Name: analyzing craniofacial morphogenesis in zebrafish using 4d confocal microscopy
Uploaded: 2014-01-30T14:00:00
Description: Watch this Scientific Journal Video about Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy at JoVE.com

我们感谢梅丽莎格里芬和詹娜Rozacky为他们的专家鱼护。 PDM感谢EGN写​​的援助，慷慨和忍耐。这项工作是由美国国立卫生研究院/ NIDCR R01DE020884到JKE支持。

1。畜牧突变等位基因<ol><li>提高和斑马鱼的繁殖所描述的21。 </li><li>在本研究中使用的斑马鱼突变体等位基因PDGFRA b1059 16，SMAD5 B1100 22，和SMO B577 23。来源这些斑马鱼品系包括ZIRC。 </li></ol> 2。解决方案及器具的制备注：所有的解决方案，并实现可预先和存储以供将来使用。 <ol><li>使胚胎培养基（EM），如前所述21。 </li><li>...

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时间推移共聚焦显微镜是一个强大的工具，用于发展的分析。在这里，我们验证了该方法的有效性学习咽弓形态中的斑马鱼突变体是采用转基因的标记的神经嵴细胞的重要信号通路。除了 ​​组织水平进行分析，时间流逝的分析也适用于分析在细胞尺度28。许多广泛使用的斑马鱼的方法也可以被掺入到时间推移显微镜实验，包括显微注射吗啉代的，mRNA表达，或转基因构建体，以及胚胎之?...

颅面形态发生是一个复杂的多步骤的过程，需要多种细胞类型之间协调的相互作用。大部分颅面骨骼是由神经嵴细胞来源，其中许多必须从背神经管迁移到被称为咽弓1瞬态结构。与许多组织中，颅面骨骼的形态是不是可以通过胚胎在发育的特定时间点的静态图像理解更复杂。虽然它是耗时的执行， 在体内时间推移显微镜提供了一个连续的外观在发育中的胚胎的细胞和组织。在延时系列中的每个图像借给上下文别人，并帮助调查员走向推断，为什么出现一种现象，而不是演绎什么是发生在那个时候。 体内成像因此是一个功能强大的工具，描述实验方法解构，引导形态的途径。斑马鱼斑马鱼是脊椎动物胚胎发育的一个流行的遗传模式，并特别适于在体内成像形态的。现代，便利的方法转基因和基因组修饰突飞猛进提供给斑马鱼研究工具的数量。这些工具提升已经非常强大的方法进行基因操纵和显微镜。在几乎任何所需的遗传背景在体内几乎所有组织的成像更接近现实比幻想。 咽弓的形态发生运动是由信号的神经嵴和邻近的上皮细胞，两者外胚层和内胚层之间的交互引导。但是也有一些必要的驱动的颅面骨骼元素的形态由上皮细胞表达大量的信号分子。在这些信号分子，刺猬（嘘）是非常重要的f或颅面部发育2-8。嘘是由口腔外胚层和内胚层咽2,6,9,10均表示。 Shh的中内胚层表达调节拱门10，神经嵴的拱门10内的图案，和颅面骨骼11的生长形态发生运动。 BMP信号也是颅面部发育12极为重要，可能会改变咽弓的形态。 BMP信号调节波峰的咽弓13,14内背/腹图案。 SMAD5在斑马鱼的破坏会导致严重的腭缺陷和麦氏软骨的破坏在15中线适当地融合。此外，该突变体也显示出降低和融合在腹侧软骨的元素，与第2 次 ，第3 次 ，有时稠的中线15第 4 次咽弓元素。这些融合强烈建议Bmp信号指示这些咽元素的形态。 PDGF信号是必要的颅面发育，但在咽弓形态发生未知的角色。小鼠和斑马鱼突变体PDGFRA有深刻的面中部的clefting 16-18。至少在斑马鱼这面中部的clefting是由于适当的神经嵴细胞迁移16的故障。神经嵴细胞继续表达PDGFRA他们已经进入了咽弓后。此外，PDGF配体是由面部上皮细胞表达和咽弓16,19,20内，从而PDGF信号也可以在下面的迁移咽弓的形态发生中发挥作用。然而，分析，并没有被执行在PDGFRA突变咽弓的形态发生。 在这里，我们展示 pharyngul的活体共焦显微镜一个阶段的转基因斑马鱼，并描述此期间内，咽弓的形态。我们进一步表明，受扰乱的BMP，PDGF和Shh信号通路的突变组织行为。

