Agradecemos a Sra. Fan Shen por seu apoio à criação de peixe-zebra. Este é apoiado por financiamentos para Dr. Bing Hu (Chinese Academy of Sciences &quot;, Bai Ren Ji Hua&quot;, NSFC 30870833 e do Programa Nacional de Ciência Básica da China, 2009CB941302). Todos os experimentos com animais foram conduzidos de acordo com as diretrizes e regulamentos estabelecidos pela Universidade de Ciência e Tecnologia da China (USTC) Centro de Recursos Animais e Cuidados Universidade Animal e Uso Comissão.

Parte I: Construir o aparato OKR Detalhes são mostrados na demonstração em vídeo. Parte II: OKR de Adultos Zebrafish Se o seu laboratório construiu um aparelho OKR, a chave de adultos zebrafish OKR é manter o peixe. Nesta parte, vamos mostrar passo a passo. Nosso aparelho compõe seguintes peças similares para os outros mencionados. A câmara de peixes circular é de 7,0 cm de diâmetro externo, 6,5 cm de diâmetro interno e 7,0 cm de altura. O tambor em torno da câmara de peixe é de 8,5 cm de diâmetro para fora, 7,5 cm de diâmetro interno e 7,0 cm de altura. Poderia girar no sentido horário e anti-horário com a velocidade de 20 ~ 70 rotações por minuto (rpm). Onze ciclos de listras pretas e brancas são desenhadas em um papel vegetal que aderiram na parte externa do tambor. O sistema de iluminação é composto por dois tubos círculo de luz em 21 cm de diâmetro. <img src="/files/ftp_upload/1742/1742fig1.jpg" alt="Figura 1" /> Figura 1. O modelo de esponja. O penhasco feitas neste meio é ter certeza de que o campo visual não estava sendo protegido. <ol><li> A esponja, como mostrado na Fig.1 composto por duas partes. A parte esquerda é tamanho 3 cm x 3 cm x largura alta 2 cm. A parte direita é o comprimento 3 cm x 3 cm x largura alto 3 cm. </li><li> Coloque esta esponja no fundo da câmara de peixe e despeje água criação em cerca de 2 cm de profundidade. Bolha de ar que se escondem na esponja deve ser expulso. </li><li> Inserir um pino na parte da frente do lado direito da esponja, cerca de 2 mm para trás do precipício. </li><li> Anestesiar um peixe-zebra adulto na MS-222. </li><li> Coloque o peixe-zebra na esponja com até dorsal e permitir que um lado da sua barbatana peitoral antes do primeiro pino. </li><li> O segundo pino é inserido atrás da barbatana peitoral outros. Poderia evitar corpo encolhendo zebrafish, cooperando com o primeiro pino (ver também na figura. 2). </li><li> Grampo sua cauda com uma artéria grampo e corrigir o clamp da artéria em um pedaço de borracha (massa de modelar), que é respeitado na câmara de peixe. </li><li> O pino de terceiro e quarto é plantada no local perto de poros cloacal do corpo de peixe. Poderia impedir a onda de zebrafish sua cauda intensamente. </li><li> O pino de quinta está situada entre o primeiro eo terceiro pino e, em seguida, o pino é sexto no outro lado do mesmo. Eles poderiam impedir zebrafish de tremer seu corpo. </li><li> O pino de sétimo e oitavo está inserido nos locais perto da brânquia para manter a orientação da cabeça. </li><li> Despeje a água na câmara de criação de peixes. </li><li> Depois que o peixe-zebra acorda, deixe-o adaptar para 5 ~ 10 minutos. </li><li> Ligar a luz deste aparelho e deixe zebrafish adaptar esta iluminação por 2 minutos. </li><li> Ligue o motor para impulsionar a grade e permitir zebrafish para se adaptar por 1 minuto. </li><li> Ligue a câmera e gravar o movimento dos olhos. </li><li> Analisar os ângulos de olho 6 e números saccade em seu computador pessoal (Um exemplo representativo da análise ângulos olho é mostrado na Fig.2). </li></ol><img src="/files/ftp_upload/1742/1742fig2.jpg" alt="Figura 2" /> Figura 2. O ângulo entre o eixo dos olhos e eixo do corpo. a) Quando a direção de ralar é nasal-to-temporal, que definimos o ângulo na frente do corpo do peixe é um ângulo negativo eo ângulo no corpo do peixe é um ângulo positivo, vice-versa (para breve revisão também ver