RESUMEN
Questioning is an important activity in teaching. In medical colleges, on-stage quiz competitions are appreciated by students as well as faculty as they are an engaging way to connect with the discipline. We organized the Physiology Quiz Competition to assess the concepts of functional mechanisms of various organ systems. It was an academic tool for teaching and learning for 200 first-year Bachelor of Medicine and Bachelor of Surgery (MBBS) course students. It was conducted in four rounds: multiple choice question-based round 1 (R1), explanatory-type round 2 (R2), rapid-fire round 3 (R3), and image-based round 4 (R4). The postquiz feedback questionnaire included 23 total questions; 10 questions used a 5-point Likert scale, another 10 questions had dichotomous options, and the remaining 3 questions were used to collect general information about all rounds. Data were collected and analyzed. The outcome of academic learning was reported by 26% of students regarding R1, 30.5% regarding R2, 18.5% regarding R3, and 25% regarding R4 round. R4 and R3 were reported by 44.5% and 23% of students and R2 and R1 by 16% of students as a source of entertainment. A total of 67% of students found the quiz to be an excellent teaching and learning method. All students endorsed the quiz as innovative and interesting. In conclusion, the Physiology Quiz Competition can be used for edutainment as an innovative teaching and learning method, especially for first-year medical students.NEW & NOTEWORTHY The Physiology Quiz Competition is a method of teaching and learning that provides education with entertainment in a medical college. It increases students' interest in the subject of Physiology and helps them to understand and learn the subject effectively.
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Fisiología , Estudiantes de Medicina , Humanos , Evaluación Educacional , Aprendizaje , Curriculum , Escolaridad , Enseñanza , Fisiología/educaciónRESUMEN
Radial glial cells are presumptive neural stem cells (NSCs) in the developing nervous system. The direct requirement of radial glia for the generation of a diverse array of neuronal and glial subtypes, however, has not been tested. We employed two novel transgenic zebrafish lines and endogenous markers of NSCs and radial glia to show for the first time that radial glia are essential for neurogenesis during development. By using the gfap promoter to drive expression of nuclear localized mCherry we discerned two distinct radial glial-derived cell types: a major nestin+/Sox2+ subtype with strong gfap promoter activity and a minor Sox2+ subtype lacking this activity. Fate mapping studies in this line indicate that gfap+ radial glia generate later-born CoSA interneurons, secondary motorneurons, and oligodendroglia. In another transgenic line using the gfap promoter-driven expression of the nitroreductase enzyme, we induced cell autonomous ablation of gfap+ radial glia and observed a reduction in their specific derived lineages, but not Blbp+ and Sox2+/gfap-negative NSCs, which were retained and expanded at later larval stages. Moreover, we provide evidence supporting classical roles of radial glial in axon patterning, blood-brain barrier formation, and locomotion. Our results suggest that gfap+ radial glia represent the major NSC during late neurogenesis for specific lineages, and possess diverse roles to sustain the structure and function of the spinal cord. These new tools will both corroborate the predicted roles of astroglia and reveal novel roles related to development, physiology, and regeneration in the vertebrate nervous system. GLIA 2016;64:1170-1189.