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			<td height="21" style="height:21px;width:243px;">6 lb. test monofilament line</td>
			<td style="width:189px;">Cortland Line Company</td>
			<td style="width:185px;">SLB16</td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Agarose I</td>
			<td style="width:189px;">Amresco</td>
			<td style="width:185px;">0710</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Argon laser</td>
			<td style="width:189px;">LASOS Lasertechnik GmbH</td>
			<td style="width:185px;">LGN 3001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Calcium chloride</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td style="width:185px;">C8106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Capillary tubing, 100 mm, 0.9 mm ID</td>
			<td style="width:189px;">FHC</td>
			<td style="width:185px;">30-31-0</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Clove oil</td>
			<td style="width:189px;">Hilltech Canada, Inc.</td>
			<td style="width:185px;">HB-102</td>
			<td></td>
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			<td height="21" style="height:21px;width:243px;">High vacuum grease</td>
			<td style="width:189px;">Dow Corning</td>
			<td style="width:185px;">2021846-0807</td>
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			<td height="21" style="height:21px;width:243px;">Isotemp dry-bath incubator</td>
			<td style="width:189px;">Fisher Scientific</td>
			<td style="width:185px;">2050FS</td>
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			<td height="21" style="height:21px;width:243px;">Laser scanning microscope</td>
			<td style="width:189px;">Carl Zeiss AG</td>
			<td style="width:185px;">LSM 710</td>
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			<td height="21" style="height:21px;width:243px;">Magnesium sulfate hexahydrate</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td style="width:185px;">230391</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Microscope cover glass, 22x22-1</td>
			<td style="width:189px;">Fisher Scientific</td>
			<td style="width:185px;">12-542-B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Microscope cover glass, 24x60-1</td>
			<td style="width:189px;">Fisher Scientific</td>
			<td style="width:185px;">12-545-M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Potassium chloride</td>
			<td style="width:189px;">Fisher Scientific</td>
			<td style="width:185px;">M-11321</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Potassium phosphate dibasic</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td style="width:185px;">P3786</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Sodium chloride</td>
			<td style="width:189px;">Fisher Scientific</td>
			<td style="width:185px;">M-11624</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Sodium phosphate dibasic</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td style="width:185px;">S7907</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">TempController 2000-2</td>
			<td style="width:189px;">PeCon GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Tricaine-S</td>
			<td style="width:189px;">Western Chemical, Inc.</td>
			<td style="width:185px;"></td>
			<td></td>
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</table>

analyzing craniofacial morphogenesis in zebrafish using 4d confocal microscopy

延时成像是一种技术，它允许直接观察形态发生的过程中，或形状的生成。由于它们的光学清晰度和顺从遗传操作，斑马鱼的胚胎已经成为一种流行的模式生物与在活胚胎进行形态的延时分析。一个活斑马鱼胚胎的共焦成像要求的感兴趣组织的持续标记有荧光标记物，如转基因或注射染料。该过程要求的胚胎被麻醉并在这里以这样一种方式，健康发展继续正常进行。成像参数必须设置为占三维增长和平衡解决单个细胞，而得到发展的快速快照的需求。我们的研究结果表明，以荧光标记的斑马鱼胚胎的体内成像进行长期检测在不同组织行为的能力颅神经嵴造成颅面畸形。造成麻醉和安装发育迟缓是微乎其微的，而且胚胎是安然无恙的过程。时移成像胚胎可以在以后的发展点返回到液体培养基中，随后成像或固定。随着转基因斑马鱼线和良好的特点命运映射和移植技术，成像越来越丰盈任何需要的组织是可能的。因此，时间推移的体内成像结合有力地与斑马鱼基因的方法，包括突变体和显微注射的胚胎的分析。

在野生型胚胎中，神经嵴人口下面，沿前/后和背/腹轴咽弓伸长移动时在一个方向喙（ 电影1）。在30小时后，受精（HPF），第一咽弓的前/后长度为1.8-1.9倍，其背/腹高度之间。背/腹伸长率稳步前进，比前/后延长至36.5 HPF更快。从这里，背/腹高高原各地的104微米至48 HPF。前/后继续延长至48个高倍视野，从低于1.5倍的背/腹高度36.5 HPF至1.95倍的背/腹高度在48 HPF整体前/后长度增加。 <p c...

Watch this Scientific Journal Video about Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy at JoVE.com

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

时间推移共焦成像是一个强大的技术表征胚胎发育有益。在这里，我们描述的方法和表征颅面形态发生在野生型，以及PDGFRA，Smad5和与SMO突变体胚胎。

使用4D共聚焦显微镜分析颅面形态的斑马鱼

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...

Research

JoVE Journal

Biology

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

双光子现场斑马鱼的胚胎干切断和时间推移共焦成像

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

科研

生物学

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

现场共聚焦显微镜对斑马鱼的胚胎脑成像

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Electroporation of Craniofacial Mesenchyme

颅面间质细胞的电

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

基于荧光测定活细胞中的钙进入商店经营：从培养的肿瘤细胞，骨骼肌纤维

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

颅面部发育中的SOX10可视化：使用枫转基因斑马鱼线时间推移共聚焦显微镜

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, ...

Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy

同时进行pH测量的内吞和胞车厢中使用共焦显微镜活细胞

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported1. Regulation of cellular pH is crucial ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

在造血干细胞和祖细胞利用共聚焦和多光子显微镜五荧光蛋白标记的活体克隆跟踪

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

4D Microscopy of Yeast

酵母的4D显微镜

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments ...

Visualizing Lymph Node Structure and Cellular Localization using Ex-Vivo Confocal Microscopy

使用前 Vivo 共聚焦显微镜可视化淋巴节点结构和细胞定位

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are ...

Analyzing the &alpha;-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy

Analyzing the &#945;-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy

利用单分子定位显微镜分析人类 iPSC 衍生肌细胞中的α-Actinin 网络

The maturation of iPSC-derived cardiomyocytes is a critical issue for their application in regenerative therapy, drug testing and disease modeling. ...