Name: probing the roles of physical forces in early chick embryonic morphogenesis
Uploaded: 2018-06-05T13:00:00
Description: Watch this Scientific Journal Video about Probing the Roles of Physical Forces in Early Chick Embryonic Morphogenesis at JoVE.com

Z.C. anerkender støtte fra Dartmouth start fond og Branco Weiss - Society for videnskab fellowship, administreret af ETH Zürich. Forfatterne takke Drs. Larry A. Taber, Benjamen A. Filas, Qiaohang Guo, og Yunfei Shi for nyttige diskussioner, såvel som de anonyme korrekturlæsere for kommentarer. Dette materiale er baseret på arbejde støttes af de National Science Foundation Graduate Research Fellowship under Grant nr. DGE-1313911. Enhver udtalelse, konstateringer og konklusioner og henstillinger udtrykt i dette materiale er dem af forfatterne (s) og afspejler ikke nødvendigvis synspunkter af National Science Foundation.

1. forberedelse af vævskultur medier
<ol><li>Bruge en 0,5 L flaske af Dulbeccos modificerede Eagle Medium (DMEM) med 4,5 g/L glukose, natriumhydrogencarbonat og L-glutamin som base for kultur medier.</li>
	<li>I en steril laminar flow hætte, tilsættes 10 mL af antibiotika til DMEM 0,5 L.</li>
	<li>Ved hjælp af en steril pipette overføres 50 mL af DMEM antibiotika til en steril 50 mL konisk slange.</li>
	<li>Der tilsættes 50 mL af chick serum til den resterende DMEM antibiotika løsning i 0,5 L flaske i sterile h?...

<ol>
	<li>Taber, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanics+of+Growth,+Remodeling,+and+Morphogenesis.">Biomechanics of Growth, Remodeling, and Morphogenesis.</a> Appl. Mech. Rev. 48, 487-545 (1995).</li><li>Wyczalkowski, M. A., Chen, Z., Filas, B. A., Varner, V. D., Taber, L. 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Eng. 136, 1-15 (2014).</li><li>Shi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bending+and+twisting+the+embryonic+heart:+A+computational+model+for+c-looping+based+on+realistic+geometry.">Bending and twisting the embryonic heart: A computational model for c-looping based on realistic geometry.</a> Front. Physiol. 5, (2014).</li><li>Taber, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphomechanics:+Transforming+tubes+into+organs.">Morphomechanics: Transforming tubes into organs.</a> Current Opinion in Genetics and Development. 27, 7-13 (2014).</li><li>Hosseini, H. S., Beebe, D. C., Taber, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+effects+of+the+surface+ectoderm+on+optic+vesicle+morphogenesis+in+the+chick+embryo.">Mechanical effects of the surface ectoderm on optic vesicle morphogenesis in the chick embryo.</a> J. Biomech. 47, 3837-3846 (2014).</li><li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chick+embryo.">A series of normal stages in the development of the chick embryo.</a> Dev. Dyn. 88, 49-92 (1951).</li><li>Shi, Y., Varner, V. D., Taber, L. a. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+is+cytoskeletal+contraction+required+for+cardiac+fusion+before+but+not+after+looping+begins?.">Why is cytoskeletal contraction required for cardiac fusion before but not after looping begins?.</a> Phys. Biol. 12, 16012 (2015).</li><li>Garcia, K. E., Okamoto, R. J., Bayly, P. V., Taber, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contraction+and+stress-dependent+growth+shape+the+forebrain+of+the+early+chicken+embryo.">Contraction and stress-dependent growth shape the forebrain of the early chicken embryo.</a> J. Mech. Behav. Biomed. Mater. 65, 383-397 (2017).</li><li>Faisst, A. M., Alvarez-Bolado, G., Treichel, D., Gruss, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rotatin+is+a+novel+gene+required+for+axial+rotation+and+left-right+specification+in+mouse+embryos.">Rotatin is a novel gene required for axial rotation and left-right specification in mouse embryos.</a> Mech. Dev. 113, 15-28 (2002).</li><li>Yalcin, H. C., Shekhar, A., Rane, A. a., Butcher, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ex-ovo+chicken+embryo+culture+system+suitable+for+imaging+and+microsurgery+applications.">An ex-ovo chicken embryo culture system suitable for imaging and microsurgery applications.</a> J. Vis. Exp. (44), (2010).</li><li>Chapman, S. C., Collignon, J., Schoenwolf, G. C., Lumsden, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+method+for+chick+whole-embryo+culture+using+a+filter+paper+carrier.">Improved method for chick whole-embryo culture using a filter paper carrier.</a> Dev. Dyn. 220, 284-289 (2001).</li><li>Camp&#224;s, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+cell-generated+mechanical+forces+within+living+embryonic+tissues.">Quantifying cell-generated mechanical forces within living embryonic tissues.</a> Nat. Methods. 11, 183-189 (2014).</li><li>Schierenberg, E., Junkersdorf, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+eggshell+and+underlying+vitelline+membrane+for+normal+pattern+formation+in+the+early+C.+elegans+embryo.">The role of eggshell and underlying vitelline membrane for normal pattern formation in the early C. elegans embryo.</a> Roux's Arch. Dev. Biol. 202, 10-16 (1992).</li><li>Chuai, M., Weijer, C. 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+Mechanisms+Underlying+Primitive+Streak+Formation+in+the+Chick+Embryo.">The Mechanisms Underlying Primitive Streak Formation in the Chick Embryo.</a> Current Topics in Developmental Biology. 81, 135-156 (2008).</li><li>Voronov, D. A., Alford, P. W., Xu, G., Taber, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+mechanical+forces+in+dextral+rotation+during+cardiac+looping+in+the+chick+embryo.">The role of mechanical forces in dextral rotation during cardiac looping in the chick embryo.</a> Dev. Biol. 272, 339-350 (2004).</li><li>Raya, A., Izpisua Belmonte, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unveiling+the+establishment+of+left-right+asymmetry+in+the+chick+embryo.">Unveiling the establishment of left-right asymmetry in the chick embryo.</a> Mechanisms of Development. 121, 1043-1054 (2004).</li><li>Voronov, D. A., Taber, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+looping+in+experimental+conditions:+Effects+of+extraembryonic+forces.">Cardiac looping in experimental conditions: Effects of extraembryonic forces.</a> Dev. Dyn. 224, 413-421 (2002).</li><li>Chen, Z., Huang, G., Trase, I., Han, X., Mei, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+Self-Assembly+of+a+Strain-Engineered+Flexible+Layer:+Wrinkling,+Rolling,+and+Twisting.">Mechanical Self-Assembly of a Strain-Engineered Flexible Layer: Wrinkling, Rolling, and Twisting.</a> Phys. Rev. Appl. 5, (2016).</li><li>Manner, J., Seidl, W., Steding, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+the+cervical+flexure:+an+experimental+study+on+chick+embryos.">Formation of the cervical flexure: an experimental study on chick embryos.</a> Acta Anat. (Basel). 152, 1-10 (1995).</li><li>Ware, M., Schubert, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+early+axon+scaffold+in+the+rostral+brain+of+the+chick+embryo.">Development of the early axon scaffold in the rostral brain of the chick embryo.</a> J. 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Mens fysiske fænomener spiller en integrerende rolle i morfogenese26,27,28,29,30, de særlige mekaniske mekanismer, samt koordinering af mekaniske og molekylære mekanismer, forbliver stort set uudforskede. Det vides at den ventrale flexure og højredrejningen torsion af den primitive hjerne er to centrale processer, der bidrager til tidlig embryonale morfoge...

