早期雏鸡胚胎形态发生中物理力的作用探讨
Summary
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。
Abstract
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。
Introduction
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。
Protocol
1. 组织培养培养基的制备 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。 在无菌层流罩中, 在 DMEM 的0.5 升中加入10毫升抗生素。 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。 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。 存储最终解决方案 (以下简称鸡文…
Representative Results
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 (<strong c…
Discussion
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,…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Fertilized Specific pathogen-free White Leghorn chicken eggs | Charles River | ||
| Optical Coherent Tomography Microscope | Thorlabs | GAN220C1 | |
| Silicone elastomer | Smooth-On, Inc. | EcoFlex 00-50 | |
| Dissecting microscope | Leica | MZ8 | |
| Dulbecco’s Modified Eagle’s Medium (DMEM) | Lonza | 12-604F | |
| Antibiotics | Sigma | P4083 | |
| Chick serum | Sigma | C5405 | |
| Micropipette puller | Sutter Instrument | Model P-30 | |
| Filter paper | Whatman | 5202-110 | |
| Phosphate buffered saline (PBS) | Corning | 21-040-CV | |
| Comsol MultiPhysics | Comsol | ||
| 3D computer graphics software | Rhino 5 | ||
| Microscope attached with OCT | Nikon | FN1 | |
| Digital single-lens reflex camera | EOS | Rebel T3i |
References
- Taber, L. A. Biomechanics of Growth, Remodeling, and Morphogenesis. Appl. Mech. Rev. 48, 487-545 (1995).
- Wyczalkowski, M. A., Chen, Z., Filas, B. A., Varner, V. D., Taber, L. A. Computational models for mechanics of morphogenesis. Birth Defects Research Part C – Embryo Today: Reviews. 96, 132-152 (2012).
- Savin, T., et al. On the growth and form of the gut. Nature. 476, 57-62 (2011).
- Gjorevski, N., Nelson, C. M. The mechanics of development: Models and methods for tissue morphogenesis. Birth Defects Research Part C – Embryo Today: Reviews. 90, 193-202 (2010).
- Shyer, A. E., et al. Villification: how the gut gets its villi. Science. 342, 212-218 (2013).
- Ambrosi, D., et al. Perspectives on biological growth and remodeling. Journal of the Mechanics and Physics of Solids. 59, 863-883 (2011).
- Chen, Z., Majidi, C., Srolovitz, D. J., Haataja, M. Tunable helical ribbons. Appl. Phys. Lett. 98, (2011).
- Gerbode, S. J., Puzey, J. R., McCormick, A. G., Mahadevan, L. How the cucumber tendril coils and overwinds. Science. 337, 1087-1091 (2012).
- Armon, S., Efrati, E., Kupferman, R., Sharon, E. Geometry and mechanics in the opening of chiral seed pods. Science. 333, 1726-1730 (2011).
- Filas, B. A., et al. A potential role for differential contractility in early brain development and evolution. Biomech. Model. Mechanobiol. 11, 1251-1262 (2012).
- Xu, G., et al. Axons pull on the brain, but tension does not drive cortical folding. J. Biomech. Eng. 132, 71013 (2010).
- Chen, Z., Guo, Q., Dai, E., Forsch, N., Taber, L. A. How the embryonic chick brain twists. J. R. Soc. Interface. 13, (2016).
- Manca, A., et al. Nerve growth factor regulates axial rotation during early stages of chick embryo development. Proc Natl Acad Sci U S A. 109, 2009-2014 (2012).
- Shi, Y., Yao, J., Xu, G., Taber, L. a. Bending of the looping heart: differential growth revisited. J. Biomech. Eng. 136, 1-15 (2014).
- Shi, Y., et al. Bending and twisting the embryonic heart: A computational model for c-looping based on realistic geometry. Front. Physiol. 5, (2014).
- Taber, L. A. Morphomechanics: Transforming tubes into organs. Current Opinion in Genetics and Development. 27, 7-13 (2014).
- Hosseini, H. S., Beebe, D. C., Taber, L. A. Mechanical effects of the surface ectoderm on optic vesicle morphogenesis in the chick embryo. J. Biomech. 47, 3837-3846 (2014).
- Hamburger, V., Hamilton, H. L. A series of normal stages in the development of the chick embryo. Dev. Dyn. 88, 49-92 (1951).
