陈竺承认达特茅斯创业基金和 Branco 的科学研究协会的支持, 由瑞士联邦管理。作者感谢 Drs. 泰伯、Benjamen a. Filas、Qiaohang 郭和云飞仕为有益的讨论, 以及匿名评论者的意见。该材料是根据国家科学基金会研究生研究金资助的工作提供的。DGE-1313911。本材料所表达的任何意见、发现和结论或建议都是作者的观点, 并不一定反映国家科学基金会的观点。

1. 组织培养培养基的制备
<ol><li>使用0.5 升瓶 Dulbecco 的改良鹰的培养基 (DMEM) 与4.5 克/升葡萄糖, 碳酸氢钠和 l-谷氨酰胺作为培养基的基础。</li>
	<li>在无菌层流罩中, 在 DMEM 的0.5 升中加入10毫升抗生素。</li>
	<li>使用无菌吸管, 将50毫升的 DMEM 抗生素溶液转移到无菌50毫升圆锥管。</li>
	<li>添加50毫升的小鸡血清到其余的 DMEM 抗生素溶液中的0.5 升瓶在消毒罩。</li>
	<li>存储最终解决方案 (以下简称鸡文化媒体 [CCM]) 在50毫升圆锥管整除数在-20 °c。</li>
</ol>2. 孵化蛋
<ol><li>使用精致的湿巾与70% 乙醇清洁受精特定病原体无白利佛诺鸡卵。在支架上排列蛋的纵向方向。</li>
	<li>打开一个鸡蛋孵化器设置一个目标温度为37.5 摄氏度, 并保持湿度在 48-55%。湿度是通过向孵化器中添加适量的水来控制的。</li>
	<li>孵化卵到 HH11-13, 大约 40-44 小时。</li>
	<li>让鸡蛋在室温下降温约 15-30 分钟之前喷洒/清洁70% 乙醇。</li>
</ol>3. 拉玻璃毛细血管
<ol><li>在微拉拔上安装10厘米长的玻璃毛细管, 外径为1.0 毫米, 内径为0.5 毫米。</li>
	<li>分别将热和拉参数设置为750和400。按下拉按钮, 将毛细管拉入细针。</li>
</ol>4. 过滤纸载体法
<ol><li>切圆直径约3厘米的过滤纸。</li>
	<li>用打孔器将矩形从圆上切约1厘米到2厘米。请确保删除任何突出或尖锐的角落。</li>
</ol>5. 胚胎的收获和准备
<ol><li>从底部裂蛋, 将壳轻轻地拉出, 小心地放入150毫米 x 15 毫米培养皿中。为了确保胚胎的一侧, 使卵在相同的方向, 他们被孵化时, 他们开裂。</li>
	<li>使用一次性的巴斯德吸管去除薄蛋白。</li>
	<li>用钝端钳将蛋黄中的浓白蛋白分离出来。通过用钝端钳轻轻刮掉蛋黄的顶部, 确保粗蛋白被除去。</li>
	<li>用细尖钳将过滤纸环放在胚胎上, 将环长轴与胚长轴相匹配。</li>
	<li>用剪刀把滤纸环周围的蛋黄切开。</li>
	<li>将环和胚从蛋黄中拉出, 朝第一个被切蛋黄的部位倾斜。</li>
	<li>用室温1x 磷酸盐缓冲盐水 (PBS) 将两个连续100毫米的盘子中的胚胎冲洗干净。</li>
	<li>先将滤纸环放入35毫米培养皿中。然后将胚背侧向上放置在35毫米培养皿中的滤纸上。</li>
	<li>将不锈钢环放在过滤纸三明治的顶端。确保不要破坏胚胎。</li>
	<li>添加3毫升以前制备的 CCM 到每个培养皿。</li>
