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. anerkjenner støtte fra Dartmouth oppstart fund og Branco Weiss - Society for Science fellesskap, administrert av ETH Zürich. Forfatterne takker Dr. Larry A. Taber, Benjamen A. Filas, Qiaohang Guo, og Yunfei Shi for nyttig diskusjoner, samt de anonyme korrekturleserne. Dette materialet er basert på arbeid støttes av National Science Foundation Graduate forskning fellesskap under Grant nr. DGE-1313911. Noen meninger, funn, og konklusjoner eller anbefalinger i dette materialet er de av forfatterne (s) og nødvendigvis gjenspeiler ikke synspunktene til National Science Foundation.

1. utarbeidelse av vev kultur medier
<ol><li>Bruke en 0,5 L flaske av Dulbeccos endret Eagle's Medium (DMEM) med 4,5 finans glukose, natriumbikarbonat og L-glutamin som grunnlag for kultur media.</li>
	<li>I sterilt laminær strømning hette, legge 10 mL av antibiotika til 0,5 L av DMEM.</li>
	<li>Bruker en steril pipette, overføre 50 mL av DMEM antibiotika løsningen til et sterilt 50 mL konisk rør.</li>
	<li>Legge til 50 mL av chick serum gjenværende DMEM antibiotika løsningen i 0,5 L flasken i sterilt panseret.<...

<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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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. 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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. Anat. 219, 203-216 (2011).</li><li>Ramasubramanian, A., 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=On+the+role+of+intrinsic+and+extrinsic+forces+in+early+cardiac+S-looping.">On the role of intrinsic and extrinsic forces in early cardiac S-looping.</a> Dev. Dyn. 242, 801-816 (2013).</li><li>Hoyle, C., Brown, N. a., Wolpert, L. <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+left/right+handedness+in+the+chick+heart.">Development of left/right handedness in the chick heart.</a> Development. 115, 1071-1078 (1992).</li><li>Nerurkar, N. L., Ramasubramanian, A., Taber, L. 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Mens fysiske fenomener spiller en vesentlig rolle i morphogenesis26,27,28,29,30, bestemte mekanisk mekanismer med samordning av mekanisk og molekylære mekanismer, forblir stort sett uutforsket. Det er kjent at ventrale flexure og rightward torsjon av primitive hjernen er to sentrale prosesser som bidrar til tidlig embryonale morphogenesis18<...

Moderne utviklingsbiologi forskning fokuserer hovedsakelig på forståelse utvikling fra perspektivet av molekylær arvelighetsforskning1,2,3,4,5,6 , 7 , 8 , 9 , 10 , 11 , 12 , 13. det er kjent at fysiske fenomener spille en sentral rolle i morphogenesis, eller generering av biologiske form14,15,16,17; imidlertid fortsatt bestemte mekanisk mekanismer for utviklingen i stor grad unstudied. Ventrale flexure og rightward torsjon av primitive hjernen rør etter Hamburger-Hamilton scenen 11 (HH 11)18 er de to viktigste prosessene som bidrar til embryonale figuren endres19,20. Spesielt fortsatt den fysisk mekanismen underliggende torsjonsmessig utviklingen i embryonale hjernen ufullstendig forstått.
Den embryonale torsjon i chick embryoet er blant de tidligste Morfogenetiske hendelsene venstre høyre (V-H) asymmetri i utvikling. Når prosessen med L-R asymmetri opprørt, skje fødselsskader som situs inversus, isomerieller heterotaxia 21. Her presenterer vi en protokoll som kombinerer ex-ovo eksperimenter22,23 med fysisk modellering å karakterisere mekaniske krefter under tidlig embryonale hjernens utvikling. Målet med metoden presentert er å identifisere de mekaniske kreftene ansvarlig for hjernen torsjon og faktorene som påvirker graden av torsjon under tidlig utvikling12. Basert på eksperimentell observasjon at eggeplomme membranen (VM) begrenser dorsal side av embryoet, hypotese vi at VM gir kraften som er nødvendig å indusere torsjon av utvikle hjernen. Derfor i denne metoden fjernet vi delen av VM som dekker området hjernen å finne ut effekter på hjernen torsjon. Videre bruk av metoden bruker væske overflatespenningen å bekrefte mekanisk rollen VM og gi et estimat over kraften som er nødvendig for hjernen torsjon, som ikke hadde blitt gjort tidligere. Måle styrker under embryonale morphogenesis er en utfordrende oppgave. Spesielt i en banebrytende studie utviklet Campàs og kolleger24 en ny metode for å kvantifisere mobilnettet stress med innsatt microdroplets. Likevel, denne metoden var begrenset til å måle styrker på cellenivå, derfor ikke aktuelt å undersøke styrker på vev - eller organisme-nivå. Protokollen presenteres i dette dokumentet ble utviklet for å delvis fylle dette gapet.

