我们感谢伯纳德 Kulemaka 和史密斯实验室的成员为他们提供有用的评论和试剂指导, Sam 康奈尔和布伦特. 雷德福3i 的成像问题和黛博拉砰, 凯伦注意和凯斯图尔特为斑马鱼护理。这项工作得到了圣母大学、伊丽莎白和迈克尔？加拉格尔家族的支持, 在巴黎圣母院, 圣母大学斑马鱼研究中心, 圣母大学的干细胞和再生医学中心。圣母.所有动物研究都是按照圣母大学 IACUC 博士的要求进行的。

所有动物研究都是由圣母大学机构动物保育和使用委员会批准的。
1. 斑马鱼标本的制备
<ol><li>将一个成年男性和一个成年女性 Tg (可转换蛋白) 放入每个标准过程的交配室13。在这篇手稿中, 使用 Tg (sox10: eos) 鱼9由于访问, 但其他转基因线与 photoconvertible 蛋白可以同样使用。设置一个以上的房间, 以防鱼不下蛋。允许鱼一夜之间留在房间里。</li>
	<li>第二天早上, 在100毫米培养皿中收集鸡蛋。在筛查前允许卵成熟到24小时后受精 (hpf)。</li>
	<li>如果上面的动物是杂合子的转基因, 屏幕 24 hpf 菜肴为 Tg (sox10: eos) + 胚胎与 488 nm 光源和 GFP 过滤器集在解剖显微镜。分离 Tg (sox10: eos) + 胚胎, 并允许成熟到 48 hpf。</li>
	<li>用针或镊子手动 Dechorionate 胚胎。</li>
	<li>准备和微波5毫升的0.8% 低熔点琼脂糖溶液。<ol>
<li>一旦琼脂糖是凉爽的触摸, 地方3-4 麻醉 Tg (sox10: eos) + 48 hpf 鱼的中心10毫米玻璃盖玻片底部培养皿。</li>
		<li>添加足够的琼脂糖, 以覆盖盖玻片的表面, 大约1毫升。用探针针在它们的两侧排列鱼。允许琼脂糖固化以确保动物的安装。可能需要不断重新安排斑马鱼, 直到琼脂固化14。凝固需要大约2分钟。</li>
	</ol>
</li>
	<li>琼脂糖凝固2分钟后, 慢慢加入含有0.02% 苯甲酸酸酯 (Tricaine) 的胚培养基, 直到琼脂和盘子的底面被淹没。这应该是大约3mL。</li>
</ol>2. 显微镜安装和预转换成像
<ol><li>打开共焦软件并选择捕获和焦点窗口 [图 1]。在 "捕获" 窗口下, 选择 "捕获设置" 下拉选项卡下的实验室特定的转换成像设置 [图 1]。在这里, 实验室特定的设置称为 "鱼映像"。</li>
	<li>将标本放在共聚焦范围内, 使用过程和微调旋钮将试样聚焦。</li>
	<li>打开焦点窗口, 找到所需的感兴趣区域 (即背根神经节)。</li>
	<li>在 "过滤器集" 菜单下选择 c488 激光器。将曝光率设置为300毫秒, 激光功率为 5, 并增强为 75 [图 2]。</li>
	<li>选中捕获类型部分下的3D 框。在3D 捕获部分中, 选择使用当前位置并在当前周围检查范围。在同一节中, 将范围设置为 35, 将平面数设为 36, 并将步长调整为1。范围可以增加或减少, 以适应成像区域的深度。对于脊髓, 35-40 栈之间的范围值通常是足够的 [图 5]。</li>
	<li>选择当前位置 [图 5]。</li>
	<li>单击 "捕获" 窗口底部的 "开始" 以获取图像。</li>
</ol>3. 单细胞 Photoconversion
<ol><li>打开共焦软件并选择捕获和焦点窗口 [图 1]。在 "捕获" 窗口下, 选择 "捕获设置" 下拉选项卡下的实验室特定的转换成像设置 [图 1]。在这里, 实验室特定的设置称为 "鱼消融全芯片。</li>
