Name: visualization and quantitative analysis of embryonic angiogenesis in xenopus tropicalis
Uploaded: 2017-05-25T14:00:00
Description: Watch this Scientific Journal Video about Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis at JoVE.com

Denne undersøgelse blev inspireret af Levine et al. , Som beskrev denne eksperimentelle metode og gav en omfattende beskrivelse af vaskulær udvikling i Xenopus laevis. Vi takker medlemmerne af vores laboratorium for deres input. Denne undersøgelse blev støttet af Yonsei Universitets Fremtidsforskningsinitiativ fra 2015 (2015-22- 0095) og Biofysisk Udviklingsprogram for National Research Foundation (NRF) finansieret af ministeriet for videnskab, IKT og fremtidig planlægning ( NRF-2013M3A9D5072551)

Alle eksperimenter overholdt protokoller godkendt af Yonsei University College of Medicine Institutionelle Dyrepleje og Brugskomitéer. 1. Fremstilling af xenopus tropicalis embryoer BEMÆRK: Xenopus tropicalis-embryoner blev fremstillet som tidligere beskrevet 10 med let modifikation. Xenopus tropicalis-embryoner blev iscenesat ifølge tabellerne i Nieuwkoop og Faber 11 . <ol><li> Induktion af ægl?...

<ol>
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Protokollen, der præsenteres her, blev først udviklet af Ali H. Brivanlou og kolleger til at undersøge udviklingshændelser under vaskulær dannelse i Xenopus laevis 4 , men som det fremgår af dette manuskript, kan det anvendes til andre små dyr. Farveinjektion i hjertet er let at udføre, og hele det vaskulære netværk kan afbildes under et fluorescensdissektionsmikroskop, såvel som et konfokalt mikroskop. Hvis farvestoffet injiceres i hjertet under skibets udvikling, kan dynamik...

Vasculogenese, dannelsen af ​​nye blodkar fra nyfødte endotelceller og angiogenese, dannelsen af ​​nye fartøjer fra eksisterende skibe, er to forskellige processer, der former embryonisk vaskulatur 1 . Enhver dysregulering i disse processer resulterer i forskellige hjertesygdomme og strukturelle abnormiteter af fartøjer. Endvidere er tumorvækst forbundet med ukontrolleret karvestigning. Som sådan er molekylære mekanismer, der ligger til grund for vaskulogenese og angiogenese, genstand for intens undersøgelse 2 . Xenopus og zebrafisk er attraktive hvirveldyrsmodeller til vaskulogenese og angiogeneseundersøgelser af flere årsager. For det første er deres embryoner små; Derfor er det relativt nemt at se hele vaskulaturen. For det andet er embryonal udvikling hurtig; Det tager kun et par dage for hele vaskulaturen at udvikle, i hvilket tidsrum den udviklende vaskul<html> Ature kan afbildes. For det tredje er genetiske og farmakologiske interventioner før og under karretdannelse nemme at udføre, såsom gennem mikroinjektion af antisense morpholino nukleotider (MOs) i det udviklende embryo eller gennem badapplikationen af ​​lægemidler 3 , 4 , 5 . Den unikke fordel ved Xenopus over zebrafisk er, at embryologiske manipulationer kan udføres, fordi Xenopus følger stereotypiske holoblastiske spaltninger, og det embryonske skæbnekort er veldefineret 6 . For eksempel er det muligt at generere et embryo, hvor kun en lateral side er genetisk manipuleret ved at indsprøjte en antisense MO til en celle i tocellefasen. Det er også muligt at transplantere hjertet primordium fra ét embryo til et andet for at bestemme, om genet udøver sin funktion ved hjælp af en celle-iboende eller -ekstruktiv mekanismeAss = &quot;xref&quot;&gt; 7. Selvom disse teknikker hovedsagelig er blevet udviklet i Xenopus laevis , som er allotetraploid og derfor ikke er ideel til genetiske undersøgelser, kan de anvendes direkte til Xenopus tropicalis , en nært beslægtet diploid art 8 . En måde at visualisere vaskulaturet på i et levende Xenopus embryo er at injicere et fluorescerende farvestof til at mærke blodkarrene. Acetyleret lavdensitetslipoprotein (AcLDL) mærket med et fluorescerende molekyle, såsom DiI, er en meget nyttig probe. I modsætning til ikke-acetyleret LDL binder AcLDL ikke til LDL-receptoren 9, men endocytteres af makrofager og endotelceller. Injektionen af ​​DiI-AcLDL i hjertet af et levende dyr resulterer i den specifikke fluorescerende mærkning af endotelceller, og hele vaskulaturen kan afbildes ved fluorescensmikroskopi i levende eller faste embryoner 4 . Her præsiderer viEnt detaljerede protokoller til visualisering og kvantificering af blodkar ved anvendelse af DiI-AcLDL i Xenopus tropicalis ( Figur 1 ). Vi giver nøgle praktiske punkter, med eksempler på succesfulde og mislykkede forsøg. Derudover giver vi en retfærdig metode til kvantitativ analyse af vaskulær kompleksitet, hvilket kan være nyttigt ved vurdering af virkningerne af genetiske og miljømæssige faktorer ved udformningen af ​​det vaskulære netværk.