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Proteína Ácida Fibrilar de la Glía/metabolismo , Neurogénesis/fisiología , Neuronas/fisiología , Médula Espinal/citología , Factores de Edad , Animales , Animales Modificados Genéticamente , Apoptosis/genética , Diferenciación Celular , Proliferación Celular/genética , Embrión no Mamífero , Desarrollo Embrionario/genética , Proteína Ácida Fibrilar de la Glía/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Locomoción/genética , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Médula Espinal/embriología , Factores de Tiempo , Pez Cebra , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Proteína Fluorescente RojaRESUMEN
Primary ciliary dyskinesia (PCD) is caused when defects of motile cilia lead to chronic airway infections, male infertility, and situs abnormalities. Multiple causative PCD mutations account for only 65% of cases, suggesting that many genes essential for cilia function remain to be discovered. By using zebrafish morpholino knockdown of PCD candidate genes as an in vivo screening platform, we identified c21orf59, ccdc65, and c15orf26 as critical for cilia motility. c21orf59 and c15orf26 knockdown in zebrafish and planaria blocked outer dynein arm assembly, and ccdc65 knockdown altered cilia beat pattern. Biochemical analysis in Chlamydomonas revealed that the C21orf59 ortholog FBB18 is a flagellar matrix protein that accumulates specifically when cilia motility is impaired. The Chlamydomonas ida6 mutant identifies CCDC65/FAP250 as an essential component of the nexin-dynein regulatory complex. Analysis of 295 individuals with PCD identified recessive truncating mutations of C21orf59 in four families and CCDC65 in two families. Similar to findings in zebrafish and planaria, mutations in C21orf59 caused loss of both outer and inner dynein arm components. Our results characterize two genes associated with PCD-causing mutations and elucidate two distinct mechanisms critical for motile cilia function: dynein arm assembly for C21orf59 and assembly of the nexin-dynein regulatory complex for CCDC65.
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Trastornos de la Motilidad Ciliar/genética , Glicoproteínas/genética , Síndrome de Kartagener/genética , Pez Cebra/genética , Animales , Chlamydomonas/genética , Cilios/genética , Análisis Mutacional de ADN/métodos , Dineínas/genética , Femenino , Humanos , Masculino , Mutación , Sistemas de Lectura Abierta , Planarias/genética , Proteoma/genéticaRESUMEN
Odd-skipped related 1 (Osr1) encodes a zinc finger transcription factor required for kidney development. Osr1 deficiency in mice results in metanephric kidney agenesis, whereas knockdown or mutation studies in zebrafish revealed that pronephric nephrons require osr1 for proximal tubule and podocyte development. osr1-deficient pronephric podocyte progenitors express the Wilms' tumor suppressor wt1a but do not undergo glomerular morphogenesis or express the foot process junctional markers nephrin and podocin. The function of osr1 in podocyte differentiation remains unclear, however. Here, we found by double fluorescence in situ hybridization that podocyte progenitors coexpress osr1 and wt1a. Knockdown of wt1a disrupted podocyte differentiation and prevented expression of osr1. Blocking retinoic acid signaling, which regulates wt1a, also prevented osr1 expression in podocyte progenitors. Furthermore, unlike the osr1-deficient proximal tubule phenotype, which can be rescued by manipulation of endoderm development, podocyte differentiation was not affected by altered endoderm development, as assessed by nephrin and podocin expression in double osr1/sox32-deficient embryos. These results suggest a different, possibly cell- autonomous requirement for osr1 in podocyte differentiation downstream of wt1a. Indeed, osr1-deficient embryos did not exhibit podocyte progenitor expression of the transcription factor lhx1a, and forced expression of activated forms of the lhx1a gene product rescued nephrin expression in osr1-deficient podocytes. Our results place osr1 in a framework of transcriptional regulators that control the expression of podocin and nephrin and thereby mediate podocyte differentiation.
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Podocitos/fisiología , Factores de Transcripción/fisiología , Proteínas WT1/fisiología , Proteínas de Pez Cebra/fisiología , Animales , Diferenciación Celular/fisiología , Femenino , Regulación del Desarrollo de la Expresión Génica , Túbulos Renales/citología , Túbulos Renales/embriología , Túbulos Renales/fisiología , Proteínas con Homeodominio LIM/genética , Proteínas con Homeodominio LIM/fisiología , Masculino , Podocitos/citología , Células Madre/citología , Células Madre/fisiología , Factores de Transcripción/genética , Transcripción Genética/fisiología , Proteínas WT1/genética , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
Tubulin post-translational modifications generate microtubule heterogeneity and modulate microtubule function, and are catalyzed by tubulin tyrosine ligase-like (TTLL) proteins. Using antibodies specific to monoglycylated, polyglycylated, and glutamylated tubulin in whole mount immunostaining of zebrafish embryos, we observed distinct, tissue-specific patterns of tubulin modifications. Tubulin modification patterns in cilia correlated with the expression of ttll3 and ttll6 in ciliated cells. Expression screening of all zebrafish tubulin tyrosine ligase-like genes revealed additional tissue-specific expression of ttll1 in brain neurons, ttll4 in muscle, and ttll7 in otic placodes. Knockdown of ttll3 eliminated cilia tubulin glycylation but had surprisingly mild effects on cilia structure and motility. Similarly, knockdown of ttll6 strongly reduced cilia tubulin glutamylation but only partially affected cilia structure and motility. Combined loss of function of ttll3 and ttll6 caused near complete loss of cilia motility and induced a variety of axonemal ultrastructural defects similar to defects previously observed in zebrafish fleer mutants, which were shown to lack tubulin glutamylation. Consistently, we find that fleer mutants also lack tubulin glycylation. These results indicate that tubulin glycylation and glutamylation have overlapping functions in maintaining cilia structure and motility and that the fleer/dyf-1 TPR protein is required for both types of tubulin post-translational modification.
