Este trabalho foi financiado pela theNIH grantNS042626. Agradecemos a Kurt Thorn e UCSF Nikon Imaging centro de assistência com a imagem.

1. Preparação de Embriões Zebrafish <ol><li> Dois dias antes da eletroporação, criada gaiolas de acasalamento de peixes do tipo selvagem usando divisores para os machos separados das fêmeas. </li><li> Um dia antes da eletroporação, puxe os divisores em gaiolas de acasalamento do dia anterior. </li><li> Coletar embriões e incubar 50 embriões fertilizados em 30 ml de meio embrionárias contendo phenylthiourea 0,003% (PTU) a 28,5 ° C. </li><li> No dia da eletroporação, dechorionate os embriões manualmente com uma pinça fina (Inox 5, Dumont Eletrônico, Suíça), quando os embriões atingem o estágio de desenvolvimento de 22hpf. Em seguida, transferir os embriões para 3 eletroporação 10ml de Ringer (180 mM NaCl, KCl 5mM, 1,8 mM CaCl2, 5 mM Hepes pH 7,2) contendo 0,003% PTU. </li><li> Adicionar 420 mL tricaina ações (4mg/ml) para o eletroporação 10ml de Ringer para anestesiar os embriões. </li></ol> 2. Preparação para Eletroporação <ol><li> Agulhas de injeção de vidro foram arrancados de capilares (1,2 mm de diâmetro externo, 0,9 mm de diâmetro, com filamento) em um Flaming-Brown P897 extrator (Sutter Instruments, Novato, CA, EUA). A ponta da agulha foi interrompido com uma pinça para obter uma ponta afiada, como mostrado na figura 1D. O diâmetro da ponta deve ser em torno de 10 mm e do ângulo do cone deve ser em torno de 30 graus. </li><li> Prepare 0,8% e 1% de baixo ponto de fusão de agarose (Shelton Scientific, Inc. catalogo IB70050) em solução de Ringer eletroporação. Alíquota da solução de agarose em 2 tubos de microcentrífuga ml. Manter as alíquotas em um bloco de calor em ~ 37 ° C. </li><li> O sistema Gal4/UAS é usada para acionar a expressão do gene em células progenitoras neurais de embriões electroporated. Dois plasmídeos são usados: plasmídeo EF1α-GFF-PT2KXIG, em que Gal4FF é impulsionado pela EF1α promotor onipresente, e plasmídeo UAS-E1B-EGFP-PT2KXIG. </li><li> Prepare-EF1α GFF-PT2KXIG e UAS-E1B-EGFP-PT2KXIG DNA plasmídeo usando GenEluteTM HP Plasmid Midiprep Kit (Sigma). </li><li> Os plasmídeos são então concentrada por precipitação de acetato de sódio / etanol para a concentração final de 1-2 mcg / mL em 10 mM Tris · Cl (pH 8,5). </li><li> Misturar os dois plasmídeos e diluir com nuclease-free H2O para uma concentração final de 500 ng / mL para cada plasmídeo. Além do vermelho de fenol (diluído 1 / 10 do volume total) para a solução é a preferida para a visualização de entrega. Manter a solução de DNA em gelo. </li><li> Direito antes de agulha eletroporação, protelar a injeção com 2 mL de solução de DNA e ajustar o volume de injecção a 2 nl. </li></ol> 3. Eletroporação <ol><li> Um microscópio de dissecção stero (Zeiss Stemi 2000, com uma ampliação máxima de 50x), um injector de pressão de ar (300 microinjetor Narishige IM), duas micromanipuladores (WPI M3301R, instrumentos de precisão Mundial, Sarasota, FL, EUA), e um estimulador elétrico (SD9 quadrados de pulso estimulador, Grass Tlefactor, West Warwick, Rhode Island, EUA) são configurados como mostrado na Figura 1A. </li><li> Para montar os embriões, mergulhe dois embriões em 1% de baixo ponto de fusão de agarose. Em seguida, retire imediatamente a embriões a partir da agarose com uma pipeta de vidro. </li><li> Coloque os embriões em uma tampa de plástico prato Petri invertida em gotas individuais de agarose. Orientar os embriões delicadamente com uma sonda de fibra para uma posição dorsal-up antes da agarose, para que se solidifica no prosencéfalo é acessível para ambos os eletrodos ea agulha microinjeção (Figura 1B). Embriões devem ser montados em cada agarose gotas. </li><li> Manipular os eletrodos com o micromanipulador esquerda. Os eléctrodos no agarose como mostrado na Figura 1B. Personalizado platina