ref. 6). Números nesta figura representam a seqüência de pinos. b) exemplo de análise OKR de um peixe no grupo normal. Um filme de um segundo foi transferido para uma série de quadros e os ângulos dos olhos de cada quadro foi medida com Image-pro software Plus. Cada ponto nesta figura representa cada quadro medimos. Notas: <ol class="lalpha"><li> Todos os pinos são utilizados apenas para evitar zebrafish de tremer seu corpo, muito apertado será prejudicial para os peixes. </li><li> Clamp da artéria pode danificar o rabo como sua área de contato é pequena. Uma vez que a cauda é livre, é muito difícil de corrigir este peixe. Enrole uma bandagem em torno da cauda é recomendado em alguma condição. </li><li> O pino de sétimo e oitavo, que são plantadas no local perto de emalhar não deve ser o abrigo de sua visão de ralar. </li></ol> Parte III. OKR de Larval Zebrafish 1. Metilcelulose Larval zebrafish podem ser imobilizadas em solução de metilcelulose a 6%, enquanto manter viva por muito tempo. Preparar a solução de metilcelulose como se segue. <ol><li> Ferva 200 ml DDH 2 O forno de microondas em cerca de 3 minutos em alto nível. </li><li> Transferir a água quente para uma placa de agitação e mexa vigorosamente. </li><li> Adicione 12 g de metilcelulose na água quente aos poucos. </li><li> Continue mexendo por 3 minutos após a metilcelulose é bem dispersa. </li><li> Esfriar a soluções com água e gelo até que fique claro, e depois colocá-lo a 4 ° C. </li></ol><pclass = &quot;jove_step&quot;&gt; 2. Larval Zebrafish <ol><li> Dois pares de macho e uma fêmea de peixe-zebra adulto em um tanque de acasalamento, às 17h00 após a alimentação. Na manhã seguinte, transferi-los para um tanque de acasalamento fresco e coletar embriões após 1 hora. </li><li> Os ovos são mantidas em meio de embriões em ± 0,5 28,5 ° C uma incubadora com a luz: ciclo escuro das 14h10min. </li><li> Os ovos serão chocados com menos 48 horas após a fertilização (hpf) eo OKR será testado em 72 hpf ou mais tarde. Neste vídeo, as larvas são menos 5 dpf. </li></ol> 3. Procedimento de OKR <ol><li> Colocar o aparelho sob o estereoscópio equipado com câmera CCD e ligar a luz deste aparelho. </li><li> Adicionar 20 ml Metilcelulosa 6% numa placa de Petri de 55 mm. </li><li> Lugar larvas várias vezes seguidas no centro deste prato. </li><li> Coloque uma bolha de ar sobre um volume de 1 ml no campo da câmera, que poderia ser como um espelho para refletir o sentido da grade. </li><li> Ligue o motor e gravar o movimento de seus olhos. </li></ol> Parte IV. OMR do Adulto Zebrafish Adulto zebrafish OMR também pode ser realizado com este aparelho, basta colocar uma coluna na câmara de peixe para prevenir peixes nadando em centro (mostrado na Fig.3, a velocidade de rotação é de 36 rpm, preto-branco ciclos são 11). <ol><li> Coloque o peixe nesta câmara de peixe. </li><li> Está uma coluna de 2 cm de diâmetro no centro desta câmara peixes. </li><li> Adaptar para um minuto, tanto na grade e ainda ralar rotação. </li><li> Registro do movimento de zebrafish. </li><li> Analisar a taxa de coincidência de tempo em seu computador pessoal (um exemplo é mostrado na Fig.3b, velocidade de rotação é de 36 rpm, preto-branco do ciclo é de 11) </li></ol><img src="/files/ftp_upload/1742/1742fig3.jpg" alt="Figura 3" /> Figura 3. OMR Zebrafish de Adultos. (A) Uma coluna de plástico branco foi colocado no centro da câmara de peixe para prevenir peixes nadando em toda ela. (B) A taxa de coincidência de zebrafish adultos após a grade. A taxa de coincidência foi definida como a proporção do tempo de ralar a seguir para o tempo de gravação total. O valor de 50% indica que o peixe está nadando aleatoriamente, enquanto 100% indica a grade seguintes peixes muito bem. Ambos os nervos ópticos foram esmagados com pinça de jóias de cerca de 10 segundos. O comportamento de OMR apareceu como uma piscina aleatória após esmagamento.

