电穿孔轴突轨迹跟踪和突触目标的后脑在鸡胚
Summary
神经网络是建立在胚胎大脑发育神经生物学的一个基本问题。在这里，我们结合电穿孔技术与新的遗传工具，如酶Cre / LOX-质粒和PiggyBac转介导的DNA换位系统在禽流后脑背中间贴上标签，并跟踪他们的轴突预测和突触在不同发育阶段的目标。
Abstract
鸡胚神经管电具有许多优点，如快速，高效的成神经细胞的外源基因表达。在这篇稿件中，我们提供了一种方法，展示了独特的电穿孔DNA禽流后脑E2.75为了明确标示神经祖细胞的一个子集，以及如何按照他们的轴突预测和突触的目标非常先进的发展阶段， E14.5。我们利用新的遗传工具，包括特异性增强子元件，酶Cre /液氧 – 基于质粒和PiggyBac转介导的DNA换位后脑细胞的一种亚型（最背的分组的interneurons，DA1）系统来驱动GFP表达。随后在早期和晚期胚胎阶段，在不同的脑干区域的轴突轨迹和DA1轴突的目标。这种策略有助于先进的技术，针对感兴趣的细胞在胚胎后脑TRA庆安电路形成多个发展阶段。
Introduction
后脑是一个关键的中继枢纽神经系统之间的通信中枢和周围神经系统神经网络通过递增和递减。它调节的基本功能，包括呼吸，意识，听觉和运动协调1-3。在早期胚胎发育过程中，脊椎动物的后脑瞬时细分成重复菱沿其前后轴（AP），不同的神经细胞类型的形成，并产生多个脑干中心4。后脑也被划分成基底和鼻翼板，在指定离散神经祖细胞变得和区分不同的DV地点3,5,6沿背腹轴（DV）。怎样早期AP和DV特定的神经元模式的脑干功能设计电路的设立在很大程度上是未知。
为了获得这个基本知识的问题，工具都需要以特定亚群的神经元在早期后脑贴上标签，以跟踪它们的轴突轨迹和连接更高级的阶段。我们先前已经利用特异性增强子元件，和一个CRE / LoxP位条件的表达背脊髓在早期鸡胚7-9的轴突轨迹跟踪系统。在当前的手稿中，我们有针对性的后脑和标记后期胚胎后脑的interneurons，轴突和其突触目标，使用修改后的电战略和PiggyBac转 – 介导DNA换位的升级实验范式。我们的新战略，可以在一侧的后脑和他们的的轴突预测和突触位点不同的胚胎阶段的跟踪标记不同的神经元亚型，从2至12天电。基于这种方法，我们贴上了最背的分组后脑的interneurons（dA1/Atoh1 + </s>细胞），并揭示了两个对侧上升轴突投射图案，分别来自不同AP的位置和拉长了一个独特的索。 DA1轴突被发现在听觉核，中脑，小脑10多层的项目，并形成突触。
小鸡电的结合，遗传神经元和先进得多的发展阶段分析预测网站跟踪研究在大脑中形成的神经网络，并阐明控制电路形成的分子机制，提供一个独特的平台。
Protocol
Representative Results
Discussion
OVO中电是一个可行的，可靠的，有效的工具来检查细胞规范和轴突指导在鸡胚神经系统的发展20。在这个协议中，我们描述了一个模式把小鸡后脑电E2.75使用增强子元件，使有条件的标签特定的interneurons。这一战略的结合换位系统PiggyBac转介导外源基因插入到鸡基因组，这使跟踪的的轴突航线，预测和突触网站在胚胎发育晚期。
以前的方法标签轴突/把小鸡?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Materials
| Name of Reagent/Equipment | Company | Catalogue Number |
| L-shaped gold Genetrodes 3 mm electrodes | BTX, Harvard Apparatus | 45-0162 |
| pulse generator, ECM 830 | BTX, Harvard Apparatus | 45-0002 |
| OCT (Optimal Cutting Temperature) Compound | Tissue-Tek Sakura | 4583 O.C.T. Compound |
| Nail Polish | From Any Commercial Supplier |
References
- Altman, J., Bayer, S. A. Development of the precerebellar nuclei in the rat: II. The intramural olivary migratory stream and the neurogenetic organization of the inferior olive. J. Comp. Neurol. 257, 490-512 (1987).