	<li>移除每个胚胎的 VM, 通过轻轻地掠过胚胎顶端的拉毛细针, 然后从前端 (右上方的前脑) 开始剥离 vm, 然后继续脊索 (图 1A)。</li>
	<li>将八35毫米的培养皿放入一个 150 mm 的盘子里, 里面衬着水饱和细腻的任务湿巾 (以保持湿度)。</li>
	<li>将150毫米的培养皿放入一个密封塑料袋中, 然后用由 95% O2和 5% CO2组成的气体混合物填充袋子。</li>
	<li>密封袋, 并把它放在一个37.5 摄氏度孵化器。</li>
	<li>孵化胚胎27小时, 直到 HH15-HH16 (图 1B)。</li>
</ol>6. 诱发表面张力
<ol><li>从孵化器中取出胚胎, 并使用光学相干层析成像 (OCT) 系统对其进行图像处理。使用 OCT 确定神经管 (NT) 的扭转角度 (图 2)。</li>
	<li>将胚胎移植到光显微镜下, 并以10X 倍的放大可视化。使用200微升吸管增量从培养皿中移除0.2 毫升培养基。</li>
	<li>每隔一段时间取 brightfield 图像, 观察培养基-空气界面对胚胎的影响。</li>
	<li>移除介质, 直到整个胚胎表面张力诱发扭转 (图 1C)。</li>
	<li>再次利用 OCT 系统对胚胎进行图像, 建立与控制胚胎的最终扭转角。 注: 使用解剖显微镜获得明亮的图像。采用附有显微镜的光学相干层析成像系统获取活胚的横断面图像栈。图像是在 3 x 10 毫米 x 3 毫米3扫描域中获得的, 然后在成像软件中进行处理。最后, 采用数字式单透镜反射相机对物理模型图像进行了分析。</li>
</ol>7. 表面张力/VM 力的物理模拟
<ol><li>开发一个简化的3D 几何, 它类似于商业建模软件中 HH14-17 之间的胚胎 (图 3A)。</li>
	<li>设计了商用3D 计算机图形学软件中3D 几何的负模。</li>
	<li>使用3D 打印机加载1.75 毫米丙烯腈丁二烯苯乙烯灯丝3D 打印设计的模具, 以 stereolithographic (. stl) 格式。</li>
	<li>铸造模具, 将硅橡胶弹性体元件 A 和 B 混合在同等部位, 并及时倒入模具中;设置铸件模具在室温下固化12小时 (图 3B)。</li>
	<li>将胚胎的物理模型标记在背侧的 NT 上, 以可视化扭转。</li>
	<li>使用盖玻片复制用于模拟 VM 或表面张力的3D 物理模型的力(图 3D)。</li>
	<li>在物理模型的侧面插入一系列相同长度的刚性导线。盖玻片在大脑模型上施加外力后, 导线就会倾斜, 这取决于位置。通过 arctan 每根导线的原始长度来确定旋转角度 (图 3E)。</li>
</ol>8. 改变心脏回路的方向
<ol><li>按照3.1 和3.2 中的步骤, 获得拉毛管。</li>
	<li>按照5.1 到5.10 的步骤准备胚胎。</li>
	<li>使用一对镊子翻转过滤纸, 使胚胎成为腹侧。</li>
	<li>用拉毛管切开 splanchnopleure (SPL) 膜中的狭缝。</li>
	<li>用毛细管施加机械力, 将心脏从右手侧推到左手边。</li>
	<li>按照5.12 到5.15 的步骤观察扭转的变化。</li>
</ol>

