Neural control of limb regeneration

火蜥蜴成年后能够完全再生被截肢的四肢，但它们如何进行这种再生的壮举仍然鲜为人知。如果正常连接肢体的神经纤维(轴突)缺失，肢体再生就不会发生。这个奖项的目的是阐明肢体再生和神经之间的关键关系，但人们对此知之甚少。传统上认为神经的作用是促进损伤部位细胞的增殖。这一提议的假设是，当神经告诉细胞增殖时，神经的缺失会导致阻止肢体再生的分子增加。墨西哥Axolotl蝾螈的新遗传学方法将被用来研究这些抗再生分子。除了展示这些研究的技术优势外，紫杉醇还是一种特别有魅力的动物，表现出强大的再生反应；它们非常适合在实验室和课堂上整合教学和研究。了解再生过程中神经-肢体相互作用的基础将对我们理解动物再生产生重大影响，并可能形成再生医学的新方法。该项目还旨在点燃人们对轴突和再生的兴趣，并招募有前途的未来科学家进入该领域。这将通过以下方式实现：制定计划，将轴突和再生引入当地的基督教青年会夏令营，包括重要的本科生和研究生研究培训，将该项目的研究整合到关于再生的本科课程中，并参与以再生为中心的多样性培训计划。切断支配再生的火蜥蜴肢体的神经束完全阻止再生。神经调节蛋白1(NRG1)最近被认为是轴突再生过程中神经轴突发出的一种有丝分裂因子，但对于为什么需要NRG1还缺乏机制上的解释。这一提议将检验这样一种假设，即轴突来源的NRG1配体不足的再生肢体将不会再生，这是因为缺乏正的有丝分裂信号，以及周围神经(雪旺细胞)来源的抑制信号增加，从而创造了一个不允许的环境。测试轴突在再生过程中发挥多方面作用的假设将改变我们对再生是如何调控的理解。目的1将通过研究神经元特异性吗啉基因敲除和空间调控的CRISPR/Cas9介导的基因失活的影响来测试轴突来源的NRG1的需求。为了测试NRG1信号在缺乏轴突的情况下支持再生的充分性，NRG1的分泌形式及其受体的结构活性形式将在轴突切除和截肢肢体中过度表达。AIM 2将利用RNA测序来识别受到NRG1抑制的特定影响的信号节点。目的3将通过对轴突切断后周围神经束分泌的分子的研究，利用候选抑制分子的功能增减实验来研究轴突的允许性质。拟议中的实验结果将增加对促进肢体再生的机制的理解，并在我们测试再生过程中的基因功能方面取得重大技术进步。

Salamanders are able to fully regenerate amputated limbs as adults, but how they perform such feats of regeneration remains poorly understood. Limb regeneration does not occur if nerve fibers (axons) that normally connect with the limb are absent. The objective of this award is to elucidate the crucial-yet-poorly-understood relationship between limb regeneration and nerves. It has traditionally been thought that the role of nerves is to promote the multiplication (proliferation) of cells located at the injury site. The hypothesis of this proposal is that, while nerves tell cells to proliferate, the absence of nerves causes an increase in molecules that stop the limb from regenerating. Novel genetic approaches in the Mexican Axolotl salamander will be used to study these anti-regenerative molecules. In addition to presenting technical advantages for these studies, Axolotls are exceptionally charismatic animals that exhibit powerful regeneration responses; they are excellent for integrating teaching and research in the laboratory and classroom. Understanding the basis of nerve-limb interactions during regeneration would have a major impact on our understanding of animal regeneration, and may shape novel approaches for regenerative medicine. This project also aims to ignite interest in axolotls and regeneration, and to recruit promising future scientists into the field. This will be carried out by developing programs introducing axolotls and regeneration to local YMCA summer camps, including significant undergraduate and graduate research training, integrating research from this project into an undergraduate course on regeneration, and participating in diversity-focused training programs that center on regeneration.Axotomy of the nerve bundles that innervate a regenerating salamander limb completely blocks regeneration. Neuregulin 1 (NRG1) has recently been implicated as a mitogenic factor emanating from nerve axons during axolotl salamander limb regeneration, but a mechanistic explanation for why NRG1 is required is lacking. This proposal will test the hypothesis that regenerating limbs with insufficient axon-derived NRG1 ligands will not regenerate due to a lack of positive mitogenic signaling as well as an increase in a peripheral nerve (Schwann cells)-derived inhibitory signals, creating a non-permissive environment. Testing the hypothesis that axons play a multifaceted role during regeneration will transform our understanding of how regeneration is regulated. Aim 1 will test the requirement of axon-derived NRG1 by studying the effects of neuron-specific morpholino knockdown and spatially controlled CRISPR/Cas9-mediated gene inactivation. To test for the sufficiency of NRG1 signaling to support regeneration in the absence of axons, a secreted form of NRG1 and a constitutively active form of its receptor will be overexpressed in axotomized and amputated limbs. Aim 2 will utilize RNA sequencing to identify signaling nodes that are specifically affected by NRG1 inhibition. Aim 3 will investigate the permissive nature of axons through the study of molecules secreted from axotomized peripheral nerve bundles using gain- and loss-of-function experiments for candidate inhibitory molecules. The findings made from the proposed experiments will increase understanding of the mechanisms promoting limb regeneration and a major technological advance in our ability to test gene function during regeneration.