	<li>在 "过滤器集" 菜单下选择 c488 和 c541 激光器。如果不使用相同的显微镜软件, 找到菜单选择不同的激光器, 并选择 488 nm 和 541 nm 激光器。将曝光设置为300毫秒, 激光功率为 5, 并增强为 75 [图 2]。这些激光设置是根据产生足够的荧光信号而不引起漂白或毒性而选择的。如果毒性或漂白可视化, 减少激光功率或曝光。</li>
	<li>打开焦点窗口, 然后单击 "焦点" 窗口中的 "photomanipulation" 选项卡。相应地调整激光参数。将激光堆栈电源更改为 2, 然后单击转到。将光栅块大小更改为 1, 然后单击设置。请将双击大小更改为4。将激光线更改为 v405 [图 3]。</li>
	<li>在 "捕获" 窗口中打开高级捕获设置。选择 "photomanipulation" 选项卡, 然后将双击重复次数更改为2。单击 "确定" (图 4)。</li>
	<li>在焦点窗口中选择 XY 选项卡。双击 photomanipulation 选项卡中的步骤2中的激光参数 [图 3,图 4]。设置激光设置, 以 photoconvert 细胞的兴趣, 没有 photoconversion 的周围细胞。<ol>
<li>如果相邻细胞 photoconversion 存在, 则减少激光功率。如果 photoconversion 细胞不发生, 激光功率可以增加。优选地, 设置激光功率 photoconvert 仅感兴趣的区域和不附近区域。</li>
	</ol>
</li>
	<li>选中捕获类型下的 timelapse 框。然后, 单击开始[图 6A]。</li>
	<li>打开 "实时 timelapse" 窗口后, 选择顶部工具栏上的 "圆圈" 工具 [图 7]</li>
	<li>在单元格的靠近中心区域中绘制一个圆。右键单击绘制的圆圈, 选择酶区域, 然后等待3秒。选定区域应变暗 [图 8]。单击停止捕获。</li>
	<li>如果成像多个动物, 请返回焦点菜单中的 XY 选项卡, 然后选择 "位置 2"。然后, 重复步骤 4.1-4.6。</li>
	<li>要转换单元格的数量, 请按照步骤 4.1-4.4 中的协议和激光参数进行操作, 但不要绘制一个圆来酶感兴趣的区域, 请使用 "线条" 工具。在感兴趣的区域上绘制一条线, 并使用上面列出的相同参数酶区域。</li>
</ol>4. photoconversion 后成像
<ol><li>一旦所有的点都 photoconverted。选择 precoversion 映像步骤3中描述的实验室特定的标准图像堆栈设置。这是在捕获窗口的 "捕获设置" 下拉选项卡下找到的 [图 5]。</li>
	<li>选择 c488 激光器并将曝光设置为 300ms, 激光功率为 5, 并加强到 75 [图 2]。</li>
	<li>选择与 c488 激光器在同一菜单下发现的 c541 激光器。将曝光率设置为500毫秒, 激光功率为 10, 并增强为 75 [图 9]。</li>
	<li>选中捕获类型部分下的3D 框。在3D 捕获部分中, 选择使用当前位置并检查当前范围。在同一节中, 将范围设置为 35, 将平面数设为 36, 并将步长调整为1。范围数可能会增加或减小以适应所需的图像深度 [图 5]。</li>
	<li>如果 "XY 焦点" 选项卡中有多个点, 请在 "捕获" 窗口中选择 "多位列表" 选项。如果没有, 请选择 "当前位置"。</li>
	<li>单击 "捕获" 窗口底部的 "开始" 以获取图像。</li>
</ol>

