Confocal Microscope-Based Laser Ablation and Regeneration Assay in Zebrafish Interneuromast Cells.
Confocal Microscope-Based Laser Ablation and Regeneration Assay in Zebrafish Interneuromast Cells.
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DOI：
10.3791/60966
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发表时间：
2020-05-20
影响因子：
1.2
通讯作者：
Steiner, Aaron B.
中科院分区：
文献类型：
--
作者：
Volpe, Bryan A.;Fotino, Teresa H.;Steiner, Aaron B.
关键词：
毛细胞是介导听觉的机械感觉细胞。这些细胞在人类损伤后不会再生，但它们在非哺乳类脊椎动物（如斑马鱼）中自然补充。斑马鱼侧线系统是表征感觉毛细胞再生的有用模型。侧线由称为神经肥大的含毛细胞的器官组成，这些器官通过一串神经间肥大细胞（INMC）连接在一起。INMC作为祖细胞在发育过程中产生新的神经瘤。INMCs可以修复细胞死亡造成的侧线系统中的缺口。本文描述了一种使用常规激光扫描共聚焦显微镜和在INMC中表达绿色荧光蛋白的转基因鱼进行选择性INMC消融的方法。然后使用延时显微镜来监测INMC再生并确定间隙闭合的速率。这代表了用于细胞消融的可访问协议，其不需要专门的设备，例如高功率脉冲紫外激光器。消融协议可以服务于更广泛的利益，因为它可以用于其他细胞类型的消融，采用许多用户已经可用的工具集。该技术将进一步使INMC再生在不同条件下，从不同的遗传背景，这将促进感觉祖细胞再生的理解的表征。
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 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.
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影响因子：
7.7
作者：
Lush ME;Piotrowski T
通讯作者：
Piotrowski T
影响因子：
--
作者：
Moya-Díaz J;Peña OA;Sánchez M;Ureta DA;Reynaert NG;Anguita-Salinas C;Marín G;Allende ML
通讯作者：
Allende ML
影响因子：
1.2
作者：
Morsch, Marco;Radford, Rowan A. W.;Chung, Roger S.
通讯作者：
Chung, Roger S.
影响因子：
5.4
作者：
Sánchez M;Ceci ML;Gutiérrez D;Anguita-Salinas C;Allende ML
通讯作者：
Allende ML
影响因子：
7.7
作者：
Viader-Llargués O;Lupperger V;Pola-Morell L;Marr C;López-Schier H
通讯作者：
López-Schier H