Name: using confocal analysis of xenopus laevis to investigate modulators of wnt and shh morphogen gradients
Uploaded: 2015-12-14T13:00:00
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

Dette arbejde blev finansieret af en BBSRC indrømmer MEP (BB / H010297 / 1), en BBSRC kvote studentship til SAR, og en MRC studentship til SWF.

Etik erklæring: Dyreforsøg blev udført under en britiske indenrigsministerium licens til MEP og forsøgene udført blev godkendt af University of York etiske komité i overensstemmelse med ankommer (Animal Research: Indberetning af in vivo forsøg) retningslinjer. (https://www.nc3rs.org.uk/arrive-guidelines). 1. Strategi at generere Fluorescerende Tagged ligander Nedenfor er et eksempel protokol til subkloning Wnt8a-HA og eGFP ind pCS2, for at producere...

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En væsentlig del af denne protokol genererer biologisk aktive ligander, der normalt forarbejdes, udskilles, og i stand til at fremkalde en reaktion i den modtagende celle, trods en fluorescerende del knyttet. Det er afgørende at fastslå, at fluorescens-mærket genprodukt er biologisk aktiv anvendelse af et passende assay. For Shh-GFP, blev evnen til at aktivere ekspressionen af PTC1 bekræftet 16. Wnt8a har en bemærkelsesværdig potent evne til at fremkalde en sekundær akse, når det udtrykkes i...

I den tidlige fosterudvikling celler gradvist forpligtet til at følge specifikke slægter af differentiering: det betyder en gruppe af totipotent (eller pluripotente) celler bliver gradvist begrænset til oprettelse populationer af stamceller bestemt at give anledning til én celletype. Celle-celle signalering er central for reguleringen af ​​afstamning specifikation under fosterudviklingen. Manipulation af disse signaler vil være forpligtet til at lede stamceller mod bestemte skæbner til at støtte nye medicinske behandlinger. Et forholdsvis lille antal signalveje er gentaget under udvikling, herunder veje reagerer på TGF overfamilien (nodals og BMP&#39;er) 1-2, FGF&#39;er 3, Wnts 4, og pindsvin 5. Disse secernerede proteiner binder receptorer til stede på cellemembranen for at aktivere signaltransduktion derved ændrer genekspression og / eller celle adfærd. Den stramme regulering af celle tegnAlling er afgørende for cellelinie specifikation og normal udvikling. Mens krydstale blandt disse veje er vigtig ved bestemmelse celleskæbnen, kan en enkelt ligand selv fremkalde distinkte reaktioner ved forskellige koncentrationer. Morfogen gradienter blev beskrevet over 100 år siden som en teori til at forklare, hvordan forskellige celletyper kan stamme fra et felt af celler 6. Signalering molekyler produceres af en gruppe af celler kan diffundere over et bestemt interval, faldende koncentrationen med en større afstand fra kilden. Celler udsat for signalet vil reagere på den lokale koncentration på deres position inden for celler, med celler ved forskellige positioner reagerer forskelligt på forskellige niveauer af signalet. Beviser for