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作者没有什么可透露的。

在复杂组织中, 不同的细胞类型组织成特定的领域。最近的技术已经被用来标记这些大型组织结构中的单个细胞1,2,3。在这里, 我们演示了两种技术, 同样可以用来可视化的单细胞相互作用和细胞群的相互作用, 在复杂的组织。photoconversion 技术的优势在于, 科学家必须对细胞进行清晰的标记。借助显微镜, 它能够在较大的成像窗口...

多个不同的细胞相互作用, 建立和维持复杂的动物和植物组织。这些细胞往往是夹层和难以区分与邻居在一个单一的细胞水平没有高分辨率显微镜, 需要固定的组织。然而, 要了解这些组织是如何形成的, 如何保持, 并成为病态, 就必须研究组织内的单个细胞在一段时间内是如何相互作用的。理想情况下, 这些实验要求在不需要固定的情况下, 用非侵入性的方式对组织内的单个细胞进行标记。科学家们现在已经开发了许多技术来完成这个任务1,2,3,4。
水母绿色荧光蛋白 (GFP) 的发现和实施是一种令人兴奋的方法, 允许在组织环境1中标记不同的细胞。使用特定于单元格的启动器, 可以从遗传学上选择标记为1的单元格子集。或者, 病毒诱导的 gfp 表达可以用于 gfp3,4的用户选择的表达式。虽然很有用, 基因介导的 GFP 表达不允许用户选择的表达在组织中的细胞子集;和病毒表达的 GFP, 虽然有利, 可侵入。随着 GFP 衍生物的出现和像 Brainbow 这样的巧妙的技术, 在组织中表达出不同的荧光蛋白更稀少, 它已经成为可能的可视化单个细胞和它们之间的相互作用在复杂的组织2,5. 但是, 这些方法以随机方式标记单元格。如果所需的实验需要对实验者定义的单个细胞或细胞的数量进行可视化, 那么它们是有限的。通过这样的实验 , 有一个基因表达的荧光蛋白 , 可以纵 , 以区别 , 在一个单一的细胞的方式 , 它从其他荧光和非荧光细胞 , 这将是有利的。
为了实现这一目标, 并可视化一个复杂的活体组织内单细胞生物学, 科学界使用的单一细胞 photoconversion 的独特的荧光蛋白6,7,8。使用 photoconvertible 蛋白的基因控制表达 (即, eos, 枫等), 当暴露于紫外线 (488 nm) 光时, 从绿色到红色荧光状态过渡, 我们可以区分单个细胞与它的荧光标记邻居6,7,8。这种方法利用连接到我们共焦显微镜的仪器, 它可以将激光栈中的光定向到感兴趣的衍射有限区域。使用此技术, 我们可以用用户定义的方式 (9、10、11) 来标记单个单元格或更大的填充。这项技术是微创与单细胞注射的病毒 GFP。作为概念的证明, 我们表明, 我们可以 photoconvert 在神经节内的单个细胞在周围神经系统和 photoconvert 更大的人群, 如位于脊髓腹侧的细胞9,10, 11,12。然后, 我们可以想象这些 photoconverted 细胞群24小时后, 以了解他们的运动和分化过程中的发展。

<table border="0" cellpadding="0" cellspacing="0" width="1846">
	<tbody>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt;width:320pt" width="427">Tg(sox10:eos) zebrafish animals</td>
			<td style="width:125pt" width="167"></td>
			<td style="width:85pt" width="113"></td>
			<td style="width:854pt" width="1139">Fish were obtained and crossed from the fish facility at the University of Notre Dame</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">100 X 15 mm petri dish</td>
			<td>VWR</td>
			<td>25384-302</td>
			<td></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">Embryo medium</td>
			<td></td>
			<td></td>
			<td>Embryo medium is made weekly and provided by the fish facility at the University of Notre Dame containing 5L RO water, 30uL methylene blue, and 200mL salt stock.</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">0.8% Low Melting Point Agarose</td>
			<td>dot scientific inc</td>
			<td>9012-36-6</td>
			<td></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">35 X 10 mm glass-coverslip bottom petri dish</td>
			<td>Ted Pella Inc.</td>
			<td>14021-20</td>
			<td></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">Needle dissecting probe</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">0.002% 3-aminobenzoic acid ester (Tricaine)</td>
			<td>Fluka analytical</td>
			<td>A5040-250G</td>
			<td></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">Fluorescent Dissecting Microscope with GFP filters</td>
			<td>Zeiss Axiozoom</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">Confocal microscope with lasers to excite GFP and RFP filter sets</td>
			<td>3i spinning disk confocal</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">UV light source (laser) for photoconversion</td>
			<td>405 nm laser</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">Slidebook software</td>
			<td>3i</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">Methylene blue</td>
			<td>Kordon</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

single-cell photoconversion in living intact zebrafish

动物和植物组织由细胞的不同的人口组成。这些细胞随着时间的推移相互作用, 以建立和维持组织, 并可能导致疾病时中断。科学家们通过在细胞子集中表达荧光蛋白, 开发出巧妙的技术来研究这些细胞在完整组织中的特性和自然动力学。然而, 有时, 实验需要更多的细胞在组织内的可视化, 有时候是在单细胞或细胞群的方式。为了实现这一目标, 并在细胞中显示单个细胞, 科学家们利用了荧光蛋白的单细胞 photoconversion。为了演示这项技术, 我们在这里展示了如何将紫外线照射到一个在完整的、活的斑马鱼身上表达出感兴趣的 Eos 细胞。然后, 我们在24小时后对那些 photoconverted Eos+单元格进行映像, 以确定它们在组织中的变化。我们描述了两种技术: 单细胞 photoconversion 和 photoconversions 的细胞群。这些技术可用于可视化细胞相互作用, 细胞命运和分化, 细胞迁移, 使其成为一种技术, 适用于许多生物学问题。

