Name: using confocal analysis of xenopus laevis to investigate modulators of wnt and shh morphogen gradients
Uploaded: 2015-12-14T13:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients at JoVE.com

Diese Arbeit wurde von einem BBSRC finanziert gewähren MdEP (BB / H010297 / 1), ein BBSRC Quoten studentship zu SAR und einen MRC studentship zu SWF.

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The authors have no competing financial interests.

Ein wesentlicher Teil dieses Protokolls erzeugt biologisch aktiven Liganden, die normalerweise bearbeitet werden, ausgeschieden wird, und fähig sind, eine Antwort in der Aufnahmezelle auslösen kann, obwohl sie eine fluoreszierende Gruppe gebunden. Es kritisch ist, nachzuweisen, dass das fluoreszenzmarkierte Genprodukt ist biologisch aktiv unter Verwendung eines geeigneten Assays. Für Shh-GFP wurde die Fähigkeit, die Expression von ptc1 aktivieren 16 bestätigt. Wnt8a hat eine bemerkenswert starke...

Während der frühen embryonalen Entwicklung werden die Zellen schrittweise verpflichtet, bestimmte Abstammungslinien der Differenzierung zu folgen: dies bedeutet eine Gruppe von totipotenten (oder pluripotenten Zellen) werden auf die Schaffung Populationen von Vorläuferzellen bestimmt Anlass zu einem Zelltyp geben allmählich beschränkt. Zell-Zell-Signalisierung ist von zentraler Bedeutung für die Regulierung der Linienspezifikation während der embryonalen Entwicklung. Manipulation dieser Signale wird benötigt, um Stammzellen in Richtung besonderen Schicksale lenken, neue medizinische Behandlungen zu unterstützen. Eine relativ kleine Anzahl von Signalwegen, werden während der Entwicklung wiederholt, einschließlich Wegen als Antwort auf die TGF-Superfamilie (nodals und BMPs) 1-2, FGFs 3, Wnt 4 und 5 Igel. Diese sekretierten Proteine ​​binden an der Zellmembran, die Signaltransduktion wodurch Genexpression und / oder das Verhalten der Zelle ändern aktivieren Rezeptoren. Die enge Regulation von ZellzeichenAlling ist essentiell für Zelllinie Spezifikation und eine normale Entwicklung. Während das Übersprechen zwischen diesen Bahnen ist bei der Bestimmung des Zellschicksals wichtig ist, kann ein einzelner Ligand selbst hervorrufen unterschiedliche Reaktionen in unterschiedlichen Konzentrationen. Morphogen Gradienten wurden vor über 100 Jahren als eine Theorie zu erklären, wie verschiedene Zelltypen können aus einem Feld von Zellen 6 abzuleiten beschrieben. Signalmoleküle durch eine Gruppe von Zellen hergestellt werden, können über einen gewissen Bereich zu diffundieren, Abnahme in der Konzentration mit einem größeren Abstand von der Quelle. Zellen auf das Signal ausgesetzt wird, um die lokale Konzentration an ihrer Position in dem Bereich der Zellen entsprechen, die sich Zellen an verschiedenen Positionen unterschiedlich reagieren auf verschiedenen Ebenen des Signals. Beweise für die Existenz von Morphogenen ergeben sich aus Studien der frühen Drosophila-Embryo 7 und der Flügelscheibe 8 sowie des Vertebraten Schenkel 9 und Neuralrohr 10. Verfahren sind in benötigtenvestigate wie Morphogen Gradienten etabliert sind und an andere Moleküle bei der Regulierung dieser Gradienten wichtig zu identifizieren. Elegante Experimente mittels Immunhistochemie auf endogenen Proteinen in vivo im Kontext von verschiedenen genetischen Hinter visualisiert wurden zur Morphogengradienten 11-12 untersuchen. , Gute Antikörpern und spezifischen Mutanten sind jedoch nicht immer zur Verfügung, so dass wir mit Überexpression von fluoreszierenden Liganden in Xenopus, um eine alternative, einfache Methode, um zu zerlegen, wie exogene Genprodukte Verteilung von Liganden, die über ein Feld von Zellen beeinflussen, bereitzustellen beschreiben hier eine Protokoll. Xenopus laevis bietet ein ausgezeichnetes System, um diese Arten von Experimenten durchzuführen, wie ihre Embryonen entwickeln, von außen, so dass sie in den frühesten Stadien zugänglich sind. Ihre Größe (1-1.5mm Durchmesser) vereinfacht die Mikroinjektion und chirurgischen Manipulation und durch Blastula Stufen die Zellen leicht zu Bild, da sie immer noch relativ groß ist (etwa 2081; m Durchmesser). Die Überexpression Studien in Xenopus sind einfach zu tun: mRNA in den frühen Embryo injiziert werden, um bestimmte Zellen gezielt und effizient umgerechnet. Die fluoreszenzmarkierten Wnt8a / Wnt11b-HA-eGFP-Konstrukte wurden mit pCS2 Wnt8a-HA 13, pCS2 Wnt11b-HA 14 und eGFP generiert. Der HA-Peptid ist wichtig, umfassen nicht nur eine zusätzliche molekulare Markierung zu schaffen, sondern auch, weil es wird angenommen, dass als Abstandshalter trennt die Wnt und eGFP-Proteinen so dass sowohl Genprodukte zu funktionieren handeln. Das zur Visualisierung von Shh verwendet Konstrukt wurde zuvor verwendet, um eine transgene Maus eine Shh-eGFP-Fusionsproteins 15 exprimieren, zu erzeugen; Dies wurde freundlicherweise von Andy McMahon zur Verfügung gestellt. Wichtig ist, dass das GFP-Tag für alle Konstrukte kloniert 3 &#39;zur Signalsequenz, so daß sie nach der Bearbeitung beibehalten. Es ist auch wichtig, um sicherzustellen, dass die endgültige Protein enthält Sequenzen, die für Änderungen erforderlich sind, wie die addition von Lipiden, wie es der Fall für Shh und Wnt ligands.The cDNAs wurden in den pCS2 + Expressionsvektor, der für die prodution synthetischer mRNA optimiert ist subkloniert; es enthält einen SP6-Promotor und Polyadenylierungssignal (http://sitemaker.umich.edu/dlturner.vectors). Die hier beschriebene Arbeit bietet ein einfaches Protokoll für den Vergleich der Sekretion und Verbreitung von fluoreszenzmarkierten Wnt und Shh getaggt Liganden. Durch Einspritzen von definierten Mengen synthetischer mRNA, umrundet das Protokoll keine Probleme mit variabler Expression aus verschiedenen Vektoren mit verschiedenen Promotoren verbunden. Diese Methoden wurden kürzlich angewendet, um die Wirkungen von Heparansulfat endosulfatase Sulf1 auf Shh-eGFP und Wnt8a / Wnt11b-HA-eGFP-Sekretion und Diffusion in Xenopus 16-17 untersuchen.

