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1.
Cells ; 13(13)2024 Jul 02.
Artículo en Inglés | MEDLINE | ID: mdl-38994990

RESUMEN

In zebrafish, like in mammals, radial glial cells (RGCs) can act as neural progenitors during development and regeneration in adults. However, the heterogeneity of glia subpopulations entails the need for different specific markers of zebrafish glia. Currently, fluorescent protein expression mediated by a regulatory element from the glial fibrillary acidic protein (gfap) gene is used as a prominent glia reporter. We now expand this tool by demonstrating that a regulatory element from the mouse Fatty acid binding protein 7 (Fabp7) gene drives reliable expression in fabp7-expressing zebrafish glial cells. By using three different Fabp7 regulatory element-mediated fluorescent protein reporter strains, we reveal in double transgenic zebrafish that progenitor cells expressing fluorescent proteins driven by the Fabp7 regulatory element give rise to radial glia, oligodendrocyte progenitors, and some neuronal precursors. Furthermore, Bergmann glia represent the almost only glial population of the zebrafish cerebellum (besides a few oligodendrocytes), and the radial glia also remain in the mature cerebellum. Fabp7 regulatory element-mediated reporter protein expression in Bergmann glia progenitors suggests their origin from the ventral cerebellar proliferation zone, the ventricular zone, but not from the dorsally positioned upper rhombic lip. These new Fabp7 reporters will be valuable for functional studies during development and regeneration.


Asunto(s)
Animales Modificados Genéticamente , Proteína de Unión a los Ácidos Grasos 7 , Pez Cebra , Animales , Pez Cebra/genética , Pez Cebra/metabolismo , Proteína de Unión a los Ácidos Grasos 7/metabolismo , Proteína de Unión a los Ácidos Grasos 7/genética , Neuroglía/metabolismo , Cerebelo/metabolismo , Cerebelo/citología , Oligodendroglía/metabolismo , Oligodendroglía/citología , Ratones , Proteínas de Unión a Ácidos Grasos/genética , Proteínas de Unión a Ácidos Grasos/metabolismo , Proteínas de Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética
2.
Nat Methods ; 20(10): 1605-1616, 2023 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-37666982

RESUMEN

Recent progress in fluorescent protein development has generated a large diversity of near-infrared fluorescent proteins (NIR FPs), which are rapidly becoming popular probes for a variety of imaging applications. However, the diversity of NIR FPs poses a challenge for end-users in choosing the optimal one for a given application. Here we conducted a systematic and quantitative assessment of intracellular brightness, photostability, oligomeric state, chemical stability and cytotoxicity of 22 NIR FPs in cultured mammalian cells and primary mouse neurons and identified a set of top-performing FPs including emiRFP670, miRFP680, miRFP713 and miRFP720, which can cover a majority of imaging applications. The top-performing proteins were further validated for in vivo imaging of neurons in Caenorhabditis elegans, zebrafish, and mice as well as in mice liver. We also assessed the applicability of the selected NIR FPs for multicolor imaging of fusions, expansion microscopy and two-photon imaging.

3.
Cell Mol Life Sci ; 80(8): 227, 2023 Jul 25.
Artículo en Inglés | MEDLINE | ID: mdl-37490159

RESUMEN

The cerebellum represents a brain compartment that first appeared in gnathostomes (jawed vertebrates). Besides the addition of cell numbers, its development, cytoarchitecture, circuitry, physiology, and function have been highly conserved throughout avian and mammalian species. While cerebellar research in avian and mammals is extensive, systematic investigations on this brain compartment in zebrafish as a teleostian model organism started only about two decades ago, but has provided considerable insight into cerebellar development, physiology, and function since then. Zebrafish are genetically tractable with nearly transparent small-sized embryos, in which cerebellar development occurs within a few days. Therefore, genetic investigations accompanied with non-invasive high-resolution in vivo time-lapse imaging represents a powerful combination for interrogating the behavior and function of cerebellar cells in their complex native environment.


