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

项目总结 在自我更新、增殖和分化之间错综复杂的动态平衡 唾液腺(SG)的干细胞/祖细胞必须受到严格的调控，以确保适当的形态发生。 动态平衡和再生。正常脑血管功能的改变，临床上常表现为 功能低下与外胚层发育不良、干燥综合征和γ- 口腔癌患者的放射治疗。目前，肺下裂的治疗选择仍然有限。 因此，识别控制干细胞/祖细胞功能的关键转录和信号网络 为了促进定向干细胞和基于再生的治疗，SG是非常必要的。干细胞Δnp63 丰富的转录因子，在上皮再生功能中起着关键作用，从 ΔNp63缺失动物的表型表现为富上皮性器官发育停滞和发育不全 包括秘书长在内。然而，我们目前对Δnp63引导基因的分子机制的了解 茎/祖细胞的承诺、维持和分化所必需的表达程序 SG中的细胞群缺乏。因此，识别p63驱动的调控网络，特别是在 全球和基因组背景下，是更好地理解SG茎/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。为了解决这些知识差距， 我们将利用多个多功能的小鼠模型来研究两个主要的独立感兴趣的领域。首先，我们将 利用条件性基因敲除小鼠模型研究ΔNp63在脑海马形态发生和成人中的作用 组织维护与修复(Aim1)。这样的系统研究是非常需要的，因为它们将为 第一次，p63在SG发育和协调干细胞/祖细胞分化中的功能作用 程序。其次，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+干细胞和祖细胞在星状细胞再生和辐射反应中的作用 损坏。此外，我们将定义依赖于p63的SG细胞恒等式以及定义的细胞和 与再生有关的分子特征，并通过执行单细胞来响应辐射 RNA测序(AIM2)。这些研究将更好地阐明ΔNp63在SG器官发生和成体发育中的作用 腺体的维持，并阐明其在SG再生和对辐射的反应中的作用 致伤。重要的是，我们的遗传和基因组研究还将发现新的ΔNp63途径 与再生和再生相关的不同细胞状态的依赖和独立的生物标志物和驱动因素 对辐射敏感。从长远来看，从我们拟议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.