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Regulación Enzimológica de la Expresión Génica/fisiología , Péptido Sintasas/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Tubulina (Proteína)/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Animales , Axonema/genética , Axonema/metabolismo , Axonema/ultraestructura , Cilios/enzimología , Cilios/genética , Cilios/ultraestructura , Técnicas de Silenciamiento del Gen , Mutación , Especificidad de Órganos/fisiología , Péptido Sintasas/genética , Tubulina (Proteína)/genética , Pez Cebra/anatomía & histología , Proteínas de Pez Cebra/genéticaRESUMEN
Cilia and basal bodies are essential organelles for a broad spectrum of functions, including the development of left-right asymmetry, kidney function, cerebrospinal fluid transport, generation of photoreceptor outer segments, and hedgehog signaling. Zebrafish fleer (flr) mutants exhibit kidney cysts, randomized left-right asymmetry, hydrocephalus, and rod outer segment defects, suggesting a pleiotropic defect in ciliogenesis. Positional cloning flr identified a tetratricopeptide repeat protein homologous to the Caenorhabditis elegans protein DYF1 that was highly expressed in ciliated cells. flr pronephric cilia were shortened and showed a reduced beat amplitude, and olfactory cilia were absent in mutants. flr cilia exhibited ultrastructural defects in microtubule B-tubules, similar to axonemes that lack tubulin posttranslational modifications (polyglutamylation or polyglycylation). flr cilia showed a dramatic reduction in cilia polyglutamylated tubulin, indicating that flr encodes a novel modulator of tubulin polyglutamylation. We also found that the C. elegans flr homologue, dyf-1, is also required for tubulin polyglutamylation in sensory neuron cilia. Knockdown of zebrafish Ttll6, a tubulin polyglutamylase, specifically eliminated tubulin polyglutamylation and cilia formation in olfactory placodes, similar to flr mutants. These results are the first in vivo evidence that tubulin polyglutamylation is required for vertebrate cilia motility and structure, and, when compromised, results in failed ciliogenesis.
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Ácido Poliglutámico/metabolismo , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Cilios/metabolismo , Secuencia Conservada , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Humanos , Microtúbulos/metabolismo , Datos de Secuencia Molecular , Mutación/genética , Bulbo Olfatorio/embriología , Bulbo Olfatorio/metabolismo , Fenotipo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Alineación de Secuencia , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/genéticaRESUMEN
The carboxamide N,N-di-ethyl-meta-toluamide (DEET) is the most effective and widely used insect repellent today. However, drawbacks concerning the efficacy and the safety of the repellent have led to efforts to design new classes of insect repellents. Through quantitative structure-activity relationships, chemists have discovered two chemical groups of novel repellents: the acylpiperidines and the carboxamides, with the acylpiperidines generally more potent in biological assays. Although the exact mechanism of action of DEET and other repellents has not yet been thoroughly elucidated, previous research shows that the activity of insect odorant receptors are inhibited in the presence of repellents. The present electrophysiological study employs two-electrode voltage clamp with Xenopus laevis oocytes expressing AgOR2/AgOrco and AgOR8/AgOrco receptors to assess the effects of the novel repellents on Anopheles gambiae Giles (Insecta: Diptera: Culicidae) mosquito odorant receptors. The novel acylpiperidines and carboxamides reversibly inhibited (12-91%) odorant-evoked currents from both AgOR2/AgOrco and AgOR8/AgOrco receptors in a dose-dependent manner at all tested concentrations (30 µM to 1 mM). Furthermore, all the novel agents were more potent inhibitors of the receptors than DEET, with the acylpiperidines producing on average greater inhibition than the carboxamides. Interestingly, there was a correlation (r2 = 0.72) between the percentage inhibition of AgOR2/AgOrco receptor currents and protection times of the acylpiperidines. Our results add to existing evidence that the repellency of a compound is linked to its ability to disrupt the insect olfactory system and that the acylpiperidines could represent a class of more effective alternatives to the current gold standard, DEET.
