Name: following endocardial tissue movements via cell photoconversion in the zebrafish embryo
Uploaded: 2018-02-20T14:00:00
Description: Watch this Scientific Journal Video about Following Endocardial Tissue Movements via Cell Photoconversion in the Zebrafish Embryo at JoVE.com

Vi vil gjerne takke Anne-Laure Duchemin, Denise Duchemin, Nathalie Faggianelli og Basile Gurchenkov for å utforme og lage mold beskrevet i denne protokollen. Dette arbeidet ble støttet av FRM (DEQ20140329553), ANR (ANR-15-CE13-0015-01, ANR-12-ISV2-0001-01), EMBO unge etterforsker Program, EU, ERC CoG N ° 682938 Evalve og av stipendet ANR-10-LABX-0030-INRT, en fransk Statens fondet forvaltes av Agence Nationale de la Recherche under ramme programmet Investissements d'Avenir merket ANR-10-IDEX-0002-02.

1. klargjøre former og montering agarose
<ol><li>Opprette en mold med dimensjoner som vist i figur 1 en 3D-skriver eller tradisjonell mekanisk bedrift verktøy.</li>
	<li>Pipetter ca 1,5 mL av smeltet 1% agarose i en 35 mm plast montering rett. Sett plast mold i de flytende agarose, ta vare for å unngå fangst luft mellom mold og agarose. Plasser rett ved 4 ° C, vente til agarose beskytter (dette tar ca 5 min), og deretter fjerner plast mold.</li>
	<li>For å lagre montering parabolen f...

