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: 10599298
• 负责人: Mimi C Sammarco
• 金额: $ 11.5万
• 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 2023-12-03
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10599298
• 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; Invaded; Knock-in Mouse; Literature; Mammals; Mesenchymal; Molecular; Natural regeneration; Nerve; Nerve Block; Nerve Growth Factors; Outcome; Pattern; Phalanx; Phosphotransferases; Physiologic Ossification; Physiologic calcification; Play; Process; Proliferating; Protocols documentation; Radiology Specialty; Regulation; Role; Sensory; Signal Pathway; Signal Transduction; Site; Skeleton; Source; Spatial Distribution; Technology; Therapeutic; Time; Tissue Engineering; Tissues; Titrations; Transgenic Mice; Transgenic Organisms; Vascularization; Vertebrates; afferent nerve; 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; pharmacologic; 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内在和外在诱导因素的共同作用 来自多种细胞类型，它们以分层和时间的方式发挥作用来控制骨骼祖细胞 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，驱动骨骼的血管化、骨化和矿化过程。相比之下 骨骼修复过程、再生，其中新的生长物取代了截断的骨骼和周围的骨骼 脊椎动物的软组织差异很大。在哺乳动物中，再生仅限于远端指骨 元素。更多的近端截肢导致肥厚愈伤组织的形成和再生失败。 人们付出了巨大的努力来剖析区分不同的信号通路 再生截肢与非再生截肢。除了促进全面再生的愿望之外，解开 这些过程可以让我们在修复和组织工程过程中利用再生机制 基于骨的治疗方法。一些先前的研究表明神经支配是一种重要的 再生的一部分，然而他们依赖于完整的坐骨神经切除，使得不可能 区分神经特异性再生结果和机械负荷引起的效应，以及两者之间的关系 神经支配和再生之间的关系仍不清楚。 使用转基因小鼠模型和药理学抑制，我们的初步结果表明严重延迟 抑制感觉神经原霉素受体激酶 A (TrkA) 后的手指再生。在这样的背景下 在之前的文献中，我们提出感觉神经 TrkA 信号传导对于正确的手指再生是必要的。 具体来说，我们建议：i）在愈合过程的早期将感觉神经募集到截肢部位 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 信号在促进胚基形成和维持细胞增殖、 成骨引发状态，因此，iii)通过转基因和/或破坏感觉神经信号传导 药理学抑制严重损害指骨再生。 具体目标 1：定义感觉神经支配的时空模式并描述感觉神经支配的影响 手指再生过程中感觉神经 TrkA 信号中断 假设：感觉神经的生长和信号传导与伤口闭合、芽基形成和 增殖，启动整体手指再生。 使用转基因敲入小鼠模型（TrkAF592A）的初步结果表明，数字存在严重缺陷 再生。在目标 1 中，我们将首先对时间和空间格局进行全面研究 手指再生早期和晚期阶段的神经营养蛋白表达和感觉神经支配。在这里，我们将 利用商业抗体以及转基因小鼠品系（Thy1-YFP、NGF-eGFP）来鉴定 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方法进行鉴定 间充质细胞和炎症细胞的标记物。然后将使用组织学和放射学方法 确定暂时滴定的 TrkA 信号传导抑制对炎症反应的影响（第 3、7 天）， 胚基的形成（第 10 天）以及随后的血管侵入和组织矿化（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传导中断相关的分子机制 数字再生 假设：感觉神经分泌因子精确作用于手指再生的十字路口，调节细胞 去分化、增殖和成骨定向。 转录数据通常来自整个胚基，而空间信息仅来自于 使用免疫组织化学方法一次确定一个目标。采用最前沿、最新 经过验证的空间转录组学（VISIUM 10X Genomics），我们将检查神经支配和 非神经支配的再生数字。虽然以前只在软组织中可行，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 截肢后再生期间的数据，在无偏的情况下，受神经支配的域抑制 TrkA 确定胚基生物学基础的神经特异性因素的时尚。结果将使用以下方式进行验证 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 尚未成功应用于成人矿化组织。使用我们新开发的协议，结果 R21将为各个阶段的感觉神经的空间定义调节提供业内首个见解 截肢后的整个再生过程，以及对其在整体再生中的作用的基本理解 维持细胞命运和可塑性。