polycystic kidney disease (PKD) mouse models, cyclin-dependent kinases (CDKs) inhibitors have been investigated as potential treatments. We have used various affinity chromatography approaches to identify the molecular targets of roscovitine and its more potent analog (S)-CR8 in human and murine polycystic kidneys. These methods revealed casein kinases 1 (CK1) as additional targets of the two drugs. CK1ε expression at the mRNA and protein levels is enhanced in polycystic kidneys of 11 different PKD mouse models as well as in human polycystic kidneys. A shift in the pattern of CK1α isoforms is observed in all PKD mouse models. Furthermore, the catalytic activities of both CK1ε and CK1α are increased in mouse polycystic kidneys. Inhibition of CK1ε and CK1α may thus contribute to the long-lasting attenuating effects of roscovitine and (S)-CR8 on cyst development. CDKs and CK1s may constitute a dual therapeutic target to develop kinase inhibitory PKD drug candidates.
Asunto(s)
Caseína Cinasa 1 épsilon/antagonistas & inhibidores , Caseína Quinasa Ialfa/antagonistas & inhibidores , Riñón/efectos de los fármacos , Enfermedades Renales Poliquísticas/prevención & control , Inhibidores de Proteínas Quinasas/farmacología , Purinas/farmacología , Piridinas/farmacología , Roscovitina/farmacología , Animales , Caseína Cinasa 1 épsilon/genética , Caseína Cinasa 1 épsilon/metabolismo , Caseína Quinasa Ialfa/genética , Caseína Quinasa Ialfa/metabolismo , Catálisis , Cromatografía de Afinidad/métodos , Modelos Animales de Enfermedad , Humanos , Riñón/enzimología , Riñón/patología , Ratones Transgénicos , Enfermedades Renales Poliquísticas/enzimología , Enfermedades Renales Poliquísticas/genética , Enfermedades Renales Poliquísticas/patología , Unión Proteica , Inhibidores de Proteínas Quinasas/metabolismo , Purinas/metabolismo , Piridinas/metabolismo , Roscovitina/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Congenital nephron number varies widely in the human population and individuals with low nephron number are at risk of developing hypertension and chronic kidney disease. The development of the kidney occurs via an orchestrated morphogenetic process where metanephric mesenchyme and ureteric bud reciprocally interact to induce nephron formation. The genetic networks that modulate the extent of this process and set the final nephron number are mostly unknown. Here, we identified a specific isoform of MITF (MITF-A), a bHLH-Zip transcription factor, as a novel regulator of the final nephron number. We showed that overexpression of MITF-A leads to a substantial increase of nephron number and bigger kidneys, whereas Mitfa deficiency results in reduced nephron number. Furthermore, we demonstrated that MITF-A triggers ureteric bud branching, a phenotype that is associated with increased ureteric bud cell proliferation. Molecular studies associated with an in silico analyses revealed that amongst the putative MITF-A targets, Ret was significantly modulated by MITF-A. Consistent with the key role of this network in kidney morphogenesis, Ret heterozygosis prevented the increase of nephron number in mice overexpressing MITF-A. Collectively, these results uncover a novel transcriptional network that controls branching morphogenesis during kidney development and identifies one of the first modifier genes of nephron endowment.
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Riñón/fisiología , Factor de Transcripción Asociado a Microftalmía/metabolismo , Nefronas/fisiología , Animales , Femenino , Humanos , Riñón/embriología , Riñón/metabolismo , Masculino , Ratones , Ratones Transgénicos , Factor de Transcripción Asociado a Microftalmía/genética , Morfogénesis , Nefronas/anatomía & histología , Nefronas/crecimiento & desarrollo , Nefronas/metabolismo , Organogénesis , Isoformas de Proteínas , Proteínas Proto-Oncogénicas c-ret/genética , Proteínas Proto-Oncogénicas c-ret/metabolismo , Uréter/metabolismo , Uréter/fisiologíaRESUMEN
Unequal centrosome maturation correlates with asymmetric division in multiple cell types. Nevertheless, centrosomal fate determinants have yet to be identified. Here, we show that the Notch pathway regulator Mindbomb1 co-localizes asymmetrically with centriolar satellite proteins PCM1 and AZI1 at the daughter centriole in interphase. Remarkably, while PCM1 and AZI1 remain asymmetric during mitosis, Mindbomb1 is associated with either one or both spindle poles. Asymmetric Mindbomb1 correlates with neurogenic divisions and Mindbomb1 is inherited by the prospective neuron. By contrast, in proliferative divisions, a supplementary pool of Mindbomb1 associated with the Golgi apparatus in interphase is released during mitosis and compensates for Mindbomb1 centrosomal asymmetry. Finally, we show that preventing Mindbomb1 centrosomal association induces reciprocal Notch activation between sister cells and promotes symmetric divisions. Thus, we uncover a link between differential centrosome maturation and Notch signaling and reveal an unexpected compensatory mechanism involving the Golgi apparatus in restoring symmetry in proliferative divisions.