<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. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computational+models+for+mechanics+of+morphogenesis.">Computational models for mechanics of morphogenesis.</a> Birth Defects Research Part C - Embryo Today: Reviews. 96, 132-152 (2012).</li><li>Savin, T., 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+growth+and+form+of+the+gut.">On the growth and form of the gut.</a> Nature. 476, 57-62 (2011).</li><li>Gjorevski, N., Nelson, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanics+of+development:+Models+and+methods+for+tissue+morphogenesis.">The mechanics of development: Models and methods for tissue morphogenesis.</a> Birth Defects Research Part C - Embryo Today: Reviews. 90, 193-202 (2010).</li><li>Shyer, A. E., 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=Villification:+how+the+gut+gets+its+villi.">Villification: how the gut gets its villi.</a> Science. 342, 212-218 (2013).</li><li>Ambrosi, D., 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=Perspectives+on+biological+growth+and+remodeling.">Perspectives on biological growth and remodeling.</a> Journal of the Mechanics and Physics of Solids. 59, 863-883 (2011).</li><li>Chen, Z., Majidi, C., Srolovitz, D. J., Haataja, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tunable+helical+ribbons.">Tunable helical ribbons.</a> Appl. Phys. Lett. 98, (2011).</li><li>Gerbode, S. J., Puzey, J. R., McCormick, A. G., Mahadevan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+the+cucumber+tendril+coils+and+overwinds.">How the cucumber tendril coils and overwinds.</a> Science. 337, 1087-1091 (2012).</li><li>Armon, S., Efrati, E., Kupferman, R., Sharon, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geometry+and+mechanics+in+the+opening+of+chiral+seed+pods.">Geometry and mechanics in the opening of chiral seed pods.</a> Science. 333, 1726-1730 (2011).</li><li>Filas, B. 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=A+potential+role+for+differential+contractility+in+early+brain+development+and+evolution.">A potential role for differential contractility in early brain development and evolution.</a> Biomech. Model. Mechanobiol. 11, 1251-1262 (2012).</li><li>Xu, G., 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=Axons+pull+on+the+brain,+but+tension+does+not+drive+cortical+folding.">Axons pull on the brain, but tension does not drive cortical folding.</a> J. Biomech. Eng. 132, 71013 (2010).</li><li>Chen, Z., Guo, Q., Dai, E., Forsch, N., 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=How+the+embryonic+chick+brain+twists.">How the embryonic chick brain twists.</a> J. R. Soc. Interface. 13, (2016).</li><li>Manca, 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=Nerve+growth+factor+regulates+axial+rotation+during+early+stages+of+chick+embryo+development.">Nerve growth factor regulates axial rotation during early stages of chick embryo development.</a> Proc Natl Acad Sci U S A. 109, 2009-2014 (2012).</li><li>Shi, Y., Yao, J., 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=Bending+of+the+looping+heart:+differential+growth+revisited.">Bending of the looping heart: differential growth revisited.</a> J. Biomech. 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. 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. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphogenetic+adaptation+of+the+looping+embryonic+heart+to+altered+mechanical+loads.">Morphogenetic adaptation of the looping embryonic heart to altered mechanical loads.</a> Dev. Dyn. 235, 1822-1829 (2006).</li><li>Levin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Left-right+asymmetry+and+the+chick+embryo.">Left-right asymmetry and the chick embryo.</a> Semin. Cell Dev. Biol. 9, 67-76 (1998).</li><li>Roebroek, A. J., 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=Failure+of+ventral+closure+and+axial+rotation+in+embryos+lacking+the+proprotein+convertase+Furin.">Failure of ventral closure and axial rotation in embryos lacking the proprotein convertase Furin.</a> Development. 125, 4863-4876 (1998).</li><li>Peebles, D. M., 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=Magnetic+resonance+proton+spectroscopy+and+diffusion+weighted+imaging+of+chick+embryo+brain+in+ovo.">Magnetic resonance proton spectroscopy and diffusion weighted imaging of chick embryo brain in ovo.</a> Dev. Brain Res. 141, 101-107 (2003).</li><li>Zhu, L., 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=Cerberus+regulates+left-right+asymmetry+of+the+embryonic+head+and+heart.">Cerberus regulates left-right asymmetry of the embryonic head and heart.</a> Curr. Biol. 9, 931-938 (1999).</li></ol>