<table><tbody><tr><td>Agarose</td><td>Melford</td><td>MB 1200</td><td></td></tr><tr><td>Ammonium acetate</td><td>Ambion</td><td>From Megascript SP6 Kit AM 1330</td><td></td></tr><tr><td>Bicarbonate</td><td>VWR International</td><td>RC-091</td><td></td></tr><tr><td>Calcium nitrate</td><td>Sigma</td><td>C13961</td><td></td></tr><tr><td>Cap analog (m7G(5&#039;))</td><td>Applied Biosystems</td><td>AM 8050</td><td></td></tr><tr><td>Chloroform</td><td>Sigma</td><td>C 2432</td><td></td></tr><tr><td>L-Cysteine hydrochloride monohydrate</td><td>Sigma</td><td>C7880</td><td></td></tr><tr><td>dNTPs</td><td>Invitrogen</td><td>18427-013</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma</td><td>O3690</td><td></td></tr><tr><td>Ethanol</td><td>VWR International</td><td>20821.33</td><td></td></tr><tr><td>Ficoll 400</td><td>Sigma</td><td>F 4375</td><td></td></tr><tr><td>Fiji image J software</td><td></td><td></td><td>Free download http://fiji.sc/Fiji</td></tr><tr><td>Gentamycin</td><td>Melford</td><td>G 0124</td><td></td></tr><tr><td>Glacial acetic acid</td><td>Fisher Scientific</td><td>A/0400/PB17</td><td></td></tr><tr><td>Glass cover slips, No.1.5&amp;nbsp;</td><td>Scientific Laboratory Supplies</td><td>22X22-SGJ3015. 22X50-SGJ3030&amp;nbsp;</td><td></td></tr><tr><td>Glass needle puller</td><td>Narishige</td><td>Narishige PC -10</td><td></td></tr><tr><td>Glass pull needles</td><td>Drummond Scientific</td><td>3-000-203-G/X</td><td></td></tr><tr><td>Human chronic gonadotropin (HCG)</td><td>Intervet</td><td></td><td></td></tr><tr><td>Isopropanol</td><td>Fisher Scientific</td><td>P/7500/PB17</td><td></td></tr><tr><td>Lithium chloride (LiCl)</td><td>Sigma</td><td>L-7026</td><td></td></tr><tr><td>LSM710 and Zen software (2008-2010)</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>Matlab software</td><td>Mathworks</td><td></td><td>http://uk.mathworks.com/</td></tr><tr><td>Molecular grade water&amp;nbsp;</td><td>Fisher Scientific</td><td>BP 2819-10</td><td></td></tr><tr><td>Nail varnish&amp;nbsp;</td><td>Boots</td><td>Bar code 3600530 373048</td><td></td></tr><tr><td>Spectrophotometer</td><td>Lab.tech International</td><td>ND-1000 / ND8000</td><td></td></tr><tr><td>Petri dish (55 mm)</td><td>VWR International</td><td>391-0865</td><td></td></tr><tr><td>Phenol-chloroform</td><td>Sigma</td><td>P3803</td><td></td></tr><tr><td>Photoshop software</td><td>Adobe</td><td></td><td>http://www.photoshop.com/products</td></tr><tr><td>High fidelity DNA polymerase and buffers&amp;nbsp;</td><td>Biolabs</td><td>M0530S&amp;nbsp; Buffer - M0531S</td><td></td></tr><tr><td>Potassium chloride (KCl)</td><td>Fisher Scientific</td><td>P/4280/53</td><td></td></tr><tr><td>PVC insulation tape</td><td>Onecall</td><td>SH5006MPK</td><td></td></tr><tr><td>Gel extraction kit&amp;nbsp;</td><td>Qiagen</td><td>S28704</td><td></td></tr><tr><td>Restriction enzymes buffers</td><td>Roche</td><td>SuRE/CUT Buffer Set 11082 035 001</td><td></td></tr><tr><td>RNAse-free DNAse</td><td>Promega</td><td>ME10A</td><td></td></tr><tr><td>Steel back single edge blades</td><td>Personna</td><td>66-0403-0000</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>Fisher Scientific</td><td>27810.364</td><td></td></tr><tr><td>SP6 transcription kit</td><td>Ambion</td><td>AM1330</td><td></td></tr><tr><td>Glass slides</td><td>Thermo Fisher</td><td>SHE 2505</td><td></td></tr><tr><td>Tris base</td><td>Invitrogen</td><td>15504-020</td><td></td></tr><tr><td>Tungsten needles</td><td></td><td></td><td>homemade</td></tr><tr><td>Zen lite software</td><td>Carl Zeiss</td><td></td><td>Free download&amp;nbsp; http://www.zeiss.co.uk/microscopy/en_gb/downloads/zen.html</td></tr></tbody></table>

