Spatiotemporal regulation of digit regeneration by sensory nerves

感觉神经对手指再生的时空调节

• 批准号: 10452887
• 负责人: Mimi C Sammarco
• 金额: $ 24.43万
• 依托单位: TULANE UNIVERSITY OF LOUISIANA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10452887
• 关键词: Adult; Amputation; Antibodies; Automobile Driving; Biology; Blood Vessels; Bone Development; Bone Regeneration; Bone Tissue; Bone callus; Cell Differentiation process; Cell Maintenance; Cells; Data; Data Set; Digit structure; Distal; Elements; Excision; Genetic Transcription; Genomics; Goals; Histologic; Impairment; In Situ Hybridization; Inflammatory; Inflammatory Response; Knock-in Mouse; Literature; Mammals; Mesenchymal; Minerals; Molecular; Natural regeneration; Nerve; Nerve Block; Nerve Growth Factors; Outcome; Pattern; Pharmacology; Phosphotransferases; Physiologic Ossification; Physiologic calcification; Play; Process; Protocols documentation; Radiology Specialty; Regulation; Role; Sensory; Signal Pathway; Signal Transduction; Site; Skeleton; Source; Spatial Distribution; Technology; Therapeutic; Time; Tissue Engineering; Tissues; Transgenic Mice; Transgenic Organisms; Vascularization; Vertebrates; afferent nerve; base; blastema; bone; bone fracture repair; bone repair; cell dedifferentiation; cell type; digit regeneration; genetic signature; healing; histological stains; innovation; insight; mechanical load; mineralization; mouse model; nerve supply; neurotransmission; neurotrophic factor; new growth; novel; novel strategies; osteogenic; receptor; recruit; regenerative; repaired; sciatic nerve; single-cell RNA sequencing; skeletal stem cell; soft tissue; spatiotemporal; stem cell proliferation; transcriptomics; wound closure

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.

具体目标 哺乳动物骨骼的再生需要固有和外在电感因子的作用 来自以分层和时间方式发挥作用的多种细胞类型来控制骨骼祖细胞 增殖和分化。感觉神经已被证明是骨断裂的组成部分 修复过程，驱动骨骼的血管化，骨化和矿化过程。与 骨修复过程，再生，新生长均取代截肢的骨骼和周围 软组织在脊椎动物中差异很大。在哺乳动物中，再生仅限于远端指向 元素。更多的近端截肢导致形成肥厚的愈伤组织和失败的再生。 为剖析区分信号通路而采取了重大努力 再生与非再生截肢。超越了促进完全再生，解开的愿望 这些过程可以使我们能够在修复和组织工程过程中利用再生机制 基于骨治疗方法。少数先前的研究已将神经视为必不可少的 再生的组成部分，但是它们依赖于完全坐骨神经切除，使得无法 区分神经特异性再生结果与机械载荷诱导的效果，并区分该关系 神经和再生之间尚不清楚。 使用转基因小鼠模型和药理抑制，我们的初步结果表明严重延迟 在抑制感觉神经对霉素受体激酶A（TRKA）的数字再生中。在 以前的文献，我们建议感觉神经TRKA信号对于正确的数字再生是必需的。 具体来说，我们建议：i）在愈合过程的早期，将感觉神经招募到截肢部位 通过我们实验室中建立的神经生长因子（NGF）-Trka信号轴，ii）感觉神经衍生 信号在促进胚泡形成和维持细胞的增殖中起着至关重要的作用 成骨的启动状态，因此，iii）通过转基因和/或破坏感觉神经信号传导 药理抑制严重损害了数字骨再生。 特定目标1：定义感觉神经的时空图案，并表征 数字再生期间的感觉神经TRKA信号传导破坏 假设：感觉神经产物的生长和信号传导与伤口闭合，胚芽形成和 增殖，启动总体数字再生。 使用转基因敲蛋白小鼠模型（TRKAF592A）的初步结果，证明了数字的大量赤字 再生。在AIM 1中，我们将首先就时间和空间图案进行全面研究 在数字再生的早期和晚期阶段，神经营养蛋白的表达和感觉神经。在这里，我们会的 利用商业抗体以及转基因小鼠系（THY1-YFP，NGF-EGFP）来识别 愈合过程中感觉神经的空间分布。 NGF的细胞来源将使用已建立的 间充质和炎性细胞的标记。然后将组织学和放射学方法用于 确定时间滴定的TRKA信号抑制对炎症反应的影响（第3、7页）， 胚芽的形成（第10天）以及随后的血管侵袭和组织矿化（第14、28天） 阐明感觉神经调节数字再生的多个方面。 特定目的2：描述与感觉神经信号传导在期间相关的分子机制 数字再生 假设：感官神经分泌因子在数字再生的十字路口，调节细胞 去分化，增殖和成骨的承诺。 转录数据通常来自整个Blastema，而空间信息仅是 使用免疫组织化学方法确定一个目标。使用尖端和新的 经过验证的空间转录组学（visium 10x基因组学），我们将检查神经支配和 非启动的再生数字。尽管以前仅在软组织中生存，但我们最近优化了 骨组织的新方法。这种创新的转录组学过程将用于收集转录 TRKA抑制后，在无偏见的神经结构域抑制TRKA后截肢后再生期间的数据 确定胚泡生物学基础神经特异性因素的时尚。结果将使用 公开可用的单细胞RNA-seq数据集，原位杂交（RNASCOPE）和组织学染色。 提议作为R21的理由：空间转录组学技术虽然非常强大，但 然而，要成功地应用于成人矿化组织。使用我们新开发的协议，结果 R21将对在各个阶段的空间定义的感觉神经调节提供第一场见解 截肢后的整个再生，以及对它们在整体中的作用的基本理解 维持细胞命运和可塑性。