Name: targeted laser ablation in the embryo of saccharina latissima
Uploaded: 2022-03-11T15:00:00
Description: Watch this Scientific Journal Video about Targeted Laser Ablation in the Embryo of Saccharina latissima at JoVE.com

S.B.的博士资助由布列塔尼大区（ARED拨款编号COH20020）和索邦大学资助。I.T.is 博士学位由布列塔尼大区（ARED拨款编号COH18020）和挪威NMBU大学资助。该项目已通过通产省跨学科项目获得CNRS的财政支持。MRic是法国国家研究机构（ANR-10-INBS-04）支持的国家基础设施法国生物成像的成员。

1. 配子体甘草的产生
<ol><li>从野外收集乳突鱼的成熟孢子植物，如前所述20，21。确保选定的孢子植物没有附生植物（在叶片表面上可见的小生物）或内部寄生虫（在叶片的漂白区域或斑点中发现）。 
</li>
	<li>使用手术刀，将刀片中心最暗的部分（可育孢子产生组织22）切成1-5个正方形（1cm²），避免?...

<ol>
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G., Charrier, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+zygotes+and+embryos+of+the+kelp+saccharina+latissima+for+cell+biology+approaches.">Preparation of zygotes and embryos of the kelp saccharina latissima for cell biology approaches.</a> Bio-protocol. 101, 4132 (2021).</li><li>L&#252;ning, K., Dring, M. 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局部细胞激光消融允许以高精度进行时间和空间消融。然而，其效率可能会受到靶细胞不可及性的阻碍;例如，所有细胞都是三维胚胎。该协议是在藻类 裂殖质的胚胎上开发的，该胚胎发育出单层层，其中所有细胞都可以很容易地用激光束单独区分和破坏。
激光功率和波长 NIR fs脉冲激光通常用于动物的发育研究24，<sup class="xref"...

激光消融已经使用了几十年来研究胚胎发育。用激光束照射胚胎细胞可以监测胚胎发生过程中细胞系的再生电位和修饰，并研究靶向消融对细胞分裂和细胞命运的影响。激光消融方法中使用的模式生物通常是动物，如昆虫1，2，线虫3，4，脊椎动物5，6，偶尔植物7，8。此外，1994年和1998年在褐藻 墨角上 使用了激光微消融方法，以证明细胞壁在早期胚胎9，10的光极化中的作用。
褐藻属于星云藻类，在16亿年前在真核生物树的根部分化。因此，它们在系统发育上独立于其他多细胞生物，例如动物和植物11。糖精属於海带目，通常被称为海带，它们是地球上最大的生物之一，大小超过30米。 13.其养殖主要在亚洲，最近在欧洲，需要在孵化场准备胚胎，然后在公海释放幼鱼。像所有海带一样，它具有一个双相生命周期，由微观配子体阶段组成，在此期间，单倍体配子体生长并产生配子用于受精，以及二倍体宏观孢子体阶段，其中大型平面叶片从附着在海底或岩石上的固定物发展而来。孢子体在成熟时释放单倍体孢子，从而完成生命周期14，15，16。
裂头孢子虫 呈现出一些有趣的形态特征17.它的胚胎发育成单层平面片15，18，19，然后获得多层结构，与不同组织类型的出现相吻合。此外，海带是仅有的褐藻类群之一，其胚胎仍然附着在母体配子体组织上（去角膜动物和孢子囊藻也这样做15）。该特征提供了研究母体组织在这一发育过程中的作用的机会，并将褐藻中的母体控制机制与动物和植物中的母体控制机制进行比较。
本文介绍了早期海带胚胎中激光消融的第一个完整的方案。该协议涉及UV ns脉冲技术，导致单个胚胎细胞的特定破坏，以研究它们在胚胎发生过程中各自的作用。该程序为研究海带胚胎发生过程中的细胞相互作用和细胞命运提供了一种可靠的方法。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>25 mm glass bottom petri dish</td>
			<td>NEST</td>
			<td>801001</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclaved sea water</td>
			<td>-</td>
			<td></td>
			<td>Collected offshore near the Astan buoy (48&#176;44.934 N 003&#176;57.702 W) close to Roscoff, France, at a depth of 20 m.</td>
		</tr>
		<tr>
			<td>Cell scraper</td>
			<td>MED 2</td>
			<td>83.3951</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer 40 &#181;m</td>
			<td>Corning / Falcon</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture cabinets</td>
			<td>Snijders Scientific Plant Growth Cabinet ECD01</td>
			<td></td>
			<td>Any other brand is suitable provided that the light intensity, the photoperiod and the temperature can be controlled.</td>
		</tr>
		<tr>
			<td>LSM 880 Zeiss confocal microscope</td>
			<td>Carl Zeiss microscopy, Jena, Germany</td>
			<td></td>
			<td>Ablation and imaging were performed using a 40x/1.2 water objective</td>
		</tr>
		<tr>
			<td>Pellet pestles</td>
			<td>Sigma Aldrich</td>
			<td>Z359947</td>
			<td>Blue polypropylene (autoclavable)</td>
		</tr>
		<tr>
			<td>Provasoli supplement</td>
			<td>-</td>
			<td></td>
			<td>Recipe is available here: http://www.sb-roscoff.fr/sites/www.sb-roscoff.fr/files/documents/station-biologique-roscoff-preparation-du-provasoli-2040.pdf</td>
		</tr>
		<tr>
			<td>Pulsed 355 laser (UGA-42 Caliburn 355/25)</td>
			<td>Rapp OptoElectronic, Wedel, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Paramount</td>
			<td>PDSS 11</td>
			<td></td>
		</tr>
		<tr>
			<td>SysCon software</td>
			<td>Rapp OptoElectronic, Wedel, Germany</td>
			<td></td>
			<td>Laser-driver software</td>
		</tr>
		<tr>
			<td>ZEN software</td>
			<td>Carl Zeiss microscopy, Jena, Germany</td>
			<td></td>
			<td>Imaging software, used together with the SysCon software; Black 2.3 version</td>
		</tr>
	</tbody>
</table>

targeted laser ablation in the embryo of saccharina latissima

在 裂殖质中，胚胎发育为称为层或叶片的单层细胞片。每个胚胎细胞都易于观察，很容易与其邻居区分开来，并且可以单独靶向。几十年来，激光消融一直用于研究胚胎发育。在这里，为褐藻狭 长链球菌的早期胚胎开发了细胞特异性激光消融方案。所介绍的工作包括：（1） 糖精 胚胎的制备，描述关键参数，包括培养条件，（2）激光消融设置，以及（3）使用延时显微镜监测受照射胚胎的后续生长。此外，还提供了有关将胚胎从成像平台运输回实验室的最佳条件的详细信息，这可能会对随后的胚胎发育产生深远的影响。属于海带目藻的藻类显示出类似于 糖精的 胚胎发生模式，因此该方案可以很容易地转移到该分类群中的其他物种。

生长 裂 头孢子体，诱导配子发生产生受精卵和胚胎。在配子发生诱导后12天，胚胎接受了激光消融。在这里，实验旨在评估特定细胞在 乳突乳酸链球菌 胚胎整体发育中的作用。靶向最顶端的细胞，最基底细胞和正中细胞。在瓷砖扫描后，整个培养皿（图2A），一个感兴趣的胚胎，被确定为适合激光射击的候选者（图2B）。选择该胚胎的特...

Watch this Scientific Journal Video about Targeted Laser Ablation in the Embryo of Saccharina latissima at JoVE.com

Targeted Laser Ablation in the Embryo of Saccharina latissima

胚胎中特定细胞的破坏是研究参与细胞命运的细胞相互作用的有力工具。本方案描述了用于在褐藻裂殖质的早期胚胎中激光消融靶细胞 的技术。

靶向激光消融术在乳酸杆菌胚胎中的应用

In Saccharina latissima, the embryo&#160;develops as a monolayered cell sheet called the lamina or the blade. Each embryo cell is easy to observe, readily ...

该协议是第一个关于早期海带胚胎的完整激光消融方案。该程序为研究褐藻胚胎发生过程中的细胞命运和相互作用提供了一种可靠的方法。局部细胞激光消融允许在细胞上以高水平的精度和组织进行时间和特殊消融。这种方法是研究褐藻早期胚胎中细胞命运和相互作用的有希望的方法。它可以应用于其他藻类系统，只需很少的变化。首先对整个培养皿进行成像，以在消融步骤中定位要选择的胚胎，并监测所选胚胎的后续发育。开始磁贴扫描。记录培养皿的四个基点的位置。输入文本手稿中定义的参数，并以低分辨率获取整个培养皿的透射或荧光图像。保存图块扫描图像，并在图像采集软件窗口中保持打开状态。在不取下培养皿的情况下更改目标。在扫描中查找感兴趣的胚胎。要校准激光器，请打开激光驱动器和图像采集软件包。然后初始化激光路径。通过单击激光驱动程序软件包中的“开始采集”来同步两个软件包。设置激光烧蚀的参数，然后单击实时。选择培养皿上的空白区域，并将载物台水平降低到培养皿顶面以下20微米处，以聚焦在玻璃底部。要查找感兴趣的区域，请单击选择AOI按钮，然后单击紫外激光驱动程序软件包中图像的边缘。单击开始校准并选择手动校准以设置烧蚀激光和成像激光轨迹。确保所有百叶窗都已打开。选择足够高的激光功率，以便在实时图像的中心看到一个黑点，对应于玻璃盖玻片上的孔。用鼠标光标单击中央黑点，然后选择软件建议的另外18个点以完成对齐过程。单击单击并触发模式。检查同一盖玻片上的校准，然后单击可见激光冲击的玻璃层。在瓷砖扫描中选择感兴趣的胚胎，然后单击它来移动舞台。启动时间序列。实时测试时间流逝参数，并在必要时进行调整。在图像采集软件中以最大速度开始延时记录。调整变焦以聚焦于感兴趣的区域并可视化整个胚胎。要在记录消融过程中跟踪胚胎，请单击按钮，获取最新图像。将参数设置为45%激光传输，对应于最大40微瓦和1毫秒脉冲持续时间。通过使用激光驱动软件点击和射击功能，对感兴趣的胚胎细胞施加破坏性照射。在688纳米以下，监测自身荧光叶绿体从细胞质中射出的情况。如果单元格内容保留在单元格中，请再次使用单击并触发功能来增加单元格中违规的大小。重复此过程，直到释放大部分细胞内容，将拍摄次数保持在最低限度。在胚胎稳定后停止延时记录，并且无法检测到进一步的细胞间运动。更新磁贴扫描图像上的批注，覆盖现有磁贴扫描，然后保存图像。通过监测激光消融后发育的胚胎数量并确定存活率，并将其与死亡胚胎进行比较。通过每天测量激光注射胚胎的长度并将其与完整胚胎进行比较来确定生长延迟。通过监测与消融细胞相邻的细胞的反应来发现相邻的损伤。感兴趣的靶向细胞是最顶端的细胞，最基底细胞和中间细胞。在对整个培养皿进行瓷砖扫描后，感兴趣的胚胎被确定为激光射击的合适候选者。当用脉冲紫外激光束以45%的功率射出时，细胞释放其内容物，而与受照射细胞相邻的细胞则扩展到细胞间空间。每24小时记录一次辐照胚胎的位置，持续10天。大多数辐照的胚胎继续发育，但显示出生长改变。相比之下，用其他激光参数测试的胚胎很快显示出严重压力的迹象，如细胞漂白，褪色或形状变化。几乎所有以这种方式拍摄的胚胎在实验后五天内死亡。某些参数（如图像格式或缩放）在校准后不应更改。需要在激光消融期间和之后进行监测，以深入了解发生了什么。激光消融正在为褐藻发育研究的新发现铺平道路，并更深入地了解不同代谢物或其细胞成分的相互作用。

targeted-laser-ablation-in-the-embryo-of-saccharina-latissima

Research

JoVE Journal

Developmental Biology

Targeted Laser Ablation in the Embryo of Saccharina latissima

Microbead Implantation in the Zebrafish Embryo

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic conservation, as well as similarities in cellular, molecular, and physiological processes, with other vertebrates including humans. During early ontogeny, zebrafish embryos are optically transparent, allowing researchers to visualize the dynamics of organogenesis using a simple stereomicroscope. Microbead implantation is a method that enables tissue manipulation through the alteration of factors in local environments. This allows researchers to assay the effects of any number of signaling molecules of interest, such as secreted peptides, at specific spatial and temporal points within the developing embryo. Here, we detail a protocol for how to manipulate and implant beads during early zebrafish development.

The zebrafish is an excellent model system for genetic and developmental studies. Bead implantation is a valuable tissue manipulation technique that can be used to interrogate developmental mechanisms by introducing alterations in local cellular environments. This protocol describes how to perform microbead implantation in the zebrafish embryo.

微珠植入斑马鱼胚胎

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic ...

科研

发育生物学

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 ...

Embryo Microinjection and Electroporation in the Chordate Ciona intestinalis

Simple model organisms are instrumental for in vivo studies of developmental and cellular differentiation processes. Currently, the evolutionary distance to man of conventional invertebrate model systems and the complexity of genomes in vertebrates are critical challenges to modeling human normal and pathological conditions. The chordate Ciona intestinalis is an invertebrate chordate that emerged from a common ancestor with the vertebrates and may represent features at the interface between invertebrates and vertebrates. A common body plan with much simpler cellular and genomic composition should unveil gene regulatory network (GRN) links and functional genomics readouts explaining phenomena in the vertebrate condition. The compact genome of Ciona, a fixed embryonic lineage with few divisions and large cells, combined with versatile community tools foster efficient gene functional analyses in this organism. Here, we present several crucial methods for this promising model organism, which belongs to the closest sister group to vertebrates. We present protocols for transient transgenesis by electroporation, along with microinjection-mediated gene knockdown, which together provide the means to study gene function and genomic regulatory elements. We extend our protocols to provide information on how community databases are utilized for in silico design of gene regulatory or gene functional experiments. An example study demonstrates how novel information can be gained on the interplay, and its quantification, of selected neural factors conserved between Ciona and man. Furthermore, we show examples of differential subcellular localization in embryonic cells, following DNA electroporation in Ciona zygotes. Finally, we discuss the potential of these protocols to be adapted for tissue specific gene interference with emerging gene editing methods. The in vivo approaches in Ciona overcome major shortcomings of classical model organisms in the quest of unraveling conserved mechanisms in the chordate developmental program, relevant to stem cell research, drug discovery, and subsequent clinical application.

Embryo Microinjection and Electroporation in the Chordate Ciona intestinalis

We present transient transgenesis and gene knockdown in Ciona intestinalis, a chordate sister group to vertebrates, using microinjection and electroporation techniques. Such methods facilitate functional genomics in this simple invertebrate that features rudimentary characteristics of vertebrates, including notochord and head sensory epithelia, and many orthologs of human disease associated genes.

胚胎显微注射和电的脊索动物<em&gt;玻璃海鞘</em

Simple model organisms are instrumental for in vivo studies of developmental and cellular differentiation processes. Currently, the evolutionary distance ...

Surgical Ablation Assay for Studying Eye Regeneration in Planarians

In the study of adult stem cells and regenerative mechanisms, planarian flatworms are a staple in vivo model system. This is due in large part to their abundant pluripotent stem cell population and ability to regenerate all cell and tissue types after injuries that would be catastrophic for most animals. Recently, planarians have gained popularity as a model for eye regeneration. Their ability to regenerate the entire eye (comprised of two tissue types: pigment cells and photoreceptors) allows for the dissection of the mechanisms regulating visual system regeneration. Eye ablation has several advantages over other techniques (such as decapitation or hole punch) for examining eye-specific pathways and mechanisms, the most important of which is that regeneration is largely restricted to eye tissues alone. The purpose of this video article is to demonstrate how to reliably remove the planarian optic cup without disturbing the brain or surrounding tissues. The handling of worms and maintenance of an established colony is also described. This technique uses a 31 G, 5/16-inch insulin needle to surgically scoop out the optic cup of planarians immobilized on a cold plate. This method encompasses both single and double eye ablation, with eyes regenerating within 1-2 weeks, allowing for a wide range of applications. In particular, this ablation technique can be easily combined with pharmacological and genetic (RNA interference) screens for a better understanding of regenerative mechanisms and their evolution, eye stem cells and their maintenance, and phototaxic behavioral responses and their neurological basis.

This protocol shows how to consistently excise planarian eyes (optic cups) without disturbing surrounding tissues. Using an insulin needle and syringe, either one or both eyes can be ablated to facilitate investigations into the mechanisms regulating eye regeneration, the evolution of visual regeneration, and the neural basis of light-induced behavior.

手术消融分析在研究涡虫再生眼

In the study of adult stem cells and regenerative mechanisms, planarian flatworms are a staple in vivo model system. This is due in large part to their ...

AFM and Microrheology in the Zebrafish Embryo Yolk Cell

Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. Various propositions claim to have been important contributions to the understanding of the mechanical properties of cells and tissues in their spatiotemporal organization in different developmental and morphogenetic processes. However, due to the lack of reliable and accessible tools to measure material properties and tensional parameters in vivo, validating these hypotheses has been difficult. Here we present methods employing atomic force microscopy (AFM) and particle tracking with the aim of quantifying the mechanical properties of the intact zebrafish embryo yolk cell during epiboly. Epiboly is an early conserved developmental process whose study is facilitated by the transparency of the embryo. These methods are simple to implement, reliable, and widely applicable since they overcome intrusive interventions that could affect tissue mechanics. A simple strategy was applied for the mounting of specimens, AFM recording, and nanoparticle injections and tracking. This approach makes these methods easily adaptable to other developmental times or organisms.

The lack of tools to measure material properties and tensional parameters in vivo prevents validating their roles during development. We employed atomic force microscopy (AFM) and nanoparticle-tracking to quantify mechanical features on the intact zebrafish embryo yolk cell during epiboly. These methods are reliable and widely applicable avoiding intrusive interventions.

斑马鱼胚胎卵黄细胞中的 AFM 和 Microrheology

Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. ...

Effectiveness of the Air Stripping in Two Salmonid Fish, Rainbow Trout (Oncorhynchus Mykiss) and Brown Trout (Salmo Trutta Morpha fario)

Egg collection is one of the most crucial procedures during fish reproduction in salmonid hatcheries. Classic methods involve the use of hand massage on fish abdomens to expel the eggs. An alternative method uses the pressure of gas injected into the body cavity, which causes the subsequent release of the eggs. This method is believed to have less negative effects on both the welfare and egg quality of the broodstocks. Herein, we compare the results of air and hand stripping methods with respect to one-year survival and egg quantity and quality in two salmonid fish, rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta morpha fario). Our results indicate that air stripping yielded a better quality of eggs and higher one-year survival rate in rainbow trout. In addition, air stripping resulted in lower mortality rate than the group subjected to hand stripping (25% vs. 35%). The pH and hatching rate of the hand stripped group was lower than those of the air stripped group. In the case of brown trout, the quality of eggs obtained by both hand and air-stripping methods was similar; however, the one-year losses in fish were higher in air stripped group (15% compared to 0% in hand stripped fish). Although the advantages of air stripping method over hand stripping in terms of egg quality might not be observed in all salmonid species, the air-stripping procedure might be a promising option to be adopted in hatcheries as it ensures a high level of reproducibility and efficiency.

Effectiveness of the Air Stripping in Two Salmonid Fish, Rainbow Trout Oncorhynchus Mykiss and Brown Trout Salmo Trutta Morpha fario

The principal aim of this study is to standardize and test the pneumatic method (air stripping) of collecting eggs in rainbow trout and brown trout. This method allows effective and simple collection of the eggs without the necessity of fish abdomen massage.

两鲑鱼鱼、虹鳟 (虹鳟鱼虹鳟鱼) 和褐鳟 (鲑鲑形态石斑鱼) 的空气剥离效果

Egg collection is one of the most crucial procedures during fish reproduction in salmonid hatcheries. Classic methods involve the use of hand massage on ...

Minimally Invasive Embryo Transfer and Embryo Vitrification at the Optimal Embryo Stage in Rabbit Model

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal and postnatal consequences. To ensure the innocuousness of ART applications, studies in animal models are necessary. In addition, as a last step, embryo development studies require evaluation of their capacity to develop full-term healthy offspring. Here, embryo transfer to the uterus is indispensable to perform any ARTs-related experiment.
The rabbit has been used as a model organism to study mammalian reproduction for over a century. In addition to its phylogenetic proximity to the human species and its small size and low maintenance cost, it has important reproductive characteristics such as induced ovulation, a chronology of early embryonic development similar to humans and a short gestation that allow us to study the consequences of ART application easily. Moreover, ARTs (such as intracytoplasmic sperm injection, embryo culture, or cryopreservation) are applied with suitable efficiency in this species.
Using the laparoscopic embryo transfer technique and the cryopreservation protocol presented in this article, we describe 1) how to transfer embryos through an easy, minimally invasive technique and 2) an effective protocol for long-term storage of rabbit embryos to provide time-flexible logistical capacities and the ability to transport the sample. The outcomes obtained after transferring rabbit embryos at different developmental stages indicate that morula is the ideal stage for rabbit embryo recovery and transfer. Thus, an oviductal embryo transfer is required, justifying the surgical procedure. Furthermore, rabbit morulae are successfully vitrified and laparoscopically transferred, proving the effectiveness of the described techniques.

Assisted reproductive techniques (ARTs) are in continuous evaluation to improve outcomes and reduce the associated risks. This manuscript describes a minimally invasive embryo transfer procedure with an efficient cryopreservation protocol that allows the use of rabbits as an ideal animal model of human reproduction.

家兔模型最佳胚胎阶段微创胚胎移植与胚胎玻璃化

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal ...

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

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a general challenge in developmental biology research. In the embryonic heart, this can be particularly difficult as regional differences in the expression of transcriptional regulators, paracrine/juxtacrine signaling cues, and hemodynamic force are all known to influence cardiomyocyte maturation. A simplified method to alter a developing cardiomyocyte's molecular and biomechanical microenvironment would, therefore, serve as a powerful technique to examine how local conditions influence cell fate and function. To address this, we have optimized a method to physically transplant juvenile cardiomyocytes into ectopic locations in the heart or the surrounding embryonic tissue. This allows us to examine how microenvironmental conditions influence cardiomyocyte fate transitions at single cell resolution within the intact embryo. Here, we describe a protocol in which embryonic myocytes can be isolated from a variety of cardiac sub-domains, dissociated, fluorescently labeled, and microinjected into host embryos with high precision. Cells can then be directly analyzed in situ using a variety of imaging and histological techniques. This protocol is a powerful alternative to traditional grafting experiments that can be prohibitively difficult in a moving tissue such as the heart. The general outline of this method can also be adapted to a variety of donor tissues and host environments, and its ease of use, low cost, and speed make it a potentially useful application for a variety of developmental studies.

In this method, embryonic cardiac tissues are surgically microdissected, dissociated, fluorescently labeled, and implanted into host embryonic tissues. This provides a platform for studying the individual or tissue level developmental organization under ectopic hemodynamic conditions, and/or altered paracrine/juxtacrine environments.

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 ...

In Vitro Reconstitution of Spatial Cell Contact Patterns with Isolated Caenorhabditis elegans Embryo Blastomeres and Adhesive Polystyrene Beads

In multicellular systems, individual cells are surrounded by the various physical and chemical cues coming from neighboring cells and the environment. This tissue complexity confounds the identification of causal link between extrinsic cues and cellular dynamics. A synthetically reconstituted multicellular system overcomes this problem by enabling researchers to test for a specific cue while eliminating others. Here, we present a method to reconstitute cell contact patterns with isolated Caenorhabditis elegans blastomere and adhesive polystyrene beads. The procedures involve eggshell removal, blastomere isolation by disrupting cell-cell adhesion, preparation of adhesive polystyrene beads, and reconstitution of cell-cell or cell-bead contact. Finally, we present the application of this method to investigate the orientation of cellular division axes that contributes to the regulation of spatial cellular patterning and cell fate specification in developing embryos. This robust, reproducible, and versatile in vitro method enables the study of direct relationships between spatial cell contact patterns and cellular responses.

In Vitro Reconstitution of Spatial Cell Contact Patterns with Isolated Caenorhabditis elegans Embryo Blastomeres and Adhesive Polystyrene Beads

Tissue complexities of multicellular systems confound the identification of causal relationship between extracellular cues and individual cellular behaviors. Here, we present a method to study the direct link between contact-dependent cues and division axes using C. elegans embryo blastomeres and adhesive polystyrene beads.

在Vitro重建空间细胞接触模式与孤立的Caenorhabditis elegans胚胎冲击器和粘性聚苯乙烯珠

In multicellular systems, individual cells are surrounded by the various physical and chemical cues coming from neighboring cells and the environment. ...

Confocal Microscope-Based Laser Ablation and Regeneration Assay in Zebrafish Interneuromast Cells

Hair cells are mechanosensory cells that mediate the sense of hearing. These cells do not regenerate after damage in humans, but they are naturally replenished in non-mammalian vertebrates such as zebrafish. The zebrafish lateral line system is a useful model for characterizing sensory hair cell regeneration. The lateral line is comprised of hair cell-containing organs called neuromasts, which are linked together by a string of interneuromast cells (INMCs). INMCs act as progenitor cells that give rise to new neuromasts during development. INMCs can repair gaps in the lateral line system created by&#160;cell death. A method is described here for selective INMC ablation using a conventional laser-scanning confocal microscope and transgenic fish that express green fluorescent protein in INMCs. Time-lapse microscopy is then used to monitor INMC regeneration and determine the rate of gap closure. This represents an accessible protocol for cell ablation that does not require specialized equipment, such as a high-powered pulsed ultraviolet laser. The ablation protocol may serve broader interests, as it could be useful for the ablation of additional cell types, employing a tool set that is already available to many users. This technique will further enable the characterization of INMC regeneration under different conditions and from different genetic backgrounds, which will advance the understanding of sensory progenitor cell regeneration.

Laser ablation is a widely applicable technology for studying regeneration in biological systems. The presented protocol describes use of a standard laser-scanning confocal microscope for laser ablation and subsequent time-lapse imaging of regenerating interneuromast cells in the zebrafish lateral line.

斑马鱼内胚细胞中基于共体显微镜的激光消融和再生测定

Hair cells are mechanosensory cells that mediate the sense of hearing. These cells do not regenerate after damage in humans, but they are naturally ...