<ol>
	<li>Chow, R. W., Vermot, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rise+of+photoresponsive+protein+technologies+applications+in+vivo:+a+spotlight+on+zebrafish+developmental+and+cell+biology.">The rise of photoresponsive protein technologies applications in vivo: a spotlight on zebrafish developmental and cell biology.</a> F1000Res. 6, (2017).</li><li>Sehnert, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+troponin+T+is+essential+in+sarcomere+assembly+and+cardiac+contractility.">Cardiac troponin T is essential in sarcomere assembly and cardiac contractility.</a> Nat Genet. 31, 106-110 (2002).</li><li>Peal, D. S., Lynch, S. N., Milan, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterning+and+development+of+the+atrioventricular+canal+in+zebrafish.">Patterning and development of the atrioventricular canal in zebrafish.</a> J Cardiovasc Transl Res. 4, 720-726 (2011).</li><li>Boselli, F., Freund, J. B., Vermot, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+flow+mechanics+in+cardiovascular+development.">Blood flow mechanics in cardiovascular development.</a> Cell Mol Life Sci. 72 (13), 2545-2559 (2015).</li><li>Steed, E., Boselli, F., Vermot, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamics+driven+cardiac+valve+morphogenesis.">Hemodynamics driven cardiac valve morphogenesis.</a> Biochim Biophys Acta. , (2015).</li><li>Boselli, F., Vermot, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+and+modeling+for+shear+stress+quantification+in+the+embryonic+zebrafish+heart.">Live imaging and modeling for shear stress quantification in the embryonic zebrafish heart.</a> Methods. , (2015).</li><li>Steed, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=klf2a+couples+mechanotransduction+and+zebrafish+valve+morphogenesis+through+fibronectin+synthesis.">klf2a couples mechanotransduction and zebrafish valve morphogenesis through fibronectin synthesis.</a> Nat Commun. 7, 11646 (2016).</li><li>Herwig, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+cellular+mechanisms+of+blood+vessel+fusion+in+the+zebrafish+embryo.">Distinct cellular mechanisms of blood vessel fusion in the zebrafish embryo.</a> Curr Biol. 21 (22), 1942-1948 (2011).</li><li>Boselli, F., Steed, E., Freund, J., Vermot, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anisotropic+shear+stress+patterns+predict+the+orientation+of+convergent+tissue+movements+in+the+embryonic+heart.">Anisotropic shear stress patterns predict the orientation of convergent tissue movements in the embryonic heart.</a> Development. , (2017).</li><li>Lombardo, V. A., Sporbert, A., Abdelilah-Seyfried, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Tracking+Using+Photoconvertible+Proteins+During+Zebrafish+Development.">Cell Tracking Using Photoconvertible Proteins During Zebrafish Development.</a> J. Vis. Exp. (67), (2012).</li><li>JoVE Science Education Database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biology II: Mouse, Zebrafish, and Chick. Zebrafish Breeding and Embryo Handling. , (2017).</li><li>Watanabe, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibitory+effect+of+2,3-butanedione+monoxime+(BDM)+on+Na(+)/Ca(2+)+exchange+current+in+guinea-pig+cardiac+ventricular+myocytes.">Inhibitory effect of 2,3-butanedione monoxime (BDM) on Na(+)/Ca(2+) exchange current in guinea-pig cardiac ventricular myocytes.</a> Br J Pharmacol. 132 (6), 1317-1325 (2001).</li><li>Jou, C. J., Spitzer, K. W., Tristani-Firouzi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blebbistatin+effectively+uncouples+the+excitation-contraction+process+in+zebrafish+embryonic+heart.">Blebbistatin effectively uncouples the excitation-contraction process in zebrafish embryonic heart.</a> Cell Physiol Biochem. 25 (4-5), 419-424 (2010).</li><li>Pestel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+3D+visualization+of+cellular+rearrangements+during+cardiac+valve+formation.">Real-time 3D visualization of cellular rearrangements during cardiac valve formation.</a> Development. 143 (12), 2217-2227 (2016).</li><li>Swinburne, I. A., Mosaliganti, K. R., Green, A. A., Megason, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+Long-Term+Imaging+of+Embryos+with+Genetically+Encoded+alpha-Bungarotoxin.">Improved Long-Term Imaging of Embryos with Genetically Encoded alpha-Bungarotoxin.</a> PLoS One. 10 (8), e0134005 (2015).</li><li>Kaufmann, A., Mickoleit, M., Weber, M., Huisken, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multilayer+mounting+enables+long-term+imaging+of+zebrafish+development+in+a+light+sheet+microscope.">Multilayer mounting enables long-term imaging of zebrafish development in a light sheet microscope.</a> Development. 139 (17), 3242-3247 (2012).</li><li>Marchant, J. S., Stutzmann, G. E., Leissring, M. A., LaFerla, F. M., Parker, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiphoton-evoked+color+change+of+DsRed+as+an+optical+highlighter+for+cellular+and+subcellular+labeling.">Multiphoton-evoked color change of DsRed as an optical highlighter for cellular and subcellular labeling.</a> Nat Biotechnol. 19 (7), 645-649 (2001).</li><li>Dempsey, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+single-cell+labeling+by+confined+primed+conversion.">In vivo single-cell labeling by confined primed conversion.</a> Nat Methods. 12 (7), 645-648 (2015).</li><li>Mohr, M. A., Pantazis, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+neuron+morphology+in+vivo+with+confined+primed+conversion.">Single neuron morphology in vivo with confined primed conversion.</a> Methods Cell Biol. 133, 125-138 (2016).</li><li>Mohr, M. A., Argast, P., Pantazis, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Labeling+cellular+structures+in+vivo+using+confined+primed+conversion+of+photoconvertible+fluorescent+proteins.">Labeling cellular structures in vivo using confined primed conversion of photoconvertible fluorescent proteins.</a> Nat Protoc. 11 (12), 2419-2431 (2016).</li><li>Mohr, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+Engineering+of+Photoconvertible+Fluorescent+Proteins+for+Dual-Color+Fluorescence+Nanoscopy+Enabled+by+a+Triplet-State+Mechanism+of+Primed+Conversion.">Rational Engineering of Photoconvertible Fluorescent Proteins for Dual-Color Fluorescence Nanoscopy Enabled by a Triplet-State Mechanism of Primed Conversion.</a> Angew Chem Int Ed Engl. 56 (38), 11628-11633 (2017).</li><li>Dempsey, W. P., Fraser, S. E., Pantazis, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PhOTO+zebrafish:+a+transgenic+resource+for+in+vivo+lineage+tracing+during+development+and+regeneration.">PhOTO zebrafish: a transgenic resource for in vivo lineage tracing during development and regeneration.</a> PLoS One. 7 (3), e32888 (2012).</li><li>Dempsey, W. P., Qin, H., Pantazis, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+cell+tracking+using+PhOTO+zebrafish.">In vivo cell tracking using PhOTO zebrafish.</a> Methods Mol Biol. 1148, 217-228 (2014).</li></ol>

