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

少数几个神经前体细胞产生了数千种执行复杂功能的不同类型的神经细胞。这些“白板岩”祖细胞通过复杂的分子网络转变为功能和空间上截然不同的成熟神经元。然而，人们对驱动神经元命运决定的分子网络知之甚少。一组重要的前体细胞表达原神经转录因子无音同源1(Atoh1)，起源于后脑的菱形唇(RL)区域。Atoh1前体细胞在发育早期由空间位置决定，在不同的迁移流中离开RL，最终形成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)的脑干核团。Atoh1前体细胞不能正确形成这些不同的核，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祖细胞通过分化阶段的分子网络仍然未知。如果Atoh1前体的转录轨迹被阐明，就可以开发再生疗法来修复导致发育不正常的遗传异常，这将减轻神经疾病的负担。这项建议的长期目标是阐明驱动Atoh1前体细胞发育的神经元多样性的分子网络，以改进治疗方法，以恢复中枢神经系统损伤后的功能。这一提议的假设是，Atoh1前体细胞经历由特定分子网络驱动的时间和空间命运决定。这项建议的目的是确定在Atoh1谱系中决定神经元命运的基因或基因集，并确定转录轨迹在后脑发育的体外模型中是否保守。具体目标1将通过指示祖细胞何时、何地和如何分化来检验分子级联驱动Atoh1谱系多样性的假设。通过分离胚胎阶段9.5至18.5的Atoh1谱系并进行单细胞RNA测序(ScRNAseq)，将确定分子级联。原位杂交将用于确定转录本的空间分布并确认scRNAseq结果。《特殊目的2》将检验这样一种假设，即在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。通过外源性添加内源性信号形态原激动剂，将建立干细胞来源的小鼠后脑模型。将通过scRNAseq阐明有机物的组成以及与活体小鼠后脑发育的比较。这些集体结果将通过阐明驱动关键大脑区域Atoh1前体细胞发育的分子网络，增加神经系统发育的基础知识。这项建议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.