iridium eletrodo bipolar paralelo 125 m de diâmetro e 500 mm espaçados separados são usados ​​para eletroporação (FHC, catalogo PBSA0575). </li><li> Inserir a agulha microinjeção no ventrículo prosencéfalo e injetar DNA mix 4NL, como fluxo de fluido vermelho eo inchaço do ventrículo podem ser observados. </li><li> Imediatamente, um pulso de 28,5 V com duração de 1 milissegundo foi aplicado manualmente. Em seguida, inverter a polaridade e aplicar mais um pulso de 28,5 V com duração de 1 milisegundo. Isso resultará em mosaic rotulagem bilateral do cérebro anterior células progenitoras neurais. </li><li> Após a eletroporação de 10 embriões, adicione meio embrionárias 5ml para cobrir os embriões e descascar a agarose com uma faca de microcirurgia para liberar os embriões. </li><li> Transferir os embriões electroporated a um prato fresco de 30 ml de meio embrionárias contendo 0,003% PTU e incubar a 28,5 ° C. </li></ol> 4. Montagem e Time-lapse de imagens ao vivo <ol><li> Em torno de 4 horas após a eletroporação, o sinal de EGFP é observável se visto sob um microscópio de epifluorescência dissecação. </li><li> Cinco horas após eletroporação, selecione os embriões com mosaico altamente mas a expressão EGFP forte na região do prosencéfalo. </li><li> Transferir os embriões selecionados para 10ml médio embrionárias contendo PTU 0,003%. </li><li> Adicionar 420 mL tricaina ações (4mg/ml) ao embrionárias 10ml médio to anestesiar os embriões. </li><li> Para montar os embriões, mergulhe um embrião em 0,8% baixo ponto de fusão de agarose. Em seguida, retire imediatamente o embrião da agarose com uma pipeta de vidro e colocar o embrião no centro de um prato de cultura 35 milímetros de vidro de fundo (Mattek corporação, Part # P35GC-1.5-10-C, <a href="http://www.glass-bottom-dishes.com/">www.glass bottom-dishes.com</a> ) . </li><li> Orientar os embriões delicadamente com uma sonda de fibra para uma posição dorsal-up antes da agarose solidifica. </li><li> Coloque o prato de forma adequada no palco temperatura controlada de um microscópio confocal, como mostrado na figura 1C (Nós usamos um microscópio Nikon C1 espectral confocal com up-direita objetivos). Ajustar a temperatura para 28,5 ° C. </li><li> Adicionar 3ml 28,5 ° C médio pré-aquecido embrionárias contendo 0,003% PTU para cobrir o embrião. O embrião está pronto para geração de imagens. </li><li> Executar lapso de tempo imagens ao vivo com um intervalo fixo utilizando objetiva de imersão de água. </li></ol> 5. Resultados representante Mostrado na Figura 2 são selecionados quadros de um lapso de tempo de imagem confocal ao vivo do embrião electroporated de EF1α-GFF + UAS-E1B-EGFP aos 22 hpf. <img src="/files/ftp_upload/2594/2594fig1.jpg" alt="Figura 1" /> Figura 1. Aparelho set-up. (A) O conjunto de equipamentos de eletroporação: the Grass SD9 quadrados de pulso estimulador (i), o micromanipulador esquerda (ii), os eletrodos (iii), o microscópio Zeiss dissecar (iv), a agulha de injeção (v), micromanipulador o direito (vi) eo Narishige IM 300 microinjetor (vii). (B) A posição relativa dos eletrodos (i), o ventrículo prosencéfalo (ii) ea agulha de injeção (iii). Observe a solução DNA de cor vermelha foi injetado no ventrículo prosencéfalo. (C) A montagem de imagens ao vivo em microscópio Nikon C1 confocal: o controlador de temperatura (i), a água molhando objetivo len (ii) eo embrião electroporated embutido no baixo ponto de fusão de agarose em um prato fundo de vidro cultura (iii) (D ) Imagem de uma agulha puxado e corte para microinjeção de DNA. <img src="/files/ftp_upload/2594/2594fig2.jpg" alt="Figura 2" /> Figura 2. Quadros selecionados de um de 18 horas de lapso de tempo de imagem confocal ao vivo do embrião electroporated com EF1α-GFF + UAS-E1B-EGFP aos 22 hpf. Superfície do ventrículo está no lado inferior de cada quadro de imagem. Barra de escala representa 10 mM.

