Department of Bioengineering, Stanford University, Stanford, CA, USA.
Department of Electrical Engineering, Stanford University, Stanford, CA, USA.
Cell Rep. 2024 Nov 26;43(11):114892. doi: 10.1016/j.celrep.2024.114892. Epub 2024 Oct 19.
Live imaging of regenerative processes can reveal how animals restore their bodies after injury through a cascade of dynamic cellular events. Here, we present a comprehensive toolkit for live imaging of tissue regeneration in the flatworm Macrostomum lignano, including a high-throughput cloning pipeline, targeted cellular ablation, and advanced microscopy solutions. Using tissue-specific reporter expression, we examine how various structures regenerate. Enabled by a custom luminescence/fluorescence microscope, we overcome intense stress-induced autofluorescence to demonstrate genetic cellular ablation and reveal the limited regenerative capacity of neurons and their essential role during wound healing, contrasting muscle cells' rapid regeneration after ablation. Finally, we build an open-source tracking microscope to continuously image freely moving animals throughout the week-long process of regeneration, quantifying kinetics of wound healing, nerve cord repair, body regeneration, growth, and behavioral recovery. Our findings suggest that nerve cord reconnection is highly robust and proceeds independently of regeneration.
实时成像的再生过程可以揭示动物如何通过一系列动态细胞事件来修复受伤后的身体。在这里，我们为扁形动物 Macrostomum lignano 的组织再生提供了一个全面的实时成像工具包，包括高通量克隆管道、靶向细胞消融和先进的显微镜解决方案。通过组织特异性报告基因表达，我们研究了各种结构的再生方式。借助定制的发光/荧光显微镜，我们克服了强烈的应激诱导的自发荧光，演示了基因细胞消融，并揭示了神经元的有限再生能力及其在伤口愈合过程中的重要作用，这与肌肉细胞在消融后的快速再生形成对比。最后，我们构建了一个开源的跟踪显微镜，在一周的再生过程中连续对自由移动的动物进行成像，量化伤口愈合、神经索修复、身体再生、生长和行为恢复的动力学。我们的研究结果表明，神经索的重新连接是高度稳健的，并且独立于再生过程进行。
