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1.
Am J Hum Genet ; 110(8): 1377-1393, 2023 08 03.
Artículo en Inglés | MEDLINE | ID: mdl-37451268

RESUMEN

Phosphoinositides (PIs) are membrane phospholipids produced through the local activity of PI kinases and phosphatases that selectively add or remove phosphate groups from the inositol head group. PIs control membrane composition and play key roles in many cellular processes including actin dynamics, endosomal trafficking, autophagy, and nuclear functions. Mutations in phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2] phosphatases cause a broad spectrum of neurodevelopmental disorders such as Lowe and Joubert syndromes and congenital muscular dystrophy with cataracts and intellectual disability, which are thus associated with increased levels of PI(4,5)P2. Here, we describe a neurodevelopmental disorder associated with an increase in the production of PI(4,5)P2 and with PI-signaling dysfunction. We identified three de novo heterozygous missense variants in PIP5K1C, which encodes an isoform of the phosphatidylinositol 4-phosphate 5-kinase (PIP5KIγ), in nine unrelated children exhibiting intellectual disability, developmental delay, acquired microcephaly, seizures, visual abnormalities, and dysmorphic features. We provide evidence that the PIP5K1C variants result in an increase of the endosomal PI(4,5)P2 pool, giving rise to ectopic recruitment of filamentous actin at early endosomes (EEs) that in turn causes dysfunction in EE trafficking. In addition, we generated an in vivo zebrafish model that recapitulates the disorder we describe with developmental defects affecting the forebrain, including the eyes, as well as craniofacial abnormalities, further demonstrating the pathogenic effect of the PIP5K1C variants.


Asunto(s)
Discapacidad Intelectual , Fosfatidilinositoles , Animales , Síndrome , Actinas , Pez Cebra/genética , Discapacidad Intelectual/genética , Monoéster Fosfórico Hidrolasas/genética , Fosfatos de Fosfatidilinositol
2.
Hum Mol Genet ; 32(3): 473-488, 2023 01 13.
Artículo en Inglés | MEDLINE | ID: mdl-36018820

RESUMEN

Kinesins are motor proteins involved in microtubule (MT)-mediated intracellular transport. They contribute to key cellular processes, including intracellular trafficking, organelle dynamics and cell division. Pathogenic variants in kinesin-encoding genes underlie several human diseases characterized by an extremely variable clinical phenotype, ranging from isolated neurodevelopmental/neurodegenerative disorders to syndromic phenotypes belonging to a family of conditions collectively termed as 'ciliopathies.' Among kinesins, kinesin-1 is the most abundant MT motor for transport of cargoes towards the plus end of MTs. Three kinesin-1 heavy chain isoforms exist in mammals. Different from KIF5A and KIF5C, which are specifically expressed in neurons and established to cause neurological diseases when mutated, KIF5B is an ubiquitous protein. Three de novo missense KIF5B variants were recently described in four subjects with a syndromic skeletal disorder characterized by kyphomelic dysplasia, hypotonia and DD/ID. Here, we report three dominantly acting KIF5B variants (p.Asn255del, p.Leu498Pro and p.Leu537Pro) resulting in a clinically wide phenotypic spectrum, ranging from dilated cardiomyopathy with adult-onset ophthalmoplegia and progressive skeletal myopathy to a neurodevelopmental condition characterized by severe hypotonia with or without seizures. In vitro and in vivo analyses provide evidence that the identified disease-associated KIF5B variants disrupt lysosomal, autophagosome and mitochondrial organization, and impact cilium biogenesis. All variants, and one of the previously reported missense changes, were shown to affect multiple developmental processes in zebrafish. These findings document pleiotropic consequences of aberrant KIF5B function on development and cell homeostasis, and expand the phenotypic spectrum resulting from altered kinesin-mediated processes.