作者声明没有利益冲突。

虽然物理现象在形态发生中扮演一个不可分割的角色26,27,28,29,30, 具体的机械机制, 以及协调机械和分子机制, 仍然主要未探索。据了解, 原脑的腹壁弯曲和向右扭转是导致早期胚胎形态发生的两个中心过程18,31,32,33, 34, 但以前没有研究讨论大脑扭转的机械起源, 这是最早的器官一级左-右不对称事件之一。
该协议的关键步骤包括删除 VM 以识别大脑扭转的机械驱动力和应用流体表面张力, 进一步证实这些结果。这项技术的故障诊断发生了, 以确定初始阶段, 应删除 VM, 以引起重大变化的扭转。
虚拟机的下行被动力被证明是生长的胚胎脑管的基本机械边界。当胚胎的 VM 被移除时, 大脑不再扭曲到正常的程度, 而是通过降低流体水平, 通过随后的表面张力的应用, 使其扭曲到控制水平。已知水在常温下的表面张力为 72.01 0.1 锰/米, 接触长度为毫米级, 可计算力。从而我们估计 VM 施加了大约10锰的力量在 HH 14-17 胚胎12。
利用该协议, 我们能够确定 VM 在胚胎脑扭转中起着关键的机械作用。使用这一新的协议的结果表明, VM 是一个关键的结构, 以促进正常的大脑扭转在胚胎形态发生, 因为它提供的几何约束和机械负荷的扭曲所需的大脑35。结果还表明, 心脏的位置决定了大脑扭转的方向。胚胎发育过程中的非对称性导致了心脏的右环形形状, 从而驱动向右扭脑36,37,38,39。值得一提的是, 取代心脏位置的机械方法不同于其他研究者所开发的化学方法13 , 更适合于描述力学在形态发生中的作用。总之, 我们的结果说明了力学在驱动扭脑在鸡胚中发生的基本作用。
今后, 该议定书可用于确定遗传和机械因素如何结合正常的胚胎扭转, 揭示这些不同因素如何协同工作, 以确保适当的形态发生。

现代发展生物学研究主要集中在从分子遗传学的角度理解发展问题1,2,3,4,5,6,7,8,9,10,11,12,13. 众所周知, 物理现象在形态发生中起着中心作用, 或产生生物形态14,15,16,17;然而, 具体的发展机械机制仍然很大程度上研究忽略。在汉堡包-汉密尔顿阶段11 (HH 11)18中, 原始脑管的腹壁弯曲和向右扭转是导致胚胎形状变化的两个主要过程1920。特别是, 胚胎大脑扭转发育的物理机制仍然不完全理解。
鸡胚的胚胎扭转是左-右 (l-R) 发育不对称的最早形态发生事件之一。当 l-R 不对称的过程受到扰动时, 诸如内脏、异构或heterotaxia等出生缺陷将发生在21中。 在这里, 我们提出一个协议, 其中结合前蛋实验22,23与物理建模, 以表征机械力在早期胚胎脑发育。所提出的方法的目的是确定在早期开发过程中负责大脑扭转的机械力和影响扭转程度的因素12。通过实验观察, 卵黄膜 (vm) 对胚胎的背侧有一定的约束, 我们假设 vm 提供了诱发发育脑扭转的必要力量。因此, 在这个方法中, 我们删除了覆盖大脑区域的 VM 的一部分, 以找出对大脑扭转的影响。此外, 采用流体表面张力的方法, 以确定 VM 的机械作用, 并提供了对大脑扭转所需的力量的估计, 这是以前没有做过的。在胚胎形态发生过程中测量力是一项艰巨的任务。值得注意的是, 在一项开创性的研究中, Campàs 和同事24开发了一种新的方法, 用注射 microdroplets 量化细胞应力。然而, 这种方法仅限于测量细胞水平的力量, 因此不适用于组织或生物体级的探针力。本文提出的协议部分填补了这一空白。