using confocal analysis of xenopus laevis to investigate modulators of wnt and shh morphogen gradients

Dieses Protokoll beschreibt ein Verfahren, um Liganden über ein Feld von Zellen verteilt visualisieren. Die Leichtigkeit der exprimieren exogene Proteine, zusammen mit der Größe ihrer Zellen in frühen Embryonen, stellen Xenopus laevis ein nützliches Modell für die Visualisierung von GFP-markierten Liganden. Synthetischer mRNAs effizient nach der Injektion in die übersetzte frühzeitig Xenopus-Embryonen und Injektionen können auf eine einzelne Zelle gezielt werden. Wenn sie mit einer Linie Tracer wie Membranbunden RFP kombiniert werden, kann der eingespritzte Zelle (und ihre Nachkommen), die Herstellung des überexprimierten Proteins leicht verfolgt werden. Dieses Protokoll beschreibt ein Verfahren zur Herstellung von fluoreszenzmarkierten Wnt und Shh Liganden aus injizierten mRNA. Die Verfahren die micro Dissektion der ektodermalen Explantate (animal caps) und der Analyse der Liganden Diffusion in mehreren Proben. Durch konfokale Bildgebung können Informationen über Ligand Sekretion und Diffusion über ein Feld von Zellen erhalten werden. Statistische Analysen der konfokalen Bilder liefern quantitative Daten über die Form des Liganden Steigungen. Diese Methoden können nützlich für Forscher, um die Auswirkungen der Faktoren, die die Form der Morphogen Gradienten regulieren kann testen wollen.

