黑皮质素受体通路介导的能量代谢调控脊椎动物断肢再生的机制研究

The central melanocortin system, including POMC and AgRP neurons in the hypothalamus, plays critical roles in regulating body weight and energy homeostasis. However, little is known whether this system regulates organ regeneration in vertebrate animals. Previous work elucidated that hormones derived from the central nervous system, such as the pituitary gland, could affect amphibian limb regeneration. However, it remains unclear whether hypothalamus-derived signals have any effect on limb regeneration. .To investigate the role of hypothalamus (and metabolism) on Xenopus limb regeneration, we have developed micro-electrocautery surgery to analyze the effect of hypothalamus removal on regeneration of Xenopus tadpole limb. We found that hypothalamus injury inhibited limb regeneration, indicating a requirement for hypothalamus-derived signals in limb regeneration. We established AgRP transgenic Xenopus animals to study amphibian energy homeostasis and showed that AgRP could regulate body weight in Xenopus, but surprisingly we noticed that AgRP overexpression inhibited limb regeneration. This led us to propose that the melanocortin pathway regulates limb regeneration, in addition to its essential role in energy balance regulation. Our hypothesis is supported by gene expression analysis showing that POMC, AgRP, MC3R and MC4R are all activated in regenerating limb, and the observation that knockdown of MC3R and MC4R blocked limb regeneration. .In this project, we aim to decipher the molecular mechanism of melanocortin receptor pathway in limb regeneration, by investigating the interaction of POMC, AgRP and MCRs, and examining possible roles of melanocortin pathway in the nerve dependency of limb regeneration. We have also found that MC4R is activated in the regenerating mouse distal digit, with specific expression in the nail matrix, a niche for nail stem cells that play instructive role in mouse digit regeneration. Thus, we will also investigate the role of MC4R and MC4R-expressing cells in digit regeneration in MC4R null and MC4R-GFP transgenic mice. .This project will establish the regulatory role of melanocortin in limb/digit regeneration, providing critical insights of regulation of limb regeneration by energy homeostasis mediated by the melanocortin receptor pathway.

下丘脑中枢黑皮质素系统在调控哺乳动物能量代谢平衡、维持正常生理机能等起重要作用。 但能量代谢与器官再生的关系尚缺乏深入研究。我们前期发现下丘脑来源信号能够影响两栖动物爪蛙的断肢再生。为研究能量代谢与器官再生，我们建立了黑皮质素系统成员AgRP转基因爪蛙模型，发现AgRP可以调控体重及能量代谢，但其过表达则抑制爪蛙断肢再生。黑皮质素系统的POMC与其受体MC4R在断肢再生中表达，MC4R功能缺失则抑制爪蛙断肢再生 。因此我们提出黑皮质素受体介导的能量代谢可以调控断肢再生的假设。本课题将深入研究爪蛙断肢及小鼠末端断趾再生中POMC、AgRP、MCRs的功能与作用机制，探讨黑皮质素系统与断肢再生神经依赖性的关系，在 MC4R突变及MC4R-GFP转基因小鼠中阐释MC4R及MC4R阳性细胞在断趾再生中的作用。本研究将明确黑皮质素系统调控断肢再生机理，为进一步研究能量代谢调控器官再生提供理论基础。

本项目着眼于能量代谢与器官再生调控的关系，研究了下丘脑来源的黑皮质素系统特别是a-MSH/MC4R通路在肢体再生中的作用及机制。结果证明了MC4R通路对两栖动物断肢再生及小鼠的趾端再生均是必要的。在两栖动物爪蛙模型中，发现MC4R通路可以调控再生芽基细胞的能量代谢，调节芽基细胞中的活性氧含量。而且MC4R通路具有类似于神经支配的营养支持功能，可以替代神经组织对肢体再生的指导作用。在小鼠模型中，详细分析了Mc4r在胚胎肢体发育及趾端再生中的表达模式，发现其与神经组织高度相关。利用Mc4r突变小鼠末端趾端再生模型，证明了Mc4r通路同样对小鼠趾端再生是必须的。而在小鼠近端趾端再生模型中，通过a-MSH激活MC4R通路可以诱导近端断趾再生。与其神经营养作用相符的是a-MSH激活MC4R通路能够拯救失神经的趾端再生。因此本项目阐明了MC4R通路调控肢体再生的作用及其机理。此外，本项目还对小鼠其它器官如心脏的再生进行了探讨，结果表明MC4R通路对器官再生具有普遍的重要性。因此，本项目的顺利完成为揭示能量代谢调控器官再生、进一步优化器官再生诱导策略奠定了基础。