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 视网膜是由100多种不同类型的细胞组成的复杂细胞组织，它们共同作用于 启用PROPRIR VISION。双极细胞是连接视杆细胞和视锥细胞的不同类型的中间神经元。 视网膜神经节细胞的光感受器，视网膜的投射神经元，然后依次发送他们的 输出到大脑。目前有15种已知的双极单元的亚型在它们的连接性上不同， 对光、形态、丰度和基因表达谱的生理反应。最近 我们实验室的发育研究进行了出生年龄分析，以确定不同的双极细胞 亚型的产生和发现双极细胞亚型的发生被安排在集中的区域中， 随着发育时间的推移发生了变化。当随着时间的推移对这些区域进行比较时，它们形成了波浪状 分布于整个视网膜的两极亚型发生的时空模式。这个结果 提出了一种双极亚型发生的分层模型，在该模型中，早期视网膜前体细胞产生 在发育时期出现的一系列亚谱系，经历了两极的时间有序的产生 子类型。为了研究这一潜在的模型，我发明了一种新的分子工具，称为SCRIBE (用于迭代条形码进化的顺序组合记录器)，其功能是一种“可进化”的谱系 录音机。通过迭代地将条形码片段添加到基因组目标位置，Scribe的目标是生成 进化的、可遗传的序列，可用于区分多个级别的姐妹子谱系 一棵克隆的谱系树。这项建议的目标是研究双极细胞亚型规格如何 在发展中取得的成就。在本建议书的指导部分(K99)中，我将进一步为 在活体视网膜中的应用，并用它来研究双极亚型产生的时间顺序和 产生双极细胞的出生后RPC之间的血统关系。中的独立部分。 建议(R00)，我将确定双极亚型规范是内在的还是外在的，并调查 双极亚型潜在分子调节剂规范。这些目标的实现将为我提供 在视网膜发育和尖端分子工具开发领域进行培训，补充 我以前的训练经验。我将在以下监督下执行本建议书的指导部分 康斯坦斯·切普科博士是视网膜发育、血统追踪和体内研究领域的领先者 分子工具开发。此外，围绕Cepko实验室的更广泛的科学环境位于 哈佛医学院将提供出色的培训机会。总而言之，这些经历将 为我提供坚实的基础，这将支持我领导自己独立的长期职业目标 学术实验室专注于研究视网膜细胞多样性的分子基础。