Timing av photoconversion: selv om Kaede er fortsatt sterkt uttrykkes i EdCs på 96 hpf, bør det bemerkes at som fosteret vokser, laserlys diffunderer mer før den når AVC, gjør begrenset photoconversion av Kaede vanskeligere. At embryonale stadier senere enn 55 hpf, ballooning av ventrikkel og atrium også betyr at fiolett laserstrålen som brukes for photoconversion ikke kan nå AVC celler uten første går gjennom enten atrium eller ventrikkel. Dette betyr at utover 55 hpf, for photoconvert EdCs av AVC, en...

Sebrafisk er en av de viktigste virveldyr modellene å studere mobilnettet og utviklingsmessige prosesser i vivo. Dette skyldes den sebrafisk optisk åpenhet og amenability til genetikk, som gjør det en effektiv modell for bruk optisk teknikker med genetisk kodet photoresponsive protein teknologier1. Spesielt til studiet av heart utvikling, sebrafisk få tilstrekkelig oksygen via diffusjon slik at selv mutanter uten hjerterytme kan overleve gjennom den første uken i utvikling, tillater analyser på virkningene av utviklingsmessige gener og plaget blodstrøm på hjertet morphogenesis ikke mulig i de fleste virveldyr2.
Sebrafisk hjertet røret er dannet av 24 timer innlegget befruktning (hpf). Kort tid etter dannelsen begynner hjertet røret aktivt slo. Med 36 hpf, en klar innsnevring skiller atrium fra ventrikkel. Dette området av innsnevring kalles atrioventrikulær kanalen (AVC), og celler i denne regionen endringen fra en squamous morfologi til kubisk morfologi3. Sebrafisk atrioventrikulær ventil morphogenesis starter rundt 48 h legge befruktning. 5 dager post befruktning, to ventil brosjyrer utvide til AVC og forhindre tilbake flyten av blod fra ventrikkel til atriet under cardiac syklus4. Spore celler under AVC og ventil formasjon er utfordrende rask juling av hjertet gjør det vanskelig å følge celler via tradisjonelle time-lapse mikroskopi5,6. Denne protokollen, tilpasset fra Steed et al., 20167, beskriver en metode som bruker vs (fli1a:gal4FFubs, UAS:kaede)8 sebrafisk transgene linjen, som photoconvertible protein Kaede uttrykkes i endotelceller, inkludert endocardium. Den narkotika 2,3-butanedione-2-monoxime (BDM) brukes til å midlertidig stoppe hjertet slo, slik at nøyaktig photoconversion av EdCs mellom 36 og 55 hpf og høy oppløsning avbildning av photoconverted EdCs på bestemte utviklingsmessige tidspunkt. Det har tidligere vært vist at EdCs photoconverted på denne måten kan forbli skilles fra sine uomvendte naboer i fem dager eller mer etter photoconversion7. Denne protokollen også detaljer en metode som brukes for bildeanalyse av photoconverted EdCs i embryoer yngre enn 48 hpf, som har blitt brukt til å følge vev bevegelser under AVC utvikling (Boselli et al., under trykking)9. Vi håper at leserne vil finne denne protokollen nyttig for å studere AVC og ventil utvikling i normal embryoer og mutanter, morphants eller narkotika-behandlet embryoer. For en mer generell protokoll knyttet til celle sporing ved hjelp av photoconvertible proteiner under sebrafisk utvikling, se artikkelen av Lombardo et al., 201210.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Necessary equipment for raising fish and collecting eggs (see the Zebrafish Book22 for details).</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Upright confocal microscope (Equipped with lasers operating at 488 nm, 561 nm, and 405 nm, a tunable multiphoton laser, and a Leica HCX IRAPO L, 25 &#215;, N.A. 0.95 objective)</td>
			<td>Leica</td>
			<td>Leica SP8</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat block</td>
			<td>ThermoScientific</td>
			<td>88870001</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm x 10 mm tissue culture dish</td>
			<td>Falcon</td>
			<td>353001</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well plate</td>
			<td>Falcon</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass pipette</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mold</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>8 mg/mL Tricaine stock solution&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 M BDM stock solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure low melting point agarose</td>
			<td>Invitrogen</td>
			<td>16520-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Lonza</td>
			<td>50004</td>
			<td></td>
		</tr>
		<tr>
			<td>PTU (1-phenyl-2-thiourea)</td>
			<td>Sigma Aldrich</td>
			<td>P7629</td>
			<td></td>
		</tr>
		<tr>
			<td>Embryo medium: 30x stock solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Matlab equipped with the Image Analysis, Curve Fitting, Bio-Formats Toolboxes</td>
			<td>MathWorks</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>8 mg/mL Tricaine stock solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>200 mg of tricaine powder (Ethyl 3-aminobenzoate methanesulfonic acid)</td>
			<td>Sigma-Aldrich</td>
			<td>E10521</td>
			<td></td>
		</tr>
		<tr>
			<td>25 mL Danieau</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adjust to pH 7, aliquot and store at -20 &#176;C&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 M BDM stock solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 g BDM</td>
			<td>Sigma-Aldrich</td>
			<td>112135</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL ddH2O</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aliquot and store at -20 &#176;C</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Embryo medium: 30x stock solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50.7 g NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr>
			<td>0.78 g KCl</td>
			<td>Sigma-Aldrich</td>
			<td>7447-40-7</td>
			<td></td>
		</tr>
		<tr>
			<td>1.47 g Magnesium sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>&#160;7487-88-9</td>
			<td></td>
		</tr>
		<tr>
			<td>2.1 g Calcium nitrite tetrahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>13477-34-4</td>
			<td></td>
		</tr>
		<tr>
			<td>19.52 g HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)</td>
			<td>Sigma-Aldrich</td>
			<td>7365-45-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Adjust to pH 7.2 and store at room temperature</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mold</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PlasCLEAR resin</td>
			<td>Asiga</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plus 39&#160;Freeform Pico 3D printer</td>
			<td>Asiga</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