Asunto(s)
Cerebelo , Pez Cebra , Animales , Encéfalo , Recuento de Células , Mamíferos
4.
Front Mol Neurosci ; 16: 1166900, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37181649

RESUMEN

Introduction: The cerebellum is organized into functional regions each dedicated to process different motor or sensory inputs for controlling different locomotor behaviors. This functional regionalization is prominent in the evolutionary conserved single-cell layered Purkinje cell (PC) population. Fragmented gene expression domains suggest a genetic organization of PC layer regionalization during cerebellum development. However, the establishment of such functionally specific domains during PC differentiation remained elusive. Methods and results: We show the progressive emergence of functional regionalization of PCs from broad responses to spatially restricted regions in zebrafish by means of in vivo Ca2+-imaging during stereotypic locomotive behavior. Moreover, we reveal that formation of new dendritic spines during cerebellar development using in vivo imaging parallels the time course of functional domain development. Pharmacological as well as cell-type specific optogenetic inhibition of PC neuronal activity results in reduced PC dendritic spine density and an altered stagnant pattern of functional domain formation in the PC layer. Discussion: Hence, our study suggests that functional regionalization of the PC layer is driven by physiological activity of maturing PCs themselves.

5.
Elife ; 122023 04 12.
Artículo en Inglés | MEDLINE | ID: mdl-37042514

RESUMEN

Zebrafish have an impressive capacity to regenerate neurons in the central nervous system. However, regeneration of the principal neuron of the evolutionary conserved cerebellum, the Purkinje cell (PC), is believed to be limited to developmental stages based on invasive lesions. In contrast, non-invasive cell type-specific ablation by induced apoptosis closely represents a process of neurodegeneration. We demonstrate that the ablated larval PC population entirely recovers in number, quickly reestablishes electrophysiological properties, and properly integrates into circuits to regulate cerebellum-controlled behavior. PC progenitors are present in larvae and adults, and PC ablation in adult cerebelli results in an impressive PC regeneration of different PC subtypes able to restore behavioral impairments. Interestingly, caudal PCs are more resistant to ablation and regenerate more efficiently, suggesting a rostro-caudal pattern of de- and regeneration properties. These findings demonstrate that the zebrafish cerebellum is able to regenerate functional PCs during all stages of the animal's life.


Asunto(s)
Células de Purkinje , Pez Cebra , Animales , Células de Purkinje/fisiología , Pez Cebra/fisiología , Animales Modificados Genéticamente , Cerebelo/fisiología , Neuronas
6.
Int J Mol Sci ; 23(21)2022 Nov 06.
Artículo en Inglés | MEDLINE | ID: mdl-36362380

RESUMEN

Neurotrophins such as nerve growth factor (ngf) and brain-derived neurotrophic factor (bdnf) play important roles in the central nervous system. They are potential therapeutic drugs for the treatment of neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. In this study, we investigated the neurotrophic properties of triterpenes isolated from fruiting bodies of Laetiporus sulphureus and a mycelial culture of Antrodia sp. MUCL 56049. The structures of the isolated compounds were elucidated based on nuclear magnetic resonance (NMR) spectroscopy in combination with high-resolution electrospray mass spectrometry (HR-ESIMS). The secondary metabolites were tested for neurotrophin (ngf and bdnf) expression levels on human astrocytoma 1321N1 cells. Neurite outgrowth activity using rat pheochromocytoma (PC-12) cells was also determined. Twelve triterpenoids were isolated, of which several potently stimulated the expression of neurotrophic factors, namely, ngf (sulphurenic acid, 15α-dehydroxytrametenolic acid, fomefficinic acid D, and 16α-hydroxyeburicoic acid) and bdnf (sulphurenic acid and 15α-dehydroxytrametenolic acid), respectively. The triterpenes also potentiated ngf-induced neurite outgrowth in PC-12 cells. This is, to the best of our knowledge, the first report on the compound class of lanostanes in direct relation to bdnf and ngf enhancement. These compounds are widespread in medicinal mushrooms; hence, they appear promising as a starting point for the development of drugs and mycopharmaceuticals to combat neurodegenerative diseases. Interestingly, they do not show any pronounced cytotoxicity and may, therefore, be better suited for therapy than many other neurotrophic compounds that were previously reported.