<ol>
	<li>Beck, J., Gilland, E., Tank, D., Baker, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+the+Ontogeny+of+Optokinetic+and+Vestibuloocular+Behaviors+in+Zebrafish+Medaka,+and+Goldfish.">Quantifying the Ontogeny of Optokinetic and Vestibuloocular Behaviors in Zebrafish Medaka, and Goldfish.</a> J Neurophysiol. 92, 3546-3561 (2004).</li><li>Brockerhoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+the+optokinetic+response+of+zebrafish+larvae.">Measuring the optokinetic response of zebrafish larvae.</a> Nature Protocols. 1, 2448-2451 (2006).</li><li>Emran, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OFF+ganglion+cells+cannot+drive+the+optokinetic+reflex+in+zebrafish.">OFF ganglion cells cannot drive the optokinetic reflex in zebrafish.</a> Proc Natl Acad Sci U S A. 104, 19126-19131 (2007).</li><li>Rinner, O., Rick, J., Neuhauss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contrast+Sensitivity,+Spatial+and+Temporal+Tuning+of+the+Larval+Zebrafish+Optokinetic+Response.">Contrast Sensitivity, Spatial and Temporal Tuning of the Larval Zebrafish Optokinetic Response.</a> Investigative Ophthalmology and Visual Science. 46, 137-142 (2005).</li><li>Roeser, T., Baier, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visuomotor+Behaviors+in+Larval+Zebrafish+after+GFP-Guided+Laser+Ablation+of+the+Optic+Tectum.">Visuomotor Behaviors in Larval Zebrafish after GFP-Guided Laser Ablation of the Optic Tectum.</a> The Journal of Neuroscience. 23, 3726-3734 (2003).</li><li>Easter, S., Nicola, G. <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+Vision+in+the+Zebrafish+(Danio+rerio).">The Development of Vision in the Zebrafish (Danio rerio).</a> Developmental Biology. 180, 646-663 (1996).</li><li>Maaswinkel, H., Li, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatio-temporal+frequency+characteristics+of+the+optomotor+response+in+zebrafish.">Spatio-temporal frequency characteristics of the optomotor response in zebrafish.</a> Vision Research. 43, 21-30 (2003).</li></ol>

De acordo com a definição de OKR, é um fenômeno que os animais se movem os olhos para seguir uma grade rotativa espontaneamente. Se esse comportamento tem sido conduzido por um longo tempo (por exemplo, acima de 6 horas), os músculos do olho pode estar cansado e OKR será atenuada. Adulto zebrafish OMR tem sido descrito completamente por outros 7. Neste vídeo, nós apenas mostrar-lhe esse comportamento com o nosso aparelho. Uma vez que os nervos ópticos são esmagados, eles...

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using the optokinetic response to study visual function of zebrafish

Resposta optocinético (OKR) é um comportamento que um animal vibra seus olhos para seguir uma grade rotativa em torno dele. Tem sido amplamente utilizado para avaliar as funções visuais de larvas zebrafish 1-5. No entanto, o protocolo padrão para larvas de peixes ainda não é facilmente aplicável em zabrafish adulto. Aqui, apresentamos como medir a OKR de peixe-zebra adulto com o nosso aparelho custom-built simples usando um novo protocolo que é estabelecido em nosso laboratório. Tanto os aparelhos como o nosso e passo-a-passo do procedimento OKR no peixe-zebra adulto são ilustrados neste vídeo. Além disso, as medidas do OKR larval, bem como a resposta optomotor (OMR) teste de peixe-zebra adulto, também são demonstradas neste video. Este ensaio OKR de peixe-zebra adulto em nosso experimento pode durar até 4 horas. Teste OKR tais aplicado em peixes adultos serão beneficiados com a investigação da função visual de forma mais eficiente quando o adulto sistema de visão dos peixes é manipulado. Su-Qi Zou e Wu Yin contribuíram igualmente para este papel.

Watch this Scientific Journal Video about Using the optokinetic response to study visual function of zebrafish at JoVE.com

Using the optokinetic response to study visual function of zebrafish

Optocinético resposta tem sido amplamente utilizada para avaliar as funções visuais de zebrafish larval. No entanto, o protocolo padrão para larvas de peixes ainda não é facilmente aplicável em adultos<sup&gt; 05/01</sup&gt;. Aqui, apresentamos como medir a OKR de zebrafish adultos usando um novo protocolo que é estabelecido em nosso laboratório.

Usando a resposta optocinético para estudar a função visual de zebrafish

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

using-the-optokinetic-response-to-study-visual-function-of-zebrafish

Research

JoVE Journal

Biology

18.2K visualizações.  University of Science and Technology of China (USTC). Optocinético resposta tem sido amplamente utilizada para avaliar as funções visuais de zebrafish larval. No entanto, o protocolo padrão para larvas de peixes ainda não é facilmente aplicável em adultos 05/01. Aqui, apresentamos como medir a OKR de zebrafish adultos usando um novo protocolo que é estabelecido em nosso laboratório.