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Proteínas de Ciclo Celular/metabolismo , Centriolos/metabolismo , Mitosis , Células-Madre Neurales/metabolismo , Neurogénesis , Ubiquitina-Proteína Ligasas/metabolismo , Animales , División Celular , Centrosoma/metabolismo , Embrión de Pollo , Aparato de Golgi/metabolismo , Receptores Notch/metabolismo , Transducción de SeñalRESUMEN
Nephron morphogenesis is a complex process that generates blood-filtration units (glomeruli) connected to extremely long and patterned tubular structures. Hepatocyte nuclear factor 1ß (HNF1ß) is a divergent homeobox transcription factor that is expressed in kidney from the first steps of nephrogenesis. Mutations in HNF1B (OMIM #137920) are frequently found in patients with developmental renal pathologies, the mechanisms of which have not been completely elucidated. Here we show that inactivation of Hnf1b in the murine metanephric mesenchyme leads to a drastic tubular defect characterized by the absence of proximal, distal and Henle's loop segments. Nephrons were eventually characterized by glomeruli, with a dilated urinary space, directly connected to collecting ducts via a primitive and short tubule. In the absence of HNF1ß early nephron precursors gave rise to deformed S-shaped bodies characterized by the absence of the typical bulge of epithelial cells at the bend between the mid and lower segments. The lack of this bulge eventually led to the absence of proximal tubules and Henle's loops. The expression of several genes, including Irx1, Osr2 and Pou3f3, was downregulated in the S-shaped bodies. We also observed decreased expression of Dll1 and the consequent defective activation of Notch in the prospective tubular compartment of comma- and S-shaped bodies. Our results reveal a novel hierarchical relationship between HNF1ß and key genes involved in renal development. In addition, these studies define a novel structural and functional component of S-shaped bodies at the origin of tubule formation.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/fisiología , Factor Nuclear 1-beta del Hepatocito/metabolismo , Nefronas/embriología , Organogénesis/fisiología , Animales , Proteínas de Unión al Calcio , Inmunoprecipitación de Cromatina , Regulación del Desarrollo de la Expresión Génica/genética , Factor Nuclear 1-beta del Hepatocito/genética , Proteínas de Homeodominio/metabolismo , Inmunohistoquímica , Hibridación in Situ , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ratones , Microscopía Electrónica , Nefronas/anomalías , Nefronas/ultraestructura , Proteínas del Tejido Nervioso/metabolismo , Organogénesis/genética , Factores del Dominio POU/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Transcripción/metabolismoRESUMEN
AIMS: Although routinely used, limited data are available regarding the long-term outcome after patent foramen ovale (PFO) closure using the HELEX® Occluder system. The aim of this study was therefore the examination of the acute and long-term outcome after transcatheter PFO closure using this system. METHODS AND RESULTS: All (n=407) patients included had undergone PFO closure with the HELEX® Occluder system for secondary prevention of stroke, transient ischaemic attack (TIA) or peripheral embolism at a single centre. Primary endpoints were residual shunts at six or 12 months (assessed by transoesophageal echocardiography) and the number of neurological and other adverse events during follow-up. Device implantation was successful in 99% of patients. Complete closure at six months was achieved in 81%. During follow-up of 1,695 patient-years, 10 neurologic events occurred (four TIA, six strokes). The annual incidence of stroke was 1.2%. Other adverse events were wire frame fractures requiring no further intervention in five (1%), device-associated thrombus formation in one (0.25%), and paroxysmal atrial fibrillation in nine patients (2%). CONCLUSIONS: PFO closure using the HELEX® Occluder system is feasible and safe. Complications and adverse events during long-term follow-up are rare. The safety profile and efficacy in prevention of recurrent events compare well to that reported with other closure devices.