Asunto(s)
Cinesinas , Animales , Humanos , Cinesinas/genética , Cinesinas/metabolismo , Mamíferos/metabolismo , Hipotonía Muscular , Neuronas/metabolismo , Fenotipo , Pez Cebra/genética , Pez Cebra/metabolismo
3.
Am J Hum Genet ; 108(6): 1126-1137, 2021 06 03.
Artículo en Inglés | MEDLINE | ID: mdl-34010604

RESUMEN

Dysregulated transforming growth factor TGF-ß signaling underlies the pathogenesis of genetic disorders affecting the connective tissue such as Loeys-Dietz syndrome. Here, we report 12 individuals with bi-allelic loss-of-function variants in IPO8 who presented with a syndromic association characterized by cardio-vascular anomalies, joint hyperlaxity, and various degree of dysmorphic features and developmental delay as well as immune dysregulation; the individuals were from nine unrelated families. Importin 8 belongs to the karyopherin family of nuclear transport receptors and was previously shown to mediate TGF-ß-dependent SMADs trafficking to the nucleus in vitro. The important in vivo role of IPO8 in pSMAD nuclear translocation was demonstrated by CRISPR/Cas9-mediated inactivation in zebrafish. Consistent with IPO8's role in BMP/TGF-ß signaling, ipo8-/- zebrafish presented mild to severe dorso-ventral patterning defects during early embryonic development. Moreover, ipo8-/- zebrafish displayed severe cardiovascular and skeletal defects that mirrored the human phenotype. Our work thus provides evidence that IPO8 plays a critical and non-redundant role in TGF-ß signaling during development and reinforces the existing link between TGF-ß signaling and connective tissue defects.


Asunto(s)
Enfermedades Óseas/etiología , Enfermedades Cardiovasculares/etiología , Enfermedades del Tejido Conjuntivo/etiología , Inmunidad Celular/inmunología , Mutación con Pérdida de Función , Pérdida de Heterocigocidad , beta Carioferinas/genética , Adolescente , Adulto , Animales , Enfermedades Óseas/patología , Enfermedades Cardiovasculares/patología , Niño , Enfermedades del Tejido Conjuntivo/patología , Femenino , Humanos , Lactante , Masculino , Persona de Mediana Edad , Linaje , Fenotipo , Transducción de Señal , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Adulto Joven , Pez Cebra , beta Carioferinas/metabolismo
4.
Genet Med ; 26(6): 101081, 2024 06.
Artículo en Inglés | MEDLINE | ID: mdl-38293907

RESUMEN

PURPOSE: Progressive inherited retinal degenerations (IRDs) affecting rods and cones are clinically and genetically heterogeneous and can lead to blindness with limited therapeutic options. The major gene defects have been identified in subjects of European and Asian descent with only few reports of North African descent. METHODS: Genome, targeted next-generation, and Sanger sequencing was applied to cohort of ∼4000 IRDs cases. Expression analyses were performed including Chip-seq database analyses, on human-derived retinal organoids (ROs), retinal pigment epithelium cells, and zebrafish. Variants' pathogenicity was accessed using 3D-modeling and/or ROs. RESULTS: Here, we identified a novel gene defect with three distinct pathogenic variants in UBAP1L in 4 independent autosomal recessive IRD cases from Tunisia. UBAP1L is expressed in the retinal pigment epithelium and retina, specifically in rods and cones, in line with the phenotype. It encodes Ubiquitin-associated protein 1-like, containing a solenoid of overlapping ubiquitin-associated domain, predicted to interact with ubiquitin. In silico and in vitro studies, including 3D-modeling and ROs revealed that the solenoid of overlapping ubiquitin-associated domain is truncated and thus ubiquitin binding most likely abolished secondary to all variants identified herein. CONCLUSION: Biallelic UBAP1L variants are a novel cause of IRDs, most likely enriched in the North African population.


Asunto(s)
Distrofias de Conos y Bastones , Linaje , Pez Cebra , Humanos , Distrofias de Conos y Bastones/genética , Distrofias de Conos y Bastones/patología , Masculino , Femenino , Pez Cebra/genética , Animales , Genes Recesivos , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología , Mutación/genética , Células Fotorreceptoras Retinianas Conos/patología , Células Fotorreceptoras Retinianas Conos/metabolismo , Retina/patología , Retina/metabolismo , Adulto , Túnez , Retinitis Pigmentosa/genética , Retinitis Pigmentosa/patología , Fenotipo , Células Fotorreceptoras Retinianas Bastones/metabolismo , Células Fotorreceptoras Retinianas Bastones/patología
5.
Cell ; 134(6): 1055-65, 2008 Sep 19.
Artículo en Inglés | MEDLINE | ID: mdl-18805097