Asunto(s)
Basidiomycota , Enfermedades Neurodegenerativas , Triterpenos , Animales , Ratas , Humanos , Factor de Crecimiento Nervioso/farmacología , Factor de Crecimiento Nervioso/metabolismo , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Triterpenos/farmacología , Triterpenos/química , Madera/metabolismo , Basidiomycota/química
8.
Sci Rep ; 12(1): 10190, 2022 06 17.
Artículo en Inglés | MEDLINE | ID: mdl-35715437

RESUMEN

Spectrally diverse fluorescent proteins (FPs) provide straightforward means for multiplexed imaging of biological systems. Among FPs fitting standard color channels, blue FPs (BFPs) are characterized by lower brightness compared to other spectral counterparts. Furthermore, available BFPs were not systematically characterized for imaging in cultured mammalian cells and common model organisms. Here we introduce a pair of new BFPs, named Electra1 and Electra2, developed through hierarchical screening in bacterial and mammalian cells using a novel dual-expression vector. We performed systematic benchmarking of Electras against state-of-art BFPs in cultured mammalian cells and demonstrated their utility as fluorescent tags for structural proteins. The Electras variants were validated for multicolor neuroimaging in Caenorhabditis elegans, zebrafish larvae, and mice in comparison with one of the best in the class BFP mTagBFP2 using one-photon and two-photon microscopy. The developed BFPs are suitable for multicolor imaging of cultured cells and model organisms in vivo. We believe that the described dual-expression vector has a great potential to be adopted by protein engineers for directed molecular evolution of FPs.


Asunto(s)
Evolución Molecular Dirigida , Pez Cebra , Animales , Línea Celular , Diagnóstico por Imagen , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Proteínas Luminiscentes/química , Proteínas Luminiscentes/genética , Mamíferos , Ratones , Pez Cebra/genética
9.
Protein Sci ; 31(3): 728-751, 2022 03.
Artículo en Inglés | MEDLINE | ID: mdl-34913537

RESUMEN

In vivo imaging of model organisms is heavily reliant on fluorescent proteins with high intracellular brightness. Here we describe a practical method for rapid optimization of fluorescent proteins via directed molecular evolution in cultured mammalian cells. Using this method, we were able to perform screening of large gene libraries containing up to 2 × 107 independent random genes of fluorescent proteins expressed in HEK cells, completing one iteration of directed evolution in a course of 8 days. We employed this approach to develop a set of green and near-infrared fluorescent proteins with enhanced intracellular brightness. The developed near-infrared fluorescent proteins demonstrated high performance for fluorescent labeling of neurons in culture and in vivo in model organisms such as Caenorhabditis elegans, Drosophila, zebrafish, and mice. Spectral properties of the optimized near-infrared fluorescent proteins enabled crosstalk-free multicolor imaging in combination with common green and red fluorescent proteins, as well as dual-color near-infrared fluorescence imaging. The described method has a great potential to be adopted by protein engineers due to its simplicity and practicality. We also believe that the new enhanced fluorescent proteins will find wide application for in vivo multicolor imaging of small model organisms.


Asunto(s)
Evolución Molecular Dirigida , Pez Cebra , Animales , Línea Celular , Colorantes Fluorescentes/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Mamíferos/genética , Ratones , Neuronas/metabolismo , Imagen Óptica , Pez Cebra/genética , Pez Cebra/metabolismo
10.
Int J Mol Sci ; 22(21)2021 Oct 21.
Artículo en Inglés | MEDLINE | ID: mdl-34768779

RESUMEN

Zebrafish have come into focus to model cerebellar diseases such as spinocerebellar ataxias (SCAs), which is caused by an expansion of translated CAG repeats in several unrelated genes. In spinocerebellar ataxia type 1 (SCA1), gain-of-function in the mutant ATXN1 contributes to SCA1's neuropathy. Human ATXN1 and its paralog ATXN1L are chromatin-binding factors, act as transcriptional repressors, and have similar expression patterns. However, little is known about atxn1 genes in zebrafish. Recently, two family members, atxn1a and atxn1b, were identified as duplicate orthologs of ATXN1, as was atxn1l, the ortholog of ATXN1L. In this study, we analyzed the phylogenetic relationship of the atxn1 family members in zebrafish, compared their genetic structures, and verified the predicted transcripts by both RT-PCR and whole-mount in situ hybridization. All three genes, atxn1a, atxn1b, and atxn1l, show overlapping, but also distinct, expression domains during embryonic and larval development. While atxn1a and atxn1l display similar spatiotemporal embryonic expression, atxn1b expression is initiated during the onset of brain development and is predominantly expressed in the cerebellum throughout zebrafish development. These results provide new insights into atxn1 genes and their expression patterns in zebrafish during embryonic and late-larval development and may contribute importantly to future experiments in disease modeling of SCAs.