Photoconversion 荧光蛋白可用于标记组织中的不同细胞6。为了证明这一点, Tg (sox10: eos)鱼9被用来表达 photoconvertible 蛋白 eos 下的调节序列sox10。Tg (sox10: eos)动物在 48 hpf 第一次安装, 然后成像, 以检测任何可能发生的非特定 photoconversion。然后, 将 eos 未转换的荧光信号与小 Eos photoconverted 荧光信号进行可视化 (<strong class="xfig...

Watch this Scientific Journal Video about Single-cell Photoconversion in Living Intact Zebrafish at JoVE.com

Single-cell Photoconversion in Living Intact Zebrafish

在这里, 我们提出了一个协议, 以显示如何实现细胞 photoconversion 是通过紫外线照射到特定区域表达荧光蛋白, Eos, 在活体动物。

活完好斑马鱼的单细胞 Photoconversion

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

single-cell-photoconversion-in-living-intact-zebrafish

Research

JoVE Journal

Developmental Biology

5.9K 次观看.  University of Notre Dame. 在这里, 我们提出了一个协议, 以显示如何实现细胞 photoconversion 是通过紫外线照射到特定区域表达荧光蛋白, Eos, 在活体动物。

视频: Single-cell Photoconversion in Living Intact Zebrafish

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion (FDAP)

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological systems. In FDAP experiments, the clearance of proteins within living organisms can be measured. A protein of interest is tagged with a photoconvertible fluorescent protein, expressed in vivo and photoconverted, and the decrease in the photoconverted signal over time is monitored. The data is then fitted with an appropriate clearance model to determine the protein half-life. Importantly, the clearance kinetics of protein populations in different compartments of the organism can be examined separately by applying compartmental masks. This approach has been used to determine the intra- and extracellular half-lives of secreted signaling proteins during zebrafish development. Here, we describe a protocol for FDAP experiments in zebrafish embryos. It should be possible to use FDAP to determine the clearance kinetics of any taggable protein in any optically accessible organism.

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion FDAP

Protein levels in cells and tissues are often tightly regulated by the balance of protein production and clearance. Using Fluorescence Decay After Photoconversion (FDAP), the clearance kinetics of proteins can be experimentally measured in vivo.

测量生活中的斑马鱼胚胎蛋白质稳定性使用荧光衰减光转化后（FDAP）

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological ...

科研

发育生物学

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Imaging Subcellular Structures in the Living Zebrafish Embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

在活斑马鱼胚胎成像的亚细胞结构

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the cells are located. Specifically, they have been developed to analyze the electrical activity of excitable cells, such as spinal neurons. In such a scenario, it is crucial to preserve the integrity of the spinal cell, but it is also important to preserve the anatomy and physiological shape of the systems involved. Indeed, the comprehension of the manner in which the nervous system-and other complex systems-works must be based on a systemic approach. For this reason, the live zebrafish embryo was chosen as a model system, and the spinal neuron membrane voltage changes were evaluated without interfering with the physiological conditions of the embryos. 
Here, an approach combining the employment of zebrafish embryos with a FRET-based biosensor is described. Zebrafish embryos are characterized by a very simplified nervous system and are particularly suited for imaging applications thanks to their transparency, allowing for the employment of fluorescence-based voltage indicators at the plasma membrane during zebrafish development. The synergy between these two components makes it possible to analyze the electrical activity of the cells in intact living organisms, without perturbing the physiological state. Finally, this non-invasive approach can co-exist with other analyses (e.g., spontaneous movement recordings, as shown here).

Protocols described here allow for the study of the electrical properties of excitable cells in the most non-invasive physiological conditions by employing zebrafish embryos in an in vivo system together with a fluorescence resonance energy transfer (FRET)-based genetically encoded voltage indicator (GEVI) selectively expressed in the cell type of interest.

