Spatiotemporal regulation of digit regeneration by sensory nerves

具体目标 哺乳动物骨骼的再生需要内在和外在诱导因子的作用 从多种细胞类型，其以分级和时间方式起作用以控制骨骼祖细胞 增殖和分化。感觉神经已被证明是骨折的一个组成部分 修复过程，驱动骨的血管化、骨化和矿化过程。相比 骨修复过程，再生，新的生长物取代了截肢的骨和周围的组织。 脊椎动物的软组织变化很大。在哺乳动物中，再生仅限于远端趾骨 元素更近端的截肢导致肥厚性骨痂的形成和再生失败。 已经做出了大量的努力来解剖出不同的信号通路， 再生性截肢和非再生性截肢除了促进完全再生的愿望， 这些过程可以让我们在修复和组织工程中利用再生机制 基于骨治疗方法。一些先前的研究已经暗示神经支配是一个必不可少的 再生的组成部分，然而，他们依赖于完整的坐骨神经切除，使其不可能 区分神经特异性再生结果与机械负荷诱导效应，以及 神经支配和再生之间的关系还不清楚。 使用转基因小鼠模型和药物抑制，我们的初步结果表明， 在抑制感觉神经原霉素受体激酶A（TrkA）后的手指再生中。背景下 在先前的文献中，我们提出感觉神经TrkA信号传导对于适当的手指再生是必要的。 具体而言，我们提出：i）感觉神经在愈合过程的早期被招募到截肢部位 通过我们实验室建立的神经生长因子（NGF）-TrkA信号轴，ii）感觉神经源性 信号在促进芽基形成和维持细胞增殖， iii）通过转基因和/或 药理学抑制严重损害趾骨再生。 具体目标1：定义感觉神经支配的时空模式，并表征 手指再生过程中感觉神经TrkA信号的破坏 假设：感觉神经的生长和信号传导与伤口闭合、芽基形成和伤口愈合一致。 增殖，启动整体数字再生。 使用转基因敲入小鼠模型（TrkAF 592 A）的初步结果表明， 再生在目标1中，我们将首先全面研究 神经营养因子的表达和感觉神经支配在早期和晚期阶段的数字再生。在这里，我们将 利用商业抗体，以及转基因小鼠系（Thy 1-YFP，NGF-eGFP）来鉴定 愈合过程中感觉神经的空间分布。神经生长因子的细胞来源将使用已建立的 间充质细胞和炎症细胞的标志物。然后将使用组织学和放射学方法， 确定时间滴定的TrkA信号传导抑制对炎症反应的影响（第3、7天）， 芽基的形成（第10天）和随后的脉管侵入和组织矿化（第14、28天）， 阐明感觉神经调节手指再生的多个方面。 具体目标2：描述与感觉神经信号中断有关的分子机制， 数字再生 假说：感觉神经分泌因子精确地作用于手指再生的十字路口，调节细胞 去分化、增殖和成骨定型。 转录数据通常来源于整个芽基，而空间信息仅来源于整个芽基。 使用免疫组织化学方法一次确定一个目标。使用最先进的和新的 验证的空间转录组学（VISIUM 10 X基因组学），我们将研究神经支配和 无神经支配的再生手指虽然以前只在软组织中可行，但我们最近优化了一种 骨组织的新方法。这一创新的转录组学过程将用于收集转录 TrkA抑制神经支配结构域后截肢后再生期间的数据， 时尚，以确定潜在的芽基生物学的神经特异性因素。将使用以下方法验证结果： 公开可用的单细胞RNA-seq数据集、原位杂交（RNAscope）和组织学染色。 作为R21提案的理由：空间转录组学技术虽然非常强大， 还没有成功应用于成人矿化组织。使用我们新开发的协议， R21将提供对不同阶段感觉神经的空间定义调节的首次现场见解 在截肢后的整个再生过程中，以及对它们在整个再生过程中的作用的基本理解， 维持细胞命运和可塑性。

Specific Aims The regeneration of the mammalian skeleton requires the action of both intrinsic and extrinsic inductive factors from multiple cell types which function in a hierarchical and temporal fashion to control skeletal progenitor cell proliferation and differentiation. Sensory nerves have been shown to be an integral part of the bone fracture repair process, driving the processes of vascularization, ossification, and mineralization of bone. In contrast to the bone repair process, regeneration, where new growth replaces both the amputated bone and surrounding soft tissue varies widely in vertebrates. In mammals, regeneration is restricted to only the distal phalangeal element. More proximal amputations result in the formation of a hypertrophic callus and failed regeneration. Significant efforts have been placed on dissecting out the distinguishing signaling pathways differentiating regenerative versus non-regenerative amputations. Beyond the desire to promote full regeneration, unraveling these processes could allow us to leverage regenerative mechanisms during repair and tissue-engineering based bone therapeutic approaches. A handful of prior studies have implicated innervation as an essential component of regeneration, however they relied on complete sciatic nerve resection, making it impossible to distinguish nerve-specific regenerative outcomes from mechanical loading-induced effects, and the relationship between innervation and regeneration remains unclear. Using transgenic mouse models and pharmacological inhibition, our preliminary results point to a severe delay in digit regeneration following inhibition of sensory nerve tropomycin receptor kinase A (TrkA). In the context of previous literature, we propose that sensory nerve TrkA signaling is necessary for proper digit regeneration. Specifically, we propose that: i) sensory nerves are recruited to the amputation