Transcriptional Regulatory Mechanisms of Salivary Gland Branching Morphogenesis

项目摘要 在胚胎发育过程中， 唾液腺的干/祖（SP）细胞必须被严格调节以维持稳态平衡， 这对正常组织发育和再生至关重要。因此，更好地了解 驱动SP细胞功能的调节机制将为研究唾液腺 形态发生，并帮助识别损伤后组织再生的关键途径， 在疾病状态下。转录因子p63，特别是Δ Np 63同种型，在大肠杆菌中高度表达。 SP细胞群的上皮丰富的组织，包括那些皮肤和腺器官，如唾液 腺（SG）。p63在SG中的关键重要性通过p63缺失小鼠的戏剧性表型而突出， 其显示组织形态发生的完全阻断。尽管p63在SG中的某些功能 我们目前对p63在早期发育中的作用的认识， SG形态发生和分化的阶段，包括分支形态发生，是相当有限的。在 特别是，p63决定细胞命运的分子机制以及它如何控制特定的 在早期SG分支的基因表达程序，是缺乏的。因此，确定Δ Np 63驱动的 调控网络，特别是在分支形态发生的背景下，将作为重要的第一步 在我们对SG干/祖细胞生物学的理解中， 用于再生和重新设计富含上皮的器官（如SG）的复杂分支结构。 为了解决这些知识缺口，我们将利用小鼠模型和复杂的基因组方法来研究 三个独立的领域。首先，我们将使用条件性基因敲除小鼠模型来确定作用 Δ Np 63在唾液腺分支形态发生（Aim 1）中的作用。这些研究是非常必要的，因为它们将确定 第一次，p63在SG分支形态发生中的功能作用。第二，我们将执行机械化 研究确定由Δ Np 63控制的关键靶基因和途径，并确定损失如何 Δ Np 63的表达改变了整体基因表达程序，并影响了分支过程中的细胞命运轨迹 单细胞分辨率下的形态发生（Aim 2）。最后，我们将利用离体组织外植体来研究 WNT信号通路，特别是Sfrp 1，介导Δ Np 63的功能，并测试Sfrp 1是否 能够恢复在p63丧失时观察到的分支缺陷。总的来说，这些研究 有可能进一步了解Δ Np 63依赖的转录和细胞网络重要 对于SG的生物学功能，特别是当它涉及分支时。我们对人类进化的认识 驱动SG分支和发展的潜在机制可以为两者的再生提供范例 小鼠和人类SG和更好地提供治疗发育障碍患者的信息， SG的疾病。

Project Summary During embryonic development, the delicate balance between proliferation and differentiation of stem/progenitor (SP) cells of the salivary gland must be tightly regulated to maintain a homeostatic balance, which is critical for normal tissue development and regeneration. Therefore, a better understanding of the regulatory mechanisms driving SP cell function will provide fundamental insights into salivary gland morphogenesis and help identify pathways that are critical for tissue regeneration following injury, damage, or during diseased states. The transcription factor p63, specifically the ΔNp63 isoform, is highly expressed in the SP cell populations of epithelial rich tissues including those of the skin and glandular organs such as the salivary gland (SG). The critical importance of p63 in the SG is highlighted by the dramatic phenotype of p63-null mice, which display a complete block in tissue morphogenesis. Although some aspects of p63 function in SG development have been reported, our current knowledge regarding the role of p63 in the early developmental stages of SG morphogenesis and differentiation, including branching morphogenesis, is rather limited. In particular, the molecular mechanisms through which p63 dictates cell fate decisions and how it controls specific gene expression programs during early SG branching, is lacking. Therefore, identifying the ΔNp63-driven regulatory networks, particularly in the context of branching morphogenesis, will serve as an essential first step in our understanding of SG stem/progenitor cell biology with the long-term goals of developing new strategies for regeneration and for re-engineering the complex branching architecture of epithelial rich organs like the SG. To address these knowledge gaps, we will utilize mouse models and sophisticated genomic approaches to study three independent areas of interest. First, we will use conditional knockout mouse models to determine the role of ΔNp63 in salivary gland branching morphogenesis (Aim1). Such studies are much needed as they will identify for the first time, the functional role of p63 in SG branching morphogenesis. Second, we will perform mechanistic studies to identify critical target genes and pathways that are governed by ΔNp63 and determine how the loss of ΔNp63 alters the global gene expression program and affects cell fate trajectories during branching morphogenesis at single cell resolution (Aim2). Finally, we will utilize ex vivo tissue explants to examine the role of the WNT signaling pathway, and specifically Sfrp1, in mediating the function of ΔNp63 and test whether Sfrp1 is able to restore the branching defects observed upon the loss of p63. Collectively, these studies have the potential to further our understanding of the ΔNp63-dependent transcriptional and cellular networks important for the biological function of the SG particularly as it pertains to branching. Advances in our understanding of the underlying mechanisms driving SG branching and development can provide a paradigm for regeneration of both mouse and human SGs and better inform therapies to treat patients with developmental dysfunctions and diseases of the SG.