Developmental regulation of tendon-bone connectivity in the jaw

项目摘要/摘要 颌骨与周围肌肉的结合对于说话和咀嚼是必不可少的。一个 颌骨整合的基本步骤始于发育，形成稳定的肌腱-骨附着体。 它们被带状组织成肌腱、纤维软骨、矿化纤维软骨和骨。的渐变 附件内的骨骼生成细胞类型来自附件祖细胞(AP)，尽管不清楚 机制，解释了软骨生成信号与张力生成信号在肌腱上获得不同的细胞命运- 骨轴。对于颌骨的AP更是知之甚少，与四肢和躯干的对应部位不同，它们是 来源于神经脊细胞(NCC)。这项研究测试了颌骨AP分化为 骨骼发生细胞类型通过一系列由NCC特定机制调节的二元开关来实现。 我们发现，JAW AP根据其沿线的位置表达SCX、Runx2和Sox9的分级水平 肌腱-骨轴。我们还发现，在AP分化过程中，一种新的中间体Scx+/Runx2+ 人口涌现。在Runx2+/-小鼠中，这种中间群体无法形成，AP分化为 软骨/骨骼上的肌腱。虽然这表明三能AP是通过血统受限来区分的 中间体，AP如何在空间上解释肌腱与软骨/骨的信号，从而决定这些细胞的命运 决定以及AP是否总是在一种或另一种命运之间做出选择(例如，腱细胞与成骨细胞)或获取 杂交特性(例如，成骨纤维)尚不清楚。我们最近发现了一个成纤维细胞生长因子-Notch信号轴 沿肌腱-骨界面区域部署，促进AP分化为肌腱 软骨/骨。这种机制似乎是NCC特有的，因为在中胚层来源的AP中FGFR2的丢失不是 改变肢体附着发育。在这项研究中，我们使用小鼠遗传学和尖端基因组学来 实验表明，在AP分化过程中，整合成纤维细胞生长因子和Notch信号可促进肌腱细胞的一系列命运 通过调节SCX、Runx2和Sox9转录水平来调节二进制开关。在Aim1中，我们将使用克隆血统 追踪和scRNA-seq以确定AP和骨骼生成细胞之间的谱系关系 肌腱-骨附着物。在AIM2中，我们将使用条件小鼠遗传学来确定 沿肌腱-骨轴的缺口信号强度改变AP细胞的命运。在Aim3中，我们将使用 结合小鼠遗传学和Cut&Run-Seq检测ERK信号整合成纤维细胞生长因子和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Notch2信号 正在启动DLL1表达式。在并联机构中，Erk和Notch2独立地激活相同的 下游针对肌腱命运的基因，包括SCX。这些目标的实现将揭示一种发展 在颌部肌腱-骨附着体中建立骨骼细胞类型梯度的机制。 所获得的知识将指导未来发展的颌骨附着体修复策略，并可能 介绍FGFR2和NOTCH2相关先天性疾病中颌骨异常是如何发展的。

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.