Wir danken Frau Fan Shen für ihre Unterstützung der Zebrafisch-Aufzucht. Dies wird durch Förderungen zu Dr. Bing Hu (Chinese Academy of Sciences &quot;Bai Ren Ji Hua&quot;, NSFC 30870833 und National Basic Science Program of China, 2009CB941302) unterstützt. Alle Tierexperimente wurden in Übereinstimmung mit den Richtlinien und Vorschriften her von der University of Science and Technology of China (USTC) Animal Resources Center und der University Animal Care und Verwenden Ausschusses durchgeführt.

Teil I: Konstruieren Sie die OKR Apparat Details sind in Video-Demonstration gezeigt. Teil II: OKR of Adult Zebrafisch Wenn Ihr Lab einen OKR Apparat konstruiert hat, ist der Schlüssel der erwachsenen Zebrafisch OKR, um die Fische zu halten. In diesem Teil zeigen wir Ihnen Schritt für Schritt. Unsere Geräte komponiert folgenden Teilen ähnlich zu anderen erwähnt. Die runde Fisch Kammer ist 7,0 cm Außendurchmesser, 6,5 cm Innendurchmesser und 7,0 cm in der Höhe. Die Trommel um Fisch Kammer ist 8,5 cm in Durchmesser, 7,5 cm Innendurchmesser und 7,0 cm in der Höhe. Es könnte im Uhrzeigersinn drehen und gegen den Uhrzeigersinn mit der Geschwindigkeit von 20 ~ 70 Umdrehungen pro Minute (rpm). Elf Zyklen von schwarzen und weißen Streifen auf einem Transparentpapier, die auf der Außenseite der Trommel eingehalten gezogen. Das Lichtsystem besteht aus zwei Kreis Leuchtstoffröhren in 21 cm Durchmesser bestehen. <img src="/files/ftp_upload/1742/1742fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Das Modell der Schwamm. Die Klippe in dieser Mitte aus ist, um sicherzustellen, dass das Gesichtsfeld nicht Geborgenheit. <ol><li> Ein Schwamm wie in Abb.1 dargestellt durch zwei Teilen zusammengesetzt. Der linke Teil ist die Länge 3 cm x Breite 3 cm x 2 cm hoch. Der rechte Teil ist die Länge 3 cm x Breite 3 cm x 3 cm hoch. </li><li> Diesen Schwamm in der unteren Kammer und Fisch gießen Aufzucht Wasser in ca. 2 cm Tiefe. Luftblase, die in den Schwamm zu verbergen sollten vertrieben werden. </li><li> Legen Sie einen Stift in der Vorderseite des rechten Teil der Schwamm, ca. 2 mm hinter der Klippe. </li><li> Anesthetize ein Erwachsener Zebrafisch in MS-222. </li><li> Setzen Sie den Zebrafisch auf den Schwamm mit dorsaler und ermöglichen einen Seite seiner Brustflosse vor dem ersten Pin. </li><li> Der zweite Stift befindet sich hinter dem anderen Brustflosse eingefügt. Es könnte Zebrafisch schrumpfenden Körper durch die Zusammenarbeit mit dem ersten Pin (siehe auch in Abb. 2). Zu vermeiden. </li><li> Clamp seinen Schwanz mit einer Arterienklemme und fixieren die Arterienklemme in einem Stück Gummi (Knetmasse), die auf die Fische Kammer eingehalten wird. </li><li> Die dritte und vierte Pin ist auf der Website der Nähe cloacal Pore Fischkörper gepflanzt. Es könnte verhindern, Zebrafische, um seinen Schwanz intensiv Welle. </li><li> Der fünfte Pin ist zwischen dem ersten und dem dritten Pin und dann die sechste Pin ist auf der anderen Seite davon gelegen. Sie könnten Zebrafische aus Schütteln seines Körpers zu verhindern. </li><li> Die siebte und achte Pin wird an den Standorten in der Nähe der Kiemen eingefügt, um den Kopf Orientierung zu behalten. </li><li> Gießen Sie Wasser in die Aufzucht der Fische Kammer. </li><li> Nach dem Zebrafisch aufwacht, lassen Sie es für 5 bis 10 Minuten anzupassen. </li><li> Schalten Sie das Licht an dieser Vorrichtung und lassen Zebrafisch anzupassen diese Beleuchtung für 2 Minuten. </li><li> Schalten Sie den Motor mit dem Gitter-Laufwerk und ermöglicht Zebrafisch für 1 Minute anzupassen. </li><li> Schalten Sie die Kamera und Aufzeichnung der Bewegung der Augen. </li><li> Analysieren Sie den Augenwinkeln 6 und Sakkade Nummern auf Ihrem PC (Ein repräsentatives Beispiel für den Augenwinkeln Analyse ist in Abb. 2 dargestellt). </li></ol><img src="/files/ftp_upload/1742/1742fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Der Winkel zwischen Auge Achse und Körper-Achse. a) Wenn die Richtung der Gitter nasal-to-zeitlich ist, definiert man den Winkel, in dem vor der Fische Körper ist ein negativer Winkel und der Winkel, in dem Fische Körper ist ein positiver Winkel, umgekehrt (für kurze Übersicht siehe auch Lit.. 6). Die Zahlen in dieser Abbildung stellen die Reihenfolge der Pins. b) OKR Analyse beispielsweise von einem Fisch in normalen Gruppe. Eine Ein-Sekunden-Film wurde zu einer Reihe Frames übertragen und den Augenwinkeln jedes Rahmens wurde mit Image-Pro Plus-Software gemessen. Jeder Punkt in dieser Abbildung repräsentiert jedes Bild haben wir gemessen. Notes: <ol class="lalpha"><li> Alle Stifte sind einfach zur Zebrafisch aus Schütteln seines Körpers zu verhindern, zu eng wird als schädlich für Fische. </li><li> Arterienklemme beschädigen könnte der Schwanz als Kontaktfläche klein ist. Sobald der Schwanz ist frei, s es sehr schwierig, diesen Fisch zu beheben. Wrap einen Verband um den Schwanz ist in eine bestimmte Bedingung empfohlen. </li><li> Die siebte und achte Stift, der in der Website in der Nähe gill gepflanzt werden, sollten keine Zuflucht sein vor ihren Augen von Gitterrosten. </li></ol> Teil III. OKR der Larven Zebrafisch 1. Methylcellulose Larven Zebrafisch kann in 6% Methylcellulose-Lösung immobilisiert werden, während für eine lange Zeit lebendig. Bereiten Methylcellulose-Lösung wie folgt. <ol><li> Kochen 200 ml ddH 2 O in der Mikrowelle ca. 3 Minuten auf hohem Niveau. </li><li> Übertragen Sie die heißen Wasser zu einer Heizplatte und rühren kräftig. </li><li> Fügen Sie 12 g Methylcellulose in das heiße Wasser nach und nach. </li><li> Keep Rühren 3 Minuten nach Methylcellulose ist gut verteilt. </li><li> Kühlen Sie die Lösungen mit Eiswasser, bis es klar wird, und dann legen Sie sie bei 4 ° C. </li></ol><pclass = &quot;jove_step&quot;&gt; 2. Larven Zebrafisch <ol><li> Paare zwei männliche und eine weibliche erwachsener Zebrafisch in einem passenden Behälter um 17:00 Uhr nach der Fütterung. Am nächsten Morgen übertragen Sie sie auf eine frische Paarung Tank sammeln und Embryonen nach 1 Stunde. </li><li> Dunkel-Zyklus von 14:10: Die Eier werden in Embryo-Medium in einem 28,5 ± 0,5 ° C-Inkubator mit dem Licht erhalten. </li><li> Eier werden bei 48 Stunden nach der Befruchtung (HPF) ausgebrütet werden und die OKR wird bei 72 hpf oder später geprüft werden. In diesem Video werden die Larven bei 5 dpf. </li></ol> 3. Vorgehensweise des OKR <ol><li> Setzen Sie das Gerät unter dem Stereoskop mit CCD-Kamera und schalten Sie das Licht dieser Apparat ausgestattet. </li><li> 20 ml 6% methycellulose in einer 55-mm-Petrischale. </li><li> Legen Sie mehrere Larven in einer Reihe in der Mitte von diesem Gericht. </li><li> Legen Sie eine Luftblase über ein Volumen von 1 ul in die Kamera Feld, könnte es als ein Spiegel sein, um die Richtung des Gitters zu reflektieren. </li><li> Schalten Sie den Motor und notieren Sie die Bewegung ihrer Augen. </li></ol> Teil IV. OMR of Adult Zebrafisch Adult Zebrafisch OMR konnte auch mit diesem Gerät durchgeführt werden, sondern wird einfach eine Spalte in der Fisch-Kammer zur Vermeidung Fische schwimmen über Mitte (gezeigt in Abb. 3a, Drehgeschwindigkeit beträgt 36 min, schwarz-weiß-Zyklen sind 11). <ol><li> Setzen Sie einen Fisch in dieser Fisch Kammer. </li><li> Stand eine Säule von 2 cm Durchmesser in der Mitte dieses Fisches Kammer. </li><li> Passen Sie für eine Minute sowohl in noch Gitter und Rotation Gitter. </li><li> Notieren Sie sich die Bewegung der Zebrafisch. </li><li> Analysieren Sie die Koinzidenz von Zeit auf Ihrem PC (ein Beispiel ist in Abb. 3b gezeigt, Drehgeschwindigkeit 36 ​​rpm ist, ist schwarz-weiß-Zyklus 11) </li></ol><img src="/files/ftp_upload/1742/1742fig3.jpg" alt="Abbildung 3" /> Abbildung 3. OMR of Adult Zebrafisch. (A) Eine weiße Kunststoff-Säule wurde in der Mitte des Fisches Kammer zur Vermeidung Fische schwimmen über sie gelegt. (B) Das Zusammentreffen von erwachsenen Zebrafisch im Anschluss an die Gitter. Der Zufall lag der Anteil der folgenden Gitter Zeit, um die gesamte Aufnahmezeit definiert. Der Wert von 50% zeigt an, dass der Fisch nach dem Zufallsprinzip schwimmen, während 100% zeigt die Fische folgenden Gitter sehr gut. Beide Sehnerven wurden mit einem Schmuck Pinzette ca. 10 Sekunden zerkleinert. Das Verhalten der OMR erschien als eine zufällige Schwimmen nach zu vernichten.