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Anopheles/metabolismo , DEET/farmacología , Repelentes de Insectos/farmacología , Receptores Odorantes/antagonistas & inhibidores , Animales , Humanos , Técnicas de Placa-Clamp , Receptores Odorantes/metabolismo , Xenopus laevisRESUMEN
Sensory cilia and intraflagellar transport (IFT), a pathway essential for ciliogenesis, play important roles in embryonic development and cell differentiation. In vertebrate photoreceptors IFT is required for the early development of ciliated sensory outer segments (OS), an elaborate organelle that sequesters the many proteins comprising the phototransduction machinery. As in other cilia and flagella, heterotrimeric members of the kinesin 2 family have been implicated as the anterograde IFT motor in OS. However, in Caenorhabditis elegans, OSM-3, a homodimeric kinesin 2 motor, plays an essential role in some, but not all sensory cilia. Kif17, a vertebrate OSM-3 homologue, is known for its role in dendritic trafficking in neurons, but a function in ciliogenesis has not been determined. We show that in zebrafish Kif17 is widely expressed in the nervous system and retina. In photoreceptors Kif17 co-localizes with IFT proteins within the OS, and co-immunoprecipitates with IFT proteins. Knockdown of Kif17 has little if any effect in early embryogenesis, including the formation of motile sensory cilia in the pronephros. However, OS formation and targeting of the visual pigment protein is severely disrupted. Our analysis shows that Kif17 is essential for photoreceptor OS development, and suggests that Kif17 plays a cell type specific role in vertebrate ciliogenesis.
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Cinesinas/metabolismo , Segmento Externo de la Célula en Bastón/embriología , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Secuencia de Aminoácidos , Animales , Axonema/metabolismo , Proteínas Portadoras/análisis , Proteínas Portadoras/metabolismo , Cilios/fisiología , Cinesinas/antagonistas & inhibidores , Cinesinas/genética , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Transporte de Proteínas , Segmento Externo de la Célula en Bastón/metabolismo , Pez Cebra/metabolismo , Proteínas de Pez Cebra/antagonistas & inhibidores , Proteínas de Pez Cebra/genéticaRESUMEN
Normal cognitive and autonomic functions require nicotinic synaptic signaling. Despite the physiological importance of these synapses, little is known about molecular mechanisms that direct their assembly during development. We show here that the tumor-suppressor protein adenomatous polyposis coli (APC) functions in localizing alpha3-nicotinic acetylcholine receptors (nAChRs) to neuronal postsynaptic sites. Our quantitative confocal microscopy studies indicate that APC is selectively enriched at cholinergic synapses; APC surface clusters are juxtaposed to synaptic vesicle clusters and colocalize with alpha3-nAChRs but not with the neighboring synaptic glycine receptors or perisynaptic alpha7-nAChRs on chick ciliary ganglion (CG) neurons. We identify PSD (postsynaptic density)-93, beta-catenin, and microtubule end binding protein EB1 as APC binding partners. PSD-93 and beta-catenin are also enriched at alpha3-nAChR postsynaptic sites. EB1 shows close proximity to and partial overlap with alpha3-nAChR and APC surface clusters. We tested the role of APC in neuronal nicotinic synapse assembly by using retroviral-mediated in vivo overexpression of an APC dominant-negative (APC-dn) peptide to block the interaction of endogenous APC with both EB1 and PSD-93 during synapse formation in CG neurons. The overexpressed APC-dn led to dramatic decreases in alpha3-nAChR surface levels and clusters. Effects were specific to alpha3-nAChR postsynaptic sites; synaptic glycine receptor and perisynaptic alpha7-nAChR clusters were not altered. In addition, APC-dn also reduced surface membrane-associated clusters of PSD-93 and EB1. The results show that APC plays a key role in organizing excitatory cholinergic postsynaptic specializations in CG neurons. We identify APC as the first nonreceptor protein to function in localizing nAChRs to neuronal synapses in vivo.