Konfokale Analyse animal cap Explantate exprimieren fluoreszent markierte Proteine ​​bietet ein wirksames System zur Visualisierung Ligand Verteilung unter unterschiedlichen Versuchsbedingungen. In einem Beispiel kann die Verteilung von GFP-markierte Shh dargestellt (Abbildung 1). In 2-Zellen-Stadium werden Xenopus-Embryonen in die beiden Zellen, die entweder mit einem Kontroll-mRNA oder mRNA, die für Sulf1, ein Enzym, das die Zelloberflächen Heparansulfat verändert und beeinflusst die S...

Watch this Scientific Journal Video about Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients at JoVE.com

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

Das Manuskript hier bietet eine einfache Reihe von Methoden für die Sekretion und Verbreitung von fluoreszierend markierten Liganden in Xenopus-Analyse. Dies bietet einen Rahmen für die Prüfung der Fähigkeit von anderen Proteinen zu Ligand Verteilung ändern und erlaubt Experimente, die Einblick in die Mechanismen, die Morphogen Gradienten geben kann.

Mittels konfokaler Analyse<em&gt; Xenopus laevis</em&gt; Um Modulatoren der Wnt und Shh Morphogen Gradienten Untersuchen

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the ...

using-confocal-analysis-xenopus-laevis-to-investigate-modulators-wnt

Research

JoVE Journal

Developmental Biology

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

11.2K Aufrufe.  The Bateson Centre, University of Sheffield.  Das Manuskript hier bietet eine einfache Reihe von Methoden für die Sekretion und Verbreitung von fluoreszierend markierten Liganden in Xenopus-Analyse. Dies bietet einen Rahmen für die Prüfung der Fähigkeit von anderen Proteinen zu Ligand Verteilung ändern und erlaubt Experimente, die Einblick in die Mechanismen, die Morphogen Gradienten geben kann. 

Serie: Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, cells from the roof of the blastocoel are pluripotent. These cells can be isolated, and programmed to generate various tissues through manipulation of genes expression or induction by morphogens. In this manuscript protocols are described for the use of Xenopus laevis blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development. These protocols allow the investigation of fate acquisition, cell migration behaviors, and cell autonomous and non-autonomous properties. The blastocoel roof explants can be cultured in a serum-free defined medium and grafted into host embryos. This transplantation into an embryo allows the investigation of the long-term lineage commitment, the inductive properties, and the behavior of transplanted cells in vivo. These assays can be exploited to investigate molecular mechanisms, cellular processes and gene regulatory networks underlying neural development. In the context of regenerative medicine, these assays provide a means to generate neural-derived cell types in vitro that could be used in drug screening.

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

In Xenopus embryos, cells from the roof of the blastocoel are pluripotent and can be programmed to generate various tissues. Here, we describe protocols to use amphibian blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development.

Stammzell-ähnlichen<em&gt; Xenopus</em&gt; Embryonale Explantate bis Früh Neurale Entwicklungs- Eigenschaften Studieren<em&gt; In-vitro-</em&gt; Und<em&gt; In-vivo-</em

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, ...

Forschung

Entwicklungsbiologie

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

Technik auf Zielmikroinjektions an die Entwicklungs<em&gt; Xenopus</em&gt; Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions

Remarkably few cell-to-cell signal transduction pathways are necessary during embryonic development to generate the large variety of cell types and tissues in the adult body form. Yet, each year more components of individual signaling pathways are discovered, and studies indicate that depending on the context there is significant cross-talk among most of these pathways. This complexity makes studying cell-to-cell signaling in any in vivo developmental model system a difficult task. In addition, efficient functional analyses are required to characterize molecules associated with signaling pathways identified from the large data sets generated by next generation differential screens. Here, we illustrate a straightforward method to efficiently identify components of signal transduction pathways governing cell fate and axis specification in sea urchin embryos. The genomic and morphological simplicity of embryos similar to those of the sea urchin make them powerful in vivo developmental models for understanding complex signaling interactions. The methodology described here can be used as a template for identifying novel signal transduction molecules in individual pathways as well as the interactions among the molecules in the various pathways in many other organisms.

The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions

This video article details a straightforward in vivo methodology that can be used to systematically and efficiently characterize components of complex signaling pathways and regulatory networks in many invertebrate embryos.

Die Kraft der Einfachheit: Seeigel Embry als<em&gt; In Vivo</em&gt; Entwicklungsmodelle für komplexe Zell-zu-Zell-Signalnetzwerk Wechselwirkungen Studieren

Remarkably few cell-to-cell signal transduction pathways are necessary during embryonic development to generate the large variety of cell types and ...