<ol>
	<li>Temple, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+neural+stem+cells.">The development of neural stem cells.</a> Nature. 414, 112-117 (2001).</li><li>G#246;tz, M., Huttner, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+biology+of+neurogenesis.">The cell biology of neurogenesis.</a> Nat. Rev. Mol. Cell Biol. 6, 777-788 (2005).</li><li>Cerda, G. A., Thomas, J. E., Allende, M. L., Karlstrom, R. O., Palma, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+of+DNA,+RNA,+and+morpholinos+into+zebrafish+embryos.">Electroporation of DNA, RNA, and morpholinos into zebrafish embryos.</a> Methods. 39, 207-211 (2006).</li><li>Hendricks, M., Jesuthasan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation-based+methods+for+in+vivo,+whole+mount+and+primary+culture+analysis+of+zebrafish+brain+development.">Electroporation-based methods for in vivo, whole mount and primary culture analysis of zebrafish brain development.</a> Neural. Dev. 2, 6-6 (2007).</li><li>Tawk, M., Bianco, I. H., Clarke, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+electroporation+in+zebrafish+embryos+and+larvae.">Focal electroporation in zebrafish embryos and larvae.</a> Methods Mol. Biol. 546, 145-151 (2009).</li><li>Tawk, M., Tuil, D., Torrente, Y., Vriz, S., Paulin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+gene+transfer+into+adult+fish:+a+new+tool+to+study+fin+regeneration.">High-efficiency gene transfer into adult fish: a new tool to study fin regeneration.</a> Genesis. 32, 27-31 (2002).</li></ol>

Não há conflitos de interesse declarados.

Neste vídeo, demonstramos um método para time-lapse imagens ao vivo de células progenitoras neurais em um nível clonal no prosencéfalo zebrafish em desenvolvimento. Nós testamos e modificou o eletroporação protocolos existentes para 4-6 geneticamente e fluorescente etiqueta individual células progenitoras neurais. Uma árvore de linhagem clonal composto de células descendentes relacionados pode ser estabelecida uma vez que apenas algumas células eram escassamente rotulados com uma tensão relativamente baixa de eletroporação. Além de EGFP, as células progenitoras neurais podem ser rotulados com subcellularly várias proteínas fluorescentes simplesmente substituindo EGFP com outros marcadores fluorescentes. Por exemplo, se EF1α-GFF foi co-electroporated com UAS-E1B-memberane-EGFP e UAS-E1B-H2B-MRFP, membranas de células progenitoras individual será marcada com a proteína verde fluorescente, enquanto núcleo será marcado com uma proteína fluorescente vermelha. A maioria das células progenitoras são saudáveis ​​rotulados como evidenciado pelo desenvolvimento geral normais dos embriões electroporated e apoptose poucos observados durante imagens ao vivo. Também é fundamental usar uma baixa concentração de agarose para montar o embrião e usar um mínimo de potência do laser durante a imagens ao vivo confocal para manter as células saudáveis. A presente técnica também pode ser aplicado a outras partes do sistema nervoso central, como o cérebro traseiras e da medula espinhal. No entanto, esta técnica não pode ser aplicado em embriões com menos de 18 hpf, uma vez que as construções de DNA têm de ser injetado no ventrículo para eletroporação.

time-lapse live imaging of clonally related neural progenitor cells in the developing zebrafish forebrain

Padrões precisos de migração, divisão e diferenciação das células progenitoras neurais são cruciais para o desenvolvimento adequado do cérebro e 1,2 função. Para entender o comportamento das células progenitoras neurais no complexo no ambiente vivo, lapso de tempo imagens ao vivo de células progenitoras neurais em um cérebro intacto é extremamente necessário. Neste vídeo, nós explorar as características únicas de embriões zebrafish para visualizar o desenvolvimento de células progenitoras neurais prosencéfalo in vivo. Usamos a eletroporação geneticamente e pouco etiqueta individual células progenitoras neurais. Resumidamente, o DNA construções de codificação para marcadores fluorescentes foram injetados no ventrículo do cérebro anterior de 22 horas após a fertilização (hpf) embriões zebrafish e pulsos elétricos foram entregues imediatamente. Seis horas depois, os embriões do zebrafish electroporated foram montadas com baixo ponto de fusão de agarose em placas de cultura de vidro de fundo. Fluorescente etiquetado células progenitoras neurais foram então fotografada por 36 horas com intervalos fixos sob um microscópio confocal utilizando água lente objetiva de imersão. O presente método fornece uma maneira de obter insights sobre o desenvolvimento in vivo de células progenitoras neurais do cérebro anterior e pode ser aplicado a outras partes do sistema nervoso central do embrião zebrafish.