Moderne udviklingsmæssige biologi forskning fokuserer i høj grad på forståelse udvikling fra perspektivet af molekylær genetik1,2,3,4,5,6 , 7 , 8 , 9 , 10 , 11 , 12 , 13. det er kendt at fysiske fænomener spiller en central rolle i morfogenese, eller generation af biologiske danne14,15,16,17; særlige mekaniske mekanismer af udviklingen er dog stadig i vid udstrækning unstudied. Ventrale flexure og højredrejningen torsion af den primitive hjerne tube efter Hamburger-Hamilton etape 11 (HH 11)18 , er de to vigtigste processer, der bidrager til embryonale form ændre19,20. Navnlig, forbliver fysisk mekanismen underliggende torsionsstivhed udviklingen i embryonale hjernen utilstrækkeligt forstået.
Den embryonale torsion i kylling embryo er blandt de tidligste morfogenetiske begivenheder af venstre-højre (L-R) asymmetri i udvikling. Når processen med L-R asymmetri er desorienterede, vil fosterskader situs inversus, isomerieller heterotaxia forekomme21. Her præsenterer vi en protokol, som kombinerer ex-ovo eksperimenter22,23 med fysisk modellering at karakterisere mekaniske kræfter i den tidlige embryonale hjerne udvikling. Målet med metoden præsenteret er at identificere de mekaniske kræfter ansvarlig for hjernen torsion og de faktorer, der påvirker graden af torsion under tidlige udvikling12. Baseret på eksperimentel observation at vitelline membranen (VM) begrænser den dorsale side af embryonet, hypotese vi at VM giver den kraft, der er nødvendig for at fremkalde torsion af udvikle hjernen. Derfor i denne metode, vi har fjernet del af VM, der dækker området hjernen til at finde ud af, virkningerne på hjerne torsion. Derudover blev metode til at anvende flydende overfladespænding brugt til at bekræfte den mekaniske rolle af VM og give et skøn over den kraft, der er nødvendig for hjerne vride, som ikke havde været gjort tidligere. Måling af kræfter under embryonale morfogenese er en udfordrende opgave. Navnlig i en banebrydende undersøgelse udviklet Campàs og co-arbejdere24 en roman metode til at kvantificere de cellulære belastninger ved hjælp af injiceres microdroplets. Ikke desto mindre, denne metode var begrænset til at måle kræfter på cellulært niveau, derfor ikke relevant at sonde styrker på væv - eller organisme-niveau. Den protokol, der præsenteres i dette dokument blev udviklet delvis udfylde hullet.