Asunto(s)
Ataxina-1/genética , Ataxina-1/fisiología , Animales , Ataxina-1/metabolismo , Cerebelo/metabolismo , Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/genética , Larva/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Filogenia , Análisis Espacio-Temporal , Ataxias Espinocerebelosas/genética , Relación Estructura-Actividad , Pez Cebra/embriología , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismo
11.
Sci Rep ; 11(1): 18408, 2021 09 15.
Artículo en Inglés | MEDLINE | ID: mdl-34526620

RESUMEN

Purkinje cells are critically involved in processing the cerebellar functions by shaping and coordinating commands that they receive. Here, we demonstrate experimentally that in the adult zebrafish valvular part of the cerebellum, the Purkinje cells exhibited variable firing and functional responses and allowed the categorization into three firing classes. Compared with the Purkinje cells in the corpus cerebelli, the valvular Purkinje cells receive weak and occasional input from the inferior olive and are not active during locomotion. Together, our findings expand further the regional functional differences of the Purkinje cell population and expose their non-locomotor functionality.


Asunto(s)
Cerebelo/citología , Cerebelo/fisiología , Células de Purkinje/citología , Pez Cebra/fisiología , Animales , Animales Modificados Genéticamente , Biomarcadores , Cerebelo/metabolismo , Fenómenos Electrofisiológicos , Técnica del Anticuerpo Fluorescente , Expresión Génica , Genes Reporteros , Inmunohistoquímica , Células de Purkinje/metabolismo
12.
Int J Mol Sci ; 22(14)2021 Jul 08.
Artículo en Inglés | MEDLINE | ID: mdl-34298970

RESUMEN

Dominant spinocerebellar ataxias (SCAs) are progredient neurodegenerative diseases commonly affecting the survival of Purkinje cells (PCs) in the human cerebellum. Spinocerebellar ataxia type 1 (SCA1) is caused by the mutated ataxin1 (Atx1) gene product, in which a polyglutamine stretch encoded by CAG repeats is extended in affected SCA1 patients. As a monogenetic disease with the Atx1-polyQ protein exerting a gain of function, SCA1 can be genetically modelled in animals by cell type-specific overexpression. We have established a transgenic PC-specific SCA1 model in zebrafish coexpressing the fluorescent reporter protein mScarlet together with either human wild type Atx1[30Q] as control or SCA1 patient-derived Atx1[82Q]. SCA1 zebrafish display an age-dependent PC degeneration starting at larval stages around six weeks postfertilization, which continuously progresses during further juvenile and young adult stages. Interestingly, PC degeneration is observed more severely in rostral than in caudal regions of the PC population. Although such a neuropathology resulted in no gross locomotor control deficits, SCA1-fish with advanced PC loss display a reduced exploratory behaviour. In vivo imaging in this SCA1 model may help to better understand such patterned PC death known from PC neurodegeneration diseases, to elucidate disease mechanisms and to provide access to neuroprotective compound characterization in vivo.


Asunto(s)
Ataxina-1/genética , Modelos Animales de Enfermedad , Ataxias Espinocerebelosas/genética , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Animales , Animales Modificados Genéticamente , Ataxina-1/fisiología , Muerte Celular , Progresión de la Enfermedad , Conducta Exploratoria , Genes Reporteros , Humanos , Larva , Proteínas Luminiscentes/genética , Células de Purkinje/patología , Transgenes , Expansión de Repetición de Trinucleótido , Pez Cebra/crecimiento & desarrollo , Proteínas de Pez Cebra/fisiología , Proteína Fluorescente Roja
13.
J Biol Chem ; 297(1): 100853, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-34090874