<table border="0" cellpadding="0" cellspacing="0" width="539">
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	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">35mm Petri dish</td>
			<td style="width:113px;">SPL</td>
			<td style="width:93px;">10035</td>
			<td style="width:180px;">Sylgard mold frame</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">60mm Petri dish</td>
			<td style="width:113px;">SPL</td>
			<td style="width:93px;">10060</td>
			<td>Embryo raising tray</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:153px;">Borosilicate Glass</td>
			<td style="width:113px;">Sutter instrument</td>
			<td style="width:93px;">B100-50-10</td>
			<td>Needle for injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">BSA</td>
			<td style="width:113px;">Sigma</td>
			<td style="width:93px;">A3059-10G</td>
			<td>Coating reagent</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:153px;">CaCl2</td>
			<td style="width:113px;">D.S.P.GR Reagent</td>
			<td style="width:93px;"></td>
			<td>0.1X MBS component</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">Coverslip</td>
			<td style="width:113px;">Superior</td>
			<td style="width:93px;">HSU-0111520</td>
			<td>For confocal imaging</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:153px;">DiI-AcLDL</td>
			<td style="width:113px;">Thermo Fisher Scientific</td>
			<td style="width:93px;">L3484</td>
			<td>Vessel staining solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">FBS</td>
			<td style="width:113px;">Hyclone</td>
			<td style="width:93px;">SH.30919.02</td>
			<td>For storage of testis</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:153px;">Fiber Optical Illuminator</td>
			<td style="width:113px;">World Precision Instruments</td>
			<td style="width:93px;">Z-LITE-Z</td>
			<td>Light</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">Ficoll</td>
			<td style="width:113px;">Sigma</td>
			<td style="width:93px;">F4375</td>
			<td>Injection buffer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:153px;">Flaming/Brown Micropipette Puller</td>
			<td style="width:113px;">Sutter instrument</td>
			<td style="width:93px;">P-97</td>
			<td>Injection needle puller</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:153px;">Forcep</td>
			<td style="width:113px;">Fine Science Tool</td>
			<td style="width:93px;">11255-20</td>
			<td style="width:180px;">For embryo hatching and 
			needle tip cutting</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">Glass Bottom dish</td>
			<td style="width:113px;">SPL</td>
			<td style="width:93px;">100350</td>
			<td>For confocal imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">hCG</td>
			<td style="width:113px;">MNS Korea</td>
			<td style="width:93px;"></td>
			<td>For priming of frogs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">HEPES</td>
			<td style="width:113px;">Sigma</td>
			<td style="width:93px;">H3375</td>
			<td>Buffering agent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">Incubator</td>
			<td style="width:113px;">Lab. Companion</td>
			<td style="width:93px;">ILP-02</td>
			<td>For raising embryos</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">KCl</td>
			<td style="width:113px;">DAEJUNG</td>
			<td style="width:93px;">6566-4400</td>
			<td>MBS component</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">L15 medium</td>
			<td style="width:113px;">Gibco</td>
			<td style="width:93px;">11415-114</td>
			<td>For storage of testis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">L-cysteine</td>
			<td style="width:113px;">Sigma</td>
			<td style="width:93px;">168149-100G</td>
			<td>De-jellying reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">MgSO4</td>
			<td style="width:113px;">Sigma</td>
			<td style="width:93px;">M7506</td>
			<td>MBS component</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">Microtube</td>
			<td style="width:113px;">Axygen</td>
			<td style="width:93px;">MCT-175-C-S</td>
			<td>For storage of testis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">MS222</td>
			<td style="width:113px;">Sigma</td>
			<td style="width:93px;">E10521</td>
			<td>Anesthetic powder</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">NaCl</td>
			<td style="width:113px;">DAEJUNG</td>
			<td style="width:93px;">7647-14-5</td>
			<td>MBS component</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">NaOH</td>
			<td style="width:113px;">Sigma</td>
			<td style="width:93px;">S-0899</td>
			<td>pH adjusting reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">Paraformaldehyde</td>
			<td style="width:113px;">Sigma</td>
			<td style="width:93px;">P6148</td>
			<td>Fixatives</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">PBS</td>
			<td style="width:113px;">BIOSESANG</td>
			<td style="width:93px;">P2007</td>
			<td>Buffer for imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">pH paper</td>
			<td style="width:113px;">Sigma</td>
			<td style="width:93px;">P4536-100EA</td>
			<td>For confirming pH</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:153px;">PICO-LITER INJECTOR</td>
			<td style="width:113px;">Waner instruments</td>
			<td style="width:93px;">PLI-100A</td>
			<td>For injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">Pin</td>
			<td style="width:113px;">Pinservice</td>
			<td style="width:93px;">26002-10</td>
			<td>For incision</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">Pinholder</td>
			<td style="width:113px;">Scitech Korea</td>
			<td style="width:93px;">26016-12</td>
			<td>For incision</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:153px;">Precision Stereo Zoom Binocular Microscope</td>
			<td style="width:113px;">World Precision Instruments</td>
			<td style="width:93px;">PZMIII</td>
			<td>For visual screening</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:153px;">Standard Manual Control Micromanipulator&#160;</td>
			<td style="width:113px;">Waner instruments</td>
			<td style="width:93px;">W4 64-0056</td>
			<td>For microinjection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">SYLGARD 184 Kit</td>
			<td style="width:113px;">Dow Corning</td>
			<td style="width:93px;"></td>
			<td>For DiI injection</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:153px;">Transfer pipette</td>
			<td style="width:113px;">Korea Ace Scientific Co.</td>
			<td style="width:93px;">YM.B78-400</td>
			<td>For eggs and 
			embryo collection</td>
		</tr>
	</tbody>
</table>