<table><tbody><tr><td>Fertilized Specific pathogen-free White Leghorn chicken eggs</td><td>Charles River</td><td></td><td></td></tr><tr><td>Optical Coherent Tomography Microscope</td><td>Thorlabs</td><td>GAN220C1</td><td></td></tr><tr><td>Silicone elastomer</td><td>Smooth-On, Inc.</td><td>EcoFlex 00-50</td><td></td></tr><tr><td>Dissecting microscope</td><td>Leica</td><td>MZ8</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium (DMEM)</td><td>Lonza</td><td>12-604F</td><td></td></tr><tr><td>Antibiotics</td><td>Sigma</td><td>P4083</td><td></td></tr><tr><td>Chick serum</td><td>Sigma</td><td>C5405</td><td></td></tr><tr><td>Micropipette puller</td><td>Sutter Instrument</td><td>Model P-30</td><td></td></tr><tr><td>Filter paper</td><td>Whatman</td><td>5202-110</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Corning</td><td>21-040-CV</td><td></td></tr><tr><td>Comsol MultiPhysics</td><td>Comsol</td><td></td><td></td></tr><tr><td>3D computer graphics software</td><td>Rhino 5</td><td></td><td></td></tr><tr><td>Microscope attached with OCT</td><td>Nikon&amp;nbsp;</td><td>FN1</td><td></td></tr><tr><td>Digital single-lens reflex camera</td><td>EOS&amp;nbsp;</td><td>Rebel T3i</td><td></td></tr></tbody></table>

probing the roles of physical forces in early chick embryonic morphogenesis

胚胎发育通常是从生物分子遗传学的角度来研究的, 但力学在形态发生中的基本重要性越来越被人们所认识。特别是, 胚胎小鸡心脏和脑管, 经过剧烈的形态学变化, 因为他们的发展, 是主要的候选者, 研究物理力量在形态发生中的作用。小管状胚胎鸡脑的渐进腹侧弯曲和向右扭转发生在鸡胚胎发育的器官水平左-右不对称的最早阶段。卵黄膜 (VM) 约束胚胎的背侧, 并有牵连提供必要的力量, 以诱发发展中的大脑扭转。在这里, 我们提出了新的前蛋实验和物理建模的组合, 以确定大脑扭转的力学。在汉堡-汉密尔顿阶段 11, 胚胎是收获和培养的前蛋 (在媒体)。然后使用拉毛管将 VM 移除。通过控制流体的水平和将胚胎置于流体-空气界面上, 介质的流体表面张力可以用来代替 VM 的机械作用。同时进行显微外科实验, 改变心脏的位置, 以发现脑扭转的手性改变。该协议的结果说明了力学在驱动形态发生中的基本作用。

在本研究中, 胚胎在 HH11 的 VM 从前端移除到胸椎弯曲。胚胎被一个 OCT 系统所成像。在这个阶段, 脑管的扭转尚未启动 (图 1A)。在孵化到 HH15-16 后, 胚胎与其 VM 删除显示减少脑管扭转, 约35度 (图 1B) 相比, 控制胚胎, 显示扭转约90度。当培养基水平降低, 以诱导胚胎的背端表面张力与其 vm 移除, 大脑扭曲的水平可比控制胚胎 (图 1C)。图 2显示了 HH 13 胚的代表性 OCT 图像, 其胸部方位角和颅骨方向角 (图 2A)。从 NT 的剖面的垂直位置 (图 2B、C) 测量角度。我们的实验结果表明, 正常的脑管扭转是由外部负荷驱动的胚胎的背端, 这一基本负载由 VM20,25提供。此外, 在正常胚胎中, 当心脏循环转到右侧时, 大脑会转向右, 而在胚胎中, 心脏循环在早期被推到左手边 (即, 在 HH 阶段12之前), 大脑也会转向左以下另一个20小时的孵化 (图3c 在参考文献 [12]), 表明心脏的位置导致大脑扭转不对称。
在实验中收集的数据使我们能够在没有 VM 的 HH14-17 (图 3A) 的情况下重建小鸡胚的简化几何。在这个计算模型中, 大脑和右环形心脏被建模为弯曲棒。一个代表 splanchnopleure (SPL) 膜的块与心脏棒接触。通过设计一个负模从这个计算模型, 3D 打印这个模具, 并铸造模具与硅胶弹性体, 我们捏造了一个物理模型从简化计算几何 (图 3B-d)。在物理模型的背端向下按下的盖玻片, 复制了 VM 提供的机械负载或我们的实验中的表面张力(图 3D)。该模型展示了可比的几何和脑扭转与实际胚胎, 培养的前蛋到 HH14-17 (图 3E)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57150/57150fig1.jpg"> 图 1: 移除 VM 的胚胎形态学和外部力量对向右脑管扭转的影响.(A)在 HH11 中移除 VM 后收获的胚胎。(B)同一胚胎为27小时的 VM 移除而孵化, 显示的是扭矩减少。(C)在应用液体表面张力时, 同一胚胎接受了脑扭转。(D)控制胚胎与正常的脑扭转在一个可比较的阶段。刻度条, (A-C) 1 毫米, (D) 1 毫米图像以10X 倍的放大率捕获。从参考文献 [12] 适应了与允许。<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57150/57150fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57150/57150fig2.jpg"> 图 2: HH13 胚胎的 OCT 图像.(a)该图的胸部方位角为 (a), 其头部方位角在 (b) 处测量。(B)、(C)NT 位置 (a) 和 (b) 的剖面。角度是从垂直位置测量的。<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57150/57150fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57150/57150fig3.jpg"> 图 3: 演示大脑扭转的胚胎的物理模型。(a) HH14-17 中没有 VM 的小鸡胚的简化几何(B)硅胶胚的有机硅弹性体物理模型。(C)模型的背景色, 心脏在右侧。(D)在盖玻片应用的外部力下模型的背景色, 开始显示向右的大脑扭转。(E)小鸡胚培养的前蛋开始扭向右在 HH14 进行比较。缩放条, (B D) 1 厘米, (E) 1 毫米从参考文献 [12] 与允许适应。<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57150/57150fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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