eksistensen af morfogener kommer fra studier af det tidlige embryo Drosophila 7 og vingen skiven 8, samt hvirveldyr lemmer 9 og neuralrøret 10. Fremgangsmåder er nødvendige ivestigate hvordan morfogen gradienter er etableret, og at identificere andre molekyler vigtige i reguleringen af ​​disse stigninger. Elegante eksperimenter under anvendelse af immunhistokemi at visualisere endogene proteiner in vivo i forbindelse med forskellige genetiske baggrunde er blevet anvendt til at undersøge morfogen gradienter 11-12. Men gode antistoffer og specifikke mutanter er ikke altid tilgængelige, så vi beskriver her en protokol ved hjælp af overekspression af fluorescerende ligander i Xenopus, at tilvejebringe en alternativ, enkel metode til at dissekere hvordan eksogene genprodukter kan påvirke fordelingen af ​​ligander over en mark af celler. Xenopus laevis giver en fremragende system til at foretage disse typer af eksperimenter som deres embryoner udvikler eksternt, så de er tilgængelige på de tidligste stadier. Deres store størrelse (1-1.5mm i diameter) forenkler mikroinjektion og kirurgisk manipulation og blastulaen stadier cellerne er nemme at billedet som stadig er forholdsvis store (ca. 2081 m på tværs). Overekspression studier i Xenopus er enkle at gøre: mRNA indsprøjtes i den tidlige embryo kan målrettes til specifikke celler og er translateres effektivt. De fluorescerende mærkede Wnt8a / Wnt11b-HA-eGFP konstruktioner blev frembragt under anvendelse pCS2 Wnt8a-HA 13, pCS2 Wnt11b-HA 14 og eGFP. HA-peptid er vigtigt at inkludere, ikke kun for at tilvejebringe en yderligere molekylemærkning, men også fordi det menes at fungere som en spacer adskille Wnt og eGFP proteiner tillader både genprodukter til at fungere. Konstruktionen anvendes til visualisering af Shh er tidligere blevet anvendt til at danne en transgen mus som udtrykker et Shh-eGFP fusionsprotein 15; dette blev venligst stillet til rådighed af Andy McMahon. Det er vigtigt, er GFP tag for alle konstruktioner klonet 3 &#39;for signalsekvensen, således at den fastholdes efter forarbejdning. Det er også vigtigt at sikre, at den endelige protein indeholder sekvenser, der kræves for modifikationer, såsom den Ydn af lipider som det er tilfældet for Shh og Wnt ligands.The cDNA&#39;er blev subklonet ind i pCS2 + ekspressionsvektor, som er optimeret til brugsdyr af syntetisk mRNA; det indeholder en SP6-promotor og polyadenyleringssignal (http://sitemaker.umich.edu/dlturner.vectors). Den her beskrevne arbejde giver en enkel protokol til at sammenligne sekretion og diffusion af fluorescens-mærkede Wnt og Shh mærkede ligander. Ved at injicere definerede mængder af syntetisk mRNA, protokollen circumnavigates problemer forbundet med variabel ekspression fra forskellige vektorer under anvendelse af forskellige promotorer. Disse metoder er for nylig blevet anvendt til at undersøge virkningerne af den heparansulfat endosulfatase Sulf1 på Shh-eGFP og Wnt8a / Wnt11b-HA-eGFP sekretion og diffusion i Xenopus 16-17.

