Name: creating avian forebrain chimeras to assess facial development
Uploaded: 2021-02-18T13:00:00
Description: Watch this Scientific Journal Video about Creating Avian Forebrain Chimeras to Assess Facial Development at JoVE.com

本出版物中报告的研究得到了美国国立卫生研究院国家牙科和颅面研究所的支持，奖励号为R01DE019648，R01DE018234和R01DE019638。

将白北京鸭（Anas platyrhynchos），白里格霍恩鸡（Gallus gallus）和日本鹌鹑（Cortunix coturnix japonica）在37°C的加湿室中孵育，直到在HH7/ 8 17进行阶段匹配。
1. 准备供体组织
注意：试剂和工具的制备以及如何打开鸡蛋进行实验操作已经描述6.
<ol><li>用中性红色、玻璃转移移液器和锋利的钨针制备 DMEM 培养...

<ol>
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所描述的方法允许检查基底前脑和相邻外胚层之间的组织相互作用。这种方法与以前的前脑移植方法不同，因为供体组织仅限于腹侧前脑。这消除了神经嵴细胞的移植，神经嵴细胞已被证明参与面部形态的图案化9，10。因此，将移植物限制在基底前脑对于评估计划的实验结果至关重要。 
在先前采用前脑移植的研究中13...

许多当代发育生物学研究都集中在基因在塑造胚胎中的作用。有很好的工具可以从遗传学的角度检查发育机制。然而，胚胎被组装并响应组织相互作用而发生形态发生。禽系统是用于评估调节发育的各种组织相互作用的经典工具，原因如下：胚胎学很好理解，胚胎很容易获得，分析鸟类系统的工具发达，胚胎便宜。
近一个世纪以来，禽类移植系统已被广泛用于谱系追踪和评估发育过程中的组织相互作用1，2，3，4。该系统用于研究调节上颌形态发生的信号中心，即额鼻外胚层区（FEZ），并发布了一段视频，描述了该技术6。除了鹌鹑雏，其他物种也被用来产生嵌合体，用于分析组织相互作用。例如，小鼠FEZ是从野生7型和突变小鼠8移植而来的，其他人使用鸭，鹌鹑和小鸡系统来评估神经嵴在面部骨骼图案化中的作用9，10，11，12。
在这项工作中，通过在鹌鹑、鸭和雏鸡胚胎中相互移植腹侧前脑，评估了前脑在调节自由经济区基因表达模式中的作用，因为需要来自前脑的信号来诱导声波刺猬在自由经济区中的表达。前脑移植在该领域并非独一无二。这些移植用于评估鹌鹑和鸭胚胎的运动发育13，尽管在这些实验中也移植了有助于非神经衍生物的组织。在其他工作中，鸟类的听觉回路已经通过前脑移植14进行评估，但这些移植含有推定的神经嵴细胞，这些细胞有助于面部形状9，10 并参与调节FEZ15中的SHH表达。因此，设计了一种在神经管闭合之前仅将腹侧前脑从一种鸟类移植到另一种鸟类的系统，以评估大脑在面部形状中的作用16 （图1A，B）。这种方法没有移植物的神经嵴污染。本文对该方法进行了说明，描述了预期结果，并讨论了面临的挑战。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1x PBS</td>
			<td>TEK</td>
			<td>TEKZR114</td>
			<td></td>
		</tr>
		<tr>
			<td>35x10 mm Petri dish</td>
			<td>Falcon</td>
			<td>1008</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermofisher</td>
			<td>11965084</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>Fine Science Tools</td>
			<td>26016-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutral Red</td>
			<td>Sigma</td>
			<td>553-24-2</td>
			<td></td>
		</tr>
		<tr>
			<td>No. 5 Dumont forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur Pipets</td>
			<td>Thermofisher</td>
			<td>13-678-6B</td>
			<td></td>
		</tr>
		<tr>
			<td>QCPN antibody</td>
			<td>Developmental Studies Hybridoma bank, Iowa University, Iowa, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Fine Science Tools</td>
			<td>14058-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Tungsten Needle</td>
			<td>Fine Science Tools</td>
			<td>26000</td>
			<td></td>
		</tr>
	</tbody>
</table>

creating avian forebrain chimeras to assess facial development

一个多世纪以来，鸟类胚胎一直被用作模型系统，并导致了对脊椎动物发育的基本理解。该模型系统的优势之一是可以直接在嵌合胚胎中评估组织的影响和相互作用。我们之前已经表明，来自前脑的信号通过调节额鼻外胚层区（FEZ）中声波刺猬（SHH）的表达域的形状来促进面部形态发生。在本文中，描述了产生前脑嵌合体的方法，并提供了这些实验结果的说明。

嵌合体和移植污染的评估 为了评估嵌合体，应解决嵌合和移植物与其他细胞类型的污染程度。通过将鹌鹑组织移植到雏鸡胚胎中来创建嵌合体允许这种类型的分析。使用QCPN抗体可以可视化鹌鹑细胞并将其与宿主组织区分开来（图1 C，D）。在这种情况下，只有来自腹侧前脑的组织被抗体染色，表明移植物没有被其他细胞类型（包括神经嵴）污染<em...

