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

项目摘要 在胚胎发育过程中和在成体中，在增殖和分化之间的微妙平衡 唾液腺(SG)中的细胞群必须受到严格的调控，以确保正常的组织发育 和动态平衡。更好地了解推动细胞命运决定和 锚定唾液腺形态发生的谱系选择将有助于识别对组织至关重要的途径 再生在受伤、损坏后或在患病状态下再生XBP-1，特别是剪接和 转录活性XBP-1的异构体与未折叠蛋白反应(UPR)、分化有关 以及许多其他生物学上重要的细胞过程。我们在SG的背景下对XBP-1的了解是 仅限于scRNA-seq数据显示XBP-1在腺泡前期细胞(腺泡细胞的前体)中高表达 细胞)，以及在XBP-1基因缺失小鼠中观察到的以前未描述的腺泡细胞表型。高度的 我们的分析显示XBP-1在SG中的丰富表达及其与超级增强子的关联 基因组和表观基因组学数据集，使我们假设XBP-1在分子上起着重要的作用 在调节SG中的细胞特性和功能方面的确定性作用。然而，分子机制 在SG期间，XBP-1通过它来决定细胞的命运并控制特定的基因表达程序 发展仍未得到探索。为了解决这些知识差距，我们将利用条件XBP-1基因敲除 小鼠模型和复杂的基因组/转录组方法来研究三个独立的领域 利息。首先，我们将确定XBP-1在不同类型的SG细胞中的时空表达模式 在发育阶段和过程中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维持成体腺体(目标1)。其次，我们将使用条件 基因敲除(CKO)小鼠模型确定XBP-1在SG发育和成体内稳态中的作用 (目标2)。这样的研究是必要的，因为他们将首次确定XBP-1在体内的功能作用。 总司令。最后，我们将进行机制研究，以确定关键的靶基因和途径 由XBP-1控制，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，我们将评估失去xbp-1对细胞命运轨迹的影响。 特别是在腺泡发育和成熟期间(目标3)。总的来说，这些研究将进一步推动我们的 了解XBP1依赖的转录和细胞网络对生物功能的重要性 特别是与分泌腺泡细胞分化有关。我们对这一现象的理解取得了进展 推动SG发展的潜在机制可以提供一个再生的范例，确定潜力 增加淋巴功能减退患者唾液流量的治疗目标，并更好地指导治疗 治疗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.