Developmental regulation of tendon-bone connectivity in the jaw

颌骨腱骨连接的发育调节

• 批准号: 10424505
• 负责人: Amy E Merrill
• 金额: $ 45.41万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-08 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10424505
• 关键词: Affect; Cartilage; Cells; Chondrocytes; Congenital Disorders; Data; Development; FGFR2 gene; Fibrocartilages; Fluorescence; Future; Gene Dosage; Genes; Genetic; Genetic Transcription; Genomics; Hybrid Cells; Hybrids; In Situ Hybridization; Jaw; Jaw Abnormalities; Knowledge; Ligands; Limb structure; Mastication; Mesoderm; Minerals; Modeling; Molecular; Morphology; Mus; Neural Crest Cell; Osteoblasts; Output; Pathology; Pattern; Population; Positioning Attribute; Property; Regulation; Repression; Series; Signal Transduction; Specific qualifier value; Speech; System; Tendon structure; Testing; Tissues; To specify; Transcriptional Regulation; Up-Regulation; Venus; bone; bone cell; cartilage cell; cell type; design; insight; jaw movement; mouse genetics; mouse model; notch protein; novel; osteogenic; progenitor; repaired; scleraxis; single molecule; single-cell RNA sequencing; skeletal; stem cells

PROJECT SUMMARY / ABSTRACT Integration of the jaw with the surrounding musculature is essential for speech and mastication. A fundamental step in jaw integration begins in development, with formation of stable tendon-bone attachments that are zonally organized into tendon, fibrocartilage, mineralized fibrocartilage, and bone. The gradient of skeletogenic cell types within the attachment arises from attachment progenitors (APs) that, through unclear mechanisms, interpret chondrogenic versus tenogenic signaling to acquire distinct cell fates along the tendon- bone axis. Even less is known about APs of the jaw which, unlike their counterparts in the limb and trunk, are derived from neural crest cells (NCC). This study tests the idea that jaw APs differentiate into a gradient of skeletogenic cell types through a series of binary switches that are regulated by an NCC-specific mechanism. We have found that jaw APs express graded levels of Scx, Runx2, and Sox9 depending on their position along the tendon-bone axis. We also found that during AP differentiation a novel intermediate Scx+/Runx2+ population emerges. In Runx2+/- mice this intermediate population fails to form, and APs differentiate into tendon over cartilage/bone. While this suggests that tripotent APs differentiate through lineage-restricted intermediates, how APs spatially interpret signals for tendon vs. cartilage/bone to make these cell fate decisions and whether APs always choose between one fate or the other (e.g. tenocyte vs. osteoblast) or acquire hybrid properties (e.g. osteofibrogenic) is unknown. We recently showed that an Fgf-Notch signaling axis is regionally deployed along the tendon-bone interface and promotes AP differentiation into tendon over cartilage/bone. This mechanism appears NCC-specific, as loss of Fgfr2 in mesoderm-derived APs does not alter limb attachment development. In this study, we use mouse genetics along with cutting-edge genomics to test that, during AP differentiation, integration of Fgf and Notch signaling promotes tendon cell fate in a series of binary switches by regulating levels of Scx, Runx2, and Sox9 transcription. In Aim1 we will use clonal lineage tracing and scRNA-seq to determine the lineage relationship between APs and the skeletogenic cells in the tendon-bone attachment. In Aim2, we will use conditional mouse genetics to determine how differences in Notch signal strength along the tendon-bone axis alter AP cell fate decisions. In Aim3, we will use a combination of mouse genetics and CUT&RUN-seq to test that Erk signaling integrates Fgf and Notch signaling through linear and parallel mechanisms. In the linear mechanism, Erk activates Notch2 signaling by initiating Dll1 expression. In the parallel mechanism, Erk and Notch2 independently activate the same downstream targets genes for tendon fate including Scx. Completion of these aims will reveal a developmental mechanism that establishes a gradient of skeletogenic cell types in tendon-bone attachments of the jaw. Knowledge gained will guide future developmentally inspired strategies for jaw attachment repair and may inform how jaw abnormalities develop in the FGFR2-and NOTCH2- related congenital disorders.

项目概要/摘要 下巴与周围肌肉组织的整合对于言语和咀嚼至关重要。一个 颌骨整合的基本步骤始于发育，形成稳定的腱骨附着物 按区域组织成肌腱、纤维软骨、矿化纤维软骨和骨。梯度为 附着内的成骨细胞类型源自附着祖细胞（AP），其通过不清楚 机制，解释软骨形成与肌腱信号传导以获得沿着肌腱的不同细胞命运 骨轴。我们对下颌的 AP 知之甚少，与肢体和躯干的 AP 不同，它是 源自神经嵴细胞（NCC）。这项研究测试了下颌 AP 分化成梯度的观点 通过一系列由 NCC 特定机制调节的二元开关来控制成骨细胞类型。 我们发现下巴 AP 表达 Scx、Runx2 和 Sox9 的分级水平，具体取决于它们在 腱骨轴。我们还发现，在 AP 分化过程中，一种新的中间体 Scx+/Runx2+ 人口出现。在 Runx2+/- 小鼠中，这种中间群体无法形成，AP 分化为 软骨/骨上的肌腱。虽然这表明三能 AP 通过谱系限制进行分化 中间体，AP如何在空间上解释肌腱与软骨/骨的信号以决定这些细胞的命运 决定以及 AP 是否总是在一种命运或另一种命运之间进行选择（例如肌腱细胞与成骨细胞）或获得 混合特性（例如骨纤维形成）尚不清楚。我们最近表明 Fgf-Notch 信号轴是 沿肌腱-骨界面局部部署，促进 AP 分化为肌腱 软骨/骨。这种机制似乎是 NCC 特异性的，因为中胚层来源的 AP 中 Fgfr2 的丢失并不影响 NCC 特异性。 改变肢体附着发育。在这项研究中，我们利用小鼠遗传学和尖端基因组学来 测试表明，在 AP 分化过程中，Fgf 和 Notch 信号传导的整合在一系列过程中促进肌腱细胞的命运 通过调节 Scx、Runx2 和 Sox9 转录水平来实现二元开关。在 Aim1 中，我们将使用克隆谱系 追踪和 scRNA-seq 以确定 AP 与骨骼中的成骨细胞之间的谱系关系 腱骨附着。在 Aim2 中，我们将使用条件小鼠遗传学来确定小鼠之间的差异 沿腱骨轴的缺口信号强度改变 AP 细胞的命运决定。在 Aim3 中，我们将使用 结合小鼠遗传学和 CUT&RUN-seq 来测试 Erk 信号传导整合 Fgf 和 Notch 通过线性和并行机制发出信号。在线性机制中，Erk 通过以下方式激活 Notch2 信号传导： 启动 Dll1 表达式。在并行机制中，Erk和Notch2独立激活相同的 下游目标肌腱命运基因，包括 Scx。完成这些目标将揭示出发展 在颌骨腱骨附着处建立成骨细胞类型梯度的机制。 获得的知识将指导未来下颌附件修复的发展策略，并可能 了解 FGFR2 和 NOTCH2 相关先天性疾病中颌骨异常如何发展。