早期雏鸡胚胎形态发生中物理力的作用探讨

在这里, 我们提出了一个协议, 介绍了一套新的前蛋实验和物理建模方法来研究早期鸡胚脑扭转的形态发生机制。

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

probing-roles-physical-forces-early-chick-embryonic

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

7.2K 次观看.  Dartmouth College. 在这里, 我们提出了一个协议, 介绍了一套新的前蛋实验和物理建模方法来研究早期鸡胚脑扭转的形态发生机制。

视频: Probing the Roles of Physical Forces in Early Chick Embryonic Morphogenesis

Grafting of Beads into Developing Chicken Embryo Limbs to Identify Signal Transduction Pathways Affecting Gene Expression

Using chicken embryos it is possible to test directly the effects of either growth factors or specific inhibitors of signaling pathways on gene expression and activation of signal transduction pathways. This technique allows the delivery of signaling molecules at precisely defined developmental stages for specific times. After this embryos can be harvested and gene expression examined, for example by in situ hybridization, or activation of signal transduction pathways observed with immunostaining.
In this video heparin beads soaked in FGF18 or AG 1-X2 beads soaked in U0126, a MEK inhibitor, are grafted into the limb bud in ovo. This shows that FGF18 induces expression of MyoD and ERK phosphorylation and both endogenous and FGF18 induced MyoD expression is inhibited by U0126. Beads soaked in a retinoic acid antagonist can potentiate premature MyoD induction by FGF18.
This approach can be used with a wide range of different growth factors and inhibitors and is easily adapted to other tissues in the developing embryo.

By grafting beads soaked in growth factors or specific inhibitors of signaling pathways into developing embryos it is possible to directly test their effects in vivo. In this protocol beads are grafted into the limb bud to determine the effects of these molecules on gene expression and signal transduction.

珠嫁接到发展鸡胚四肢识别信号转导途径影响基因表达

Using chicken embryos it is possible to test directly the effects of either growth factors or specific inhibitors of signaling pathways on gene expression ...

科研

发育生物学

Application of Impermeable Barriers Combined with Candidate Factor Soaked Beads to Study Inductive Signals in the Chick

