Innate immune system regulation of retinal regeneration

项目摘要 退化性视力障碍是由视网膜细胞的丧失引起的。在斑马鱼中，视网膜Müller神经胶质（MG）行为 作为干细胞，产生MG衍生的祖细胞（MGPC），其替代丢失的视网膜细胞。哺乳动物 MG/MGPCs也可以产生新的视网膜神经元，然而，增殖能力是有限的，疾病- 相关细胞类型（例如，光感受器）很少产生。我们的目标是了解为什么再生的 与再生物种（即斑马鱼）相比，MG/MGPC在哺乳动物中的潜力有限。 在斑马鱼中，广泛的视网膜细胞损失触发了“发育”再生，产生所有主要的视网膜细胞， 班相反，选择性视网膜细胞损失导致“命运偏向”的过程，其中MGPC引起视网膜细胞凋亡。 丢失的细胞类型。然而，MG如何感受到损失的程度尚不清楚。由于免疫系统在免疫系统中 在视网膜再生过程中的作用，我们研究了小胶质细胞反应对斑马鱼MG激活的影响。 我们观察到MG干细胞活化需要小胶质细胞，并且免疫抑制可以抑制或 根据处理时间增强再生动力学。我们假设小胶质细胞的反应性 水平与视网膜细胞丢失的程度、持续时间和/或特异性成比例，以协调MG 活化、MGPC增殖率和脂肪决定了损伤的类型。来测试我们 假设，这里我们提出将改进靶向细胞消融系统联合收割机组合， 用标记免疫细胞类型的资源/方法，活体内， 时间推移成像、谱系追踪和单细胞转录组学。具体来说，我们将定义 (Aim 1）、视锥光感受器或视网膜神经节细胞丢失的持续时间（Aim 2）和特异性（Aim 3）的影响 免疫细胞反应性和视网膜再生。这些实验将有助于确定特定免疫的作用 细胞亚型在斑马鱼再生过程中塑造MGPC增殖和细胞命运中发挥作用。

PROJECT SUMMARY Degenerative vision disorders are caused by the loss of retinal cells. In zebrafish, retinal Müller glial (MG) act as stem cells, generating MG-derived progenitor cells (MGPCs) which replace lost retinal cells. Mammalian MG/MGPCs can also produce new retinal neurons, however, proliferative capacity is limited and disease- relevant cell types (e.g., photoreceptors) are rarely generated. Our goal is to understand why the regenerative potential of MG/MGPCs are limited in mammals in comparison to a regenerative species (i.e. zebrafish). In zebrafish, widespread retinal cell loss triggers “developmental” regeneration, producing all major retinal cell classes. Conversely, selective retinal cell loss results in a “fate-biased” process where MGPCs give rise to the lost cell type. How MG sense the extent of loss, however, is unknown. As the immune system plays critical roles during retinal regeneration, we examined the impact of microglia reactivity on MG activation in zebrafish. We observed that microglia are required for MG stem cell activation and that immunosuppression can inhibit or enhance regeneration kinetics depending on the treatment timing. We hypothesize that microglia reactivity levels scale to the extent, duration, and/or specificity of retinal cell loss in order to coordinate MG activation, MGPC proliferation rates and fat decisions to the type of injury incurred. To test our hypothesis, here we propose to combine an improved targeted cell ablation system enabling titratable, sustainable, and selective retinal cell loss with resources/methods for labeling of immune cell types, intravital timelapse imaging, lineage tracing, and single cell transcriptomics. Specifically we will define how the extent (Aim 1), the duration (Aim 2) and specificity (Aim 3) of cone photoreceptor or retinal ganglion cell loss impact immune cell reactivity and retinal regeneration. These experiments will serve to define roles specific immune cell subtypes play in shaping MGPC proliferation and cell fates during regeneration in zebrafish.