Investigating β-cell Functional Heterogeneity in Zebrafish Using Single-Cell Optogenetics

越来越多的证据表明，胰岛β细胞是异质性的，形成具有鲜明特征的亚群。据信，单个β细胞专门在胰岛执行特定的任务(例如，增殖与功能)，这可能会增加细胞群落的整体适合性。然而，细胞异质性在胰岛生理学和病理生理学中的起源和作用尚不清楚。最近，我们确定了一个关键的β细胞亚群，称为领导细胞，它指导其余细胞对葡萄糖的反应。当我们用光消融法精确摧毁前导细胞时，协调的胰岛活动立即受到损害。在与伦敦帝国理工学院Guy Rutter教授的密切合作下，我们应用了体内高速三维钙成像和新颖的分析方法，揭示了领先的β-细胞是斑马鱼和小鼠胰岛中第一个对葡萄糖做出反应的细胞。关键的是，取自T2 DM受试者的人胰岛协调异常。这导致我们提出了这样的概念，即在糖尿病进展过程中，领导细胞受到选择性的影响，导致胰岛功能障碍。因此，目前的工作模型是，β细胞的一个关键亚群的丧失可能会通过破坏钙离子反应来损害胰岛的功能。为了了解这种功能异质性对胰岛素分泌的作用，在之前的资助期间，我的团队开发了独特的最先进的工具来研究和操纵β细胞的功能。我们最新的工具包括具有光遗传效应器的β细胞特异性表达的转基因株，以及随着时间的推移对胰岛中的单个细胞进行光标记和跟踪的方法。利用我们的新技术，我们建议解决与胰岛内领导细胞的发育、其稳定性和代谢特征相关的基本问题，以及定义新的领导细胞和跟随细胞的分子标记。此外，我们将应用新的工具来研究β-细胞应激(代谢或炎症)如何影响领导细胞，并找出在这种条件下领导细胞是否会死亡或失去功能。解决这些基本问题对于理解为什么2型糖尿病胰岛表现出正常的β细胞群，但细胞协调和胰岛功能受损是至关重要的。一种可能的解释是，前导细胞的丢失阻碍了胰岛的正常功能。如果事实证明是这样，那么旨在保护和恢复领导细胞的新战略将是必要的。

There is emerging evidence that the pancreatic beta-cells are heterogeneous and form subpopulations with distinct characteristics. It is believed that individual beta-cells specialize towards performing specific tasks in the islet (e.g. proliferation versus function), which might increase the overall fitness of the cellular community. However, the origins and the role of cell heterogeneity in islet physiology and pathophysiology are yet to be defined. Recently, we identified a critical subpopulation of β-cells, called leader cells, which guide the response of the rest of cells to glucose. When we destroyed precisely the leader cells using photo-ablation, the coordinated islet activity was immediately impaired. In a close collaboration with Prof. Guy Rutter (Imperial College London), we applied high-speed in vivo 3D Ca2+ imaging and novel analytical approaches to reveal that the leader β-cells are the first cells to respond to glucose in both zebrafish and mouse islets. Critically, islet coordination was aberrant in human islets taken from subjects with T2DM. This led us to put forward the concept that leader cells are selectively affected during diabetes progression, leading to islet dysfunction. Thus, the current working model is that the loss of a critical subpopulation of β-cells may impair the function of islets trough disrupted Ca2+ responses. To understand the role of this functional heterogeneity for insulin secretion, during the previous funding period, my group has developed unique state-of-the-art tools to study and manipulate β-cell function. Among our newest tools are transgenic lines with β-cell-specific expression of optogenetic effectors, as well as methods to photo-label and track over time single cells in the islet. Using our new technologies, we propose to address fundamental questions related to the development of leader cells within islets, their stability and metabolic signatures, as well as to define novel molecular markers of leader and follower cells. Moreover, we will apply the new tools to study how leader cells are affected by β-cell stress, either metabolic or inflammatory, and to find out if leader-cells can die or lose their function under such conditions. Addressing these fundamental questions will be paramount to understand why type 2 diabetic islets show normal β-cell mass but impaired cell-coordination and islet function. A possible explanation is that the loss of leader cells prevents the proper functioning of the islets. If this turns out to be the case, new strategies aiming to protecting and restoring leader cells will be necessary.