Watch this Scientific Journal Video about Creating Avian Forebrain Chimeras to Assess Facial Development at JoVE.com

Creating Avian Forebrain Chimeras to Assess Facial Development

本文介绍了一种组织移植技术，旨在测试颅面发育过程中基底前脑的信号传导和模式特性。

创建鸟类前脑嵌合体以评估面部发育

The avian embryo has been used as a model system for more than a century and has led to fundamental understanding of vertebrate development. One of the ...

这种方法允许研究人员评估基底前脑和面部之间的组织相互作用。该技术的主要优点是它可以分离基底前脑进行评估，并防止神经嵴细胞污染，这会混淆分析。该技术的困难部分是从宿主中取出前脑并用供体组织替换。演示该程序的将是来自Ralph Marcucio博士实验室的研究科学家Diane Hu。首先，用中性红色、玻璃转移移液器和锋利的钨制备 DMEM 培养基。使用 10 毫升注射器和 18 号针头，从蛋壳的尖端取出 0.5 毫升白蛋白。用剪刀尖在壳的顶部开一个小孔。然后将一块胶带放在孔上，并切开一个圆形开口以暴露胚胎。用中性红色染色胚胎。从第七或第八阶段胚胎的基底前脑左侧收获组织移植物。使用弯曲的锋利钨针轻轻切开前脑的一部分。为了不包括下面的内胚层，将针头滑到前脑下方并平行于神经管的轴。使用玻璃移液管从供体胚胎中取出移植物，并将其转移到含有中性红色的DMEM中两分钟以使其染色。然后将染色的移植物放入不含中性红色的DMEM中，直到准备植入。将白勒霍恩鸡的受精卵在 37 摄氏度的加湿室中孵化，直到汉堡-汉密尔顿第 7 至 8 阶段。如前所述暴露胚胎。使用锋利的钨针，通过切割然后去除0.3×0.2毫米的基底前脑片来准备移植部位以容纳移植物。避免过度破坏底层内胚层，这将很明显，因为蛋黄颗粒将开始通过任何撕裂泄漏。将移植物转移到宿主后，放置移植物以替换已切除的宿主基底前脑。将胶带紧紧地贴在孔上，然后将胚胎放回37摄氏度的培养箱中，直到它们准备好进行分析。最初，嵌合体是通过将鹌鹑组织移植到雏鸡胚胎中而产生的。使用QCPN抗体，可视化鹌鹑细胞并将其与宿主组织区分开来。鹌鹑雏系统证实，所有移植物仅由神经组织组成，没有被其他细胞类型污染。将鹌鹑组织移植到鸭胚胎中，由于鹌鹑大脑发育较快，导致嵌合体严重畸形。因此，将鸭组织移植到鸡胚胎中。由此产生的鸭雏嵌合体表明大脑参与调节形态。采用全山C2杂交法评估声波刺猬在嵌合体中的表达情况。与形态相似，嵌合体鸭侧的索尼克刺猬表情看起来更像鸭子。准备主机站点需要练习。大量的胚胎不能存活，因此创建大量的嵌合体可以帮助确保适当的样品进行分析。这种方法使得评估来自大脑的信号如何塑造额鼻外胚层区域的索尼克刺猬表达域成为可能。

Research

JoVE Journal

Developmental Biology

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

优化<em&gt;前OVO</em&gt;鸡胚培养，以发展的高级阶段

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos ...

科研

发育生物学

Immunostaining to Visualize Murine Enteric Nervous System Development

免疫组化可视化小鼠肠神经系统的发展

The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Following colonization, neural crest cells must ...

Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development

采用轻质板材荧光显微镜到图像斑马鱼眼睛发育

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM ...

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells

人诱导多能干细胞的前脑型脑 Organoids 标准化和可再生性的生成

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. ...

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

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 ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Microinjection-based System for In Vivo Implantation of Embryonic Cardiomyocytes in the Avian Embryo

基于微注射的免疫胚胎心肌细胞体内植入系统

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a ...

Fast and Efficient Expression of Multiple Proteins in Avian Embryos Using mRNA Electroporation

使用mRNA电穿孔快速有效地表达Avian胚胎中的多种蛋白质

We report that mRNA electroporation permits fluorescent proteins to label cells in living quail embryos more quickly and broadly than DNA electroporation. ...

A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult

A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult

一种直接而简单的方法，用于评估从胚胎到成人的果蝇黑色素的生存能力

In Drosophila melanogaster, viability assays are used to determine the fitness of certain genetic backgrounds. Allelic variations can result in partial or ...

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

斑马鱼胚胎全山原位杂交和管状测定在iPSC-ECs中研究恩多林在血管发育中的作用

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...