Watch this Scientific Journal Video about Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain at JoVE.com

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

O vídeo atual demonstra um método que tira proveito da combinação de eletroporação e microscopia confocal para realizar imagens ao vivo em células individuais progenitoras neurais no cérebro anterior zebrafish em desenvolvimento.<em&gt; In vivo</em&gt; Análise do desenvolvimento de células progenitoras do prosencéfalo neural em um nível clonal pode ser alcançada dessa forma.

Lapso de tempo de imagens ao vivo de relação clonal células progenitoras neurais no desenvolvimento Zebrafish Encéfalo anterior

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

time-lapse-live-imaging-clonally-related-neural-progenitor-cells

Nanyang Technological University

Research

JoVE Journal

Neuroscience

10.2K visualizações.  University of California San Francisco. O vídeo atual demonstra um método que tira proveito da combinação de eletroporação e microscopia confocal para realizar imagens ao vivo em células individuais progenitoras neurais no cérebro anterior zebrafish em desenvolvimento. In vivo Análise do desenvolvimento de células progenitoras do prosencéfalo neural em um nível clonal pode ser alcançada dessa forma.

Vídeo: Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Em células vivas, imagens de células precursoras de órgãos sensoriais na Intact Drosophila As pupas

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Neurociência

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

De alta resolução de imagem ao vivo de comportamento de células no desenvolvimento neuroepitélio

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells in restricted areas of the adult mammalian brain. Newborn neuroblasts from the subventricular zone (SVZ) migrate along the rostral migratory stream (RMS) to their final destination in the olfactory bulb (OB)1. In the RMS, neuroblasts migrate tangentially in chains ensheathed by astrocytic processes2,3 using blood vessels as a structural support and a source of molecular factors required for migration4,5. In the OB, neuroblasts detach from the chains and migrate radially into the different bulbar layers where they differentiate into interneurons and integrate into the existing network1, 6.
In this manuscript we describe the procedure for monitoring cell migration in acute slices of the rodent brain. The use of acute slices allows the assessment of cell migration in the microenvironment that closely resembling to in vivo conditions and in brain regions that are difficult to access for in vivo imaging. In addition, it avoids long culturing condition as in the case of organotypic and cell cultures that may eventually alter the migration properties of the cells. Neuronal precursors in acute slices can be visualized using DIC optics or fluorescent proteins. Viral labeling of neuronal precursors in the SVZ, grafting neuroblasts from reporter mice into the SVZ of wild-type mice, and using transgenic mice that express fluorescent protein in neuroblasts are all suitable methods for visualizing neuroblasts and following their migration. The later method, however, does not allow individual cells to be tracked for long periods of time because of the high density of labeled cells. We used a wide-field fluorescent upright microscope equipped with a CCD camera to achieve a relatively rapid acquisition interval (one image every 15 or 30 sec) to reliably identify the stationary and migratory phases. A precise identification of the duration of the stationary and migratory phases is crucial for the unambiguous interpretation of results. We also performed multiple z-step acquisitions to monitor neuroblasts migration in 3D. Wide-field fluorescent imaging has been used extensively to visualize neuronal migration7-10. Here, we describe detailed protocol for labeling neuroblasts, performing real-time video-imaging of neuroblast migration in acute slices of the adult mouse forebrain, and analyzing cell migration. While the described protocol exemplified the migration of neuroblasts in the adult RMS, it can also be used to follow cell migration in embryonic and early postnatal brains.

We describe a protocol for real-time videoimaging of neuronal migration in the mouse forebrain. The migration of virally-labeled or grafted neuronal precursors was recorded in acute live slices using wide-field fluorescent imaging with a relatively rapid acquisition interval to study the different phases of cell migration, including the durations of the stationary and migration phases and the speed of migration.

Lapso de tempo de imagem de migração neuroblastos em fatias agudas do cérebro anterior Rato Adulto

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells ...