Neural Explant Cultures from Xenopus laevis

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During axon outgrowth, the growth cone must integrate multiple sources of guidance cue information to modulate its cytoskeleton in order to propel the growth cone forward and accurately navigate to find its specific targets1. How this integration occurs at the cytoskeletal level is still emerging, and examination of cytoskeletal protein and effector dynamics within the growth cone can allow the elucidation of these mechanisms. Xenopus laevis growth cones are large enough (10-30 microns in diameter) to perform high-resolution live imaging of cytoskeletal dynamics (e.g.2-4 ) and are easy to isolate and manipulate in a lab setting compared to other vertebrates. The frog is a classic model system for developmental neurobiology studies, and important early insights into growth cone microtubule dynamics were initially found using this system5-7 . In this method8, eggs are collected and fertilized in vitro, injected with RNA encoding fluorescently tagged cytoskeletal fusion proteins or other constructs to manipulate gene expression, and then allowed to develop to the neural tube stage. Neural tubes are isolated by dissection and then are cultured, and growth cones on outgrowing neurites are imaged. In this article, we describe how to perform this method, the goal of which is to culture Xenopus laevis growth cones for subsequent high-resolution image analysis. While we provide the example of +TIP fusion protein EB1-GFP, this method can be applied to any number of proteins to elucidate their behaviors within the growth cone.

Neural Explant Cultures from Xenopus laevis

Culturing neural explants from dissected Xenopus laevis embryos that express fluorescent fusion proteins allows for imaging of growth cone cytoskeletal dynamics.

Neural Explantatkulturen aus<em&gt; Xenopus laevis</em

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During ...

Neurowissenschaften

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation1-16. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and M&uuml;ller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates 12,14-18.
While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells 7,19-23. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,19-22,24-33. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,27-30,33-39. Xenopus laevis, a classic model system for the study of early neural development 19,27,29,31-32,40-42, serves as a particularly suitable system for retinal primary cell culture 10,38,43-45.
Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction 25,38,43. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products 10,24,44-45.
However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1).

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Xenopus laevis provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.

Dissection, Kultur und Analyse der<em&gt; Xenopus laevis</em&gt; Embryonic Netzhautgewebe

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

Dissection and Lateral Mounting of Zebrafish Embryos: Analysis of Spinal Cord Development

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene knockdown approaches can be utilized to examine the molecular mechanisms underlying nervous system development. Second, large clutches of developmentally synchronized embryos provide large experimental sample sizes. Third, the optical clarity of the zebrafish embryo permits researchers to visualize progenitor, glial, and neuronal populations. Although zebrafish embryos are transparent, specimen thickness can impede effective microscopic visualization. One reason for this is the tandem development of the spinal cord and overlying somite tissue. Another reason is the large yolk ball, which is still present during periods of early neurogenesis. In this article, we demonstrate microdissection and removal of the yolk in fixed embryos, which allows microscopic visualization while preserving surrounding somite tissue. We also demonstrate semipermanent mounting of zebrafish embryos. This permits observation of neurodevelopment in the dorso-ventral and anterior-posterior axes, as it preserves the three-dimensionality of the tissue.

Developmental processes such as proliferation, patterning, differentiation, and axon guidance can be readily modeled in the zebrafish spinal cord. In this article, we describe a mounting procedure for zebrafish embryos, which optimizes visualization of these events.

Dissection und seitlicher Anbau von Zebrafisch-Embryonen: Analyse der Rückenmarksentwicklung

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene ...

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the development of neuronal connectivity. During establishment of the retino-tectal projection, optic axons extend from the eye and navigate through distinct regions of the brain to reach their target tissue, the optic tectum. Once optic axons enter the tectum, they elaborate terminal arbors that function to increase the number of synaptic connections they can make with target interneurons in the tectum. Here, we describe a method to express DNA encoding green fluorescent protein (GFP), and gain- and loss-of-function gene constructs, in optic neurons (retinal ganglion cells) in Xenopus embryos. We explain how to microinject a combined DNA/lipofection reagent into eyebuds of one day old embryos such that exogenous genes are expressed in single or small numbers of optic neurons. By tagging genes with GFP or co-injecting with a GFP plasmid, terminal axonal arbors of individual optic neurons with altered molecular signaling can be imaged directly in brains of intact, living Xenopus tadpoles several days later, and their morphology can be quantified. This protocol allows for determination of cell-autonomous molecular mechanisms that underlie the development of optic axon arborization in vivo.