- Rose, M. F., Ahmad, K. A., Thaller, C., Zoghbi, H. Y. Excitatory neurons of the proprioceptive, interoceptive, and arousal hindbrain networks share a developmental requirement for Math1. Proc. Natl. Acad. Sci. U.S.A. 106, 22462-22467 (2009).
- Storm, R., et al. The bHLH transcription factor Olig3 marks the dorsal neuroepithelium of the hindbrain and is essential for the development of brainstem nuclei. Development. 136, 295-305 (2009).
- Lumsden, A. The cellular basis of segmentation in the developing hindbrain. Trends Neurosci. 13, 329-335 (1990).
- Liu, Z., et al. Control of precerebellar neuron development by Olig3 bHLH transcription factor. J. Neurosci. 28, 10124-10133 (2008).
- Muller, T., et al. The bHLH factor Olig3 coordinates the specification of dorsal neurons in the spinal cord. Genes Dev. 19, 733-743 (2005).
- Avraham, O., et al. Motor and dorsal root ganglion axons serve as choice points for the ipsilateral turning of dI3 axons. J. Neurosci. 30, 15546-15557 (2010).
- Avraham, O., et al. Transcriptional control of axonal guidance and sorting in dorsal interneurons by the Lim-HD proteins Lhx9 and Lhx1. Neural Dev. 4, 21 (2009).
- Avraham, O., Zisman, S., Hadas, Y., Vald, L., Klar, A. Deciphering axonal pathways of genetically defined groups of neurons in the chick neural tube utilizing in ovo electroporation. J. Vis. Exp. (39), e1792 (2010).
- Kohl, A., Hadas, Y., Klar, A., Sela-Donenfeld, D. Axonal Patterns and Targets of dA1 Interneurons in the Chick Hindbrain. J. Neurosci. 32, 5757-5771 (2012).
- Vogel, J., Mobius, C., Kuschinsky, W. Early delineation of ischemic tissue in rat brain cryosections by high-contrast staining. Stroke. 30, 1134-1141 (1999).
- Helms, A. W., Abney, A. L., Ben-Arie, N., Zoghbi, H. Y., Johnson, J. E. Autoregulation and multiple enhancers control Math1 expression in the developing nervous system. Development. 127, 1185-1196 (2000).
- Lumpkin, E. A., et al. Math1-driven GFP expression in the developing nervous system of transgenic mice. Gene Expr. Patterns. 3, 389-395 (2003).
- Lu, Y., Lin, C., Wang, X. PiggyBac transgenic strategies in the developing chicken spinal cord. Nucleic Acids Res. 37, e141 (2009).
- Wang, J., et al. piggyBac-like elements in the pink bollworm, Pectinophora gossypiella. Insect Mol. Biol. 19, 177-184 (2010).
- Alsina, B., Vu, T., Cohen-Cory, S. Visualizing synapse formation in arborizing optic axons in vivo: dynamics and modulation by BDNF. Nat. Neurosci. 4, 1093-1101 (2001).
- Leal-Ortiz, S., et al. Piccolo modulation of Synapsin1a dynamics regulates synaptic vesicle exocytosis. J. Cell Biol. 181, 831-846 (2008).
- Gardzinski, P., et al. The role of synaptotagmin I C2A calcium-binding domain in synaptic vesicle clustering during synapse formation. J. Physiol. 581, 75-90 (2007).
- Nowack, A., Yao, J., Custer, K. L., Bajjalieh, S. M. SV2 regulates neurotransmitter release via multiple mechanisms. Am. J. Physiol. Cell Physiol. 299, C960-C967 (2010).
- Itasaki, N., Sharpe, J., Morrison, A., Krumlauf, R. Reprogramming Hox expression in the vertebrate hindbrain: influence of paraxial mesoderm and rhombomere transposition. Neuron. 16, 487-500 (1996).
- Clarke, J. D., Lumsden, A. Segmental repetition of neuronal phenotype sets in the chick embryo hindbrain. Development. 118, 151-162 (1993).
- Diaz, C., Glover, J. C., Puelles, L., Bjaalie, J. G. The relationship between hodological and cytoarchitectonic organization in the vestibular complex of the 11-day chicken embryo. J. Comp. Neurol. 457, 87-105 (2003).
- Marin, F., Puelles, L. Morphological fate of rhombomeres in quail/chick chimeras: a segmental analysis of hindbrain nuclei. Eur. J. Neurosci. 7, 1714-1738 (1995).
- Niwa, H., Yamamura, K., Miyazaki, J. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene. 108, 193-199 (1991).