Vídeo: Using the optokinetic response to study visual function of zebrafish

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Teste de sensibilidade visual para a velocidade e direção do movimento em Lagartos

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Biologia

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

Gerados por computador Modelo Estímulos animal

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Microinjection of Zebrafish Embryos to Analyze Gene Function

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic oligonucleotides with antisense complementarity to target RNAs, can be added to the embryo to reduce the expression of a particular gene product.  Conversely, processed mRNA can be added to the embryo to increase levels of a gene product.  The vehicle for adding either mRNA or morpholino to an embryo is microinjection.  Microinjection is efficient and rapid, allowing for the injection of hundreds of embryos per hour.  This video shows all the steps involved in microinjection.  Briefly, eggs are collected immediately after being laid and lined up against a microscope slide in a Petri dish.  Next, a fine-tipped needle loaded with injection material is connected to a microinjector and an air source, and the microinjector controls are adjusted to produce a desirable injection volume.  Finally, the needle is plunged into the embryo's yolk and the morpholino or mRNA is expelled.

This video shows how morpholino or mRNA can be injected into zebrafish embryos at the one-cell stage to decrease or increase the level of specific gene products during subsequent development.

Microinjeção de embriões Zebrafish para analisar a função do gene

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic ...

Methods for the Study of the Zebrafish Maxillary Barbel

Barbels are skin sensory appendages found in fishes, reptiles and amphibians. The zebrafish, Danio rerio, develops two pairs of barbels- a short nasal pair and a longer maxillary pair. Barbel tissue contains cells of ectodermal, mesodermal and neural crest origin, including skin cells, glands, taste buds, melanocytes, circulatory vessels and sensory nerves. Unlike most adult tissue, the maxillary barbel is optically clear, allowing us to visualize the development and maintenance of these tissue types throughout the life cycle. 
This video shows early development of the maxillary barbel (beginning approximately one month post-fertilization) and demonstrates a surgical protocol to induce regeneration in the adult appendage (&gt;3 months post-fertilization). Briefly, the left maxillary barbel of an anesthetized fish is elevated with sterile forceps just distal to the caudal edge of the maxilla. A fine, sterile spring scissors is positioned against the forceps to cut the barbel shaft at this level, establishing an anatomical landmark for the amputation plane. Regenerative growth can be measured with respect to this plane, and in comparison to the contralateral barbel. Barbel tissue regenerates rapidly, reaching maximal regrowth within 2 weeks of injury.
Techniques for analyzing the regenerated barbel include dissecting and embedding matched pairs of barbels (regenerate and control) in the wells of a standard DNA electrophoresis gel. Embedded specimens are conveniently photographed under a stereomicroscope for gross morphology and morphometry, and can be stored for weeks prior to downstream applications such as paraffin histology, cryosectioning, and/or whole mount immunohistochemistry. These methods establish the maxillary barbel as a novel in vivo tissue system for studying the regenerative capacity of multiple cell types within the genetic context of zebrafish.

The zebrafish maxillary barbel is an integumentary sense organ containing ectodermal, mesodermal and neural crest derivatives. Importantly, the adult barbel can regenerate after proximal amputation. This video introduces maxillary barbel development and demonstrates a surgical protocol to induce regeneration, followed by collection, embedding and downstream imaging of barbel specimens.

Métodos para o Estudo do maxilar Zebrafish Barbel

Barbels are skin sensory appendages found in fishes, reptiles and amphibians.  The zebrafish, Danio rerio, develops two pairs of barbels- a short nasal ...

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

Oi-C: um método para estudar a arquitetura tridimensional de Genomas.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &amp;#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

Quantificação de γH2AX Focos em resposta à radiação ionizante

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Laser Ablation of the Zebrafish Pronephros to Study Renal Epithelial Regeneration