Forebrain Electrophysiological Recording in Larval Zebrafish

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked seizures, epilepsy can be acquired as a result of an insult to the brain or a genetic mutation. Efforts to model seizures in animals have primarily utilized acquired insults (convulsant drugs, stimulation or brain injury) and genetic manipulations (antisense knockdown, homologous recombination or transgenesis) in rodents. Zebrafish are a vertebrate model system1-3 that could provide a valuable alternative to rodent-based epilepsy research. Zebrafish are used extensively in the study of vertebrate genetics or development, exhibit a high degree of genetic similarity to mammals and express homologs for ~85% of known human single-gene epilepsy mutations. Because of their small size (4-6 mm in length), zebrafish larvae can be maintained in fluid volumes as low as 100 &mu;l during early development and arrayed in multi-well plates. Reagents can be added directly to the solution in which embryos develop, simplifying drug administration and enabling rapid in vivo screening of test compounds4. Synthetic oligonucleotides (morpholinos), mutagenesis, zinc finger nuclease and transgenic approaches can be used to rapidly generate gene knockdown or mutation in zebrafish5-7. These properties afford zebrafish studies an unprecedented statistical power analysis advantage over rodents in the study of neurological disorders such as epilepsy. Because the "gold standard" for epilepsy research is to monitor and analyze the abnormal electrical discharges that originate in a central brain structure (i.e., seizures), a method to efficiently record brain activity in larval zebrafish is described here. This method is an adaptation of conventional extracellular recording techniques and allows for stable long-term monitoring of brain activity in intact zebrafish larvae. Sample recordings are shown for acute seizures induced by bath application of convulsant drugs and spontaneous seizures recorded in a genetically modified fish.

A simple method to record extracellular field potentials in the larval zebrafish forebrain is described. The method provides a robust in vivo read-out of seizure-like activity. This technique can be used with genetically modified zebrafish larvae carrying epilepsy-related genes or seizures evoked by administration of convulsant drugs.

Prosencéfalo gravação eletrofisiológico em larval Zebrafish

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked ...

Motor Nerve Transection and Time-lapse Imaging of Glial Cell Behaviors in Live Zebrafish

The nervous system is often described as a hard-wired component of the body even though it is a considerably fluid organ system that reacts to external stimuli in a consistent, stereotyped manner, while maintaining incredible flexibility and plasticity. Unlike the central nervous system (CNS), the peripheral nervous system (PNS) is capable of significant repair, but we have only just begun to understand the cellular and molecular mechanisms that govern this phenomenon. Using zebrafish as a model system, we have the unprecedented opportunity to couple regenerative studies with in vivo imaging and genetic manipulation. Peripheral nerves are composed of axons surrounded by layers of glia and connective tissue. Axons are ensheathed by myelinating or non-myelinating Schwann cells, which are in turn wrapped into a fascicle by a cellular sheath called the perineurium. Following an injury, adult peripheral nerves have the remarkable capacity to remove damaged axonal debris and re-innervate targets. To investigate the roles of all peripheral glia in PNS regeneration, we describe here an axon transection assay that uses a commercially available nitrogen-pumped dye laser to axotomize motor nerves in live transgenic zebrafish. We further describe the methods to couple these experiments to time-lapse imaging of injured and control nerves. This experimental paradigm can be used to not only assess the role that glia play in nerve regeneration, but can also be the platform for elucidating the molecular mechanisms that govern nervous system repair.

Although the peripheral nervous system (PNS) is capable of significant repair after injury, little is known about the cellular and molecular mechanisms that govern this phenomenon. Using live, transgenic zebrafish and a reproducible nerve transection assay, we can study dynamic glial cell behaviors during nerve degeneration and regeneration.

Motor Nerve transecção e Time-lapso de Comportamentos de células gliais em Zebrafish ao vivo

The  nervous system is often described as a hard-wired component of the body even  though it is a considerably fluid organ system that reacts to external ...

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to neuroblasts able to move along the rostral migratory stream (RMS) towards the olfactory bulb (OB). This long-distance migration is required for the subsequent maturation of newborn neurons in the OB, but the molecular mechanisms regulating this process are still unclear. Investigating the signaling pathways controlling neuroblast motility may not only help understand a fundamental step in neurogenesis, but also have therapeutic regenerative potential, given the ability of these neuroblasts to target brain sites affected by injury, stroke, or degeneration.
In this manuscript we describe a detailed protocol for in vivo postnatal electroporation and subsequent time-lapse imaging of neuroblast migration in the mouse RMS. Postnatal electroporation can efficiently transfect SVZ progenitor cells, which in turn generate neuroblasts migrating along the RMS. Using confocal spinning disk time-lapse microscopy on acute brain slice cultures, neuroblast migration can be monitored in an environment closely resembling the in vivo condition. Moreover, neuroblast motility can be tracked and quantitatively analyzed. As an example, we describe how to use in vivo postnatal electroporation of a GFP-expressing plasmid to label and visualize neuroblasts migrating along the RMS. Electroporation of shRNA or CRE recombinase-expressing plasmids in conditional knockout mice employing the LoxP system can also be used to target genes of interest. Pharmacological manipulation of acute brain slice cultures can be performed to investigate the role of different signaling molecules in neuroblast migration. By coupling in vivo electroporation with time-lapse imaging, we hope to understand the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair.