RESUMEN

The highly conserved dual-specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1A) plays crucial roles during central nervous system development and homeostasis. Furthermore, its hyperactivity is considered responsible for some neurological defects in individuals with Down syndrome. We set out to establish a zebrafish model expressing human Dyrk1A that could be further used to characterize the interaction between Dyrk1A and neurological phenotypes. First, we revealed the prominent expression of dyrk1a homologs in cerebellar neurons in the zebrafish larval and adult brains. Overexpression of human dyrk1a in postmitotic cerebellar Purkinje neurons resulted in a structural misorganization of the Purkinje cells in cerebellar hemispheres and a compaction of this cell population. This impaired Purkinje cell organization was progressive, leading to an age-dependent dispersal of Purkinje neurons throughout the cerebellar molecular layer with larval swim deficits resulting in miscoordination of swimming and reduced exploratory behavior in aged adults. We also found that the structural misorganization of the larval Purkinje cell layer could be rescued by pharmacological treatment with Dyrk1A inhibitors. We further reveal the in vivo efficiency of a novel selective Dyrk1A inhibitor, KuFal194. These findings demonstrate that the zebrafish is a well-suited vertebrate organism to genetically model severe neurological diseases with single cell type specificity. Such models can be used to relate molecular malfunction to cellular deficits, impaired tissue formation, and organismal behavior and can also be used for pharmacological compound testing and validation.


Asunto(s)
Cerebelo/metabolismo , Síndrome de Down/genética , Neuronas/metabolismo , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/genética , Proteínas de Pez Cebra/genética , Animales , Encéfalo/metabolismo , Encéfalo/patología , Cerebelo/patología , Modelos Animales de Enfermedad , Síndrome de Down/patología , Humanos , Neuronas/patología , Fosforilación/genética , Células de Purkinje/metabolismo , Células de Purkinje/patología , Pez Cebra/genética , Quinasas DyrK
14.
J Vis Exp ; (168)2021 02 10.
Artículo en Inglés | MEDLINE | ID: mdl-33645565

RESUMEN

Understanding the ephemeral changes that occur during brain development and maturation requires detailed high-resolution imaging in space and time at cellular and subcellular resolution. Advances in molecular and imaging technologies have allowed us to gain numerous detailed insights into cellular and molecular mechanisms of brain development in the transparent zebrafish embryo. Recently, processes of refinement of neuronal connectivity that occur at later larval stages several weeks after fertilization, which are for example control of social behavior, decision making or motivation-driven behavior, have moved into focus of research. At these stages, pigmentation of the zebrafish skin interferes with light penetration into brain tissue, and solutions for embryonic stages, e.g., pharmacological inhibition of pigmentation, are not feasible anymore. Therefore, a minimally invasive surgical solution for microscopy access to the brain of awake zebrafish is provided that is derived from electrophysiological approaches. In teleosts, skin and soft skull cartilage can be carefully removed by micro-peeling these layers, exposing underlying neurons and axonal tracts without damage. This allows for recording neuronal morphology, including synaptic structures and their molecular contents, and the observation of physiological changes such as Ca2+ transients or intracellular transport events. In addition, interrogation of these processes by means of pharmacological inhibition or optogenetic manipulation is feasible. This brain exposure approach provides information about structural and physiological changes in neurons as well as the correlation and interdependence of these events in live brain tissue in the range of minutes or hours. The technique is suitable for in vivo brain imaging of zebrafish larvae up to 30 days post fertilization, the latest developmental stage tested so far. It, thus, provides access to such important questions as synaptic refinement and scaling, axonal and dendritic transport, synaptic targeting of cytoskeletal cargo or local activity-dependent expression. Therefore, a broad use for this mounting and imaging approach can be anticipated.


Asunto(s)
Encéfalo/diagnóstico por imagen , Encéfalo/crecimiento & desarrollo , Imagenología Tridimensional , Piel/diagnóstico por imagen , Cráneo/diagnóstico por imagen , Pez Cebra/crecimiento & desarrollo , Anestesia , Animales , Encéfalo/irrigación sanguínea , Larva/fisiología , Neuronas/fisiología , Vigilia/fisiología
15.
Biomolecules ; 10(10)2020 10 14.
Artículo en Inglés | MEDLINE | ID: mdl-33066380