following endocardial tissue movements via cell photoconversion in the zebrafish embryo

Under embryogenesis, celler gjennomgå dynamiske endringer i celle atferd og tyde mobilnettet logikken bak disse endringene er et grunnleggende mål innen utviklingsbiologi. Oppdagelsen og utvikling av photoconvertible proteiner har sterkt hjulpet vår forståelse av disse dynamiske endringer ved å tilby en metode for å optisk markere celler og vev. Men mens photoconversion, time-lapse mikroskopi og påfølgende bildeanalyser har vist seg for å være svært vellykket i å avdekke mobilnettet dynamikk i organer som hjernen eller øyet, brukes denne tilnærmingen ikke vanligvis i utviklingsland hjertet grunn utfordringer som rask bevegelse av hjertet under cardiac syklus. Denne protokollen består av to deler. Første del beskriver en metode for photoconverting og senere sporing endocardial celler (EdCs) under sebrafisk atrioventrikulær kanalen (AVC) og atrioventrikulær hjertet ventil utvikling. Metoden innebærer timelig stoppe hjertet med et medikament for at nøyaktig photoconversion finne sted. Hjerter er tillatt å gjenoppta juling på fjerning av stoffet, og embryonale utvikling fortsetter som normalt til hjertet stoppes igjen ved høy oppløsning avbildning av photoconverted EdCs på en senere utviklingsmessige tidspunkt. Den andre delen av protokollen beskriver en bildet analysemetode for å kvantifisere lengden på et photoconverted eller ikke-photoconverted område i AVC i unge embryoer ved å tilordne fluorescerende signalet fra tredimensjonale strukturen til et todimensjonalt kart . Sammen de to delene av protokollen tillater en å undersøke opprinnelsen og oppførsel av celler som utgjør sebrafisk AVC og atrioventrikulær hjerteklaffen, og kan potensielt bli brukt for å studere mutanter, morphants eller embryo som har blitt behandlet med reagenser som forstyrrer AVC og/eller ventil utvikling.

