转录因子CEH-31/BARHL1在线虫神经前体细胞命运决定中的功能研究

An important question in developmental neurogenesis is how neuronal precursor cells acquire the neuronal fate and differentiate into specific types of mature neuronal cells. The simplicity, clarity, and invariance of C. elegans developmental cell lineage and cell fate make it a leading model organism to elucidate the molecular mechanism of neuronal differentiation in vivo. Although the mechanism controlling terminal differentiation of neuronal cells has been extensively studied and partially revealed, the regulation of earlier events of neural differentiation, that is, the determination of neuronal fates in multiple precursor cells, is poorly understood. One major challenge in studying fate determination is how to continuously assay and trace series differentiated cell states during in vivo development at high spatiotemporal resolution. To this end, this proposal plans to take state-of-the-art technology combining high-resolution live imaging, automated cell tracing and single-cell quantitative analysis to systematically investigate the function and mechanism of CEH-31/BARHL1, a conserved homeobox transcription factor, in fate determination in multiple C. elegans neuronal precursor cells. First, we plan to quantify the protein expression levels and dynamic pattern of CEH-31 in every cell at high temporal resolution during embryogenesis (Aim 1). On the basis of the single-cell expression pattern, we further plan to determine the regulatory role of CEH-31 in neuronal fate determination based on gene knockout, over-expression, and single-cell phenotypic analysis of neuronal differentiation (Aim 2). Finally, we plan to determine the interactors, upstream regulators, and downstream direct targets of CEH-31 to construct the regulatory pathway and reveal the molecular mechanism (Aim 3). Together, the proposed study applies single-cell techniques to address an important and unresolved question in neurogenesis, which is expected to provide important insights into the molecular regulation of neuronal lineage differentiation in vivo.

发育神经生物学的一个重要问题是：多个神经前体细胞的命运如何被决定，并经过逐级定向分化产生多类成熟神经细胞？秀丽线虫具有简单、清晰和固定的发育细胞谱系和命运，是解析神经分化调控机制的重要模型。虽然神经细胞终末分化的机制目前了解较多，但是关于神经分化的早期事件，即神经前体细胞的命运决定过程知之甚少，主要原因在于难以快速准确地判定并追踪发育过程中神经前体细胞的一系列分化状态。为此，本课题申请拟基于大量前期工作，集成先进的活体成像、自动化细胞追踪、单细胞基因表达和表型定量分析、基因编辑等方法，通过对神经分化的高时空分辨率连续、动态分析开展如下研究：1.绘制进化保守转录因子CEH-31/BARHL1随胚胎发育的单细胞蛋白动态表达图谱；2.解析CEH-31在神经前体细胞谱系命运决定中的调控功能；3.解析CEH-31的时空特异性调控通路和作用机制。本工作的开展有助于阐明神经细胞谱系建立的分子机制。

神经系统发育早期，复杂多样的神经细胞经由细胞谱系有序产生，但是形态相似的大量神经前体细胞其发育潜能和命运决定过程是如何被调控的一直是发育神经生物学领域的研究热点和难点。本研究以发育谱系清晰明确的秀丽线虫胚胎为研究模型，基于项目负责人前期构建完成的胚胎期266个转录因子蛋白表达图谱资源，聚焦功能未知的转录因子CEH-31及其调控网络来研究这一个问题。首先，我们利用CRISPR/Cas9方法构建了转录因子CEH-31的内源荧光蛋白融合品系，并基于活体成像、细胞追踪和单细胞荧光定量分析体系，在单细胞、每分钟的分辨率定量了CEH-31/BARHL1在谱系身份明确的90%胚胎期单细胞的动态表达水平，发现CEH-31在神经细胞谱系特异表达。进一步，基于266个转录因子蛋白表达图谱资源，预测了CEH-31所在的转录因子调控网络（包括LIN-32、UNC-86、VAB-3等多个重要的神经发育调控基因），发现表皮命运决定因子ELT-1调控了多个神经前体细胞中LIN-32的表达，提示神经前体细胞命运决定过程具有显著的细胞特异性。同时，利用多个转录因子的组合表达定义神经细胞所处的调控状态，从系统生物学角度分析发现终末分化神经细胞的异质性依赖其谱系来源，揭示了神经细胞分化过程中细胞状态转变呈现非定向轨迹。最后，基于CEH-31表达的时空特性和新开发的低样本量组学技术，探究了CEH-31在神经发育中的功能。综上，本项目从系统生物学角度勾勒了复杂的神经分化调控规律，初步揭示了CEH-31及其调控通路在神经细胞谱系逐级分化中的功能，进一步加深了领域内对神经前体细胞命运决定调控机制的理解。