<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>

Nach der Definition des OKR, ist es ein Phänomen, dass die Tiere ihre Augen bewegen, um ein rotierendes Gitter spontan zu verfolgen. Wenn dieses Verhalten für eine lange Zeit (zB über 6 Stunden) durchgeführt, kann das Auge Muskeln müde werden und der OKR werden gedämpft. Adult Zebrafisch OMR wurde komplett von anderen 7 beschrieben. In diesem Video zeigen wir Ihnen nur dieses Verhalten mit unserem Gerät. Sobald die Sehnerven zerkleinert sind, könnten sie sich nicht an diese Gitter sehr gut. Wir haben OKR Verhalten zu testen in der Erwachsenenbildung, Larven Zebrafisch und OMR bei Erwachsenen durchgeführt mit unserem einfachen custom-built Apparat, um die visuelle Funktion des Zebrafisch zu bewerten. Diese OKR-Assay von erwachsenen Zebrafisch in unserem Experiment halten konnte stark genug, innerhalb eines Zeitraums von 4 Stunden, ohne zu schaden. Dies kann eröffnen OKR-Test bei erwachsenen Fischen Funktion Studie effizienter.

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using the optokinetic response to study visual function of zebrafish

Optokinetischen Antwort (OKR) ist ein Verhalten, dass ein Tier schwingt seine Augen auf eine rotierende Gitter um ihm zu folgen. Es wurde weithin verwendet, um die visuellen Funktionen von Larven Zebrafisch 1-5 zu bewerten. Dennoch ist das Standardprotokoll für die Fischlarven noch nicht ohne weiteres anwendbar bei erwachsenen zabrafish. Hier stellen wir Ihnen, wie Sie die OKR der erwachsenen Zebrafisch mit unserem einfachen custom-built Gerät mit einem neuen Protokoll, das in unserem Labor etabliert ist, zu messen. Sowohl unsere Apparate-und Schritt-für-Schritt-Verfahren der OKR bei erwachsenen Zebrafisch sind in diesem Video dargestellt. Darüber hinaus sind die Messungen der Larven OKR, sowie die optomotorischen Ansprechen (OMR) Test der erwachsenen Zebrafisch, auch in diesem Video demonstriert. Diese OKR-Assay von erwachsenen Zebrafisch in unserem Experiment kann für bis zu 4 Stunden dauern. Solche OKR-Test bei erwachsenen Fischen angewendet werden, um die Sehfunktion Untersuchung effizienter nutzen, wenn die erwachsenen Fische Vision-System manipuliert wird. Su-Qi Zou und Wu Yin trugen gleichermaßen zu dieser Veröffentlichung.

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

Optokinetischen Reaktionen waren weit verbreitet, um die visuellen Funktionen von Larven Zebrafisch zu beurteilen. Dennoch ist das Standardprotokoll für die Fischlarven noch nicht ohne weiteres anwendbar bei Erwachsenen<sup&gt; 1-5</sup&gt;. Hier stellen wir Ihnen, wie Sie die OKR der erwachsenen Zebrafisch mit einem neuen Protokoll, das in unserem Labor etabliert ist, zu messen.

Mit der optokinetischen Reaktion auf visuelle Funktion des Zebrafisch-Studie

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

Nanyang Technological University

Research

JoVE Journal

Biology

18.3K Aufrufe.  University of Science and Technology of China (USTC). Optokinetischen Reaktionen waren weit verbreitet, um die visuellen Funktionen von Larven Zebrafisch zu beurteilen. Dennoch ist das Standardprotokoll für die Fischlarven noch nicht ohne weiteres anwendbar bei Erwachsenen 1-5. Hier stellen wir Ihnen, wie Sie die OKR der erwachsenen Zebrafisch mit einem neuen Protokoll, das in unserem Labor etabliert ist, zu messen.

Serie: 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.

Testen visueller Sensibilität für die Geschwindigkeit und Richtung der Bewegung in Lizards

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

Forschung

Biologie

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.

Computer-Generated-Tiermodell Stimuli

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.

Mikroinjektion von Zebrafisch-Embryos, um Gene Function Analyze

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.

Methoden zur Untersuchung des Zebrafisch 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 ...

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.

Hallo-C: eine Methode, um die dreidimensionale Architektur des Genoms Study.

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 &#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.

Die Quantifizierung der γH2AX Foci in Response to ionisierender Strahlung

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.

Laser Ablation des Zebrafisch Vorniere auf Study Renal Epithelregeneration

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.

Kraftmessung während der Kontraktion zu Muscle Funktion in Zebrafisch-Larven bewerten

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.

Hohe Auflösung Ganze Berge<em&gt; In Situ</em&gt; Hybridisierung innerhalb Zebrafischembryonen Gene Expression und Funktion zu untersuchen

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.

Analyse der Nephron Zusammensetzung und Funktion im erwachsenen Zebrafisch Nieren

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