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Proteína de la Poliposis Adenomatosa del Colon/fisiología , Proteínas del Tejido Nervioso/fisiología , Receptores Nicotínicos/química , Sinapsis/fisiología , Proteína de la Poliposis Adenomatosa del Colon/análisis , Proteína de la Poliposis Adenomatosa del Colon/genética , Animales , Embrión de Pollo , Fibras Colinérgicas/química , Fibras Colinérgicas/ultraestructura , Proteínas del Citoesqueleto/análisis , ADN Complementario/genética , Ganglios Parasimpáticos/citología , Ganglios Parasimpáticos/embriología , Genes APC , Interneuronas/química , Interneuronas/ultraestructura , Microscopía Confocal , Microscopía Fluorescente , Proteínas Asociadas a Microtúbulos/análisis , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/química , Unión Proteica , Receptores de Glicina/análisis , Receptores Nicotínicos/análisis , Receptores Nicotínicos/fisiología , Proteínas Recombinantes de Fusión/fisiología , Sinapsis/química , Sinapsis/ultraestructura , Transactivadores/análisis , Técnicas del Sistema de Dos Híbridos , beta CateninaRESUMEN
Glutamylation is a functionally important tubulin posttranslational modification enriched on stable microtubules of neuronal axons, mitotic spindles, centrioles, and cilia. In vertebrates, balanced activities of tubulin glutamyl ligase and cytoplasmic carboxypeptidase deglutamylase enzymes maintain organelle- and cell type-specific tubulin glutamylation patterns. Tubulin glutamylation in cilia is regulated via restricted subcellular localization or expression of tubulin glutamyl ligases (ttlls) and nonenzymatic proteins, including the zebrafish TPR repeat protein Fleer/Ift70. Here we analyze the expression patterns of ccp deglutamylase genes during zebrafish development and the effects of ccp gene knockdown on cilia formation, morphology, and tubulin glutamylation. The deglutamylases ccp2, ccp5, and ccp6 are expressed in ciliated cells, whereas ccp1 expression is restricted to the nervous system. Only ccp5 knockdown increases cilia tubulin glutamylation, induces ciliopathy phenotypes, including axis curvature, hydrocephalus, and pronephric cysts, and disrupts multicilia motility, suggesting that Ccp5 is the principal tubulin deglutamylase that maintains functional levels of cilia tubulin glutamylation. The ability of ccp5 knockdown to restore cilia tubulin glutamylation in fleer/ift70 mutants and rescue pronephric multicilia formation in both fleer- and ift88-deficient zebrafish indicates that tubulin glutamylation is a key driver of ciliogenesis.
