The Translational Regulatory Mechanisms of Neural Precursor Cell Maintenance and Differentiation

在哺乳动物中，神经干细胞/前体细胞(NPC)产生数十亿个神经元来构建大脑。为了确保适当的神经发生，鼻咽癌维持和分化之间的平衡受到严格控制。我的研究计划的长期目标是了解鼻咽癌维持和分化的基因调控机制。在过去的几十年里，人们已经做出了相当大的努力来了解调控NPC的转录机制。然而，基因调控的其他层面如何控制鼻咽癌在很大程度上仍未被探索。使用发育中的小鼠皮质作为我们的模型系统，我们最近发现许多促进分化的基因在皮质NPC中普遍转录，但这些转录产物没有翻译。因此，翻译调控在鼻咽癌的维持和分化中起着关键作用，尽管具体机制尚不清楚。因此，我的短期目标是了解翻译控制机制。我们鉴定了一组含有核心抑制蛋白4E-T的翻译抑制复合体。这些复合体在维持皮质神经前体细胞的干细胞状态方面起着关键作用。在寻找4E-T复合体的其他成分时，我们在鼻咽癌中发现了许多与4E-T相互作用的靶向特异性RNA结合蛋白，如细胞质多聚腺苷化元件结合蛋白4(CPEB4)。我们假设，通过与不同的靶标特异性RNA结合蛋白形成复合物，4E-T控制着不同的mRNAs亚群，以调控NPC的维持和分化。在这项提案中，我们将以CPEB4为例，说明从长远来看，我们将如何剖析各种RNA结合蛋白在鼻咽癌动态平衡中的作用。在三个特定的目标中，我们将：1)通过应用功能丧失和功能获得的方法，在体外和体内确定RNA结合蛋白在鼻咽癌维持和分化中的作用；2)使用全基因组和候选基因的方法，确定RNA结合蛋白的靶mRNAs；以及3)表征4E-T和RNA结合蛋白之间的相互作用，以评估不同的复合体如何调节鼻咽癌的稳态。建议的研究将阐明鼻咽癌内稳态的翻译控制机制，并为我的长期研究目标全面揭示鼻咽癌基因调控格局奠定基础。通过提高我们对基因表达如何在多个水平上进行控制以协调干细胞动态平衡的理解，我的研究将引发干细胞制造和组织工程应用的新技术。重要的是，通过学习最先进的高分辨率成像技术、基因调控和干细胞生物学，我研究项目中的HQP将为学术界内外的职业生涯培养专业技能。

In mammals, neural stem/precursor cells (NPCs) give rise to billions of neurons to build the brain. To ensure the appropriate neurogenesis the balance between NPC maintenance and differentiation is tightly controlled. The long-term objective of my research program is to understand gene regulatory mechanisms that underlie NPC maintenance and differentiation. In the past few decades, considerable efforts have been directed towards understanding transcriptional mechanisms that regulate NPCs. Nevertheless, how other layers of gene regulation control NPCs remains largely unexplored. Using the developing mouse cortex as our model system, we have recently found that many genes that promote differentiation are transcribed ubiquitously in cortical NPCs, but those transcripts are not translated. Thus, translational regulation plays a critical role in NPC maintenance and differentiation, though the specific mechanisms are poorly understood. Therefore, my short-term objective is focused on understanding translational control mechanisms. We identified a group of translational repression complexes that contain the core repressor protein 4E-T. The complexes play a key role in maintaining the stem cell state of cortical NPCs. In searching the other components of the 4E-T complexes, we identified many target-specific RNA-binding proteins that interact with 4E-T in NPCs, such as the cytoplasmic polyadenylation element binding protein 4 (CPEB4). We hypothesize that by forming complexes with distinct target-specific RNA-binding proteins, 4E-T controls different subsets of mRNAs to regulate NPC maintenance and differentiation. In this proposal, we will use CPEB4 as an example to illustrate how in the long term we will dissect the role of various RNA-binding proteins in NPC homeostasis. In three specific aims, we will: 1) Determine the roles of RNA-binding proteins in NPC maintenance and differentiation both in vitro and in vivo, by applying loss- and gain-of-function approaches; 2) Identify target mRNAs for the RNA-binding proteins, using both genome-wide and candidate-gene approaches; and 3) Characterize the interactions between 4E-T and RNA-binding proteins to assess how the different complexes regulate NPC homeostasis. The proposed studies will elucidate translational control mechanisms of NPC homeostasis and serve as a foundation for my long-term research goal to fully reveal the gene regulatory landscape in NPCs. By advancing our understanding of how gene expression is controlled at multiple levels to coordinate stem cell homeostasis, my research will spark new technologies for stem cell manufacturing and tissue engineering applications. Importantly, by learning state-of-the-art techniques for high-resolution imaging, gene regulation, and stem cell biology, HQP in my research program will develop professional skills for a career within or outside academia.