Identifying the core transcriptional regulatory network initiating a tooth program

项目概述：由于口腔疾病、创伤、衰老或先天性缺陷而导致的牙齿缺失是 最常见的器官衰竭牙齿发育的第一个形态学标志是牙本质的形成。 层（DL）。如何DL-启动牙齿发育计划的关键结构-指定从 幼稚的上颌和下颌外胚层，仍然知之甚少。我们应用激光显微切割技术 偶联时空RNA-seq和单细胞Multiome-seq来描绘小鼠下颌骨外胚层在 DL规范和齿起始（E9.5-E12.5）。与非DL上皮（即，未来的皮肤），我们 鉴定了一组用于DL特化的潜在驱动转录因子（TF），包括Pitx 1、Pitx 2、Sox 2， 福克斯1相反，另一组TF在DL中显著减少，尽管在背口/腹侧中富集， 外胚层，包括Tfap 2a、Tfap 2b、Irx 3和Irx 4。而Tfap 2a或Tfap 2b的单外胚层敲除（KO）则是一种新的表达。 Tfap 2b导致正常DL和非DL上皮发育，我们发现两者的外胚层双KO 导致严重的非DL上皮缺陷，DL规格的空间扩展和异位牙齿。 同样，我们和其他研究小组发现，Pitx 1、Pitx 2或Sox 2的单个KO不影响DL 尽管这些基因在DL规范和牙齿缺陷的后期阶段之前表达。 在这里，我们建议，像非DL上皮的冗余网络存在，以指定DL上皮。的 这项研究的长期目标是开发基于干细胞的牙齿修复和再生方法。 这项研究的总体目标是剖析牙齿的分子和细胞机制， 启动，特别是核心转录调控网络（TRN），驱动DL的规范 在上颌骨和下颌骨外胚层内，并启动牙齿发育程序。我们的中央 有一种假说认为，一些关键TF--包括PITX 1、PITX 2、SOX 2、FOXE 1等--冗余驱动核心 TRN调节DL形成和牙齿起始程序。此外，异位表达的组合， 这些TF可以转化非DL上皮（例如，皮肤）到DL上皮并诱导异位牙齿发育。 目的1：通过双敲除技术，确定Pitx 1、Pitx 2和Sox 2的遗传冗余度和特异性 模型和基于CUT& RUN的TF目标识别。在目标2中，我们将定义PITX 1/PITX 2驱动的 用Pitx 1/Pitx 2基因敲除小鼠研究牙齿形成过程中DL上皮和间充质之间的通讯 模型最后，在目标3中，我们将确定足以驱动DL沿袭规范的核心TF集， 非DL上皮重编程。总的来说，使用一组强大的体内动物模型，离体外植体， 和全基因组分析，这些研究将填补我们目前对DL的理解中的关键知识空白 规范及其在牙齿再生中的应用。这种实验设计和方法，沿着 具有互补专业知识的PI/CoI以及本提案中概述的强大环境提供了 催化剂，以改善口腔和颅面健康。

PROJECT SUMMARY: Loss of teeth due to oral disease, trauma, aging or congenital defects is one of the most common organ failures. The first morphological sign of tooth development is the formation of the dental lamina (DL). How the DL—the critical structure for initiation of a tooth development program—is specified from naïve maxillary and mandibular ectoderm, remains poorly understood. We applied Laser Microdissection coupled spatiotemporal RNA-seq and single cell Multiome-seq to profile mouse mandibular ectoderm during DL specification and tooth initiation (E9.5-E12.5). In comparison to the non-DL epithelium (i.e., future skin), we identified a set of potential driver transcription factors (TFs) for DL specification including Pitx1, Pitx2, Sox2, and Foxe1. In contrast, another set of TFs was notably reduced in the DL, although enriched in aboral/ventral ectoderm, including Tfap2a, Tfap2b, Irx3, and Irx4. While single ectodermal knock-out (KO) of Tfap2a or Tfap2b resulted in normal DL and non-DL epithelial development we found that ectodermal double KO of both resulted in severe non-DL epithelial defects, a spatial expansion of DL specification, and an ectopic tooth. Similarly, we and other groups have found that the single KO of Pitx1, Pitx2 or Sox2 does not affect DL specification, despite expression of these genes prior to DL specification and tooth defects at later stages. Here, we propose that like the non-DL epithelium a redundant network exists to specify the DL epithelium. The long-term goal of this study is development of stem cell-based approaches for tooth repair and regeneration. The overall objective of the proposed research is to dissect the molecular and cellular mechanisms of tooth initiation, particularly the core transcriptional regulatory networks (TRNs) that drive specification of the DL within maxillary and mandibular ectoderm and initiation of a tooth development program. Our central hypothesis is that a few key TFs—including PITX1, PITX2, SOX2, FOXE1, etc.—redundantly drive the core TRNs that regulate DL formation and a tooth initiation program. Further, ectopic expression of a combination of these TFs could convert non-DL epithelium (e.g., skin) to DL epithelium and induce ectopic tooth development. In Aim 1, we will determine genetic redundancy and specificity of Pitx1, Pitx2 and Sox2 through double KO models and CUT&RUN-based TF target identification. In Aim 2, we will define the PITX1/PITX2-driven communication between DL epithelium and mesenchyme during tooth initiation using a Pitx1/Pitx2 KO mouse model. Finally, in Aim 3, we will determine the core set of TFs sufficient to drive DL lineage specification and non-DL epithelial reprogramming. Collectively, using a strong set of in vivo animal models, ex vivo explants, and genome-wide assays, these studies will fill a critical knowledge-gap in our current understanding of DL specification and its application to tooth regeneration. This experimental design and approach, along with PI’s/CoI’s with complementary expertise, and the strong environment outlined in this proposal provide the catalyst to improve oral and craniofacial health.