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Carboxipeptidasas/fisiología , Cilios/fisiología , Ácido Glutámico/metabolismo , Tubulina (Proteína)/metabolismo , Proteínas de Pez Cebra/fisiología , Animales , Técnicas de Silenciamiento del Gen , Larva/citología , Larva/enzimología , Microtúbulos/metabolismo , Pronefro/citología , Pronefro/enzimología , Procesamiento Proteico-Postraduccional , Pez CebraRESUMEN
BACKGROUND: Obesity is rapidly escalating in India in all age groups. School-based data indicate a prevalence rate between 5.6% and 24% in children and adolescents. Adolescent obesity is associated with a greater long-term risk of hypertension and type 2 diabetes mellitus in adulthood. However, studies investigating pulmonary functions in obese adolescents are few. The present study assesses pulmonary functions in obese adolescent boys from a school in Baroda city, Gujarat. AIMS: (i) To assess the dynamic lung functions in obese adolescent boys. (ii) To determine the predominant lung function impairment associated with obesity in adolescence. MATERIALS AND METHODS: Dynamic lung functions were measured in 30 obese adolescent boys and an equal number of age-matched controls using MEDI:SPIRO software (Maestros Mediline Systems Ltd., Navi Mumbai, India). RESULTS: Forced expiratory volume in the 1(st) second (FEV(1))/forced vital capacity (FVC) and maximum voluntary ventilation (MVV) were significantly decreased in the obese group (P < 0.001). Pulmonary functions in the study population correlated negatively with various indices of obesity, viz. weight, body mass index (BMI), waist circumference, hip circumference, and waist-to-hip ratio. The strongest negative correlation was between BMI and FEV(1)/FVC (P < 0.001) and between BMI, MVV, and Forced Expiratory Flow (FEF(25-75%)) (P < 0.001). Waist-to-hip ratio in the study population correlated negatively with MVV (P < 0.01), but not with FEV(1)/FVC. CONCLUSIONS: Lung function impairment, particularly decreased MVV and reduced FEV(1)/FVC ratio, is associated with obesity in adolescence. In addition, pulmonary functions deteriorate with increasing obesity in adolescence and correlate negatively with various indices of obesity, viz. weight, BMI, waist circumference, hip circumference, and waist-to-hip ratio. This study reveals another health hazard associated with obesity and highlights the need to aggressively reduce weight at a younger age.
RESUMEN
Organ development leads to the emergence of organ function, which in turn can impact developmental processes. Here we show that fluid flow-induced collective epithelial migration during kidney nephron morphogenesis induces cell stretch that in turn signals epithelial proliferation. Increased cell proliferation was dependent on PI3K signaling. Inhibiting epithelial proliferation by blocking PI3K or CDK4/Cyclin D1 activity arrested cell migration prematurely and caused a marked overstretching of the distal nephron tubule. Computational modeling of the involved cell processes predicted major morphological and kinetic outcomes observed experimentally under a variety of conditions. Overall, our findings suggest that kidney development is a recursive process where emerging organ function "feeds back" to the developmental program to influence fundamental cellular events such as cell migration and proliferation, thus defining final organ morphology.
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Células Epiteliales/citología , Túbulos Renales/citología , Fenómenos Mecánicos , Morfogénesis , Fosfatidilinositol 3-Quinasas/metabolismo , Pronefro/embriología , Pez Cebra/embriología , Animales , Fenómenos Biomecánicos , Movimiento Celular , Proliferación Celular , Túbulos Renales/embriología , Modelos Biológicos , Pronefro/citología , Transducción de SeñalRESUMEN
Cilia are essential for fertilization, respiratory clearance, cerebrospinal fluid circulation and establishing laterality. Cilia motility defects cause primary ciliary dyskinesia (PCD, MIM244400), a disorder affecting 1:15,000-30,000 births. Cilia motility requires the assembly of multisubunit dynein arms that drive ciliary bending. Despite progress in understanding the genetic basis of PCD, mutations remain to be identified for several PCD-linked loci. Here we show that the zebrafish cilia paralysis mutant schmalhans (smh(tn222)) encodes the coiled-coil domain containing 103 protein (Ccdc103), a foxj1a-regulated gene product. Screening 146 unrelated PCD families identified individuals in six families with reduced outer dynein arms who carried mutations in CCDC103. Dynein arm assembly in smh mutant zebrafish was rescued by wild-type but not mutant human CCDC103. Chlamydomonas Ccdc103/Pr46b functions as a tightly bound, axoneme-associated protein. These results identify Ccdc103 as a dynein arm attachment factor that causes primary ciliary dyskinesia when mutated.