Asunto(s)
Cateterismo Cardíaco/instrumentación , Foramen Oval Permeable/terapia , Prevención Secundaria/instrumentación , Dispositivo Oclusor Septal , Adulto , Anciano , Anciano de 80 o más Años , Cateterismo Cardíaco/efectos adversos , Distribución de Chi-Cuadrado , Supervivencia sin Enfermedad , Ecocardiografía Transesofágica , Embolia/mortalidad , Embolia/prevención & control , Femenino , Foramen Oval Permeable/diagnóstico por imagen , Foramen Oval Permeable/mortalidad , Alemania/epidemiología , Humanos , Incidencia , Ataque Isquémico Transitorio/mortalidad , Ataque Isquémico Transitorio/prevención & control , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Diseño de Prótesis , Accidente Cerebrovascular/mortalidad , Accidente Cerebrovascular/prevención & control , Factores de Tiempo , Resultado del Tratamiento , Adulto JovenRESUMEN
Heterozygous deletion or mutation in hepatocyte nuclear factor 1 homeobox B/transcription factor 2 (HNF1B/TCF2) causes renal cyst and diabetes syndrome (OMIM #137920). Mice with homozygous liver-specific deletion of Hnf1ß revealed that a complete lack of this factor leads to ductopenia and bile duct dysplasia, in addition to mild hepatocyte defects. However, little is known about the hepatic consequences of deficient HNF1B function in humans. Three patients with heterozygous HNF1B deficiency were found to have normal bile duct formation on radiology and routine liver pathology. Electron microscopy revealed a paucity or absence of normal primary cilia. Therefore, heterozygous HNF1B deficiency is associated with ciliary anomalies in cholangiocytes, and this may cause cholestasis.
Asunto(s)
Conductos Biliares/citología , Cilios , Células Epiteliales/patología , Factor Nuclear 1-beta del Hepatocito/deficiencia , Adulto , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Hepatocyte nuclear factor-1beta (HNF-1beta) is a transcription factor required for the expression of several renal cystic genes and whose prenatal deletion leads to polycystic kidney disease (PKD). We show here that inactivation of Hnf1b from postnatal day 10 onward does not elicit cystic dilations in tubules after their proliferative morphogenetic elongation is over. Cystogenic resistance is intrinsically linked to the quiescent state of cells. In fact, when Hnf1b deficient quiescent cells are forced to proliferate by an ischemia-reperfusion injury, they give rise to cysts, owing to loss of oriented cell division. Remarkably, in quiescent cells, the transcription of crucial cystogenic target genes is maintained even in the absence of HNF-1beta. However, their expression is lost as soon as cells proliferate and the chromatin of target genes acquires heterochromatin marks. These results unveil a previously undescribed aspect of gene regulation. It is well established that transcription is shut off during the mitotic condensation of chromatin. We propose that transcription factors such as HNF-1beta might be involved in reprogramming gene expression after transcriptional silencing is induced by mitotic chromatin condensation. Notably, HNF-1beta remains associated with the mitotically condensed chromosomal barrels. This association suggests that HNF-1beta is a bookmarking factor that is necessary for reopening the chromatin of target genes after mitotic silencing.
Asunto(s)
Factor Nuclear 1-beta del Hepatocito/fisiología , Enfermedades Renales Poliquísticas/genética , Animales , División Celular/genética , Cromatina/fisiología , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/fisiología , Factor Nuclear 1-beta del Hepatocito/genética , Túbulos Renales/crecimiento & desarrollo , Ratones , Mitosis/fisiología , Enfermedades Renales Poliquísticas/etiología , Activación Transcripcional/genética , Activación Transcripcional/efectos de la radiaciónRESUMEN
In the last few years, evidence has come to light suggesting that planar cell polarity signaling in vertebrates may be controlled and modulated by primary cilia, subcellular organelles that emerge from the plasma membrane of most cell types. This characteristic distinguishes vertebrate planar cell polarity signaling from that in insects. We review here some of the experimental evidence contributing to this finding. These observations have begun to suggest molecular and cellular mechanisms of the so-called ciliopathies, important human diseases characterized by defective ciliary functions.