RESUMEN

Medicinal mushrooms of the genus Hericium are known to produce secondary metabolites with homeostatic properties for the central nervous system. We and others have recently demonstrated that among these metabolites cyathane diterpenoids and in particular erinacine C possess potent neurotrophin inducing properties in astrocytic cells. Yet, the signaling events downstream of erinacine C induced neurotrophin acitivity in neural-like adrenal phaeochromocytoma cells (PC12) cells have remained elusive. Similar, signaling events activated by erinacine C in astrocytic cells are unknown. Using a combination of genetic and pharmacological inhibitors we show that erinacine C induced neurotrophic activity mediates PC12 cell differentiation via the TrkA receptor and likely its associated PLCγ-, PI3K-, and MAPK/ERK pathways. Furthermore, a small library of transcriptional activation reporters revealed that erinacine C induces transcriptional activation mediated by DNA consensus binding sites of selected conserved transcription factor families. Among these, transcription is activated from an ETS consensus in a concentration dependent manner. Interestingly, induced ETS-consensus transcription occurs in parallel and independent of neurotrophin induction. This finding helps to explain the many pleiotropic functions of cyathane diterpenoids. Moreover, our studies provide genetic access to cyathane diterpenoid functions in astrocytic cells and help to mechanistically understand the action of cyathanes in glial cells.


Asunto(s)
Astrocitos/efectos de los fármacos , Diterpenos/farmacología , Activación Transcripcional/efectos de los fármacos , Animales , Astrocitos/fisiología , Sitios de Unión/genética , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Células Cultivadas , Secuencia Conservada/efectos de los fármacos , Secuencia Conservada/genética , Motivo ETS , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/genética , Factor de Crecimiento Nervioso/genética , Factor de Crecimiento Nervioso/metabolismo , Células PC12 , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Ratas , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Activación Transcripcional/genética , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética
16.
PLoS One ; 15(8): e0237167, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32764780

RESUMEN

The zebrafish Danio rerio is a valuable and common model for scientists in the fields of genetics and developmental biology. Since zebrafish are also amenable to genetic manipulation, modelling of human diseases or behavioral experiments have moved into the focus of zebrafish research. Consequently, gene expression data beyond embryonic and larval stages become more important, yet there is a dramatic knowledge gap of gene expression beyond day four of development. Like in other model organisms, the visualization of spatial and temporal gene expression by whole mount in situ hybridization (ISH) becomes increasingly difficult when zebrafish embryos develop further and hence the growing tissues become dense and less permeable. Here we introduce a modified method for whole mount ISH, which overcomes these penetration and detection problem. The method is an all in one solution that enables the detection and visualization of gene expression patterns up to the late larval stage in a 3D manner without the need for tissue sectioning and offers a valuable extension for whole mount ISH by immunohistochemistry in the zebrafish field.


Asunto(s)
Biología Evolutiva/métodos , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica , Pez Cebra/crecimiento & desarrollo , Animales , Embrión no Mamífero , Inmunohistoquímica , Hibridación in Situ , Larva/genética , Larva/crecimiento & desarrollo , Modelos Animales , Pez Cebra/genética
17.
J Cell Biol ; 219(10)2020 10 05.
Artículo en Inglés | MEDLINE | ID: mdl-32668451

RESUMEN

Neuronal migration during development is necessary to form an ordered and functional brain. Postmitotic neurons require microtubules and dynein to move, but the mechanisms by which they contribute to migration are not fully characterized. Using tegmental hindbrain nuclei neurons in zebrafish embryos together with subcellular imaging, optogenetics, and photopharmacology, we show that, in vivo, the centrosome's position relative to the nucleus is not linked to greatest motility in this cell type. Nevertheless, microtubules, dynein, and kinesin-1 are essential for migration, and we find that interference with endosome formation or the Golgi apparatus impairs migration to a similar extent as disrupting microtubules. In addition, an imbalance in the traffic of the model cargo Cadherin-2 also reduces neuronal migration. These results lead us to propose that microtubules act as cargo carriers to control spatiotemporal protein distribution, which in turn controls motility. This adds crucial insights into the variety of ways that microtubules can support successful neuronal migration in vivo.


