Mechanisms of skeletal patterning during limb development and regeneration

项目摘要 肢体先天性缺陷是美国残疾的最常见原因之一， 0.8/1000新生儿最近实现的合理再生医学疗法， 人类越来越努力地了解动物再生的例子， 推动这一进程的机制。在本提案中，我们的目标是深入了解 通过研究一种临床相关的信号分子，视黄酸， 蝾螈的再生蝾螈（Ambystoma mexicanum）是特别有用的， 因为他们在截肢后通过产生异质的血统来再生整个肢体- 被称为芽基的限制性祖细胞。芽基使用位置性的 这些信息保留在结缔组织细胞中，以再生适当的肢体区域。这个目标 本文的建议是对这一过程中的内源性作用进行研究，并研究位置信息是如何建立的 并维持在结缔组织细胞中，这对我们理解肢体再生至关重要。 我们将使用无偏的单细胞RNAseq和空间转录组学方法来比较基因组。 在整个再生过程中以单细胞分辨率表达。我们还将确定 视黄酸的转录靶点，特别是在肢体结缔组织细胞中， 从远端到近端细胞身份的重编程。最后，结缔组织的位置记忆 将在体内对细胞进行实验编程，以证明位置记忆是如何维持的。 最后，这个项目的主要成果将是了解蝾螈的肢体是如何知道什么肢体 结构重新生长，以刺激哺乳动物系统中新的再生方法。

PROJECT SUMMARY Limb congenital defects are among the most common causes of disability in the United States affecting 0.8/1000 children born each year. Recent realizations of plausible regenerative medicine therapies in humans has increased efforts towards understanding examples of animal regeneration and the mechanisms that drive the process. In this proposal, we aim to provide insight into the mechanisms that facilitate regeneration by studying a clinically relevant signaling molecule, retinoic acid, during limb regeneration in the salamander. Axolotl salamanders (Ambystoma mexicanum) are particularly useful, because they regenerate entire limbs after amputation by generating a heterogeneous mass of lineage- restricted progenitors called a blastema at the amputation site. The blastema uses positional information retained in connective tissue cells to regenerate the proper limb regions. The goal of this proposal is to the endogenous role in this process and study how positional information is established and maintained in connective tissue cells, which is critical to our understanding of limb regeneration. We will use unbiased single cell RNAseq and spatial transcriptomic approaches to compare gene expression at single-cell resolution throughout the regeneration process. We will also determine the transcriptional targets of retinoic acid specifically in limb connective tissue cells that are undergoing reprogramming from distal to proximal cell identity. Lastly, the positional memory of connective tissue cells will be experimentally programmed in vivo to demonstrate how positional memory is maintained. In the end, the major outcome of this project will be to know how a salamander limb knows what limb structures to grow back in order to stimulate new regenerative approaches in mammalian systems.