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Polaridad Celular , Cilios/metabolismo , Transducción de Señal , Animales , División Celular , Humanos , Riñón/citología , Riñón/metabolismoRESUMEN
Hepatocyte nuclear factor-1beta (HNF-1beta) is a transcription factor that regulates gene expression in the kidney, liver, pancreas, and other epithelial organs. Mutations of HNF-1beta lead to a syndrome of inherited renal cysts and diabetes and are also a common cause of sporadic renal dysplasia. The full complement of target genes responsible for the functions of HNF-1beta, however, is incompletely defined. Using a functional genomics approach involving chromatin immunoprecipitation and promoter arrays, combined with gene expression profiling, we found that an HNF-1beta target gene in the kidney is kinesin family member 12 (Kif12), a gene previously identified as a candidate modifier gene in the cpk mouse model of polycystic kidney disease. Mutations of HNF-1beta inhibited Kif12 transcription in both cultured cells and knockout mice by altering co-factor recruitment and histone modification. Because kinesin-12 family members participate in orienting cell division, downregulation of Kif12 may underlie the abnormal planar cell polarity observed in cystic kidney diseases.
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Factor Nuclear 1-beta del Hepatocito/fisiología , Cinesinas/genética , Animales , Proteína de Unión a CREB/fisiología , Línea Celular , Inmunoprecipitación de Cromatina , Regulación de la Expresión Génica , Túbulos Renales Colectores/metabolismo , Ratones , Ratones Endogámicos C57BL , Regiones Promotoras Genéticas , Transcripción GenéticaRESUMEN
The genetic program controlled by transcription factors can be modulated by multiple mechanisms. Binding of coactivators or corepressors, for example, can modulate the transcription of target genes. Dudziak and colleagues identified novel HNF1beta-interacting proteins that, when overexpressed, affect nephrogenesis. These results could improve our understanding of the way HNF1beta controls kidney development.
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Regulación del Desarrollo de la Expresión Génica , Factor Nuclear 1-beta del Hepatocito/metabolismo , Riñón/embriología , Organogénesis , Animales , Factor Nuclear 1-beta del Hepatocito/genética , Riñón/metabolismo , Ratones , Organogénesis/genética , RatasRESUMEN
Tissue organization in Drosophila is regulated by the core planar cell polarity (PCP) proteins Frizzled, Dishevelled, Prickle, Van Gogh and Flamingo. Core PCP proteins are conserved in mammals and function in mammalian tissue organization. Recent studies have identified another group of Drosophila PCP proteins, consisting of the protocadherins Fat and Dachsous (Ds) and the transmembrane protein Four-jointed (Fj). In Drosophila, Fat represses fj transcription, and Ds represses Fat activity in PCP. Here we show that Fat4 is an essential gene that has a key role in vertebrate PCP. Loss of Fat4 disrupts oriented cell divisions and tubule elongation during kidney development, leading to cystic kidney disease. Fat4 genetically interacts with the PCP genes Vangl2 and Fjx1 in cyst formation. In addition, Fat4 represses Fjx1 expression, indicating that Fat signaling is conserved. Together, these data suggest that Fat4 regulates vertebrate PCP and that loss of PCP signaling may underlie some cystic diseases in humans.