Et eksempel på et embryo photoconverted på 48 hpf og fotografert igjen på 80 hpf er vist i figur 2, filmer 1 og 2. Utsette Kaede 405 nm lys irreversibelt konverterer fra protein fra sin fluorescerende grønne form til fluoriserende rødt form, aktivere virkemåten til celler merket med grønn eller rød skjemaet skal følges med hensyn til deres ulikt fargede naboer i ventil formasjon. Det kan sees at de photoconverted cel...

Watch this Scientific Journal Video about Following Endocardial Tissue Movements via Cell Photoconversion in the Zebrafish Embryo at JoVE.com

Following Endocardial Tissue Movements via Cell Photoconversion in the Zebrafish Embryo

Denne protokollen beskriver en metode for photoconversion av Kaede fluorescerende proteinet i endocardial cellene levende sebrafisk embryo som muliggjør sporing av endocardial celler under atrioventrikulær kanalen og atrioventrikulær hjertet ventil utvikling .

Følgende Endocardial vev bevegelser via celle Photoconversion i sebrafisk Embryo

During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the ...

The overall goal of this procedure is to follow endocardial cells during atrioventricular canal and heart valve formation. This method can help to address key question in the field of cardiac development, such as to understand how endocardial cells behave in different types of mutants. Tracking cells during AVC and valve formation is challenging because of the rapid beat of the heart.It makes it difficult to follow cells via traditional time-lapse microscopy. The main advantage of this technique is that it bypasses some of these difficulties. Demonstrating the procedure will be Renee, a postdoc in my lab.After creating a mold according to the text protocol, pipette about 1.5 milliliters of melted 1%agarose into a 35-millimeter plastic mounting dish. Then, position the plastic mold in the liquid agarose, taking care to avoid trapping air between the mold and the agarose. Place the dish at four degrees Celsius until the agarose hardens.Then, remove the plastic mold from the agarose. Next, add two milliliters of previously prepared mounting medium to the mounting dish, and allow 15 to 30 minutes for the solution to diffuse into the agarose. In the meantime, melt a one-milliliter aliquot of 0.7%low melting point, or LMP, agarose by placing the tube in a 70-degree Celsius heat block for about five minutes.Pipette the LMP agarose up and down to cool it slightly. Then, add 25 microliters of eight milligram per milliliter tricaine and 40 microliters of one molar BDM solutions to the tube. Mix by pipetting up and down.Then, transfer the tube to a 38-degree Celsius heat block to keep the LMP agarose in its liquid state. Transfer the embryos to a separate dish containing two milliliters of mounting medium. Then, when the hearts have stopped beating, transfer the embryos to the mounting dish and use forceps to arrange them in the wells so that they lie at a 25-degree angle, tails pointing toward the deeper part of the well, with the yolk facing up.When the embryos are roughly in place, remove the medium, and embed them in approximately 200 microliters of 0.7%LMP agarose containing tricaine and BDM. Then, readjust the embryo positions, tilting them to the left or right as necessary. Press down on the walls of the indented square to allow excess agarose to overflow to the side areas of the mounting dish.Wait approximately five minutes for the LMP agarose to set, then add mounting medium to the dish. The embryos are now ready to be photoconverted. To carry out photoconversion, on an upright confocal microscope equipped with a 405, 488, and 561-nanometer laser sources, use transmitted white light and an objective lens to locate the embryo's heart.Using the 561-nanometer laser, check for red Kaede fluorescence. Then, use the 488-nanometer laser to visualize the endocardium. Next, on the microscope acquisition software, enter FRAP mode.Then, select approximately 1%laser power for both the 488 and the 561-nanometer lasers. While focusing on the plane of interest, select a region of interest, or ROI. Then, using the 405-nanometer diode laser at 25%laser power, photoconvert the cells, scanning over the ROI three times.Should insufficient Kaede be photoconverted in the ROI, scan the 405-nanometer laser over the area three times again. Return to the TCS SP8 mode on the software, and using the 561 and 488-nanometer lasers sequentially, in Bidirectional mode, acquire a Z-stack. After photoconversion, use a glass pipette to press down slightly just above the head of the embryo to break the LMP agarose.Then, gently suck up the embryo. To wash away the BDM containing medium, eject the embryo from the glass pipette into an 85-millimeter Petri dish containing embryo medium with PTU. Transfer the embryo to a well in a six-well plate containing embryo medium with PTU.During this step, make sure to keep note of which embryo goes into which well, as this is essential to correlate the position of photoconverted cells at later developmental stages. Now that the embryos have been removed from the BDM containing medium, their hearts should start beating again in about five minutes. Return embryos to the dark at 28.5 degrees Celsius to allow them to continue to develop normally.At the desired stage, stop the heart and re-embed the embryos, as demonstrated earlier in this video, with the important difference that the embryos must be treated with BDM in separate, marked dishes to keep track of them. For embryos up to 62 hpf, acquire Z-stacks of the endocardium using 561 and 488 nanometers for red and green fluorescent signals, respectively. For embryos older than 62 hpf, use a multiphoton laser set to 940 nanometers to image green Kaede instead of the 488-nanometer laser.For embryos younger than 48 hpf, it is possible to project the three-dimensional AVC onto a two-dimensional image for analysis. First, threshold the image. Then, erase points that lie outside of the heart.To perform manual segmentation, orient the image on the left to match the image on the right. Segment the side of the heart in which the orange vector passes through, and add points in the direction of the blue vector. Next, interpolate the cross-sections, and project the color intensity onto the surface.Finally, adjust the orientation of the AVC, and project it onto a 2D surface. An example of an embryonic heart just after photoconversion at 48 hpf is shown here. Cytoplasmic Kaede in several cells of the superior side of the AVC has been converted from green to red.In the same embryo at 80 hpf, it's evident that the photoconverted cells have proliferated and have migrated to the inner side of the superior atrioventricular valve. An example of a 36 hpf embryo where the EdCs in the atrium and ventricle have been photoconverted is shown here. The AVC is then projected into two dimensions for easier visualization and analysis and allows one to quantify the distance between the two photoconverted regions.While attempting this procedure, it's important to keep the time the heart is stopped to a minimum to avoid affecting normal development. With practice, photoconversion can be completed within about 30 minutes provided that everything is prepared in advance. After watching this video, you should have a good understanding of how to track endocardial cells after photoconversion in live zebrafish embryo and analyze your data sets.