<table border="0" cellpadding="0" cellspacing="0" width="751">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:288px;">Fertilized Specific pathogen-free White Leghorn chicken eggs</td>
			<td style="width:177px;">Charles River</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:288px;">Optical Coherent Tomography Microscope</td>
			<td style="width:177px;">Thorlabs</td>
			<td style="width:119px;">GAN220C1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:288px;">Silicone elastomer</td>
			<td style="width:177px;">Smooth-On, Inc.</td>
			<td style="width:119px;">EcoFlex 00-50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Dissecting microscope</td>
			<td style="width:177px;">Leica</td>
			<td style="width:119px;">MZ8</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:288px;">Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)</td>
			<td style="width:177px;">Lonza</td>
			<td style="width:119px;">12-604F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Antibiotics</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:119px;">P4083</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Chick serum</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:119px;">C5405</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Micropipette puller</td>
			<td style="width:177px;">Sutter Instrument</td>
			<td style="width:119px;">Model P-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Filter paper</td>
			<td style="width:177px;">Whatman</td>
			<td style="width:119px;">5202-110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Phosphate buffered saline (PBS)</td>
			<td style="width:177px;">Corning</td>
			<td style="width:119px;">21-040-CV</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:288px;">Comsol MultiPhysics</td>
			<td style="width:177px;">Comsol</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">3D computer graphics software</td>
			<td style="width:177px;">Rhino 5</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Microscope attached with OCT</td>
			<td style="width:177px;">Nikon&#160;</td>
			<td style="width:119px;">FN1</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;width:288px;">Digital single-lens reflex camera</td>
			<td style="width:177px;">EOS&#160;</td>
			<td style="width:119px;">Rebel T3i</td>
			<td></td>
		</tr>
	</tbody>
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probing the roles of physical forces in early chick embryonic morphogenesis

Fosterudviklingen er traditionelt studerede fra perspektivet af Biomolekylær genetik, men den grundlæggende betydning af mekanik i morfogenese er ved at blive stadig mere anerkendt. Især embryonale chick hjerte og hjerne tube, som undergår drastiske morfologiske ændringer, som de udvikler, er blandt de vigtigste kandidater til at studere fysiske kræfter i morfogenese rolle. Progressive ventrale bøjning og højredrejningen torsion af rørformede embryonale chick hjernen ske på det tidligste stadium af orgel-niveau højre-asymmetri i chick embryonale udvikling. Den vitelline membran (VM) begrænser den dorsale side af fosteret og har været impliceret i leverer den kraft, der er nødvendig for at fremkalde torsion af udvikle hjernen. Her præsenterer vi en kombination af nye ex-ovo eksperimenter og fysiske modellering for at identificere mekanikken i hjernen torsion. På Hamburger-Hamilton Stadium 11, embryoner er høstet og kulturperler ex ovo (i medierne). VM fjernes efterfølgende ved hjælp af en trak kapillarrør. Ved at kontrollere niveauet af væske og underkaste embryonet et væske-air interface, kan flydende overfladespænding medier bruges til at erstatte den mekaniske rolle af VM. Mikrokirurgi eksperimenter blev også udført for at ændre placeringen af hjertet for at finde den deraf følgende ændring i chiralitet af hjernen torsion. Resultaterne fra denne protokol illustrere mekanik i kørsel morfogenese grundlæggende roller.

I denne undersøgelse, blev VM af embryo på HH11 fjernet fra den forreste ende af thorax flexure. Embryoner blev afbildet af en OCT system. På dette stadium startet torsion af hjernen rør ikke (figur 1A). Efter at være udruget til HH15-16, udstillet embryoner med deres VM fjernet reducerede hjerne tube torsion, ca 35 grader (figur 1B) i forhold til at styre embryoner, som udviser torsion af omkring 90 grader. Når media nivea...

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Probing the Roles of Physical Forces in Early Chick Embryonic Morphogenesis

Sondering roller af fysiske kræfter i tidlige Chick embryonale morfogenese

Vi præsenterer her, en protokol, at indføre en række nye ex-ovo eksperimenter og fysiske modellering tilgange til at studere mekanikken i morfogenese under tidlige chick embryonale hjernen torsion.

Embryonic development is traditionally studied from the perspective of biomolecular genetics, but the fundamental importance of mechanics in morphogenesis ...

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