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Cadherinas/metabolismo , División Celular , Polaridad Celular , Enfermedades Renales Quísticas/metabolismo , Riñón/embriología , Animales , Cadherinas/genética , Drosophila melanogaster , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Regulación del Desarrollo de la Expresión Génica , RatonesRESUMEN
Hepatocyte nuclear factor-1beta (HNF-1beta) is a Pit-1, Oct-1/2, Unc-86 (POU) homeodomain-containing transcription factor expressed in the kidney, liver, pancreas, and other epithelial organs. Mutations of HNF-1beta cause maturity-onset diabetes of the young, type 5 (MODY5), which is characterized by early-onset diabetes mellitus and congenital malformations of the kidney, pancreas, and genital tract. Knockout of HNF-1beta in the mouse kidney results in cyst formation. However, the signaling pathways and transcriptional programs controlled by HNF-1beta are poorly understood. Using genome-wide chromatin immunoprecipitation and DNA microarray (ChIP-chip) and microarray analysis of mRNA expression, we identified SOCS3 (suppressor of cytokine signaling-3) as a previously unrecognized target gene of HNF-1beta in the kidney. HNF-1beta binds to the SOCS3 promoter and represses SOCS3 transcription. The expression of SOCS3 is increased in HNF-1beta knockout mice and in renal epithelial cells expressing dominant-negative mutant HNF-1beta. Increased levels of SOCS-3 inhibit HGF-induced tubulogenesis by decreasing phosphorylation of Erk and STAT-3. Conversely, knockdown of SOCS-3 in renal epithelial cells expressing dominant-negative mutant HNF-1beta rescues the defect in HGF-induced tubulogenesis by restoring phosphorylation of Erk and STAT-3. Thus, HNF-1beta regulates tubulogenesis by controlling the levels of SOCS-3 expression. Manipulating the levels of SOCS-3 may be a useful therapeutic approach for human diseases induced by HNF-1beta mutations.
Asunto(s)
Factor Nuclear 1-beta del Hepatocito/fisiología , Túbulos Renales/crecimiento & desarrollo , Morfogénesis/genética , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Animales , Inmunoprecipitación de Cromatina , Células Epiteliales/fisiología , Factor Nuclear 1-beta del Hepatocito/genética , Túbulos Renales/metabolismo , Ratones , Ratones Noqueados , Ratones Mutantes , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/antagonistas & inhibidores , Proteínas Supresoras de la Señalización de Citocinas/genéticaRESUMEN
Morphogenesis involves coordinated proliferation, differentiation and spatial distribution of cells. We show that lengthening of renal tubules is associated with mitotic orientation of cells along the tubule axis, demonstrating intrinsic planar cell polarization, and we demonstrate that mitotic orientations are significantly distorted in rodent polycystic kidney models. These results suggest that oriented cell division dictates the maintenance of constant tubule diameter during tubular lengthening and that defects in this process trigger renal tubular enlargement and cyst formation.
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Polaridad Celular , Túbulos Renales/patología , Enfermedades Renales Poliquísticas/patología , Animales , Modelos Animales de Enfermedad , Factor Nuclear 1-beta del Hepatocito/genética , Túbulos Renales/efectos de los fármacos , Ratones , Ratones Mutantes , Mitosis , Mucoproteínas/metabolismo , Enfermedades Renales Poliquísticas/genética , Ratas , Ratas Sprague-Dawley , Huso Acromático/fisiología , Tamoxifeno/farmacología , UromodulinaAsunto(s)
Enfermedades Renales Quísticas/genética , Animales , Cilios/fisiología , Proteínas de Unión al ADN/genética , Modelos Animales de Enfermedad , Factor Nuclear 1-beta del Hepatocito , Humanos , Ratones , Ratones Endogámicos , Fenotipo , Riñón Poliquístico Autosómico Recesivo/genética , Riñón Poliquístico Autosómico Recesivo/fisiopatología , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
Mutations in cystic kidney disease genes represent a major genetic cause of end-stage renal disease. However, the molecular cascades controlling the expression of these genes are still poorly understood. Hepatocyte Nuclear Factor 1beta (HNF1beta) is a homeoprotein predominantly expressed in renal, pancreatic and hepatic epithelia. We report here that mice with renal-specific inactivation of HNF1beta develop polycystic kidney disease. We show that renal cyst formation is accompanied by a drastic defect in the transcriptional activation of Umod, Pkhd1 and Pkd2 genes, whose mutations are responsible for distinct cystic kidney syndromes. In vivo chromatin immunoprecipitation experiments demonstrated that HNF1beta binds to several DNA elements in murine Umod, Pkhd1, Pkd2 and Tg737/Polaris genomic sequences. Our results uncover a direct transcriptional hierarchy between HNF1beta and cystic disease genes. Interestingly, most of the identified HNF1beta target gene products colocalize to the primary cilium, a crucial organelle that plays an important role in controlling the proliferation of tubular cells. This may explain the increased proliferation of cystic cells in MODY5 patients carrying autosomal dominant mutations in HNF1beta.