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

少数神经祖细胞产生数千种不同的神经元细胞类型，执行复杂的功能。这些“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知之甚少。一组重要的祖细胞表达前神经转录因子，Atoh1（Atoh1），并起源于后脑的菱形唇（RL）区域。Atoh1祖细胞在发育的早期阶段由空间位置定义，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（例如平衡，听力和呼吸）的脑干核。Atoh1祖细胞不能正确形成这些不同的细胞核可能对生命有害。尽管Atoh1谱系对健康和疾病有多重要，但指导Atoh1祖细胞分化阶段的分子网络仍然未知。如果Atoh1祖细胞的转录轨迹得到阐明，再生疗法可以被开发出来，以修复导致发育不正常的遗传畸变，这将减轻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方法。该提议的假设是Atoh1祖细胞经历由定义的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基因或基因组驱动神经元的命运决定Atoh1谱系，并确定是否转录轨迹是保守的后脑发育的体外模型。具体目标1将测试的假设，即分子级联驱动Atoh1谱系多样性的指示祖细胞何时，何地，以及如何分化。将通过从胚胎期9.5至18.5分离Atoh1谱系并进行单细胞RNA测序（scRNAseq）来确定分子级联。原位杂交将用于鉴定转录物的空间分布并确认scRNAseq结果。具体目标2将检验体外衍生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。将通过外源性添加内源性信号传导形态发生素的激动剂来开发干细胞衍生的小鼠后脑模型。将通过scRNAseq阐明类器官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Atoh1祖细胞在关键大脑区域发育的分子网络来增加神经系统发育的基础知识。该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.