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video tracking.

In vivo Eletroporação pós-natal e tempo-lapso de neuroblasto Migração na aguda fatias do cérebro do rato

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to ...

Assessing Cell Cycle Progression of Neural Stem and Progenitor Cells in the Mouse Developing Brain after Genotoxic Stress

Neurons of the cerebral cortex are generated during brain development from different types of neural stem and progenitor cells (NSPC), which form a pseudostratified epithelium lining the lateral ventricles of the embryonic brain. Genotoxic stresses, such as ionizing radiation, have highly deleterious effects on the developing brain related to the high sensitivity of NSPC. Elucidation of the cellular and molecular mechanisms involved depends on the characterization of the DNA damage response of these particular types of cells, which requires an accurate method to determine NSPC progression through the cell cycle in the damaged tissue. Here is shown a method based on successive intraperitoneal injections of EdU and BrdU in pregnant mice and further detection of these two thymidine analogues in coronal sections of the embryonic brain. EdU and BrdU are both incorporated in DNA of replicating cells during S phase and are detected by two different techniques (azide or a specific antibody, respectively), which facilitate their simultaneous detection. EdU and BrdU staining are then determined for each NSPC nucleus in function of its distance from the ventricular margin in a standard region of the dorsal telencephalon. Thus this dual labeling technique allows distinguishing cells that progressed through the cell cycle from those that have activated a cell cycle checkpoint leading to cell cycle arrest in response to DNA damage.
An example of experiment is presented, in which EdU was injected before irradiation and BrdU immediately after and analyzes performed within the 4 hr&#160;following irradiation. This protocol provides an accurate analysis of the acute DNA damage response of NSPC in function of the phase of the cell cycle at which they have been irradiated. This method is easily transposable to many other systems in order to determine the impact of a particular treatment on cell cycle progression in living tissues.

Administration of two analogs of thymidine, EdU and BrdU, in pregnant mice allows the analysis of cell cycle progression in neural and progenitor cells in the embryonic mouse brain. This method is useful to determine the effects of genotoxic stress, including ionizing radiation, during brain development.

Avaliando a progressão do ciclo celular de tronco neurais e as células progenitoras no desenvolvimento do cérebro do rato após estresse genotóxico

Neurons of the cerebral cortex are generated during brain development from different types of neural stem and progenitor cells (NSPC), which form a ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Imagens ao vivo da mitose no desenvolvimento Cortex embrionárias de camundongos

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

Derivação sem alimentador de células da crista neural progenitoras pluripotentes humanas de células-tronco

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Long-term Time Lapse Imaging of Mouse Cochlear Explants

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building the highly ordered, complex structure of the mammalian cochlea proceeds for around ten days. In order to study changes in gene expression over this period and their response to pharmaceutical or genetic manipulation, long-term imaging is necessary. Previously, live imaging has typically been limited by the viability of explanted tissue in a humidified chamber atop a standard microscope. Difficulty in maintaining optimal conditions for culture growth with regard to humidity and temperature has placed limits on the length of imaging experiments. A microscope integrated into a modified tissue culture incubator provides an excellent environment for long term-live imaging. In this method we demonstrate how to establish embryonic mouse cochlear explants and how to use an incubator microscope to conduct time lapse imaging using both bright field and fluorescent microscopy to examine the behavior of a typical embryonic day (E) 13 cochlear explant and Sox2, a marker of the prosensory cells of the cochlea, over 5 days.

Live imaging of the embryonic mammalian cochlea is challenging because the developmental processes at hand operate on a temporal gradient over ten days. Here we present a method for culturing and then imaging embryonic cochlear explant tissue taken from a fluorescent reporter mouse over five days.

Longo prazo Time Lapse Imagem do mouse cocleares explantes

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building ...