RESUMEN

The different cell types in the central nervous system develop from a common pool of progenitor cells. The nuclei of progenitors move between the apical and basal surfaces of the neuroepithelium in phase with their cell cycle, a process termed interkinetic nuclear migration (INM). In the retina of zebrafish mikre oko (mok) mutants, in which the motor protein Dynactin-1 is disrupted, interkinetic nuclei migrate more rapidly and deeply to the basal side and more slowly to the apical side. We found that Notch signaling is predominantly activated on the apical side in both mutants and wild-type. Mutant progenitors are, thus, less exposed to Notch and exit the cell cycle prematurely. This leads to an overproduction of early-born retinal ganglion cells (RGCs) at the expense of later-born interneurons and glia. Our data indicate that the function of INM is to balance the exposure of progenitor nuclei to neurogenic versus proliferative signals.


Asunto(s)
Núcleo Celular/metabolismo , Células Neuroepiteliales/citología , Organogénesis , Retina/embriología , Animales , Tipificación del Cuerpo , Ciclo Celular , Diferenciación Celular , Complejo Dinactina , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas Asociadas a Microtúbulos/genética , Mutación , Células Neuroepiteliales/metabolismo , Receptores Notch/metabolismo , Retina/citología , Células Ganglionares de la Retina/metabolismo , Células Madre/citología , Células Madre/metabolismo , Pez Cebra , Proteínas de Pez Cebra/genética
6.
Nat Methods ; 15(11): 977-983, 2018 11.
Artículo en Inglés | MEDLINE | ID: mdl-30323353

RESUMEN

Understanding how distributed neuronal circuits integrate sensory information and generate behavior is a central goal of neuroscience. However, it has been difficult to study neuronal networks at single-cell resolution across the entire adult brain in vertebrates because of their size and opacity. We address this challenge here by introducing the fish Danionella translucida to neuroscience as a potential model organism. This teleost remains small and transparent even in adulthood, when neural circuits and behavior have matured. Despite having the smallest known adult vertebrate brain, D. translucida displays a rich set of complex behaviors, including courtship, shoaling, schooling, and acoustic communication. In order to carry out optical measurements and perturbations of neural activity with genetically encoded tools, we established CRISPR-Cas9 genome editing and Tol2 transgenesis techniques. These features make D. translucida a promising model organism for the study of adult vertebrate brain function at single-cell resolution.


Asunto(s)
Conducta Animal , Encéfalo/anatomía & histología , Encéfalo/fisiología , Cyprinidae/anatomía & histología , Cyprinidae/fisiología , Procesamiento de Imagen Asistido por Computador/métodos , Neuronas/fisiología , Animales , Edición Génica , Técnicas de Transferencia de Gen , Modelos Animales , Red Nerviosa , Fenómenos Fisiológicos del Sistema Nervioso
7.
Nat Methods ; 15(12): 1126, 2018 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-30397327

RESUMEN

The version of this paper originally published contained errors in reference citations: in the first paragraph of the Results section, the text "This extent of optical clarity probably results from the absence of skull above the brain22. In our specimens, Nissl-stained coronal sections through the head showed that the skull surrounds the brain only laterally and ventrally" should have read "This extent of optical clarity probably results from the absence of skull above the brain21. In our specimens, Nissl-stained coronal sections through the head22 showed that the skull surrounds the brain only laterally and ventrally." In addition, the unit abbreviation "µm" was incorrectly divided at a line break in the third paragraph of the Discussion, which might have led to some confusion. These errors have been corrected in the PDF and HTML versions of the article.

8.
Nat Methods ; 15(11): 969-976, 2018 11.
Artículo en Inglés | MEDLINE | ID: mdl-30377377

RESUMEN

Currently available inhibitory optogenetic tools provide short and transient silencing of neurons, but they cannot provide long-lasting inhibition because of the requirement for high light intensities. Here we present an optimized blue-light-sensitive synthetic potassium channel, BLINK2, which showed good expression in neurons in three species. The channel is activated by illumination with low doses of blue light, and in our experiments it remained active over (tens of) minutes in the dark after the illumination was stopped. This activation caused long periods of inhibition of neuronal firing in ex vivo recordings of mouse neurons and impaired motor neuron response in zebrafish in vivo. As a proof-of-concept application, we demonstrated that in a freely moving rat model of neuropathic pain, the activation of a small number of BLINK2 channels caused a long-lasting (>30 min) reduction in pain sensation.