<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&#39;))</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>&#160;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>N/A</td>
			<td>N/A</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&#160;</td>
			<td>Scientific Laboratory Supplies</td>
			<td>22X22-SGJ3015. 22X50-SGJ3030&#160;</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&#160;</td>
			<td>Fisher Scientific</td>
			<td>BP 2819-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Nail varnish&#160;</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 (55mm)</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>N/A</td>
			<td>http://www.photoshop.com/products</td>
		</tr>
		<tr>
			<td>High fidelity DNA polymerase and buffers&#160;</td>
			<td>Biolabs</td>
			<td>M0530S&#160; 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&#160;</td>
			<td>Qiagen</td>
			<td>S28704</td>
			<td></td>
		</tr>
		<tr>
			<td>Restriction enzymes buffers</td>
			<td>Roche</td>
			<td colspan="2">SuRE/CUT Buffer Set 11082 035 001</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>N/A</td>
			<td>N/A</td>
			<td>homemade</td>
		</tr>
		<tr>
			<td>Zen lite software</td>
			<td>Carl Zeiss</td>
			<td>N/A</td>
			<td>Free download&#160; 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

Denne protokol beskriver en fremgangsmåde til at visualisere ligander fordelt på et felt af celler. Den lethed at udtrykke eksogene proteiner, sammen med den store størrelse af deres celler i tidlige embryoner, gør Xenopus laevis en nyttig model til at visualisere GFP-mærkede ligander. Syntetiske mRNA&#39;er translateres effektivt efter injektion i det tidlige stadie Xenopus embryoner og injektioner kan målrettes til en enkelt celle. Når det kombineres med en slægt sporstof såsom membran tøjret RFP, kan den injicerede celle (og dens efterkommere), der producerer det overudtrykte protein let følges. Denne protokol beskriver en fremgangsmåde til fremstilling af fluorescens mærkede Wnt og Shh-ligander fra injiceret mRNA. Metoderne involverer miCRO dissektion af ektodermale eksplantater (animalsk hætter) og analyse af ligand diffusion i flere prøver. Ved at anvende konfokal billeddannelse, kan information om ligand sekretion og diffusion over et felt af celler opnås. Statistiske analyser af konfokal billeder giver kvantitative data om formen af ​​ligand gradienter. Disse metoder kan være nyttige for forskere, der ønsker at afprøve virkningerne af faktorer, der kan regulere formen af ​​morfogen gradienter.

Konfokal analyse af animalsk cap eksplantater udtrykker fluorescens mærkede proteiner giver et effektivt system til visualisering ligand fordeling under forskellige eksperimentelle betingelser. I et eksempel fordelingen af GFP mærkede Shh er vist (figur 1). Ved 2-celle stadiet, er Xenopus embryoner injiceret i begge celler med enten med en kontrol mRNA eller med mRNA, der koder for Sulf1, et enzym, der modificerer celleoverflade heparansulfat og har indflydelse på Shh morfogenet gradient <su...

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

Manuskriptet her giver et simpelt sæt af metoder til analyse sekretion og udbredelse af fluorescens mærkede ligander i Xenopus. Dette giver en kontekst for at afprøve, om andre proteiner for at modificere ligand distribution og tillade eksperimenter, der kan give indsigt i mekanismer, der regulerer morfogen gradienter.

Brug af Konfokal Analyse af<em&gt; Xenopus laevis</em&gt; At Undersøg Modulatorer af Wnt og Shh Morfogen Gradienter

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

Research

JoVE Journal

Developmental Biology

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

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Forståelse Tidlig Organogenesis efter en forenklet<em&gt; In Situ</em&gt; Hybridization protokol<em&gt; Xenopus</em

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development.  The Xenopuslaevis embryo ...

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

Manipulation og<em&gt; In vitro</em&gt; Modning af<em&gt; Xenopus laevis</em&gt; Oocytter, efterfulgt af ICSI, for at studere fosterudviklingen

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during ...

Functional Cloning Using a Xenopus Oocyte Expression System

Functional Cloning Using a Xenopus Oocyte Expression System

Funktionel kloning ved anvendelse af en<em&gt; Xenopus</em&gt; Oocyt Expression System

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in ...

Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

Underskudsgivende og Gain-of-funktion tilgang til Undersøge Tidlig Cell Fate Determinanter i præimplantationsperioden Mouse Embryoner

 Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene ...

Technique to Target Microinjection to the Developing Xenopus Kidney

Technique to Target Microinjection to the Developing Xenopus Kidney

Teknik Target Mikroinjektion til udviklingslandene<em&gt; Xenopus</em&gt; Nyre

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 ...

Studying Wnt Signaling During Patterning of Conducting Airways

Studere Wnt Signaling Under mønster af ledende Airways

Wnt signaling pathways play critical roles during development of the respiratory tract. Defining precise mechanisms of differentiation and morphogenesis ...

Building Finite Element Models to Investigate Zebrafish Jaw Biomechanics

Opbygning Finite Element Modeller til undersøgelse Zebrafisk Jaw Biomekanik

Skeletal morphogenesis occurs through tightly regulated cell behaviors during development; many cell types alter their behavior in response to mechanical ...

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

Visualisering og kvantitativ analyse af embryonisk angiogenese i<em&gt; Xenopus tropicalis</em

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The ...

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

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

Mikroinjektion af DNA i øjen knopper i xenopus laevis embryoner og billeddannelse af Gfp udtrykker optiske Axonal Arbors i intakt, levende 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

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

Fluorescerende calciumimaging og efterfølgende In Situ Hybridisering for neuronal precursor karakterisering i 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 ...