Elucidating the role of p63 and transcriptional control mechanisms in progenitor cells of the salivary gland

项目总结 唾液干/祖细胞增殖和分化之间的微妙平衡 腺体(SG)必须受到严格调控，以确保适当的形态发生、动态平衡和再生。 正常SG功能的改变，临床上常表现为肝功能减退，与疾病有关 例如Sjögren综合征和头颈部鳞癌患者的γ放射治疗。 目前，肺功能减退的治疗选择和恢复正常脑白质功能的机制仍然存在 有限的。因此，开发新的工具和战略旨在识别转录和 SG的干细胞/祖细胞功能的潜在信号网络，最终目标是用于干细胞 以再生为基础的疗法和组织工程方法是至关重要的。因此，我们的目标是检查 SG的干/祖细胞的转录组图谱，并鉴定和表征调控 这个细胞群体的网络。众所周知，ΔNp63在上皮细胞再生中起关键作用 作为ΔNp63缺失的动物不能发育包括SG在内的几个富含上皮的器官。然而，我们的 目前对ΔNp63如何调节SG的干/祖细胞中的基因表达缺乏了解。 因此，研究p63驱动的调控网络是更好地理解生物学的关键一步。 以及指导治疗SG功能障碍的新策略。要解决这些问题 知识缺口，我们将利用从新型Δnp63-gfp转基因小鼠获得的SG干/祖细胞来 研究两个主要感兴趣的领域。首先，我们将进行功能分析来比较ΔNp63- GFPhi和ΔNP63-GFP使SG细胞既保留了祖细胞能力，又具有分化为 SG有机球中的成熟细胞谱系(Aim1A)。此外，我们将在体内进行转录 在ΔNp63-GFPhi SG的纯群体中产生基因表达谱(RNA-seq) 祖细胞和ΔNp63-GFP低SG细胞(Aim 1B)。这样的体内研究很重要，因为它们将 首次在广泛和动态的尺度上鉴定SG祖细胞的基因表达谱。 第二，我们将进行体内芯片序列研究，以确定关键的调控网络和调控途径。 ΔNp63在SG细胞中的表达。总的来说，我们的方法使用了基因定义的模型系统和尖端技术 下一代测序技术将更好地阐明SG祖细胞的转录图景 并揭示了ΔNp63调控的转录调控网络和信号通路。既然这样 特别缺乏对SG细胞的基因组研究，我们拟议的实验可能会揭示新的 在这个器官中，干细胞功能的生物标记物和重要的调节器和驱动因素。从长远来看，这种知识 将对患有SG功能障碍疾病的人类患者具有临床和治疗意义。

PROJECT SUMMARY The delicate balance between 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 Sjögren’s Syndrome and with γ-irradiation therapy of Head and Neck Squamous Carcinoma patients. Currently, treatment options for hyposalivation and mechanisms for restoring normal SG function remain limited. Therefore, the development of new tools and strategies directed at identifying transcriptional and signaling networks underlying stem/progenitor cell function of the SG with the ultimate goal for use in stem cell regenerative-based therapies and tissue engineering approaches, are critical. Hence, our goal is to examine the transcriptome landscape of the stem/progenitor cells of the SG and identify and characterize the regulatory networks of this cell population. It is well established that ΔNp63, plays a critical role in epithelial regenerative function as ΔNp63-null animals fail to develop several epithelial-rich organs including the SG. However, our current knowledge of how ΔNp63 regulates gene expression in the stem/progenitor cells of the SG is lacking. Thus examining the p63-driven regulatory networks is a key step towards a better understanding of the biology of the SG progenitor cells and in directing new strategies in treating SG dysfunction. To address these knowledge gaps, we will utilize SG stem/progenitor cells obtained from novel ΔNp63-GFP transgenic mice to study two major areas of interest. First, we will perform functional assays to compare the abilities of ΔNp63- GFPhi and ΔNp63-GFPlow SG cells to retain both their progenitor capacity and their ability to differentiate into mature cell lineages in SG organospheres (Aim1A). Furthermore, we will perform in vivo transcriptomic profiling (RNA-seq) to generate the mRNA expression profile in a pure population of ΔNp63-GFPhi SG progenitor cells and ΔNp63-GFPlow SG cells (Aim 1B). Such in vivo studies are important, since they will identify for the first time the gene expression profile of SG progenitor cells on a broad and dynamic scale. Second, we will perform in vivo ChIP-seq studies to identify key regulatory networks and pathways regulated by ΔNp63 in SG cells. Collectively, our approach using a genetically-defined model system and cutting-edge next generation sequencing technology will better elucidate the transcriptomic landscape of SG progenitor cells and shed light on the ΔNp63-governed transcriptional regulatory network and signaling pathways. Since such genomic studies are particularly lacking for SG cells, our proposed experiments will likely reveal new biomarkers and important regulators and drivers of stem cell function in this organ. Long term, such knowledge will have clinical and therapeutic implications for human patients who suffer from SG dysfunction diseases.