Asunto(s)
Potenciales de Acción , Hiperalgesia/fisiopatología , Neuronas/fisiología , Optogenética , Dolor/fisiopatología , Enfermedades del Sistema Nervioso Periférico/fisiopatología , Proteínas Recombinantes de Fusión/metabolismo , Animales , Femenino , Luz , Masculino , Ratones Endogámicos C57BL , Neuronas/citología , Paclitaxel/toxicidad , Dolor/inducido químicamente , Enfermedades del Sistema Nervioso Periférico/inducido químicamente , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/genética , Pez Cebra
9.
Cell Mol Life Sci ; 77(1): 161-177, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-31161284

RESUMEN

Peripheral nervous system development involves a tight coordination of neuronal birth and death and a substantial remodelling of the myelinating glia cytoskeleton to achieve myelin wrapping of its projecting axons. However, how these processes are coordinated through time is still not understood. We have identified engulfment and cell motility 1, Elmo1, as a novel component that regulates (i) neuronal numbers within the Posterior Lateral Line ganglion and (ii) radial sorting of axons by Schwann cells (SC) and myelination in the PLL system in zebrafish. Our results show that neuronal and myelination defects observed in elmo1 mutant are rescued through small GTPase Rac1 activation. Inhibiting macrophage development leads to a decrease in neuronal numbers, while peripheral myelination is intact. However, elmo1 mutants do not show defective macrophage activity, suggesting a role for Elmo1 in PLLg neuronal development and SC myelination independent of macrophages. Forcing early Elmo1 and Rac1 expression specifically within SCs rescues elmo1-/- myelination defects, highlighting an autonomous role for Elmo1 and Rac1 in radial sorting of axons by SCs and myelination. This uncovers a previously unknown function of Elmo1 that regulates fundamental aspects of PNS development.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Vaina de Mielina/metabolismo , Neurogénesis , Neuronas/citología , Proteínas de Pez Cebra/metabolismo , Pez Cebra/crecimiento & desarrollo , Proteína de Unión al GTP rac1/metabolismo , Animales , Apoptosis , Axones/metabolismo , Axones/ultraestructura , Movimiento Celular , Neuronas/metabolismo , Neuronas/ultraestructura , Nervios Periféricos/crecimiento & desarrollo , Nervios Periféricos/ultraestructura , Células de Schwann/citología , Células de Schwann/metabolismo , Células de Schwann/ultraestructura
10.
Dev Biol ; 455(2): 393-408, 2019 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-31323192

RESUMEN

The cerebellum and the cerebellum-like structure in the mesencephalic tectum in zebrafish contain multiple cell types, including principal cells (i.e., Purkinje cells and type I neurons) and granule cells, that form neural circuits in which the principal cells receive and integrate inputs from granule cells and other neurons. It is largely unknown how these cells are positioned and how neural circuits form. While Reelin signaling is known to play an important role in cell positioning in the mammalian brain, its role in the formation of other vertebrate brains remains elusive. Here we found that zebrafish with mutations in Reelin or in the Reelin-signaling molecules Vldlr or Dab1a exhibited ectopic Purkinje cells, eurydendroid cells (projection neurons), and Bergmann glial cells in the cerebellum, and ectopic type I neurons in the tectum. The ectopic Purkinje cells and type I neurons received aberrant afferent fibers in these mutants. In wild-type zebrafish, reelin transcripts were detected in the internal granule cell layer, while Reelin protein was localized to the superficial layer of the cerebellum and the tectum. Laser ablation of the granule cell axons perturbed the localization of Reelin, and the mutation of both kif5aa and kif5ba, which encode major kinesin I components in the granule cells, disrupted the elongation of granule cell axons and the Reelin distribution. Our findings suggest that in zebrafish, (1) Reelin is transported from the granule cell soma to the superficial layer by axonal transport; (2) Reelin controls the migration of neurons and glial cells from the ventricular zone; and (3) Purkinje cells and type I neurons attract afferent axons during the formation of the cerebellum and the cerebellum-like structure.


