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

少量的神经祖细胞产生了数千种不同的神经细胞类型，这些细胞具有复杂的功能。这些“空白板”祖细胞通过复杂的分子网络转变为功能和空间上不同的成熟神经元。然而，驱动神经元命运决定的分子网络却知之甚少。一个重要的祖细胞群表达前神经转录因子Atoh1，起源于后脑的菱形唇区。Atoh1祖细胞在发育早期由空间位置确定，并以不同的迁移流从RL迁移，最终产生所有小脑兴奋性神经元和数十个负责关键功能（如平衡、听力和呼吸）的脑干核。Atoh1祖细胞不能正确形成这些独特的细胞核可能对生命有害。尽管Atoh1谱系对健康和疾病有多么重要，但指导Atoh1祖细胞分化阶段的分子网络仍然未知。如果Atoh1祖细胞的转录轨迹被阐明，再生疗法可以被开发来修复导致不正常发育的遗传畸变，这将减轻神经系统疾病的负担。这项提议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，以改善治疗方法，恢复中枢神经系统创伤后的功能。这一提议的假设是，Atoh1祖细胞经历了一个确定的分子网络驱动的时间和空间命运决定。本提案的目的是确定Atoh1谱系中驱动神经元命运决定的基因或基因组，并确定转录轨迹在后脑发育的体外模型中是否保守。特异性目标1将验证分子级联通过指示祖细胞何时、何地以及如何分化来驱动Atoh1谱系多样性的假设。分子级联将通过分离胚胎期9.5至18.5的Atoh1谱系并进行单细胞RNA测序（scRNAseq）来确定。原位杂交将用于鉴定转录本的空间分布并确认scRNAseq结果。特异性目标2将验证体外来源的小鼠后脑类器官经历与体内发育相似的转录轨迹的假设。通过外源添加内源性信号形成因子的激动剂，建立干细胞来源的小鼠后脑模型。类器官组成及其与小鼠后脑发育的比较将通过scRNAseq来阐明。通过阐明在一个关键的大脑区域驱动Atoh1祖细胞发育的分子网络，这些集体结果将增加神经系统发育的基础知识。这项提议中使用的策略将广泛适用于研究大脑其他区域的祖细胞发育。

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.