生物传感运动神经元膜电位在活斑马鱼胚胎

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and zebrafish embryos. Our system comprises a chamber featuring a simple setup that is nevertheless robust to induce and maintain a specific oxygen concentration and temperature in any experimental solution of choice. The presented system is very cost-effective but highly functional, it allows induction and sustainment of hypoxia for direct experiments in vivo and for various time periods up to 48 h.
To monitor and study the effects of hypoxia, we have employed two methods - measurement of levels of hypoxia-inducible factor 1alpha (HIF-1&#945;) in whole embryos or specific tissues and determination of retinal stem cell proliferation by 5-ethynyl-2&#8242;-deoxyuridine (EdU) incorporation into the DNA. HIF-1&#945; levels can serve as a general hypoxia marker in the whole embryo or tissue of choice, here embryonic retina. EdU incorporation into the proliferating cells of embryonic retina is a specific output of hypoxia induction. Thus, we have shown that hypoxic embryonic retinal progenitors decrease proliferation within 1 h of incubation under 5% oxygen of both frog and zebrafish embryos.
Once mastered, our setup can be employed for use with small aquatic model organisms, for direct in vivo experiments, any given time period and under normal, hypoxic or hyperoxic oxygen concentration or under any other given gas mixture.

We introduce a novel hypoxic chamber system for use with aquatic organisms such as frog and zebrafish embryos. Our system is simple, robust, cost-effective and allows the induction and sustainment of hypoxia in vivo and for up to 48 h. We present 2 reproducible methods to monitor the effectiveness of hypoxia.

诱导生活青蛙和斑马鱼胚胎缺氧

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

Following Endocardial Tissue Movements via Cell Photoconversion in the Zebrafish Embryo

During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the field of developmental biology. The discovery and development of photoconvertible proteins have greatly aided our understanding of these dynamic changes by providing a method to optically highlight cells and tissues. However, while photoconversion, time-lapse microscopy, and subsequent image analysis have proven to be very successful in uncovering cellular dynamics in organs such as the brain or the eye, this approach is generally not used in the developing heart due to challenges posed by the rapid movement of the heart during the cardiac cycle. This protocol consists of two parts. The first part describes a method for photoconverting and subsequently tracking endocardial cells (EdCs) during zebrafish atrioventricular canal (AVC) and atrioventricular heart valve development. The method involves temporally stopping the heart with a drug in order for accurate photoconversion to take place. Hearts are allowed to resume beating upon removal of the drug and embryonic development continues normally until the heart is stopped again for high-resolution imaging of photoconverted EdCs at a later developmental time point. The second part of the protocol describes an image analysis method to quantify the length of a photoconverted or non-photoconverted region in the AVC in young embryos by mapping the fluorescent signal from the three-dimensional structure onto a two-dimensional map. Together, the two parts of the protocol allows one to examine the origin and behavior of cells that make up the zebrafish AVC and atrioventricular heart valve, and can potentially be applied for studying mutants, morphants, or embryos that have been treated with reagents that disrupt AVC and/or valve development.

This protocol describes a method for the photoconversion of Kaede fluorescent protein in endocardial cells of the living zebrafish embryo that enables the tracking of endocardial cells during atrioventricular canal and atrioventricular heart valve development.

在斑马鱼胚胎中通过细胞 Photoconversion 的心内膜组织运动后

During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the ...

Single-cell Transcriptomic Analyses of Mouse Pancreatic Endocrine Cells

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, including insulin-secreting &#946; cells, are differentiated from common endocrine progenitors during the embryonic stage. Immature endocrine cells expand via cell proliferation and mature during a long postnatal developmental period. However, the mechanisms underlying these processes are not clearly defined. Single-cell RNA-sequencing is a promising approach for the characterization of distinct cell populations and tracing cell lineage differentiation pathways. Here, we describe a method for the single-cell RNA-sequencing of isolated pancreatic &#946; cells from embryonic, neonatal and postnatal pancreases.

We describe a method for the isolation of endocrine cells from embryonic, neonatal and postnatal pancreases followed by single-cell RNA sequencing. This method allows analyses of pancreatic endocrine lineage development, cell heterogeneity and transcriptomic dynamics.

小鼠胰腺内分泌细胞的单细胞 Transcriptomic 分析

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, ...