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in renal failure1. AKI can be caused by a number of factors including ischemia, drug-based toxicity, or obstructive injury1. This results in an inability to maintain fluid and electrolyte homeostasis. While AKI has been observed for decades, effective clinical therapies have yet to be developed. Intriguingly, some patients with AKI recover renal functions over time, a mysterious phenomenon that has been only rudimentally characterized1,2. Research using mammalian models of AKI has shown that ischemic or nephrotoxin-injured kidneys experience epithelial cell death in nephron tubules1,2, the functional units of the kidney that are made up of a series of specialized regions (segments) of epithelial cell types3. Within nephrons, epithelial cell death is highest in proximal tubule cells. There is evidence that suggests cell destruction is followed by dedifferentiation, proliferation, and migration of surrounding epithelial cells, which can regenerate the nephron entirely1,2. However, there are many unanswered questions about the mechanisms of renal epithelial regeneration, ranging from the signals that modulate these events to reasons for the wide variation of abilities among humans to regenerate injured kidneys.
The larval zebrafish provides an excellent model to study kidney epithelial regeneration as its pronephric kidney is comprised of nephrons that are conserved with higher vertebrates including mammals4,5. The nephrons of zebrafish larvae can be visualized with fluorescence techniques because of the relative transparency of the young zebrafish6. This provides a unique opportunity to image cell and molecular changes in real-time, in contrast to mammalian models where nephrons are inaccessible because the kidneys are structurally complex systems internalized within the animal. Recent studies have employed the aminoglycoside gentamicin as a toxic causative agent for study of AKI and subsequent renal failure: gentamicin and other antibiotics have been shown to cause AKI in humans, and researchers have formulated methods to use this agent to trigger kidney damage in zebrafish7,8. However, the effects of aminoglycoside toxicity in zebrafish larvae are catastrophic and lethal, which presents a difficulty when studying epithelial regeneration and function over time. Our method presents the use of targeted cell ablation as a novel tool for the study of epithelial injury in zebrafish. Laser ablation gives researchers the ability to induce cell death in a limited population of cells. Varying areas of cells can be targeted based on morphological location, function, or even expression of a particular cellular phenotype. Thus, laser ablation will increase the specificity of what researchers can study, and can be a powerful new approach to shed light on the mechanisms of renal epithelial regeneration. This protocol can be broadly applied to target cell populations in other organs in the zebrafish embryo to study injury and regeneration in any number of contexts of interest.

Acute kidney injury (AKI) in humans is a common clinical problem caused by damage to the epithelial cells that comprise kidney nephrons, and AKI is associated with high mortality rates of 50-70%1. Following epithelial cell destruction, nephrons have a limited ability to regenerate, though the mechanisms and limitations that guide this phenomenon remain poorly understood. In this video article, we describe our technique for targeted laser ablation of kidney nephron cells in the zebrafish embryo kidney, or pronephros. Our new method can be used to complement nephrotoxicity-induced models of AKI and gain a high-resolution understanding of the cell and molecular alterations that are associated with epithelial regeneration in the kidney nephron.

Ablação a laser da Pronephros Zebrafish para estudo da regeneração epitelial renal

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in ...

Force Measurement During Contraction to Assess Muscle Function in Zebrafish Larvae

Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional measures of muscle health. In this video article, we demonstrate a method to measure force generation during contraction of zebrafish larval trunk muscle. Force measurements are accomplished by placing an anesthetized larva into a chamber filled with a salt solution. The anterior end of the larva is tied to a force transducer and the posterior end of the larva is tied to a length controller. An isometric twitch contraction is elicited by electric field stimulation and the force response is recorded for analysis. Force generation during contraction provides a measure of overall muscle health and specifically provides a measure of muscle function. Although we describe this technique for use with wild-type larvae, this method can be used with genetically modified larvae or with larvae treated with drugs or toxicants, to characterize muscle disease models and evaluate treatments, or to study muscle development, injury, or chemical toxicity.

Force measurements can be used to demonstrate changes in muscle function due to development, injury, disease, treatment or chemical toxicity. In this video, we demonstrate a method to measure force during a maximal contraction of zebrafish larval trunk muscle.

Medição de força durante a contração para avaliar a função muscular em larvas do peixe

Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional ...

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression both in terms of tissue distribution and developmental stage. Protocols are described for the use of WISH of zebrafish embryos using antisense RNA probes labeled with digoxigenin. Probes are generated by incorporating digoxigenin-linked nucleotides through in vitro transcription of gene templates that have been cloned and linearized. The chorions of embryos harvested at defined developmental stages are removed before incubation with specific probes. Following a washing procedure to remove excess probe, embryos are incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase. By employing a chromogenic substrate for alkaline phosphatase, specific gene expression can be assessed. Depending on the level of gene expression the entire procedure can be completed within 2-3 days.

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

The zebrafish, a small tropical fish, has become a popular model for studying gene function during vertebrate development and disease. The temporal and spatial expression of target genes can be determined by in situ hybridization. Our improved protocol allows for the detection of low abundant transcripts with low non-specific background signal.

Alta resolução Monte Whole<em&gt; In Situ</em&gt; Hibridização dentro embriões Zebrafish para estudar a expressão gênica e função

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression ...

Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.

The zebrafish adult kidney is an excellent system for renal regeneration and disease studies. An essential aspect of such research is the assessment of nephron structure and function. This protocol describes several methodologies that can be implemented to assess nephron tubule composition and to evaluate renal reabsorption.

Análise de Composição Nephron e função no rim adulto Zebrafish

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys ...