靶向激光消融术在乳酸杆菌胚胎中的应用
Summary
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。
Abstract
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。
Introduction
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。
Protocol
1. 配子体甘草的产生 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）。 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?…
Representative Results
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…
Discussion
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"…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 25 mm glass bottom petri dish | NEST | 801001 | |
| Autoclaved sea water | – | Collected offshore near the Astan buoy (48°44.934 N 003°57.702 W) close to Roscoff, France, at a depth of 20 m. | |
| Cell scraper | MED 2 | 83.3951 | |
| Cell strainer 40 µm | Corning / Falcon | 352340 | |
| Culture cabinets | Snijders Scientific Plant Growth Cabinet ECD01 | Any other brand is suitable provided that the light intensity, the photoperiod and the temperature can be controlled. | |
| LSM 880 Zeiss confocal microscope | Carl Zeiss microscopy, Jena, Germany | Ablation and imaging were performed using a 40x/1.2 water objective | |
| Pellet pestles | Sigma Aldrich | Z359947 | Blue polypropylene (autoclavable) |
| Provasoli supplement | – | Recipe is available here: http://www.sb-roscoff.fr/sites/www.sb-roscoff.fr/files/documents/station-biologique-roscoff-preparation-du-provasoli-2040.pdf | |
| Pulsed 355 laser (UGA-42 Caliburn 355/25) | Rapp OptoElectronic, Wedel, Germany | ||
| Scalpel | Paramount | PDSS 11 | |
| SysCon software | Rapp OptoElectronic, Wedel, Germany | Laser-driver software | |
| ZEN software | Carl Zeiss microscopy, Jena, Germany | Imaging software, used together with the SysCon software; Black 2.3 version |
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Diesen Artikel zitieren
Boscq, S., Dutertre, S., Theodorou, I., Charrier, B. Targeted Laser Ablation in the Embryo of Saccharina latissima. J. Vis. Exp. (181), e63518, doi:10.3791/63518 (2022).