visualization and quantitative analysis of embryonic angiogenesis in xenopus tropicalis

Blodkar leverer ilt og næringsstoffer i hele kroppen, og dannelsen af ​​det vaskulære netværk er under tæt udviklingskontrol. Den effektive in vivo visualisering af blodkar og den pålidelige kvantificering af deres kompleksitet er nøglen til forståelsen af ​​biologi og sygdom i det vaskulære netværk. Her giver vi en detaljeret metode til visualisering af blodkar med et kommercielt tilgængeligt fluorescerende farvestof, humanplasmaacetyleret lavdensitetslipoprotein DiI-kompleks (DiI-AcLDL) og kvantificering af deres kompleksitet i Xenopus tropicalis . Blodkar kan mærkes ved en simpel injektion af DiI-AcLDL ind i et embryos slagende hjerte, og blodkar i hele embryoet kan afbildes i levende eller faste embryoner. Kombineret med genforstyrrelse ved målrettet mikroinjektion af nukleinsyrer og / eller badapplikationen af ​​farmakologiske reagenser kan et gens eller en signalvejsstats rolle på vaskulær udvikling være invektetSteget inden for en uge uden at ty til sofistikerede genetisk manipulerede dyr. På grund af det veldefinerede venesystem af Xenopus og dets stereotype angiogenese kan spiring af allerede eksisterende skibe, fartøjskompleksitet kvantificeres effektivt efter perturbationsforsøg. Denne relativt enkle protokol skal fungere som et let tilgængeligt værktøj inden for forskellige områder inden for kardiovaskulær forskning.

Tidslinje for eksperimenter (figur 1 og 2) Kort efter befrugtning kan målrettet mikroinjektion udføres for at modulere genekspression. For eksempel kan en antisense MO, der specifikt binder til initieringskodonet af det endogene Tie2-mRNA, injiceres, hæmme oversættelsen af ​​Tie2-målmRNA ved sterisk hindring. En MO kan konjugeres til fluorescein til den nemme visuelle screening af vellykkede injicerede embryoner. Alterna...

Watch this Scientific Journal Video about Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis at JoVE.com

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

Denne protokol demonstrerer en fluorescensbaseret metode til visualisering af vaskulaturen og kvantificering af dets kompleksitet i Xenopus tropicalis . Blodkar kan billedbehandles minutter efter injektion af et fluorescerende farvestof i et embryos slaghalve efter genetisk og / eller farmakologisk manipulation for at studere kardiovaskulær udvikling in vivo .

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 ...

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JoVE Journal

Developmental Biology

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

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 ...

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

Analyse af Cardiomyocyte Development hjælp Immunofluorescens i embryonale Mouse Hjerte

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated ...

Visualization of Chondrocyte Intercalation and Directional Proliferation via Zebrabow Clonal Cell Analysis in the Embryonic Meckel&#8217;s Cartilage

Visualization of Chondrocyte Intercalation and Directional Proliferation via Zebrabow Clonal Cell Analysis in the Embryonic MeckelÃƒÂ¢Ã¢â€šÂ¬Ã¢â€žÂ¢s Cartilage

Visualisering af Chondrocyt Intercalation og Directional spredning via Zebrabow Klonal Cell Analysis i Embryonale Meckel s Brusk

Development of the vertebrate craniofacial structures requires precise coordination of cell migration, proliferation, adhesion and differentiation. ...

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

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

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 ...

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

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

Stamcelle-lignende<em&gt; Xenopus</em&gt; Embryonale Eksplantater at studere Tidlig Neural Udviklingsmæssige Egenskaber<em&gt; In vitro</em&gt; Og<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, ...

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Transkriptom profilering<em&gt; In-vivo</em&gt; Produceret Bovine præimplantationsembryoner Brug To farve Microarray Platform

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo ...

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 ...

Analysis of Coronary Vessels in Cleared Embryonic Hearts

Analyse af koronarkar i Cleared Embryonale hjerter

Whole mount visualization of the embryonic coronary plexus from which the capillary and arterial networks will form is rendered problematic using standard ...

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

Lysmedieret reversibel modulering af den mitogenaktiverede proteinkinasevej under celledifferentiering og<em&gt; Xenopus</em&gt; Embryonudvikling

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early ...

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

Kvantitative hele-mount immunofluorescens analyse af Cardiac stamfader populationer i mus embryoner

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...