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Dineínas/metabolismo , Síndrome de Kartagener/genética , Animales , Cilios/metabolismo , Femenino , Humanos , Masculino , Mutación , Linaje , Pez CebraRESUMEN
Epithelial tubules consist of multiple cell types that are specialized for specific aspects of organ function. In the zebrafish pronephros, multiciliated cells (MCCs) are specialized for fluid propulsion, whereas transporting epithelial cells recover filtered-blood solutes. These cell types are distributed in a ;salt-and-pepper' fashion in the pronephros, suggesting that a lateral inhibition mechanism may play a role in their differentiation. We find that the Notch ligand Jagged 2 is expressed in MCCs and that notch3 is expressed in pronephric epithelial cells. Morpholino knockdown of either jagged 2 or notch3, or mutation in mind bomb (in which Notch signaling is impaired), dramatically expands ciliogenic gene expression, whereas ion transporter expression is lost, indicating that pronephric cells are transfated to MCCs. Conversely, ectopic expression of the Notch1a intracellular domain represses MCC differentiation. Gamma-secretase inhibition using DAPT demonstrated a requirement for Notch signaling early in pronephric development, before the pattern of MCC differentiation is apparent. Strikingly, we find that jagged 2 knockdown generates extra cilia and is sufficient to rescue the kidney cilia mutant double bubble. Our results indicate that Jagged 2/Notch signaling modulates the number of multiciliated versus transporting epithelial cells in the pronephros by way of a genetic pathway involving repression of rfx2, a key transcriptional regulator of the ciliogenesis program.
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Proteínas de Homeodominio/metabolismo , Nefronas/embriología , Proteínas del Tejido Nervioso/metabolismo , Receptor Notch1/metabolismo , Receptores Notch/fisiología , Proteínas de Pez Cebra/metabolismo , Proteínas de Pez Cebra/fisiología , Pez Cebra/embriología , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Proteínas de Unión al Calcio/antagonistas & inhibidores , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Diferenciación Celular , Cilios/metabolismo , Cilios/fisiología , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Epitelio/metabolismo , Proteínas de Homeodominio/genética , Proteína Jagged-2 , Nefronas/citología , Nefronas/metabolismo , Proteínas del Tejido Nervioso/genética , Receptor Notch1/genética , Receptor Notch3 , Receptores Notch/genética , Transducción de Señal , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/antagonistas & inhibidores , Proteínas de Pez Cebra/genéticaAsunto(s)
Ácido Poliglutámico/metabolismo , Procesamiento Proteico-Postraduccional , Tubulina (Proteína)/metabolismo , Proteínas de Pez Cebra/genética , Animales , Cilios/metabolismo , Cilios/ultraestructura , Genes , Humanos , Inmunohistoquímica/instrumentación , Inmunohistoquímica/métodos , Morfogénesis/fisiología , Tubulina (Proteína)/genética , Pez Cebra/anatomía & histología , Pez Cebra/embriología , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismoRESUMEN
G proteins modulate synaptic transmission. Regulators of G protein signaling (RGS) proteins accelerate the intrinsic GTPase activity of Galpha subunits, and thus terminate G protein activation. Whether RGS proteins themselves are under cellular control is not well defined, particularly in native cells. In dorsal root ganglion neurons overexpressing RGS3, we find that G protein signaling is rapidly terminated (or "desensitized") by calcium influx through voltage-gated channels. This rapid desensitization is most likely mediated by direct binding of calcium to RGS3, as deletion of an EF-hand domain in RGS3 abolishes both the desensitization (observed physiologically) and a calcium-RGS3 interaction (observed in a gel-shift assay). A naturally occurring variant of RGS3 that lacks the EF hand neither binds calcium nor produces rapid desensitization, giving rise instead to a slower calcium-dependent desensitization that is attenuated by a calmodulin antagonist. Thus, activity-evoked calcium entry in sensory neurons may provide differential control of G protein signaling, depending on the isoform of RGS3 expressed in the cells. In complex neural circuits subjected to abundant synaptic inhibition by G proteins (as occurs in dorsal spinal cord), rapid termination of inhibition by electrical activity by EF hand-containing RGS3 may ensure the faithful transmission of information from the most active sensory inputs.