The chick embryo provides a superb vertebrate model that can be used to dissect developmental questions in a direct way. Its accessibility and robustness following surgical intervention are key experimental strengths. Mica plates were the first barriers used to prevent chick limb bud initiation1. Protocols that use aluminum foil as an impermeable barrier to wing bud or leg bud induction and or initiation are described. We combine this technique with bead placement lateral to the barrier to exogenously supply candidate endogenous factors that have been blocked by the barrier. The results are analyzed using in situ hybridization of subsequent gene expression. Our main focus is on the role of retinoic acid signaling in the induction and later initiation of the chick embryo fore and hindlimb. We use BMS 493 (an inverse agonist of retinoic acid receptors (RAR)) soaked beads implanted in the lateral plate mesoderm (LPM) to mimic the effect of a barrier placed between the somites (a source of retinoic acid (RA)) and the LPM from which limb buds grow. Modified versions of these protocols could also be used to address other questions on the origin and timing of inductive cues. Provided the region of the chick embryo is accessible at the relevant developmental stage, a barrier could be placed between the two tissues and consequent changes in development studied. Examples may be found in the developing brain, axis extension and in organ development, such as liver or kidney induction.

Protocols using impermeable barriers to block induction events between tissues of the main body axis and flank in the chick embryo required for limb formation are described. Beads soaked in candidate inductive signals are used to overcome the effect of barrier placement and analysis of gene expression confirms this.

在小鸡学习感应信号与候选因子浸泡珠结合防渗屏障中的应用

The chick embryo provides a superb vertebrate model that can be used to dissect developmental questions in a direct way. Its accessibility and robustness ...

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

双整装原位杂交技术在早期鸡胚

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

生物学

Assay for Neural Induction in the Chick Embryo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying the formation and initial patterning of the nervous system. This video demonstrates how to graft organizer tissue into a host, a method by which Hensen s node (the organizer in the chick embryo) is grafted to a host competent ectoderm. The organizer graft instructs overlying na ve tissue to adopt a neural fate via neural inducing signals. This mechanism is referred to as neural induction, and constitutes the initial step in the formation of brain and spinal cord in amniotes. This method is essentially used for the characterization of putative neural inducing molecules in chick. This video demonstrates the different steps in the assay for neural induction; First, the donnor embryo is explanted and pinned on a dish. Then, the host embryo is prepared for New culture. The graft is excised and transplanted to the host area pellucida margin. The host is cultured for 18-22 hrs. The assembly is fixed and processed for further applications (e.g. in situ hybridization). This method was originally devised by Waddington 1,2 and Gallera 3,4.

Neural induction is the first step in the formation of the brain. It is a mechanism by which Hensen's node (organizer), instructs adjacent tissue to adopt a neural fate, i.e. to give rise to the nervous system. This video demonstrates an assay for neural induction in chick embryo.

在鸡胚神经诱导的含量为

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ideal for experimentation. Currently, the study of these developmental pathways in the chicken embryo is conducted by applying inhibitors and drugs at localized sites and at low concentrations using a variety of methods. In vitro tissue&#160;culturing is a technique that enables the study of tissues separated from the host organism, while simultaneously bypassing many of the physical limitations present when working with whole embryos, such as the susceptibility of embryos to high doses of potentially lethal chemicals. Here, we present an organotypic culturing protocol for culturing the embryonic chicken half head in vitro, which presents new opportunities for the examination of developmental processes beyond the currently established methods.

Here, we present an organotypic culturing protocol to grow embryonic chicken organs in vitro. Using this method, the development of embryonic chicken tissue can be studied, while maintaining a high degree of control over the culture environment.

脊髓培养法研究胚胎鸡组织的发展

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...

Chicken Recombinant Limbs Assay to Understand Morphogenesis, Patterning, and Early Steps in Cell Differentiation

Cell differentiation is the fine-tuned process of cell commitment leading to the formation of different specialized cell types during the establishment of developing tissues and organs. This process is actively maintained in adulthood. Cell differentiation is an ongoing process during the development and homeostasis of organs. Understanding the early steps of cell differentiation is essential to know other complex processes such as morphogenesis. Thus, recombinant chicken limbs are an experimental model that allows the study of cell differentiation and pattern generation under embryonic patterning signals. This experimental model imitates an in vivo environment; it assembles reaggregated cells into an ectodermal cover obtained from an early limb bud. Later, ectoderms are transferred and implanted in a chick embryo receptor to allow its development. This assay was mainly used to evaluate mesodermal limb bud cells; however, it can be applied to other stem or progenitor cells from other organisms.

