Genomic and functional investigations of the transcriptional regulatory network of salivary gland morphogenesis and stem cell fate choices in defined genetic models

项目摘要 细胞的自我更新、增殖和分化之间的复杂动态平衡， 唾液腺（SG）的干/祖细胞必须被严格调节以确保适当的形态发生， 体内平衡和再生。正常SG功能的改变，临床上通常表现为 唾液分泌不足，与诸如外胚层发育不良、舍格伦综合征和γ- 口腔癌患者的放射治疗。目前，唾液过少的治疗选择仍然有限。 因此，确定控制干/祖细胞功能的关键转录和信号网络， 非常需要SG来促进靶向干细胞和基于再生治疗。Δ Np 63，干细胞 富集的转录因子，在上皮再生功能中起着关键作用，这一点从 Δ Np 63缺失动物表现出发育停滞和富上皮器官发育不全的表型 包括SG。然而，我们目前对Δ Np 63指导基因表达的分子机制的认识， 干/祖细胞定型、维持和分化所必需的表达程序 SG中的细胞群缺乏。因此，确定p63驱动的调控网络，特别是在 全球和基因组背景下，是朝着更好地了解SG干/祖细胞生物学的关键一步 细胞，并最终指导治疗SG功能障碍的新策略。为了弥补这些知识差距， 我们将利用多种多样的小鼠模型来研究两个主要的独立领域。一是 使用条件性基因敲除小鼠模型来研究Δ Np 63在SG形态发生和成年中的作用。 组织维护和修复（目标1）。这种系统的研究是非常必要的，因为它们将确定 第一次，p63在SG发育和协调干/祖细胞分化中的功能作用 程序.第二，我们将使用p63基因敲除小鼠模型和谱系追踪实验来确定p63基因敲除小鼠的基因表达。 p63+干细胞和祖细胞在SG再生过程中的作用以及对辐射诱导的 损害此外，我们将定义p63依赖性SG细胞身份和所定义的细胞和 与再生相关的分子特征以及通过执行单细胞对辐射的响应 RNA测序（Aim 2）。这些研究将更好地阐明Δ Np 63在SG器官发生中的作用， 腺体的维护，并阐明其对SG再生的贡献，并在辐射响应 诱导损伤。重要的是，我们的遗传学和基因组研究还将揭示新的Δ Np 63通路。 与再生相关的不同细胞状态的依赖性和独立性生物标志物和驱动因素， 辐射敏感性从长远来看，从我们提出的机制研究中获得的知识将具有临床和 对患有SG功能障碍疾病的人类患者的治疗意义。

PROJECT SUMMARY The intricate and dynamic balance between self-renewal, proliferation and differentiation of stem/progenitor cells of the salivary gland (SG) must be tightly regulated to ensure proper morphogenesis, homeostasis and regeneration. Alterations to normal SG function, clinically often manifested with hyposalivation, are associated with diseases such as Ectodermal Dysplasias, Sjögren’s Syndrome and with γ- irradiation therapy of oral cancer patients. Currently, treatment options for hyposalivation remain limited. Therefore, identifying crucial transcriptional and signaling networks that govern stem/progenitor cell function of the SG are much needed to facilitate targeted stem cell and regenerative-based therapies. ΔNp63, a stem cell enriched transcription factor, plays a critical role in epithelial regenerative function as evident from the phenotype of ΔNp63-null animals which exhibit developmental arrest and agenesis of epithelial-rich organs including the SG. However, our current knowledge of the molecular mechanisms by which ΔNp63 directs gene expression programs necessary for the commitment, maintenance and differentiation of the stem/progenitor cell population in the SG is lacking. Thus, identifying the p63-driven regulatory networks, particularly in the global and genomic context, is a key step towards a better understanding of the biology of SG stem/progenitor cells and ultimately in directing new strategies in treating SG dysfunction. To address these knowledge gaps, we will utilize multiple versatile mouse models to study two major independent areas of interest. First, we will use conditional knockout mouse models to examine the role of ΔNp63 in SG morphogenesis and in adult tissue maintenance and repair (Aim1). Such systematic studies are much needed as they will identify for the first time, the functional role of p63 in SG development and in orchestrating stem/progenitor cell differentiation programs. Second, we will use p63 knockout mouse models and lineage tracing experiments to determine the contribution of p63+ stem and progenitor cells during SG regeneration and in response to irradiation induced damage. Furthermore, we will define p63 dependent SG cellular identities and the defined cellular and molecular signature that is associated with regeneration and in response to irradiation by performing single cell RNA-sequencing (Aim2). These studies will better elucidate the role of ΔNp63 in SG organogenesis, and adult gland maintenance, and elucidate its contribution towards SG regeneration and in response to irradiation induced injury. Importantly, our genetic and genomic studies will also uncover novel ΔNp63-pathways dependent and independent biomarkers and drivers of the distinct cell states associated with regeneration and radiosensitivity. Long term, knowledge garnered from our proposed mechanistic studies will have clinical and therapeutic implications for human patients who suffer from SG dysfunction diseases.