Identifying the core transcriptional regulatory network initiating a tooth program

识别启动牙齿计划的核心转录调控网络

• 批准号: 10710770
• 负责人: Huojun Cao
• 金额: $ 43.81万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2028-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10710770
• 关键词: Adult; Aging; Animal Model; Biological Assay; Cell Lineage; Cells; Communication; Congenital Abnormality; Coupled; Defect; Dental; Development; Ectoderm; Ectoderm Cell; Ectopic Expression; Elderly; Environment; Epithelium; Experimental Designs; Fibroblast Growth Factor; Future; Gene Expression; Genes; Genetic; Genetic Transcription; Goals; Health; Human; Incisor; Knock-out; Knockout Mice; Knowledge; Lasers; Lead; Life; Mandible; Maxilla; Mesenchyme; Microdissection; Modeling; Molecular; Morphology; Mouth Diseases; Mus; Natural regeneration; Oral; Organ failure; Outcome Study; Population; Program Development; Repression; Reptiles; Research; SHH gene; Shark; Side; Signal Pathway; Skin; Specific qualifier value; Specificity; Stem Cell Development; Structure; Supernumerary Tooth; Testing; Time; Tissue Recombination; Tooth Loss; Tooth regeneration; Tooth structure; Trauma; Up-Regulation; catalyst; craniofacial; experimental study; genome-wide; improved; in vivo; mouse model; multiple omics; oral cavity epithelium; overexpression; permanent tooth; programs; regenerative therapy; repaired; single-cell RNA sequencing; spatiotemporal; stem cell based approach; transcription factor; transcription regulatory network; transcriptome; transcriptome sequencing

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.

项目简介：因口腔疾病、创伤、衰老或先天缺陷导致的牙齿缺失是 最常见的器官衰竭。牙齿发育的第一个形态标志是牙齿的形成 叶片(Dl)。牙齿发育计划的关键结构--牙齿发育的关键结构--是如何从 幼稚的上颌外胚层和下颌外胚层，仍然知之甚少。我们应用了激光显微解剖 时空RNA-SEQ和单细胞Multiome-SEQ联用研究小鼠下颌外胚层发育 DL规范和齿根(E9.5-E12.5)。与非DL上皮(即未来的皮肤)相比，我们 确定了一组潜在的驱动转录因子(TF)，包括PITX1、PITX2、SOX2、 和Foxe1。相反，另一组TF在DL中显著减少，尽管在流产/腹侧丰富 外胚层，包括TFAP2a、Tfap2b、IRX3和Irx4。而TFAP2A或TFAP2A的单个外胚层敲除(KO Tfap2b导致正常的DL和非DL的上皮发育，我们发现两者的外胚层双KO 导致严重的非DL上皮缺陷，DL规格的空间扩张，以及异位牙齿。 类似地，我们和其他研究小组发现，PITX1、PITX2或SOX2的单个KO不影响DL 规范，尽管这些基因在DL规范之前表达，并在后期出现牙齿缺陷。 在这里，我们提出，像非DL上皮一样，存在一个冗余网络来指定DL上皮。这个 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。 这项拟议研究的总体目标是剖析牙齿的分子和细胞机制。 启动，特别是驱动DL规范的核心转录调控网络(TRN) 在上颌和下颌外胚层内，并启动牙齿发育计划。我们的中央 假设几个关键的TF--包括PITX1、PITX2、SOX2、FOXE1等--冗余地驱动核心 调节DL形成的TRN和牙齿启动程序。此外，异位表达的组合 这些因子可以将非DL上皮(如皮肤)转化为DL上皮，并诱导异位牙齿发育。 在目标1中，我们将通过两次KO来确定Pitx1、Pitx2和Sox2的遗传冗余度和特异性 模型和基于切割和运行的TF目标识别。在目标2中，我们将定义PITX1/PITX2驱动 Pitx1/Pitx2 KO小鼠牙齿起始过程中DL上皮与间充质间的通讯 模特。最后，在目标3中，我们将确定足以驱动DL谱系规范和 非DL上皮细胞重编程。总而言之，使用一套强大的体内动物模型，体外植体， 和全基因组分析，这些研究将填补我们目前对DL理解中的一个关键知识空白 规范及其在牙齿再生中的应用。此实验设计和方法，以及 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 改善口腔和头面部健康的催化剂。