Elucidating the molecular basis of Atoh1 lineage diversity in the developing hindbrain

阐明发育中的后脑 Atoh1 谱系多样性的分子基础

基本信息

批准号：
10320332
负责人：
Jessica Christine Butts
金额：
$ 4万
依托单位：
BAYLOR COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-21 至 2022-04-20
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10320332
关键词：
Agonist Animal Model Arousal Benchmarking Biological Models Brain Stem Brain region Breathing Cell Nucleus Cells Cerebellum Cerebral cortex Complement Complex Cues Development Developmental Process Disease Embryo Equilibrium Event Failure Fluorescence Genes Genetic Genetic Transcription Goals Health Hearing Homologous Gene In Situ Hybridization In Vitro Injury Knock-in Mouse Knowledge Lead Life Lip structure Location Malignant Neoplasms Mediating Modeling Molecular Mus Nervous System Trauma Neuraxis Neurologic Dysfunctions Neurons Organoids Outcome Pattern Population Process Proprioception Protocols documentation Publishing Reporter Series Signal Transduction Spatial Distribution Specific qualifier value Spinal Cord Stream System Testing Therapeutic Time Tissue-Specific Gene Expression Tissues Training Transcript Trauma Work base cell type developmental disease embryonic stem cell excitatory neuron functional restoration hindbrain human pluripotent stem cell improved in vitro Model in vivo migration morphogens mouse genetics nerve stem cell nervous system development nervous system disorder neural circuit neuron development prevent progenitor programs regenerative therapy relating to nervous system repaired respiratory single-cell RNA sequencing stem cell model stem cells transcription factor

项目摘要

A handful of neural progenitors give rise to thousands of diverse neuronal cell types that perform complex functions. These “blank slate” progenitors’ transition through a complex molecular network to become functionally and spatially distinct mature neurons. However, the molecular networks that drive neuronal fate decisions are poorly understood. One important group of progenitors express the proneural transcription factor, Atonal homolog 1 (Atoh1), and originate at the rhombic lip (RL) region of the hindbrain. Atoh1 progenitors are defined by spatial location at early stages of development and migrate away from the RL in distinct migration streams to ultimately give rise to all cerebellar excitatory neurons and dozens of brainstem nuclei responsible for critical functions (e.g. balance, hearing, and breathing). Failure of Atoh1 progenitors to properly form these distinct nuclei can be detrimental to life. Despite how important the Atoh1 lineage is to health and disease, the molecular network that directs Atoh1 progenitors through stages of differentiation remains unknown. If the transcriptional trajectories of Atoh1 progenitors were elucidated, regenerative therapies could be developed to repair genetic aberrations that lead to improper development, which would reduce the burden of neurological disease. The long-term objective of this proposal is to elucidate the molecular networks that drive the neuronal diversity of Atoh1 progenitor development to improve therapeutics to restore function following trauma to the central nervous system. The hypothesis of this proposal is that Atoh1 progenitors undergo temporal and spatial fate decisions driven by a defined molecular network. The objectives of this proposal are to identify the gene or sets of genes that drive neuronal fate decisions in the Atoh1 lineage and to determine if transcriptional trajectories are conserved in in vitro models of hindbrain development. Specific Aim 1 will test the hypothesis that a molecular cascade drives Atoh1 lineage diversity by instructing progenitors when, where, and how to differentiate. The molecular cascade will be determined by isolating the Atoh1 lineage from embryonic stage 9.5 to 18.5 and performing single cell RNA sequencing (scRNAseq). In situ hybridization will be used to identify spatial distribution of transcripts and confirm scRNAseq results. Specific Aim 2 will test the hypothesis that in vitro-derived mouse hindbrain organoids undergo similar transcriptional trajectories to in vivo development. A stem cell-derived mouse hindbrain model will be developed by exogenously adding agonists of endogenous signaling morphogens. Organoid composition and comparison to in vivo mouse hindbrain development will be elucidated though scRNAseq. The collective results will add to the fundamental knowledge of nervous system development by elucidating the molecular network that drives development of Atoh1 progenitors in a critical brain region. The strategies used in this proposal would be broadly applicable to studying progenitor development in other brain regions.

少数神经元的祖细胞产生了成千上万种具有复杂功能的神经元细胞类型。这些“空白的板岩”祖细胞通过复杂的分子网络的过渡，在功能和空间上变为成熟的神经元。但是，驱动神经元脂肪决策的分子网络知之甚少。一组重要的祖细胞表达了胸腔转录因子Atonal同源物1（ATOH1），并起源于后脑的龙骨唇（RL）区域。 ATOH1祖细胞是由发育初期的空间位置定义的，并在不同的迁移流中从RL迁移，最终引起了所有小脑令人兴奋的神经元和数十个脑干核负责关键功能（例如平衡，听力和呼吸）。 ATOH1祖细胞正确形成这些独特的核可能对生命有害。尽管ATOH1谱系对健康和疾病的重要性有多重要，但指导ATOH1祖细胞通过分化阶段的分子网络仍然未知。如果阐明了ATOH1祖细胞的转录轨迹，则可以开发再生疗法来修复导致发育不当的遗传畸变，从而减少神经系统疾病的负担。该提案的长期目标是阐明驱动ATOH1祖细胞发育的神经元多样性的分子网络，以改善中枢神经系统创伤后恢复功能的治疗。该提议的假设是ATOH1祖细胞执行由定义的分子网络驱动的临时和空间脂肪决策。该提案的目标是确定在ATOH1谱系中驱动神经元脂肪决策的基因或基因集，并确定在后脑发育的体外模型中转录轨迹是否保存。具体目标1将检验以下假设：分子级联反应通过指示祖细胞，何时何地和如何区分来驱动ATOH1谱系多样性。分子级联反应将通过从胚胎阶段9.5至18.5分离并进行单细胞RNA测序（SCRNASEQ）来确定。原位杂交将用于识别转录本的空间分布并确认scrnaseq结果。具体目标2将检验以下假设：体外衍生的小鼠后脑器官经历与体内发育相似的转录轨迹。干细胞来源的小鼠后脑模型将通过外源添加内源性信号形态的激动剂来开发。通过scrnaseq阐明了器官的组成和与体内小鼠后脑发育的比较。集体结果将通过阐明驱动关键大脑区域中ATOH1祖细胞发展的分子网络来增加神经系统发育的基本知识。该提案中使用的策略将广泛适用于研究其他大脑区域的祖细胞发展。