Genetic and molecular mechanisms of Xbp-1 mediated salivary gland development and differentiation

项目摘要 在胚胎发生和成体中，不同的细胞的增殖和分化之间的微妙平衡， 唾液腺（SG）中的细胞群必须严格调节以确保正常的组织发育 和体内平衡。更好地理解驱动细胞命运决定的调控机制， 锚唾液腺形态发生的谱系选择将有助于确定组织关键的途径， 再生后受伤，损坏，或在疾病状态。Xbp-1，特别是剪接和 转录活性的Xbp-1，同种型，已经涉及未折叠蛋白反应（UPR），分化 以及其他生物学上重要的细胞过程。我们在SG背景下对Xbp-1的了解是 限于scRNA-seq数据显示Xbp-1在前腺泡细胞（腺泡细胞的前体）中高度表达 细胞）以及先前在Xbp-1缺失小鼠中观察到的未表征的腺泡细胞表型。高度 Xbp-1在SG中的富集表达及其与超级增强子的相关性， 基因组学和表观基因组学数据集，使我们假设Xbp-1在分子水平上起着重要的作用， 在调节SG中的细胞身份和功能中的确定性作用。然而，分子机制 Xbp-1通过其决定细胞命运并控制SG期间的特定基因表达程序 发展尚未探索。为了解决这些知识缺口，我们将利用条件性Xbp-1敲除， 小鼠模型和复杂的基因组/转录组学方法来研究三个独立的领域， 兴趣首先，我们将鉴定Xbp-1在不同SG细胞类型中的时空表达模式， 通过发育阶段和发育期间测量未剪接和剪接异构体的相对丰度， 利用免疫染色和qRT-PCR的成体腺体维持（目的1）。其次，我们将使用条件 基因敲除（cKO）小鼠模型，以确定Xbp-1在SG发育和成年稳态中的作用 (Aim 2）。这些研究是必要的，因为它们将首次鉴定Xbp-1在体内的功能作用， 的SG。最后，我们将进行机制研究，以确定关键的靶基因和途径， 通过Xbp-1的调控，并确定Xbp-1的缺失如何改变SG的基因表达程序。 重要的是，通过实施scRNA-seq研究，我们将评估Xbp-1缺失对细胞命运轨迹的影响。 特别是在腺泡发育和成熟期间（目的3）。总的来说，这些研究将进一步促进我们的 了解Xbp 1依赖的转录和细胞网络对生物学功能的重要性 特别是因为它涉及分泌腺泡细胞分化。我们对人类进化的认识 驱动SG发展的潜在机制可以为再生提供一个范例， 治疗目标是增加唾液分泌不足患者的唾液流量，并更好地为治疗提供信息 用于治疗患有发育障碍和唾液腺疾病的患者。

Project Summary During embryogenesis and in adult, the delicate balance between proliferation and differentiation of the diverse cellular populations in the Salivary Gland (SG) must be tightly regulated to ensure normal tissue development and homeostasis. A better understanding of the regulatory mechanisms driving the cell fate decisions and lineage choices that anchor salivary gland morphogenesis will help identify pathways that are critical for tissue regeneration following injury, damage, or during diseased states. Xbp-1, specifically the spliced and transcriptionally active Xbp-1, isoform, has been implicated in Unfolded Protein Response (UPR), differentiation and a host of other biologically important cellular processes. Our knowledge of Xbp-1 in context of the SG is limited to scRNA-seq data showing Xbp-1 to be highly expressed in pro-acinar cells (the precursors to acinar cells) as well as a previously uncharacterized acinar cell phenotype observed in Xbp-1 null mice. The highly enriched expression of Xbp-1 in the SG and its association with super-enhancers as revealed by our analysis of genomic and epigenomics datasets, has led us to hypothesize that Xbp-1 plays an important and molecularly deterministic role in regulating cellular identity and function in the SG. However, the molecular mechanisms through which Xbp-1, dictates cell fate decisions and controls specific gene expression programs during SG development remains unexplored. To address these knowledge gaps, we will utilize conditional Xbp-1 knockout mouse models and sophisticated genomic/transcriptomic approaches to study three independent areas of interest. First, we will identify the spatiotemporal expression pattern of Xbp-1 in diverse SG cell types and measure the relative abundance of unspliced and spliced isoforms through developmental stages and during adult gland maintenance utilizing immunostainings and qRT-PCR (Aim 1). Second, we will use a conditional knockout (cKO) mouse model to determine the role of Xbp-1, during SG development and adult homeostasis (Aim 2). Such studies are necessary as they will identify, for the first time, the in vivo functional role of Xbp-1 in the SG. Finally, we will perform mechanistic studies to identify critical target genes and pathways that are governed by Xbp-1 and determine how the loss of Xbp-1 alters the gene expression program of the SG. Importantly, by implementing scRNA-seq studies, we will evaluate effects of loss of Xbp-1 on cell fate trajectories specifically during acinar development and maturation (Aim 3). Collectively, these studies will further our understanding of the Xbp1-dependent transcriptional and cellular networks important for the biological function of the SG particularly as it pertains to secretory acinar cell differentiation. Advances in our understanding of the underlying mechanisms driving SG development can provide a paradigm for regeneration, identify potential therapeutic targets to increase salivary flow in patients suffering from hyposalivation, and better inform therapies to treat patients with developmental dysfunctions and diseases of the salivary gland.