Asunto(s)
Moléculas de Adhesión Celular Neuronal/fisiología , Cerebelo/embriología , Proteínas de la Matriz Extracelular/fisiología , Proteínas del Tejido Nervioso/fisiología , Serina Endopeptidasas/fisiología , Proteínas de Pez Cebra/fisiología , Pez Cebra/embriología , Animales , Sistemas CRISPR-Cas , Moléculas de Adhesión Celular Neuronal/genética , Movimiento Celular , Cerebelo/citología , Proteínas de la Matriz Extracelular/genética , Cinesinas/metabolismo , Mutación , Proteínas del Tejido Nervioso/genética , Células de Purkinje/citología , Proteína Reelina , Serina Endopeptidasas/genética , Transducción de Señal , Pez Cebra/anatomía & histología , Proteínas de Pez Cebra/genética
11.
Genome Res ; 26(5): 681-92, 2016 05.
Artículo en Inglés | MEDLINE | ID: mdl-26957310

RESUMEN

CRISPR/Cas9-mediated targeted mutagenesis allows efficient generation of loss-of-function alleles in zebrafish. To date, this technology has been primarily used to generate genetic knockout animals. Nevertheless, the study of the function of certain loci might require tight spatiotemporal control of gene inactivation. Here, we show that tissue-specific gene disruption can be achieved by driving Cas9 expression with the Gal4/UAS system. Furthermore, by combining the Gal4/UAS and Cre/loxP systems, we establish a versatile tool to genetically label mutant cell clones, enabling their phenotypic analysis. Our technique has the potential to be applied to diverse model organisms, enabling tissue-specific loss-of-function and phenotypic characterization of live and fixed tissues.


Asunto(s)
Sistemas CRISPR-Cas , Silenciador del Gen , Organismos Modificados Genéticamente , Pez Cebra , Animales , Organismos Modificados Genéticamente/genética , Organismos Modificados Genéticamente/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismo
12.
PLoS Genet ; 12(11): e1006459, 2016 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-27902705

RESUMEN

Axon ensheathment by specialized glial cells is an important process for fast propagation of action potentials. The rapid electrical conduction along myelinated axons is mainly due to its saltatory nature characterized by the accumulation of ion channels at the nodes of Ranvier. However, how these ion channels are transported and anchored along axons is not fully understood. We have identified N-myc downstream-regulated gene 4, ndrg4, as a novel factor that regulates sodium channel clustering in zebrafish. Analysis of chimeric larvae indicates that ndrg4 functions autonomously within neurons for sodium channel clustering at the nodes. Molecular analysis of ndrg4 mutants shows that expression of snap25 and nsf are sharply decreased, revealing a role of ndrg4 in controlling vesicle exocytosis. This uncovers a previously unknown function of ndrg4 in regulating vesicle docking and nodes of Ranvier organization, at least through its ability to finely tune the expression of the t-SNARE/NSF machinery.


Asunto(s)
Proteínas Musculares/genética , Proteínas Sensibles a N-Etilmaleimida/biosíntesis , Nódulos de Ranvier/genética , Proteína 25 Asociada a Sinaptosomas/biosíntesis , Proteínas de Pez Cebra/biosíntesis , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Animales , Axones/metabolismo , Exocitosis/genética , Regulación de la Expresión Génica , Humanos , Proteínas Musculares/metabolismo , Proteínas Sensibles a N-Etilmaleimida/genética , Neuroglía/metabolismo , Neuronas/metabolismo , Nódulos de Ranvier/metabolismo , Células de Schwann , Canales de Sodio/genética , Canales de Sodio/metabolismo , Transmisión Sináptica/genética , Proteína 25 Asociada a Sinaptosomas/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismo
13.
Development ; 142(5): 832-9, 2015 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-25655700

