Developmental regulation of tendon-bone connectivity in the jaw

颌骨腱骨连接的发育调节

• 批准号: 10209547
• 负责人: Amy E Merrill
• 金额: $ 47.3万
• 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/10209547
• 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.

项目总结/摘要 颌骨与周围肌肉组织的整合对于语言和咀嚼是必不可少的。一 颌骨整合的基本步骤始于发育，形成稳定的肌腱-骨附着 它们被带状组织成肌腱、纤维软骨、矿化纤维软骨和骨。的梯度 附件中的骨骼发生细胞类型来自附件祖细胞（AP），通过不清楚的 机制，解释软骨形成与肌腱形成的信号传导，以获得不同的细胞命运沿着肌腱- 骨轴更少的是已知的AP的下巴，不像他们的同行在四肢和躯干， 来源于神经嵴细胞（NCC）。这项研究测试的想法，下巴AP分化成梯度， 通过一系列由NCC特异性机制调节的二元开关，调节成骨细胞类型。 我们已经发现，颌骨AP表达Scx，Runx 2和Sox 9的分级水平取决于它们沿沿着的位置。 肌腱-骨轴我们还发现，在AP分化过程中， 人口出现。在Runx 2 +/-小鼠中，该中间群体未能形成，并且AP分化为 软骨/骨上的肌腱。虽然这表明三能AP通过谱系限制性 中间体，AP如何在空间上解释肌腱与软骨/骨的信号，以使这些细胞的命运 决定以及AP是否总是在一种命运或另一种命运之间选择（例如，肌腱细胞与成骨细胞）或获得 杂交特性（例如骨纤维形成）是未知的。我们最近发现Fgf-Notch信号轴是 沿着腱-骨界面局部展开，并促进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Fgfr 2的丢失并不 改变肢体附着发育。在这项研究中，我们使用小鼠遗传学沿着尖端基因组学， 试验表明，在AP分化过程中，Fgf和Notch信号的整合以一系列的方式促进肌腱细胞的命运， 通过调节Scx、Runx 2和Sox 9转录水平来实现二元开关。在Aim 1中，我们将使用克隆谱系 追踪和scRNA-seq，以确定AP和骨骼发生细胞之间的谱系关系。 腱骨附着在Aim 2中，我们将使用条件小鼠遗传学来确定 沿着腱-骨轴的Notch信号强度改变AP细胞命运决定。在Aim 3中，我们将使用 小鼠遗传学和CUT&RUN-seq的组合以测试Erk信号传导整合Fgf和Notch 通过线性和并行机制发送信号。在线性机制中，Erk通过以下方式激活Notch 2信号传导： 启动Dll 1表达。在并联机构中，Erk和Notch 2独立地激活相同的 下游靶向肌腱命运的基因，包括Scx。这些目标的实现将揭示一个发展的 在颌骨的肌腱-骨附着中建立成骨细胞类型梯度的机制。 获得的知识将指导未来的发展启发的策略，颌骨附件修复， 告知颌骨异常如何在FGFR 2和NOTCH 2相关的先天性疾病中发展。