Asunto(s)
Cadherinas/genética , Desarrollo Embrionario/genética , Cinesinas/genética , Neuronas/metabolismo , Animales , Movimiento Celular/genética , Dineínas/genética , Embrión no Mamífero , Aparato de Golgi/genética , Proteínas de Microtúbulos/genética , Microtúbulos/genética , Proteínas Motoras Moleculares/genética , Pez Cebra/genética , Pez Cebra/crecimiento & desarrollo
18.
Proc Natl Acad Sci U S A ; 117(29): 17330-17337, 2020 07 21.
Artículo en Inglés | MEDLINE | ID: mdl-32632015

RESUMEN

Purkinje cells, the principal neurons of cerebellar computations, are believed to comprise a uniform neuronal population of cells, each with similar functional properties. Here, we show an undiscovered heterogeneity of adult zebrafish Purkinje cells, revealing the existence of anatomically and functionally distinct cell types. Dual patch-clamp recordings showed that the cerebellar circuit contains all Purkinje cell types that cross-communicate extensively using chemical and electrical synapses. Further activation of spinal central pattern generators (CPGs) revealed unique phase-locked activity from each Purkinje cell type during the locomotor cycle. Thus, we show intricately organized Purkinje cell networks in the adult zebrafish cerebellum that encode the locomotion rhythm differentially, and we suggest that these organizational properties may also apply to other cerebellar functions.


Asunto(s)
Locomoción/fisiología , Células de Purkinje/fisiología , Pez Cebra/fisiología , Potenciales de Acción , Animales , Conducta Animal , Encéfalo , Generadores de Patrones Centrales/fisiología , Cerebelo/fisiología , Análisis por Conglomerados , Fenómenos Electrofisiológicos , Femenino , Masculino , Modelos Animales , Médula Espinal
19.
J Vis Exp ; (156)2020 02 14.
Artículo en Inglés | MEDLINE | ID: mdl-32116293

RESUMEN

Clostridioides difficile infection (CDI) is considered to be one of the most common healthcare-associated gastrointestinal infections in the United States. The innate immune response against C. difficile has been described, but the exact roles of neutrophils and macrophages in CDI are less understood. In the current study, Danio rerio (zebrafish) larvae are used to establish a C. difficile infection model for imaging the behavior and cooperation of these innate immune cells in vivo. To monitor C. difficile, a labeling protocol using a fluorescent dye has been established. A localized infection is achieved by microinjecting labeled C. difficile, which actively grows in the zebrafish intestinal tract and mimics the intestinal epithelial damage in CDI. However, this direct infection protocol is invasive and causes microscopic wounds, which can affect experimental results. Hence, a more noninvasive microgavage protocol is described here. The method involves delivery of C. difficile cells directly into the intestine of zebrafish larvae by intubation through the open mouth. This infection method closely mimics the natural infection route of C. difficile.


Asunto(s)
Clostridioides difficile/fisiología , Pez Cebra/microbiología , Animales , Clostridioides difficile/crecimiento & desarrollo , Infecciones por Clostridium/microbiología , Modelos Animales de Enfermedad , Colorantes Fluorescentes/química , Vida Libre de Gérmenes , Intestinos/microbiología , Intestinos/patología , Larva/microbiología , Esporas Bacterianas/fisiología
20.
J Exp Neurosci ; 13: 1179069519880515, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31666796

RESUMEN

The cerebellum integrates sensory information and motor actions. Increasing experimental evidence has revealed that these functions as well as the cerebellar cytoarchitecture are highly conserved in zebrafish compared with mammals. However, the potential of zebrafish for modelling human cerebellar diseases remains to be addressed. Spinocerebellar ataxias (SCAs) represent a group of genetically inherited cerebellar diseases leading to motor discoordination that is most often caused by affected cerebellar Purkinje cells (PCs). Towards modelling SCAs in zebrafish we identified a small-sized PC-specific regulatory element that was used to develop coexpression vectors with tunable expression strength. These vectors allow for in vivo imaging of SCA-affected PCs by high-resolution fluorescence imaging. Next, zebrafish with SCA type 13 (SCA13) transgene expression were established, revealing that SCA13-induced cell-autonomous PC degeneration results in eye movement deficits. Thus, SCA13 zebrafish mimic the neuropathology of an SCA-affected brain as well as the involved loss of motor control and hence provide a powerful approach to unravel SCA13-induced cell biological pathogenic and cytotoxic mechanisms.

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