RESUMEN

Divisions that generate one neuronal lineage-committed and one self-renewing cell maintain the balance of proliferation and differentiation for the generation of neuronal diversity. The asymmetric inheritance of apical domains and components of the cell division machinery has been implicated in this process, and might involve interactions with cell fate determinants in regulatory feedback loops of an as yet unknown nature. Here, we report the dynamics of Anillin - an essential F-actin regulator and furrow component - and its contribution to progenitor cell divisions in the developing zebrafish retina. We find that asymmetrically dividing retinal ganglion cell progenitors position the Anillin-rich midbody at the apical domain of the differentiating daughter. anillin hypomorphic conditions disrupt asymmetric apical domain inheritance and affect daughter cell fate. Consequently, the retinal cell type composition is profoundly affected, such that the ganglion cell layer is dramatically expanded. This study provides the first in vivo evidence for the requirement of Anillin during asymmetric neurogenic divisions. It also provides insights into a reciprocal regulation between Anillin and the ganglion cell fate determinant Ath5, suggesting a mechanism whereby the balance of proliferation and differentiation is accomplished during progenitor cell divisions in vivo.


Asunto(s)
Proteínas Contráctiles/metabolismo , Células Ganglionares de la Retina/citología , Células Ganglionares de la Retina/metabolismo , Células Madre/citología , Células Madre/metabolismo , Animales , Proteínas Contráctiles/genética , Técnica del Anticuerpo Fluorescente , Hibridación in Situ , Microscopía Confocal , Pez Cebra , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo
14.
Methods ; 121-122: 77-85, 2017 05 15.
Artículo en Inglés | MEDLINE | ID: mdl-28300641

RESUMEN

With its variety of applications, the CRISPR/Cas9 genome editing technology has been rapidly evolving in the last few years. In the zebrafish community, knock-out reports are constantly increasing but insertion studies have been so far more challenging. With this review, we aim at giving an overview of the homologous directed repair (HDR)-based knock-in generation in zebrafish. We address the critical points and limitations of the procedure such as cutting efficiency of the chosen single guide RNA, use of cas9 mRNA or Cas9 protein, homology arm size etc. but also ways to circumvent encountered issues with HDR insertions by the development of non-homologous dependent strategies. While imprecise, these homology-independent mechanisms based on non-homologous-end-joining (NHEJ) repair have been employed in zebrafish to generate reporter lines or to accurately edit an open reading frame by the use of intron-targeting modifications. Therefore, with higher efficiency and insertion rate, NHEJ-based knock-in seems to be a promising approach to target endogenous loci and to circumvent the limitations of HDR whenever it is possible and appropriate. In this perspective, we propose new strategies to generate cDNA edited or tagged insertions, which once established will constitute a new and versatile toolbox for CRISPR/Cas9-based knock-ins in zebrafish.


Asunto(s)
Proteínas Bacterianas/genética , Sistemas CRISPR-Cas , Endonucleasas/genética , Edición Génica/métodos , Técnicas de Sustitución del Gen , Técnicas de Transferencia de Gen , ARN Guía de Kinetoplastida/genética , Alelos , Animales , Animales Modificados Genéticamente , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Proteína 9 Asociada a CRISPR , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Embrión no Mamífero , Endonucleasas/metabolismo , Marcación de Gen/métodos , Genoma , Microinyecciones , ARN Guía de Kinetoplastida/metabolismo , Reparación del ADN por Recombinación , Pez Cebra/genética
15.
Dev Biol ; 414(2): 133-41, 2016 06 15.
Artículo en Inglés | MEDLINE | ID: mdl-27158028

RESUMEN

It is now becoming evident that hydrogen peroxide (H2O2), which is constantly produced by nearly all cells, contributes to bona fide physiological processes. However, little is known regarding the distribution and functions of H2O2 during embryonic development. To address this question, we used a dedicated genetic sensor and revealed a highly dynamic spatio-temporal pattern of H2O2 levels during zebrafish morphogenesis. The highest H2O2 levels are observed during somitogenesis and organogenesis, and these levels gradually decrease in the mature tissues. Biochemical and pharmacological approaches revealed that H2O2 distribution is mainly controlled by its enzymatic degradation. Here we show that H2O2 is enriched in different regions of the developing brain and demonstrate that it participates to axonal guidance. Retinal ganglion cell axonal projections are impaired upon H2O2 depletion and this defect is rescued by H2O2 or ectopic activation of the Hedgehog pathway. We further show that ex vivo, H2O2 directly modifies Hedgehog secretion. We propose that physiological levels of H2O2 regulate RGCs axonal growth through the modulation of Hedgehog pathway.