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the development of neuronal connectivity. During establishment of the retino-tectal projection, optic axons extend from the eye and navigate through distinct regions of the brain to reach their target tissue, the optic tectum. Once optic axons enter the tectum, they elaborate terminal arbors that function to increase the number of synaptic connections they can make with target interneurons in the tectum. Here, we describe a method to express DNA encoding green fluorescent protein (GFP), and gain- and loss-of-function gene constructs, in optic neurons (retinal ganglion cells) in Xenopus embryos. We explain how to microinject a combined DNA/lipofection reagent into eyebuds of one day old embryos such that exogenous genes are expressed in single or small numbers of optic neurons. By tagging genes with GFP or co-injecting with a GFP plasmid, terminal axonal arbors of individual optic neurons with altered molecular signaling can be imaged directly in brains of intact, living Xenopus tadpoles several days later, and their morphology can be quantified. This protocol allows for determination of cell-autonomous molecular mechanisms that underlie the development of optic axon arborization in vivo.

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

This protocol aims to demonstrate how to microinject a DNA/DOTAP mixture into eyebuds of one day old Xenopus laevis embryos, and how to image and reconstruct individual green fluorescent protein (GFP) expressing optic axonal arbors in tectal midbrains of intact, living Xenopus tadpoles.

将DNA微注射到异种胚胎的眼蕾中，以及GFP表达光学轴状轴向轴向的成像，活的Xenopus大白鼠

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the ...

Visualizing the Developing Brain in Living Zebrafish using Brainbow and Time-lapse Confocal Imaging

Development of the vertebrate nervous system requires a precise coordination of complex cellular behaviors and interactions. The use of high resolution in vivo imaging techniques can provide a clear window into these processes in the living organism. For example, dividing cells and their progeny can be followed in real time as the nervous system forms. In recent years, technical advances in multicolor techniques have expanded the types of questions that can be investigated. The multicolor Brainbow approach can be used to not only distinguish among like cells, but also to color-code multiple different clones of related cells that each derive from one progenitor cell. This allows for a multiplex lineage analysis of many different clones and their behaviors simultaneously during development. Here we describe a technique for using time-lapse confocal microscopy to visualize large numbers of multicolor Brainbow-labeled cells over real time within the developing zebrafish nervous system. This is particularly useful for following cellular interactions among like cells, which are difficult to label differentially using traditional promoter-driven colors. Our approach can be used for tracking lineage relationships among multiple different clones simultaneously. The large datasets generated using this technique provide rich information that can be compared quantitatively across genetic or pharmacological manipulations. Ultimately the results generated can help to answer systematic questions about how the nervous system develops.

In vivo imaging is a powerful tool that can be used to investigate the cellular mechanisms underlying nervous system development. Here we describe a technique for using time-lapse confocal microscopy to visualize large numbers of multicolor Brainbow-labeled cells in real time within the developing zebrafish nervous system.

使用"彩虹"和"延时共聚焦成像"可视化活斑马鱼中发育中的脑

Development of the vertebrate nervous system requires a precise coordination of complex cellular behaviors and interactions. The use of high resolution in ...

Eye Removal in Living Zebrafish Larvae to Examine Innervation-dependent Growth and Development of the Visual System

Zebrafish exhibit remarkable life-long growth and regenerative abilities. For example, specialized stem cell niches established during embryogenesis support continuous growth of the entire visual system, both in the eye and the brain. Coordinated growth between the retinae and the optic tectum ensures accurate retinotopic mapping as new neurons are added in the eyes and brain. To address whether retinal axons provide crucial information for regulating tectal stem and progenitor cell behaviors such as survival, proliferation, and/or differentiation, it is necessary to be able to compare innervated and denervated tectal lobes within the same animal and across animals. 
Surgical removal of one eye from living larval zebrafish followed by observation of the optic tectum achieves this goal. The accompanying video demonstrates how to anesthetize larvae, electrolytically sharpen tungsten needles, and use them to remove one eye. It next shows how to dissect brains from fixed zebrafish larvae. Finally, the video provides an overview of the protocol for immunohistochemistry and a demonstration of how to mount stained embryos in low-melting-point agarose for microscopy.

The article explains how to surgically remove eyes from living zebrafish larvae as the first step toward investigating how retinal input influences optic tectum growth and development. In addition, the article provides information about larval anesthetization, fixation, and brain dissection, followed by immunohistochemistry and confocal imaging.

活斑马鱼幼虫的眼睛去除以检查视觉系统的神经支配依赖性生长和发育

Zebrafish exhibit remarkable life-long growth and regenerative abilities. For example, specialized stem cell niches established during embryogenesis ...