Why Don’t Lizards Regenerate Perfect Tails Like Salamanders?

 描述(申请人提供)：增强再生能力是医学的一个基本目标。到目前为止，火蜥蜴再生以增强哺乳动物愈合的原理还不能直接适用。在这里，我们建议使用蜥蜴作为一系列旨在操纵骨骼再生能力的研究的模式生物。蜥蜴与哺乳动物关系更密切，但具有显著的再生能力。虽然火蜥蜴和蜥蜴都会再生尾巴，但前者再生的是原始尾巴的近乎完美的副本，后者被称为“不完美复制”，与原始尾巴相比有几个关键的解剖学差异，其中最引人注目的是再生的尾巴骨架。我们最近对再生尾巴发育的比较分析发现，与(1)背腹模式信号、(2)干细胞群和(3)分割信号有关的3个主要差异。在骨骼再生过程中，蜥蜴在尾轴的腹侧形成软骨棒(CR)，而再生的蜥蜴尾巴缺乏背腹骨骼图案，形成软骨管(CT)。我们的初步发现表明，再生的脊髓是蜥蜴和蜥蜴尾巴形成软骨图案的原因。蜥蜴脊髓产生抑制和诱导软骨形成的因子，而蜥蜴脊髓只产生软骨诱导因子；此外，它们的神经干细胞群也不同。蜥蜴干细胞能够分化为背侧和腹侧血统，而蜥蜴 干细胞只能分化成腹型血统。一旦形成，火蜥蜴CR经历分割，新的软骨形成在不同的区域，由大量增殖的软骨细胞和骨膜细胞组成。这些区域在蜥蜴CT中是检测不到的，可能是由于缺乏分子增殖信号而没有分割。我们假设，这些模式形成和调控网络的差异是蜥蜴和火蜥蜴之间不同的再生结果的基础。基于这种比较分析，我们假设了机械干预的可行性，将“不完美”的再生蜥蜴尾巴转变为“完美”的再生蜥蜴尾巴。其目的是：(1)操纵在再生的蜥蜴尾巴中存在的背腹信号，但在蜥蜴尾巴中没有；(2)引入在蜥蜴尾巴中发现的干细胞群体，而不是蜥蜴尾巴；以及(3) 确定和操纵再生的蜥蜴尾巴中的增殖信号，而蜥蜴尾巴中没有这种信号。提出了一种综合的方法，包括转录切分、蜥蜴干细胞的CRIPSR/Cas9基因组编辑、分子和细胞分析、体内手术操作以及细胞和生物活性物质的输送。我们相信，这种方法将产生第一条蜥蜴尾巴，其骨骼显示出与表观复制再生的蜥蜴尾巴相同的图案和分割。这些研究将有助于从机制上理解脊椎动物的再生过程，并可能导致改善包括人类在内的非再生生物的愈合，特别是与骨骼发育和修复有关的愈合。

 DESCRIPTION (provided by applicant): Enhancing regenerative capacities is a fundamental goal in medicine. As yet, the principles of salamander regeneration to augment mammalian healing are not directly applicable. Here we propose using lizards, more closely related to mammals yet exhibiting remarkable regenerative capabilities, as a model organism in a set of studies aimed at manipulating skeletal regeneration capacities. While both salamanders and lizards regenerate their tails, the former regenerate a near-perfect copy of the original tail, whie the latter is known as an "imperfect replicate" with several key anatomical differences compared to the original tail, most striking of which concerns the regenerated tail skeleton. Our recent comparative analyses of regenerated tail development have identified 3 main differences related to (1) dorsoventral patterning signals, (2) stem cell populations, and (3) segmentation signals. During skeletal regeneration, salamanders form a cartilage rod (CR) ventral to the tail axis, whereas the regenerated lizard tail lacks dorsoventral skeletal patterning and forms a cartilage tube (CT). Our initial findings suggest that the regenerated spinal cord is responsible for cartilage patterning in both salamander and lizard tails. The salamander spinal cord produces factors that both inhibit and induce cartilage formation, while the lizard spinal cord produces cartilage inductive factors only; furthermore, they differ in their neural stem cell populations. Salamander stem cells are able to differentiate into both dorsal and ventral lineages, while lizard stem cells differentiate into ventral lineages only. Once formed, the salamander CR undergoes segmentation marked by new cartilage formed at distinct regions by populations of proliferating chondrocytes and periosteal cells. These regions are not detectable in the lizard CT, which does not segment, likely due to lack of molecular proliferative signals. We hypothesize that these differences in pattern formation and regulatory networks underlie the divergent regenerative outcomes between lizards and salamanders. Based on this comparative analysis, we hypothesize the feasibility of mechanistically based intervention to shift the "imperfectly" regenerating lizard tail to phenocopy the "perfectly" regenerating salamander tail. The Aims are: (1) Manipulate the dorsoventral signals present in regenerating salamander tails but absent in lizard tails; (2) Introduce stem cell populations found in salamander but not lizard tails; and (3) Determine and manipulate the proliferative signals in regenerating salamander tails that are absent in lizard tails. An integrated approach is proposed, incorporating transcriptomics, CRIPSR/Cas9 genome editing of lizard stem cells, molecular and cellular analyses, in vivo surgical manipulations, and delivery of cell and bioactive agents. We believe that this approach will produce the first lizard tails with skeletons exhibiting patterning and segmentation that phenocopy regenerated salamander tails. These studies will contribute towards mechanistic understanding of a vertebrate regenerative process, and may lead to improving healing in non-regenerative organisms, including humans, specifically related to skeletal development and repair.