Asunto(s)
Orientación del Axón/efectos de los fármacos , Proteínas Hedgehog/fisiología , Peróxido de Hidrógeno/metabolismo , Neurogénesis/fisiología , Células Ganglionares de la Retina/fisiología , Proteínas de Pez Cebra/fisiología , Pez Cebra/embriología , Animales , Animales Modificados Genéticamente , Orientación del Axón/fisiología , Axones/metabolismo , Catalasa/metabolismo , Cisteína/metabolismo , Células HeLa , Humanos , Proteínas Luminiscentes/análisis , Transporte de Proteínas/efectos de los fármacos , Células Ganglionares de la Retina/ultraestructura , Transducción de Señal/fisiología , Superóxido Dismutasa/metabolismo , Pez Cebra/metabolismo
16.
Genome Res ; 24(1): 142-53, 2014 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-24179142

RESUMEN

Sequence-specific nucleases like TALENs and the CRISPR/Cas9 system have greatly expanded the genome editing possibilities in model organisms such as zebrafish. Both systems have recently been used to create knock-out alleles with great efficiency, and TALENs have also been successfully employed in knock-in of DNA cassettes at defined loci via homologous recombination (HR). Here we report CRISPR/Cas9-mediated knock-in of DNA cassettes into the zebrafish genome at a very high rate by homology-independent double-strand break (DSB) repair pathways. After co-injection of a donor plasmid with a short guide RNA (sgRNA) and Cas9 nuclease mRNA, concurrent cleavage of donor plasmid DNA and the selected chromosomal integration site resulted in efficient targeted integration of donor DNA. We successfully employed this approach to convert eGFP into Gal4 transgenic lines, and the same plasmids and sgRNAs can be applied in any species where eGFP lines were generated as part of enhancer and gene trap screens. In addition, we show the possibility of easily targeting DNA integration at endogenous loci, thus greatly facilitating the creation of reporter and loss-of-function alleles. Due to its simplicity, flexibility, and very high efficiency, our method greatly expands the repertoire for genome editing in zebrafish and can be readily adapted to many other organisms.


Asunto(s)
Proteínas Asociadas a CRISPR/metabolismo , Reparación del ADN , Técnicas de Sustitución del Gen , Ingeniería Genética/métodos , Pez Cebra/genética , Animales , Proteínas Asociadas a CRISPR/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Roturas del ADN de Doble Cadena , Genoma , Mutagénesis , Reparación del ADN por Recombinación , Pez Cebra/embriología , ARN Pequeño no Traducido
17.
EMBO J ; 30(9): 1676-7, 2011 May 04.
Artículo en Inglés | MEDLINE | ID: mdl-21540882

RESUMEN

Interkinetic nuclear migration (INM) is a common feature of developing neuroepithelia, consisting of the periodic movement of the cell nucleus in phase with cell-cycle progression. In this issue of The EMBO Journal, Kosodo et al provide a first molecular mechanism to couple nuclear migration and cell cycle: the microtubule-associated protein Tpx2 redistributes from the nucleus to the apical process during the S-G2 transition, modulating microtubule organization to promote apical nuclear migration.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/fisiología , Núcleo Celular/fisiología , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Células Neuroepiteliales/fisiología , Proteínas Nucleares/metabolismo , Microtúbulos/fisiología , Modelos Biológicos
18.
Methods ; 69(2): 142-50, 2014 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-24704174

RESUMEN

The targeted introduction of mutations utilizing sequence specific transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system (RNA-guided nucleases, RGNs) has revolutionized reverse genetic approaches in numerous model organisms. In zebrafish, both systems were successfully applied to generate loss-of-function alleles by targeting open reading frames or deletion and inversion of whole chromosomal regions. In addition to the production of these loss-of-function alleles, genomic engineering by insertion of short sequences utilizing single stranded DNA oligonucleotides as templates for homology based repair was made possible, enabling effective insertion of loxP sites or tags for protein coding genes. Recent studies based on homologous recombination and non-homologous end joining have also broadened the repertoire for genome editing. These approaches allow the targeted insertion of open reading frames or even whole donor vectors. In this review we summarize the use of TALENs and RNA-guided nucleases in the field of zebrafish genetics with a special focus on knock-in approaches.


