Development and validation of genetic cell ablation systems in axolotl

摘要： Axolotl是一种经典且健壮的组织再生模型。Axolotl对肢体的研究是众所周知的 而且还能再生他们的眼睛、肺、心脏、肾脏、脊髓、大脑，并提供一个系统来 研究机体各组织器官再生的免疫调节。再生机制的研究 为在哺乳动物中设计类似的再生机制提供了路线图，并最终 人类。再生生物学中仍未完全了解的两个关键问题是：(1)。什么是 损伤修复过程中替代细胞的来源？和(2)。细胞如何在再生过程中相互作用以创造 复杂的组织和原始器官一样发挥功能？我们在产生转基因紫杉醇方面的工作已经 将这一经典系统带入了现代基因组时代，在这个时代，我们可以传承痕迹细胞， 有助于再生，并开始梳理不同器官中特定的再生机制。 然而，为了充分了解不同细胞类型和谱系对再生、细胞消融的贡献 方法是必需的。细胞消融允许我们提出这样的问题：特定细胞的作用是什么 在再生过程中输入复杂的细胞混合物？器官/附件需要某种细胞类型吗？ 再生？当正常参与 再生过程缺失了？不同类型的细胞如何对器官的整体构图做出贡献 组织会再生吗？为了回答这些重要的问题，我们建议产生广泛适用的转基因 用于谱系特异性细胞消融研究的axolotl工具。这些工具要么利用产生毒素的酶，要么利用 利用蛋白质/毒素来激活/诱导细胞凋亡。具体地说，我们将产生硝基还原酶 (NTR)或白喉毒素受体(DTR)在loxP控制下的转基因轴突并与组织杂交 我们实验室已有的特异表达DTR或NTR的可诱导Cre品系 感兴趣的组织。这将为我们提供对细胞消融的空间控制。进一步通过提供甲硝唑 (药物)或白喉(毒素)分别到NTR和DTR线，我们将能够实现暂时控制。 如果可诱导Cre线是 可用于感兴趣的组织。因此，我们认为遗传细胞消融系统的发展 紫杉醇将为研究各种器官和附件打开大门，例如四肢、尾巴、眼睛、肺、 心、肾、脊髓、脑及免疫细胞在轴突组织再生中的作用。

Abstract: The axolotl is a classical and robust model of tissue regeneration. Axolotls are well known for studies of limb regeneration but also regenerate their eyes, lungs, heart, kidney, spinal cord, brain and provide a system to study immune regulation of regeneration of all body tissues and organs. Studying mechanisms of regeneration in axolotl provides a roadmap for engineering similar regeneration mechanisms in mammals and ultimately humans. Two key questions in regeneration biology that remain incompletely understood are: (1). What are the sources of replacement cells during injury repair? and (2). How do cells interact during regeneration to create complex tissues that function as well as the original organs? Our work on generating transgenic axolotl has brought this classical system into the modern genomic era where we can now lineage trace cells that contribute to regeneration and start to tease apart specific regeneration mechanisms in different organs. However, to fully understand the contribution of different cell types and lineages to regeneration, cell ablation approaches are required. Cell ablation allows us to ask questions such as: What is the role of a specific cell type in a complex mixture of cells during regeneration? Is a certain cell type necessary for organ/appendage regeneration? Can other cells act as a reserve pool when the cell type that normally participates in the regenerative process is missing? How do different cell types contribute to the overall patterning of the organ or tissue regenerate? To answer these important questions, we propose generating broadly applicable transgenic axolotl tools for lineage specific cell ablation studies. These tools either utilize enzymes that produces toxins or make use of proteins/toxins that activate/induce cell apoptosis. Specifically, we will generate nitroreductase (NTR) or Diphtheria toxin receptor (DTR) transgenic axolotls under LoxP control and cross them with tissue specific inducible Cre lines that are already available in our laboratory to specifically express DTR or NTR in the tissue of interest. This will provide us spatial control over cell ablation. Further by providing metronidazole (drug) or diphtheria (toxin) respectively to NTR and DTR lines, we will be able to achieve temporal control. Such spatiotemporal control of cell ablation can be achieved over any cell type if an inducible Cre line is available for the tissue of interest. Hence, we believe that the development of genetic cell ablation systems in axolotl will open doors for studying a wide variety of organs and appendages for example, limb, tail, eye, lung, heart, kidney, spinal cord, brain and the role of immune cells in axolotl tissue regeneration.