Uncovering the Cellular and Molecular Mechanisms Driving B-cell Neogenesis During Regeneration

项目总结/摘要 糖尿病是一个日益严重的全球健康问题，需要改善终身治疗。一个更普遍 治疗1型糖尿病（T1 D）的方法是诱导内源性细胞恢复丢失的胰岛细胞。因此，我们认为， 我的工作目标是发现驱动胰岛细胞新生的细胞和分子机制， 这些途径可以用来恢复糖尿病患者的胰岛细胞。我们使用斑马鱼作为模型系统， 具有称为中央腺泡细胞（CAC）的特化的导管细胞，其表现为祖细胞。所有CAC都是 Notch反应，但具有较低Notch活性的细胞更有可能分化。所有的CAC都表达sox 9 b， 我们的实验室已经表明，斑马鱼中的sox 9 b单倍不足加速了β细胞再生。更好地 为了了解CAC的祖细胞行为，我们的实验室使用批量RNA-seq来揭示CAC转录组。 批量RNA-seq的一个主要问题是，它掩盖了CAC异质性，不能用于揭示细胞内的 以及在单细胞水平上驱动CAC分化的分子机制。我建议更好地理解 CACs的细胞和分子机制，解决两个主要目标：（1）表征细胞 使用单细胞RNA-seq研究CAC群体内的异质性，以及（2）研究转录组变化 因为CAC在再生过程中转变为ACC细胞。(1)为了表征CAC群体中的变异，CAC 将使用荧光激活细胞分选（FACS）从野生型（WT）成年斑马鱼胰腺中分离。 分离的CAC将进行scRNA-seq，并应用聚类分析来分离CAC 转录组分化为不同的亚群。(2)我将用转基因斑马鱼模型来诱导胰岛细胞消融。 在再生过程的中途，我将通过FACS分离CAC，并将这些CAC用于scRNA-seq。 聚类分析和伪时间分析将被应用于对CAC进行排序，并确定CAC的机制。 再生对于这两个目标，我将进行适当的原位实验，以可视化CAC亚群， 确认从我的伪时间模型中鉴定出的“新”基因是否与细胞分化有关。我 该项目将阐明存在于CAC种群中的自然变异，并将揭示分子生物学特征。 驱动胰岛细胞再生的机制。我的实验室和其他人将能够使用我验证的数据来获得更好的 了解可用于改善T1 D患者治疗的潜在治疗途径。

Project Summary/Abstract Diabetes is a growing global health problem that requires improved life-long treatments. A more universal approach to treat type one diabetes (T1D) would be inducing endogenous cells to restore lost cells. Therefore, my proposed work aims to discover the cellular and molecular mechanism driving -cell neogenesis to identify pathways that can be exploited to restore  cells in diabetics. We use zebrafish as a model system because they have specialized ducal cells called centroacinar cells (CACs) that behave as -cell progenitors. All CACs are Notch responsive, but cells that have lower Notch activity are more likely to differentiate. All CACs express sox9b, and our lab has shown that sox9b haploinsufficiency in zebrafish accelerates -cell regeneration. To better understand the progenitor behavior of CACs, our lab used bulk RNA-seq to uncover the CAC transcriptome. One major issue with bulk RNA-seq is that it masks CAC heterogeneity and cannot be used to reveal cellular and molecular mechanisms driving CAC differentiation at the single-cell level. I propose to better understand the cellular and molecular mechanisms of CACs by addressing two major aims: (1) Characterize cellular heterogeneity within the CAC population using single-cell RNA-seq and (2) Investigate transcriptome changes as CACs transition into  cells during regeneration. (1) To characterize the variation in the CAC population, CACs will be isolated from wild type (WT) adult zebrafish pancreata using fluorescent activated cell sorting (FACS). Isolated CACs will be subject to scRNA-seq and cluster analysis will be applied to separate the CAC transcriptomes into distinct subpopulations. (2) I will use a transgenic zebrafish model to induce -cell ablation. At a halfway-point during regeneration, I will isolate CACs by FACS and will use these CACs for scRNA-seq. Cluster analysis and pseudotime analysis will be applied to order CACs and identify a mechanism for regeneration. For both aims, I will perform appropriate in situ experiments to visualize CAC subpopulations and confirm whether ‘new’ genes identified from my pseudotime model are associated with -cell differentiation. My project will elucidate the natural variation that exists within the CAC population and will uncover molecular mechanisms driving -cell regeneration. My lab and others will be able to use my validated data to gain a better understanding of the potential therapeutic routes that can be exploited to improve the treatment of T1D patients.