CRCNS US-German Research Proposal - The diversification of retinal ganglion cells: A combined transcriptomic, genome engineering and imaging approach

大脑包含多种类型的神经元细胞，它们组装成复杂的回路，构成感觉、知觉和行为的基础。神经科学中的一个基本问题是，发育中的大脑如何产生如此令人印象深刻的各种神经元类型。具体来说，最大的挑战是确定基因网络，其活性限制未成熟的“前体”神经元采取特定的终端命运。神经视网膜是位于眼睛后部的大脑的前哨，是解决这个问题的理想系统，因为它的实验可及性和几个物种中神经元多样性的明确普查。20世纪80年代开始的开创性研究使用当时可用的青蛙，啮齿动物和鱼类实验工具，以获得对视网膜细胞局限于广义“类”的过程的有价值的见解。然而，一些视网膜类别包含几种（20-50）不同的神经元类型，并且细胞如何在类别内致力于特定类型是未知的，并且不能使用经典工具进行研究。该项目汇集了一位实验神经科学家和计算研究人员来解决这个问题，结合几种最近开发的技术，以前所未有的细节研究斑马鱼视网膜中细胞类型规范的过程。在这项工作中开发的方法将有助于了解其他大脑区域和物种的神经元发育和成熟，并查明可能导致发育异常的基因。该项目将结合联合收割机高通量单核RNA测序（snRNA-seq），先进的计算方法，基因组工程和实时成像，以了解约35-50种视网膜神经节细胞（RGC），眼睛的输出神经元，如何在发育和生长的斑马鱼视网膜中出现。通过利用斑马鱼的实验优势，它允许在体内直接对发育中的细胞进行实时成像，研究人员将研究有丝分裂后神经元分化如何在小鼠（最常用的脊椎动物模型）中详细展开。我们将使用在斑马鱼的六个发育阶段收集的snRNA-seq配置文件绘制不同RGC的转录景观，并使用计算方法重建整个发育过程中转录簇之间的谱系关系。为了测试基因组分析的预测，研究人员将通过基于CRISPR/Cas9的基因组工程标记分子定义的未成熟RGCs，并对其进行成像，以可视化它们从胚胎阶段到成年的初始分化和转分化。这两项工作将为复杂神经生物系统的模式提供新的见解。该项目由生物科学理事会的神经系统集群和工程理事会的工程生物学和健康集群共同资助。德国联邦教育和研究部（BMBF）正在资助一个配套项目。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

The brain contains a multitude of types of neuronal cells that assemble into elaborate circuits that underlie sensation, perception, and behavior. A fundamental question in neuroscience is how the developing brain generates such an impressive array of diverse neuronal types. Specifically, the grand challenge is to determine the networks of genes whose activity restricts immature “precursor” neurons to adopt specific terminal fates. The neural retina, which is an outpost of the brain residing in the back of the eye, is an ideal system for addressing this question due to its experimental accessibility and well-defined census of neuronal diversity in several species. Pioneering studies beginning in the 1980s used the then available experimental tools in frogs, rodents and fish to gain valuable insights into the process by which retinal cells become restricted to broadly defined “classes”. However, some retinal classes contain several (20-50) distinct neuronal types, and how cells commit to specific types within a class is unknown, and cannot be studied using classical tools. This project brings together an experimental neuroscientist and computational researcher to address this question combining several recently developed technologies to study the process of cell-type specification in the retina of the zebrafish in unprecedented detail. The approaches developed in this work will be useful for understanding neuronal development and maturation in other brain regions and species, and pinpoint the genes whose dysregulation may lead to developmental abnormalities. The project will combine high-throughput single-nucleus RNA-sequencing (snRNA-seq), advanced computational methods, genome engineering and live imaging to understand how ~35-50 types of retinal ganglion cells (RGCs), the output neurons of the eye, emerge in the developing and growing zebrafish retina. By leveraging the experimental advantages of zebrafish, which permits direct live imaging of developing cells in vivo, the researchers will study how postmitotic neuronal differentiation unfolds at a level of detail that is not possible in mice, the most commonly used vertebrate model. We will map the transcriptional landscapes of differentiating RGCs using snRNA-seq profiles collected at six developmental stages of zebrafish,and use computational methods to reconstruct lineage relationships among transcriptional clusters across development. To test predictions from genomic analysis, the researchers will label molecularly defined immature RGCs by CRISPR/Cas9-based genome engineering and image them to visualize their initial differentiation and transdifferentiation from embryonic stages into adulthood. Taken together, the efforts will lead to new insights into the patterning of a complex neurobiological system.This project is funded jointly by the Neural Systems Cluster in the Directorate for Biological Sciences and the Engineering Biology and Health Cluster in the Directorate for Engineering. A companion project is being funded by the German Federal Ministry of Education and Research (BMBF).This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.