Asunto(s)
Sistemas CRISPR-Cas/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Técnicas de Sustitución del Gen/métodos , Proteínas de Pez Cebra/genética , Secuencia de Aminoácidos , Animales , Datos de Secuencia Molecular , Pez Cebra
19.
Nature ; 461(7262): 407-10, 2009 Sep 17.
Artículo en Inglés | MEDLINE | ID: mdl-19759620

RESUMEN

Locomotion relies on neural networks called central pattern generators (CPGs) that generate periodic motor commands for rhythmic movements. In vertebrates, the excitatory synaptic drive for inducing the spinal CPG can originate from either supraspinal glutamatergic inputs or from within the spinal cord. Here we identify a spinal input to the CPG that drives spontaneous locomotion using a combination of intersectional gene expression and optogenetics in zebrafish larvae. The photo-stimulation of one specific cell type was sufficient to induce a symmetrical tail beating sequence that mimics spontaneous slow forward swimming. This neuron is the Kolmer-Agduhr cell, which extends cilia into the central cerebrospinal-fluid-containing canal of the spinal cord and has an ipsilateral ascending axon that terminates in a series of consecutive segments. Genetically silencing Kolmer-Agduhr cells reduced the frequency of spontaneous free swimming, indicating that activity of Kolmer-Agduhr cells provides necessary tone for spontaneous forward swimming. Kolmer-Agduhr cells have been known for over 75 years, but their function has been mysterious. Our results reveal that during early development in zebrafish these cells provide a positive drive to the spinal CPG for spontaneous locomotion.


Asunto(s)
Luz , Locomoción/fisiología , Médula Espinal/fisiología , Pez Cebra/genética , Pez Cebra/fisiología , Animales , Animales Modificados Genéticamente , Axones/fisiología , Cilios/fisiología , Femenino , Larva/genética , Larva/fisiología , Larva/efectos de la radiación , Locomoción/genética , Locomoción/efectos de la radiación , Masculino , Modelos Neurológicos , Neuronas/fisiología , Neuronas/efectos de la radiación , Médula Espinal/citología , Médula Espinal/efectos de la radiación , Natación/fisiología , Cola (estructura animal)/fisiología , Pez Cebra/embriología , Pez Cebra/crecimiento & desarrollo
20.
Commun Biol ; 7(1): 615, 2024 May 22.
Artículo en Inglés | MEDLINE | ID: mdl-38777862

RESUMEN

Deficiency of adenosine deaminase 2 (DADA2) is an inborn error of immunity caused by loss-of-function mutations in the adenosine deaminase 2 (ADA2) gene. Clinical manifestations of DADA2 include vasculopathy and immuno-hematological abnormalities, culminating in bone marrow failure. A major gap exists in our knowledge of the regulatory functions of ADA2 during inflammation and hematopoiesis, mainly due to the absence of an ADA2 orthologue in rodents. Exploring these mechanisms is essential for understanding disease pathology and developing new treatments. Zebrafish possess two ADA2 orthologues, cecr1a and cecr1b, with the latter showing functional conservation with human ADA2. We establish a cecr1b-loss-of-function zebrafish model that recapitulates the immuno-hematological and vascular manifestations observed in humans. Loss of Cecr1b disrupts hematopoietic stem cell specification, resulting in defective hematopoiesis. This defect is caused by induced inflammation in the vascular endothelium. Blocking inflammation, pharmacological modulation of the A2r pathway, or the administration of the recombinant human ADA2 corrects these defects, providing insights into the mechanistic link between ADA2 deficiency, inflammation and immuno-hematological abnormalities. Our findings open up potential therapeutic avenues for DADA2 patients.


Asunto(s)
Adenosina Desaminasa , Hematopoyesis , Células Madre Hematopoyéticas , Inflamación , Pez Cebra , Animales , Pez Cebra/genética , Adenosina Desaminasa/genética , Adenosina Desaminasa/metabolismo , Adenosina Desaminasa/deficiencia , Células Madre Hematopoyéticas/metabolismo , Inflamación/genética , Inflamación/metabolismo , Hematopoyesis/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Humanos , Transducción de Señal , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo
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