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

CRCNS 美国-德国研究提案 - 视网膜神经节细胞的多样化：转录组学、基因组工程和成像相结合的方法

• 批准号: 2309039
• 负责人: Karthik Shekhar
• 金额: $ 73.28万
• 依托单位: University of California-Berkeley
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-11-01 至 2028-10-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2309039&HistoricalAwards=false
• 关键词: CRCNS; US; German; Research; Proposal

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.

大脑包含多种类型的神经细胞，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，发育中的大脑是如何产生如此令人印象深刻的各种神经元类型的。具体地说，最大的挑战是确定其活性限制未成熟的“前体”神经元采用特定终端命运的基因网络。神经视网膜是大脑的前哨，位于眼睛后面，由于其实验可及性和对几个物种神经元多样性的明确普查，是解决这一问题的理想系统。始于20世纪80年代的开创性研究使用了当时可用的青蛙、啮齿动物和鱼类实验工具，以获得对视网膜细胞被限制在广泛定义的“类”中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，细胞如何致力于类别中的特定类型是未知的，也不能使用经典工具进行研究。该项目汇集了一位实验神经学家和计算研究人员来解决这个问题，并结合了几项最新开发的技术，以前所未有的详细研究斑马鱼视网膜细胞类型指定的过程。这项工作中开发的方法将有助于了解其他脑区和物种的神经元发育和成熟，并准确定位其失调可能导致发育异常的基因。该项目将结合高通量单核RNA测序(SnRNA-seq)、先进的计算方法、基因组工程和实时成像，以了解大约35-50种视网膜神经节细胞(RGC)-眼睛的输出神经元-是如何在发育和生长的斑马鱼视网膜中出现的。利用斑马鱼的实验优势，可以直接在体内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是如何在细节水平上展开的，这在小鼠身上是不可能的，小鼠是最常用的脊椎动物模型。我们将使用从斑马鱼六个发育阶段收集的SnRNA-seq图谱来绘制分化RGC的转录图谱，并使用计算方法重建不同发育阶段转录簇之间的谱系关系。为了验证基因组分析的预测，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RGC，并对它们进行成像，以可视化它们从胚胎阶段到成年的初始分化和转分化。综上所述，这些努力将带来对复杂神经生物系统模式的新见解。该项目由生物科学局的神经系统组和工程局的工程生物学和健康组共同资助。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智力优势和更广泛的影响审查标准进行评估，被认为值得支持。