<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>
</table>

probing the roles of physical forces in early chick embryonic morphogenesis

Embryonale utvikling er tradisjonelt studerte fra perspektivet til biomolecular genetikk, men den grunnleggende betydningen av mekanikken i morphogenesis er blitt stadig mer anerkjent. Spesielt embryonale chick hjertet og hjernen røret, som gjennomgår morfologiske Retningsendringer mens de utvikler, er blant de førsteklasses kandidatene å studere rollen til fysiske kreftene i morphogenesis. Progressiv ventrale bøying og rightward torsjon av rørformede embryonale chick hjernen skje på tidligste scenen organ-nivå venstre høyre asymmetri i chick embryonale utviklingen. Eggeplomme membranen (VM) begrenser dorsal side av embryoet og har vært innblandet i å gi kraft nødvendig å indusere torsjon av utvikle hjernen. Her presenterer vi en kombinasjon av nye ex-ovo eksperimenter og fysisk modellering for å identifisere mekanikken i hjernen torsjon. Hamburger-Hamilton etappe 11, embryoer er høstet og kultivert ex ovo (i media). VM fjernes senere ved hjelp av en trakk kapillær rør. Kontrollere nivået av væsken og utsette embryoet til en væske-air-grensesnitt, kan flytende overflaten spenningen av media brukes til å erstatte mekanisk rollen av VM. Mikrokirurgi eksperimentene ble også utført for å endre plasseringen av hjertet å finne resulterende endringen i chiralitet av hjernen torsjon. Resultater fra denne protokollen illustrerer grunnleggende rollene mekanikk drive morphogenesis.

I denne studien var VM av embryoet på HH11 fjernet fra den fremre delen av thorax flexure. Embryoene ble fotografert av en OCT-system. På dette stadiet startet torsjon av hjernen ikke (figur 1A). Etter blir ruges HH15-16, utstilt embryo med sine VM fjernet redusert hjernen tube torsjon, ca 35 grader (figur 1B) sammenlignet med kontrollere embryoer, som viser torsjon rundt 90 grader. Når media nivået var senket å indusere ove...

Watch this Scientific Journal Video about Probing the Roles of Physical Forces in Early Chick Embryonic Morphogenesis at JoVE.com

Probing the Roles of Physical Forces in Early Chick Embryonic Morphogenesis

Verifiserer rollene fysiske kreftene i tidlig Chick embryonale Morphogenesis

Her presenterer vi en protokoll introduserer et sett med nye ex-ovo eksperimenter og fysisk modellering tilnærminger for å studere mekanikken i morphogenesis under tidlig chick embryonale hjernen torsjon.