Research

JoVE Journal

Developmental Biology

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion (FDAP)

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion FDAP

Måling Protein Stabilitet på Living Sebrafisk embryo med fluoresens Decay Etter Photoconversion (FDAP)

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological ...

Microbead Implantation in the Zebrafish Embryo

Mikro Implantasjon i sebrafisk Embryo

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic ...

Imaging Subcellular Structures in the Living Zebrafish Embryo

Imaging subcellulære strukturer i Living Sebrafisk Embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

Observing Mitotic Division and Dynamics in a Live Zebrafish Embryo

Observerer Mitotisk divisjon og Dynamics i en Live Sebrafisk Embryo

Mitosis is critical for organismal growth and differentiation. The process is highly dynamic and requires ordered events to accomplish proper chromatin ...

Visualizing the Interrenal Steroidogenic Tissue and Its Vascular Microenvironment in Zebrafish

Visualisere Interrenal Steroidogenic Tissue og dens Vaskulær mikromiljøet i Sebrafisk

This protocol introduces how to detect differentiated interrenal steroidogenic cells through a simple whole-mount enzymatic activity assay. Identifying ...

Use of Immunolabeling to Analyze Stable, Dynamic, and Nascent Microtubules in the Zebrafish Embryo

Bruk av Immunolabeling å analysere stabil, dynamisk og gryende piskehale som henger i sebrafisk Embryo

Microtubules (MTs) are dynamic and fragile structures that are challenging to image in vivo, particularly in vertebrate embryos. Immunolabeling methods ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

AFM and Microrheology in the Zebrafish Embryo Yolk Cell

AFM og Microrheology i sebrafisk Embryo eggeplomme cellen

Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. ...

Single-cell Photoconversion in Living Intact Zebrafish

Encellede Photoconversion i levende intakt sebrafisk

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Microinjection-based System for In Vivo Implantation of Embryonic Cardiomyocytes in the Avian Embryo

Microinjection-basert System for i Vivo implantering av embryonale Cardiomyocytes i Avian Embryo

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a ...