Recombinant limbs are a powerful experimental model that allows for studying the process of cell differentiation and the generation of patterns under the influence of embryonic signals. This protocol presents a detailed method for generating recombinant limbs with chicken limb-mesodermal cells, adaptable to other cell types obtained from different organisms.

鸡重组肢体测定，以了解细胞分化的形态发生，图案化和早期步骤

Cell differentiation is the fine-tuned process of cell commitment leading to the formation of different specialized cell types during the establishment of ...

Analysis of Cell Differentiation, Morphogenesis, and Patterning During Chicken Embryogenesis Using the Soaked-Bead Assay

A multitude of genetic programs is activated during embryonic development that orchestrates cell differentiation to generate an astounding diversity of somatic cells, tissues, and organs. The precise activation of these genetic programs is regulated by morphogens, diffusible molecules that direct cell fate at different thresholds. Understanding how genetic activation coordinates morphogenesis requires the study of local interactions triggered by morphogens during development. The use of beads soaked in proteins or drugs implanted into distinct regions of the embryo enables studying the role of specific molecules in the establishment of digits and other developmental processes. This experimental technique provides information on the control of cell induction, cell fate, and pattern formation. Thus, this soaked bead assay is an extremely powerful and valuable experimental tool applicable to other embryonic models.

The soaked bead assay involves targeted delivery of test reagent at any developmental time point to study the regulation of cell differentiation and morphogenesis. A detailed protocol, applicable to any experimental animal model, for preparing three different types of soaked beads and implanting these in the interdigit of a chicken embryo is presented.

使用浸泡珠测定分析鸡胚胎发生过程中的细胞分化，形态发生和图案化

A multitude of genetic programs is activated during embryonic development that orchestrates cell differentiation to generate an astounding diversity of ...

禽胚胎皮肤外植体培养方案的全面回顾

Using Avian Skin Explants to Study Tissue Patterning and Organogenesis

The developing avian skin during embryogenesis is a unique model that can provide valuable insights into tissue patterning. Here three variations on skin explant cultures to examine different aspects of skin development are described. First, ex vivo organ cultures and manipulations offer researchers opportunities to observe and study the development of feather buds directly. Skin explant culture can grow for 7 days enabling direct analysis of cellular behavior and 4D imaging at intervals during this growth period. This also allows for physical and molecular manipulations of culture conditions to visualize tissue response. For example, growth factor-coated beads can be applied locally to induce changes in feather patterning in a limited area. Alternatively, viral transduction can be delivered globally in the culture media to up or downregulate gene expression. Second, the skin recombination protocol allows researchers to investigate tissue interactions between the epidermis and mesenchyme that are derived from different skin regions, different life stages, or different species. This affords an opportunity to test the time window in which the epithelium is competent to respond to signals and its ability to form different skin appendages in response to signals from different mesenchymal sources. Third, skin reconstitution using dissociated dermal cells overlaid with intact epithelium resets skin development and enables the study of the initial processes of periodic patterning. This approach also enhances our ability to manipulate gene expression among the dissociated cells before creating the reconstituted skin explant. This paper provides the three culture protocols and exemplary experiments to demonstrate their utility.

作者聚焦:深入了解当前的实验性禽皮外植体方法

Here we describe protocols for three types of avian embryonic skin explant cultures that can be used to examine tissue interactions, 4D imaging timelapse movie (3D plus time), global or local perturbation of molecular function, and systems biology characterization.

使用禽皮肤外植体研究组织模式和器官发生

The developing avian skin during embryogenesis is a unique model that can provide valuable insights into tissue patterning. Here three variations on skin ...

早期雏鸡胚胎形态发生中物理力的作用探讨

摘要

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鸡重组肢体测定，以了解细胞分化的形态发生，图案化和早期步骤

使用浸泡珠测定分析鸡胚胎发生过程中的细胞分化，形态发生和图案化

使用禽皮肤外植体研究组织模式和器官发生

使用禽皮肤外植体研究组织模式和器官发生

使用禽皮肤外植体研究组织模式和器官发生