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

The overall goal of this experimental protocol is to study the role of mechanical forces in early embryonic brain development through ex ovo experiments. This method can help answer key questions in developmental biology and biomechanics, such as the roles of mechanical cues in morphogenesis, which is the generation of biological form. The main advantage of this technique is that it enables the characterization of the role of mechanical forces in morphogenesis at the tissue and organism level.To begin this procedure, use delicate wipes with 70%ethanol to clean the fertilized specific-pathogen-free White Leghorn chicken eggs. Then, arrange the eggs in a longitudinal orientation on a holder. After that, set the egg incubator at a target temperature of 37.5 degrees Celsius, and maintain the humidity at 48 to 55%The humidity is controlled by adding an appropriate amount of water to the incubator.Incubate the eggs for approximately 40 to 44 hours to HH11 to 13. Afterward, let the eggs cool down at room temperature for approximately 15 to 30 minutes prior to cleaning with 70%ethanol. In this step, crack the eggs from the bottom, pull apart the shell gently, and carefully deposit the contents into a 150 by 15 millimeter Petri dish.To ensure the embryo side is up, keep the eggs in the same orientation as they were incubated while cracking them. Remove the thin albumin using a disposable Pasteur pipette. Separate the thick albumin from the yolk using the blunt-ended forceps.Ensure that the thick albumin has been removed by lightly scraping the top of the yolk. Then, using fine-tipped forceps, place a filter paper ring over the embryo and center it, matching the long axis of the ring with the long axis of the embryo. Subsequently, cut the yolk surrounding the filter paper ring with scissors.Following that, pull the ring and embryo off the yolk in an oblique direction towards the site where the yolk was first cut. Then, rinse the embryo in two sequential 100-millimeter dishes with room temperature PBS. Next, place a filter paper ring into a 35-millimeter Petri dish.Place the embryo dorsal side up onto the other filter paper in the 35-millimeter Petri dish. After that, place a stainless-steel ring on top of the filter paper sandwich. Take care not to damage the embryo.Now, add three milliliters of the previously prepared CCM to each Petri dish. Remove the vitelline membrane of each embryo by lightly skimming the pulled capillary needle across the top of the embryo. Then, peel the vitelline membrane away, starting from the anterior end and proceeding to the notochord.Following that, place eight 35-millimeter Petri dishes into one 150-millimeter dish lined with water-saturated wipes to maintain humidity. Then, place the 150-millimeter Petri dish into a sealable plastic bag, and fill the bag with a gas mixture comprised of 95%oxygen and 5%CO2. Seal the bag, and place it in a 37.5 degree Celsius incubator.Now, remove the embryo from the incubator, and use an OCT system to image them and to determine the torsional angle of the neural tube. Transfer the embryos to a light microscope, and visualize at 10X magnification. Use a 200-microliter pipette to incrementally remove 0.2 milliliters of media from the Petri dish.Take bright-field images following each round of media removal to observe the effect of the media-air interface on the embryo. Continue removing media until the surface tension across the embryo induces torsion. Image the embryo using the OCT system once again to establish a final torsional angle for comparison to control embryos.To alter the direction of the heart loop, use a pair of forceps to flip the filter paper so that the embryo becomes ventral side up. Then, use the pulled capillary tube to cut open a slit in the splanchnopleure membrane and to exert a mechanical force to push the heart from the right-hand side to the left-hand side. Apply the fluid surface tension to keep the heart on the left-handed side.Then, incubate the embryo for another 20 hours to see the change in chirality of the torsion. Shown here is a harvested embryo with VM removed at HH11. The same embryo incubated for 27 hours post VM removal showing reduced torsion.Here is the same embryo that underwent brain torsion upon application of fluid surface tension, and here is the control embryo with normal brain torsion at a comparable stage. While attempting this procedure, it's important to be careful not to damage the embryo. Following this procedure, other methods like finite element analysis can be performed in order to computationally model how physical forces can influence morphology.Therefore, this technique developed here paved the way for future studies on mechanics of morphogenesis in chick embryos. After watching this video, you should understand how to harvest chick embryos for in vitro culture, micro-surgically remove the vitelline membrane, recapitulate the force provided by the vitelline membrane, and monitor embryonic morphology through OCT and bright-field imaging.

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