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

This protocol aims to demonstrate how to microinject a DNA/DOTAP mixture into eyebuds of one day old Xenopus laevis embryos, and how to image and reconstruct individual green fluorescent protein (GFP) expressing optic axonal arbors in tectal midbrains of intact, living Xenopus tadpoles.

Mikroinjektion von DNA in Augenknospen in Xenopus laevis Embryonen und Bildgebung von GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the ...

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological processes. In particular, appropriate regulation of calcium activity patterns during embryogenesis is necessary for many aspects of vertebrate neural development, including proper neural tube closure, synaptogenesis, and neurotransmitter phenotype specification. While the observation that calcium activity patterns can differ in both frequency and amplitude suggests a compelling mechanism by which these fluxes might transmit encoded signals to downstream effectors and regulate gene expression, existing population-level approaches have lacked the precision necessary to further explore this possibility. Furthermore, these approaches limit studies of the role of cell-cell interactions by precluding the ability to assay the state of neuronal determination in the absence of cell-cell contact. Therefore, we have established an experimental workflow that pairs time-lapse calcium imaging of dissociated neuronal explants with a fluorescence in situ hybridization assay, allowing the unambiguous correlation of calcium activity pattern with molecular phenotype on a single-cell level. We were successfully able to use this approach to distinguish and characterize specific calcium activity patterns associated with differentiating neural cells and neural progenitor cells, respectively; beyond this, however, the experimental framework described in this article could be readily adapted to investigate correlations between any time-series activity profile and expression of a gene or genes of interest.

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

We present a two-part protocol that combines fluorescent calcium imaging with in situ hybridization, allowing the experimenter to correlate patterns of calcium activity with gene expression profiles on a single-cell level.

Fluoreszierende Calcium-Bildgebung und anschließende In-Situ-Hybridisierung zur neuronalen Vorläufercharakterisierung bei Xenopus laevis

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

Live-Cell-Imaging und Quantitative Analyse der embryonalen Epithelzellen in Xenopus laevis</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Biologie

Flow Cytometry and Confocal Imaging Analysis of Low Wnt Expression in Axin2-mTurquoise2 Reporter Thymocytes

Measuring Wnt expression levels is essential when trying to identify or test new Wnt therapeutic targets. Previous studies have shown that canonical Wnt signaling operates via a dosage-driven mechanism, motivating the need to study and measure Wnt signaling in various cell types. Although several reporter models have been proposed to represent physiological Wnt expression, either the genetic context or the reporter protein highly influenced the validity, accuracy, and flexibility of these tools. This paper describes methods for acquiring and analyzing data obtained with the Axin2-mTurquoise2 mouse Wnt reporter model, which contains a mutated Axin2em1Fstl allele. This model facilitates the study of endogenous canonical Wnt signaling in individual cells over a wide range of Wnt activity.
This protocol describes how to fully appreciate Axin2-mTurquoise2 reporter activity using cell population analysis of the hematopoietic system, combined with cell surface markers or &#946;-catenin intracellular staining. These procedures serve as a base for implementation and reproduction in other tissues or cells of interest. By combining fluorescence-activated cell sorting and confocal imaging, distinct canonical Wnt expression levels can be visualized. The recommended measurement and analysis strategies provide quantitative data on the fluorescent expression levels for precise assessment of canonical Wnt signaling. These methods will be useful for researchers who want to use the Axin2-mTurquise2 model for canonical Wnt expression patterns.

Signaling levels are known to regulate cell fate, indicating that regulation of Wnt signaling constitutes an interesting therapeutic target. Here, we describe flow cytometry and confocal microscopy analysis methods for a robust murine canonical Wnt signaling reporter model that measures distinct Wnt signaling levels.

Measuring Wnt expression levels is essential when trying to identify or test new Wnt therapeutic targets. Previous studies have shown that canonical Wnt ...

Mittels konfokaler Analyse

Zusammenfassung

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Mikroinjektion von DNA in Augenknospen in Xenopus laevis Embryonen und Bildgebung von GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

Fluoreszierende Calcium-Bildgebung und anschließende In-Situ-Hybridisierung zur neuronalen Vorläufercharakterisierung bei Xenopus laevis

Live-Cell-Imaging und Quantitative Analyse der embryonalen Epithelzellen in Xenopus laevis

Flow Cytometry and Confocal Imaging Analysis of Low Wnt Expression in Axin2-mTurquoise2 Reporter Thymocytes