- Shi, Y., Varner, V. D., Taber, L. a. Why is cytoskeletal contraction required for cardiac fusion before but not after looping begins?. Phys. Biol. 12, 16012 (2015).
- Garcia, K. E., Okamoto, R. J., Bayly, P. V., Taber, L. A. Contraction and stress-dependent growth shape the forebrain of the early chicken embryo. J. Mech. Behav. Biomed. Mater. 65, 383-397 (2017).
- Faisst, A. M., Alvarez-Bolado, G., Treichel, D., Gruss, P. Rotatin is a novel gene required for axial rotation and left-right specification in mouse embryos. Mech. Dev. 113, 15-28 (2002).
- Yalcin, H. C., Shekhar, A., Rane, A. a., Butcher, J. T. An ex-ovo chicken embryo culture system suitable for imaging and microsurgery applications. J. Vis. Exp. (44), (2010).
- Chapman, S. C., Collignon, J., Schoenwolf, G. C., Lumsden, A. Improved method for chick whole-embryo culture using a filter paper carrier. Dev. Dyn. 220, 284-289 (2001).
- Campàs, O., et al. Quantifying cell-generated mechanical forces within living embryonic tissues. Nat. Methods. 11, 183-189 (2014).
- Schierenberg, E., Junkersdorf, B. The role of eggshell and underlying vitelline membrane for normal pattern formation in the early C. elegans embryo. Roux’s Arch. Dev. Biol. 202, 10-16 (1992).
- Chuai, M., Weijer, C. J. The Mechanisms Underlying Primitive Streak Formation in the Chick Embryo. Current Topics in Developmental Biology. 81, 135-156 (2008).
- Voronov, D. A., Alford, P. W., Xu, G., Taber, L. A. The role of mechanical forces in dextral rotation during cardiac looping in the chick embryo. Dev. Biol. 272, 339-350 (2004).
- Raya, A., Izpisua Belmonte, J. C. Unveiling the establishment of left-right asymmetry in the chick embryo. Mechanisms of Development. 121, 1043-1054 (2004).
- Voronov, D. A., Taber, L. A. Cardiac looping in experimental conditions: Effects of extraembryonic forces. Dev. Dyn. 224, 413-421 (2002).
- Chen, Z., Huang, G., Trase, I., Han, X., Mei, Y. Mechanical Self-Assembly of a Strain-Engineered Flexible Layer: Wrinkling, Rolling, and Twisting. Phys. Rev. Appl. 5, (2016).
- Manner, J., Seidl, W., Steding, G. Formation of the cervical flexure: an experimental study on chick embryos. Acta Anat. (Basel). 152, 1-10 (1995).
- Ware, M., Schubert, F. R. Development of the early axon scaffold in the rostral brain of the chick embryo. J. Anat. 219, 203-216 (2011).
- Ramasubramanian, A., et al. On the role of intrinsic and extrinsic forces in early cardiac S-looping. Dev. Dyn. 242, 801-816 (2013).
- Hoyle, C., Brown, N. a., Wolpert, L. Development of left/right handedness in the chick heart. Development. 115, 1071-1078 (1992).
- Nerurkar, N. L., Ramasubramanian, A., Taber, L. A. Morphogenetic adaptation of the looping embryonic heart to altered mechanical loads. Dev. Dyn. 235, 1822-1829 (2006).
- Levin, M. Left-right asymmetry and the chick embryo. Semin. Cell Dev. Biol. 9, 67-76 (1998).
- Roebroek, A. J., et al. Failure of ventral closure and axial rotation in embryos lacking the proprotein convertase Furin. Development. 125, 4863-4876 (1998).
- Peebles, D. M., et al. Magnetic resonance proton spectroscopy and diffusion weighted imaging of chick embryo brain in ovo. Dev. Brain Res. 141, 101-107 (2003).
- Zhu, L., et al. Cerberus regulates left-right asymmetry of the embryonic head and heart. Curr. Biol. 9, 931-938 (1999).
Cite This Article
Li, Y., Grover, H., Dai, E., Yang, K., Chen, Z. Probing the Roles of Physical Forces in Early Chick Embryonic Morphogenesis. J. Vis. Exp. (136), e57150, doi:10.3791/57150 (2018).