The role of lineage in the temporospatial genesis of retinal bipolar cell subtypes

谱系在视网膜双极细胞亚型时空发生中的作用

• 批准号: 10571329
• 负责人: Ryan Delgado
• 金额: $ 12.36万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10571329
• 关键词: Back; Bar Codes; Benchmarking; Birth; Brain; CRISPR/Cas technology; Cell Survival; Cell division; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Competence; Complement; Complex; Cues; DNA; DNA Sequence; Data; Data Set; Development; Distant; Environment; Evolution; Eye; Foundations; Gene Expression; Generations; Genes; Genetic Transcription; Genomics; Goals; Guide RNA; Heritability; Individual; Inherited; Interneurons; Investigation; Knockout Mice; Laboratories; Light; Mentors; Modeling; Molecular; Morphology; Neurons; Output; Pattern; Phase; Physiological; Process; Production; Publishing; Retina; Retinal Cone; Retinal Ganglion Cells; Role; Series; Shapes; Sister; Site; Solid; Specific qualifier value; Supervision; Surface; Testing; Time; Tissues; Tracer; Training; Transgenic Organisms; Trees; Vertebrate Photoreceptors; Vision; Work; base; career; cell type; combinatorial; daughter cell; design; experience; in vivo; invention; medical schools; novel; postnatal; prime editor; progenitor; programs; response; retinal neuron; retinal progenitor cell; single-cell RNA sequencing; small molecule inhibitor; spatiotemporal; tool; tool development; training opportunity

PROJECT SUMMARY / ABSTRACT The retina is a cellularly complex tissue comprised of over 100 different cell types which work together to enable proprer vision. Bipolar cells are a diverse class of interneurons that connect rod and cone photoreceptors to retinal ganglion cells, the projection neurons of the retina, which then in turn send their output to the brain. There are currently 15 known subtypes of bipolar cells that differ in their connectivity, physiological responses to light, morphology, abundance, and gene expression profiles. A recent developmental study from our lab performed birthdating analysis to determine when different bipolar cell subtypes were born and found that bipolar cell subtype genesis was arranged into concentrated domains that changed over developmental time. When these domains were compared over time, they formed a wave-like temporospatial pattern of bipolar subtype genesis that spread over the entire extent of the retina. This result suggests a hierarchical model of bipolar subtype genesis in which early retinal progenitor cells generate a series of sub-lineages off-set in developmental time, that undergo a temporally ordered production of bipolar subtypes. To investigate this potential model, I have invented a novel molecular tool called SCRIBE (Sequential Combinatorial Recorder for Iterative Barcode Evolution) that functions as an “evolvable” lineage recorder. By iteratively adding barcode fragments to a genomic target site, SCRIBE aims to generate an evolving, heritable sequence that can be used to distinguish between sister sub-lineages across multiple levels of a clonal lineage tree. The goal of this proposal is to investagate how bipolar cell subtype specification is achieved in development. During the mentored portion of this proposal (K99), I will further develop SCRIBE for use in the in vivo retina and use it to investagate the temporal ordering of bipolar subtype production and the lineage relationships between postnatal RPCs that generate bipolar cells. In the independent portion of the proposal (R00), I will determine whether bipolar subtype specification is intrinsic or extrinsic, and investigate potential molecular regulators of bipolar subtypes specification. The completion of these aims will provide me with training in the fields of retinal development and cutting-edge molecular tool development, complementing my previous training experiences. I will perform the mentored portion of this proposal under the supervision of Dr. Constance Cepko who has been a leader in the fields of retinal development, lineage tracing, and in vivo molecular tool development. Furthermore, the broader scientific environment surrounding the Cepko lab at Harvard Medical School will provide outstanding training opportunities. Together, these experiences will provide me with a solid foundation that will support my long-term career goal of leading my own independent academic laboratory focused on studying the molecular bases of cellular diversity in the retina.

项目摘要 /摘要 视网膜是一种填充了100多种不同细胞类型的细胞复杂组织，它们一起起作用 启用前列人的视野。双极细胞是连接杆和锥的潜水中间神经元类 视网膜神经节细胞，视网膜的投影神经元的感光器，然后又发送其 输出大脑。目前有15个已知的双极细胞亚型，其连通性不同， 对光，形态，抽象和基因表达谱的生理反应。最近 我们实验室的发展研究进行了生日分析，以确定何时不同的双极细胞 亚型诞生，发现双极细胞亚型的创世纪被布置到集中域中 在发育时间内发生了变化。随着时间的推移比较这些域时，它们形成了波浪状 双极亚型创世纪的时间空间模式分布在视网膜的整个范围内。这个结果 提出了双极亚型发生的分层模型，其中残留的祖细胞产生A 一系列在发育时间中的亚线局部偏离现场，该临时订购了双极的生产 亚型。为了研究这种潜在模型，我发明了一种新型的分子工具，称为Scribe （迭代条形码演化的顺序组合记录仪）作为“可进化”谱系的作用 录音机。通过迭代将条形码片段添加到基因组目标位点，Scribe旨在生成一个 不断发展的遗传序列，可用于区分多个层次 克隆谱系树。该提案的目的是投资双极单元格亚型规范的方式 在发展中实现。在此提案的修订部分（K99）期间，我将进一步制作抄写员 用于体内视网膜，并使用它来投资双极亚型生产的临时排序和 产生双极细胞的产后RPC之间的谱系关系。在独立部分 提案（R00），我将确定双极亚型规范是固有的还是外部的，并研究 双极亚型规范的潜在分子调节剂。这些目标的完成将为我提供 在剩余开发和尖端分子工具开发领域进行培训，完成 我以前的培训经验。我将在 康斯坦斯·塞普科（Constance Cepko）博士一直是剩余发展，谱系跟踪和体内领域的领导者 分子工具开发。此外，围绕Cepko实验室的更广泛的科学环境 哈佛医学院将提供出色的培训机会。这些经历将在一起 为我提供坚实的基础，该基础将支持我领导自己独立的长期职业目标 学术实验室的重点是研究视网膜细胞多样性的分子碱基。