site early in the healing process through the nerve growth factor (NGF)-TrkA signaling axis established in our lab, ii) sensory nerve-derived signals play an essential role in promoting blastema formation and maintaining cells in a proliferative, osteogenically primed state, and thus, iii) disruption of sensory nerve signaling through transgenic and/or pharmacological inhibition severely impairs digit bone regeneration. Specific Aim 1: Define the spatiotemporal patterning of sensory innervation and characterize the effects of sensory nerve TrkA signaling disruption during digit regeneration Hypothesis: Sensory nerve outgrowth and signaling coincides with wound closure, blastema formation and proliferation, initiating overall digit regeneration. Preliminary results using a transgenic knockin mouse model (TrkAF592A), demonstrate a substantial deficit in digit regeneration. In Aim 1 we will first conduct a comprehensive study on the temporal and spatial patterning of neurotrophin expression and sensory innervation during early and late stages of digit regeneration. Here, we will make use of commercial antibodies, as well as transgenic mouse lines (Thy1-YFP, NGF-eGFP) to identify the spatial distribution of sensory nerves during healing. Cellular sources of NGF will be identified using established markers of mesenchymal and inflammatory cells. Histological and radiological approaches will then be used to determine the effects of temporally titrated TrkA signaling inhibition on the inflammatory response (day 3, 7), formation of the blastema (day 10) and subsequent vascular invasion and tissue mineralization (day 14, 28) to elucidate the multiple facets through which sensory nerves regulate digit regeneration. Specific Aim 2: Delineate the molecular mechanisms affiliated with sensory nerve signaling disruption during digit regeneration Hypothesis: Sensory nerves secrete factors to precisely act at the crossroad of digit regeneration, regulating cell dedifferentiation, proliferation and osteogenic commitment. Transcriptional data is typically derived from the whole blastema, while spatial information has only been determined using immunohistochemical approaches one target at a time. Using the cutting-edge and newly validated spatial transcriptomics (VISIUM 10X Genomics), we will examine the gene signature in innervated and non-innervated regenerating digits. Though previously only viable in soft tissues, we have recently optimized a novel approach for bone tissue. This innovative transcriptomics process will be used to gather transcriptional data during regeneration after amputation following TrkA inhibition from innervated domains in an unbiased fashion to determine the nerve-specific factors underlying blastema biology. Results will be validated using publicly available single-cell RNA-seq data sets, in situ hybridization (RNAscope) and histological staining. Justification for proposal as an R21: The technology of spatial transcriptomics, though extremely powerful, is yet to be successfully applied to adult mineralized tissue. Using our newly developed protocol, the results of this R21 will provide first-in-field insights into the spatially-defined regulation of sensory nerves at various stages throughout regeneration after amputation, as well as fundamental understandings of their role in the overall maintenance of cell fate and plasticity.