miR-29b对人胚胎干细胞向神经管-神经嵴分化命运调控的机制研究

The neural crest (NC) is a transient embryonic cell population in vertebrates and gives rise to numerous cell types. Problems in NC development are the basis of many human syndromes and birth defects known collectively as neurocristopathies. During neurulation, in response to both intrinsic and extrinsic influences, the neural crest is induced at the neural plate border whereas the adjacent neural plate is fused to form the neural tube. As these two neighbor tissues give rise to totally distinguished cell types, the NC development has became a good system for studying the molecular mechanism underlining the cell fate choice in neurulation, and also for highlighting the pathologic research in neurocristopathies. The gene regulatory network that orchestrates NC cells development has been extensively studied, however the role of miRNAs in neural crest specification is largely unknown. In our previous studies, we have successes differentiated human ES cells to neural tube epithelia(NTE) and neural crest cell(NCC), and discovered a group of miRNAs showed significant expression changes between NTE and NCC. Base on these data, we choose miR-29b, which enriched expressing in NTE, for further research. The reporter gene expression system and bioinformatics analysis indicated that DNMT3a is probably a miR-29b target gene. On this basis, in this project, we will first demonstrate the functions of miR-29b in cell fate choice during neural differentiation by loss- and gain-of-function experiments. We will then use the Tet-On inducible transgenic system to elucidate the miR-29b “functional window” during the differentiation. Finally, signaling pathways chip and reporter gene expression system will be uesd to further explore and validate regulatory signaling pathways of miR-29b. Our study will not only reveal the mechanisms and functional roles of miR-29b in cell fate determining during neural differentiation of human ES cells, but also provide a target for the optimization of in vitro neural crest differentiation system and therapies for neurocristopathies.

神经发育中，来源于神经外胚层的神经板发育成神经管，神经板两侧缘则发育成为神经嵴，神经嵴发育缺陷可导致严重的先天性疾病——神经嵴病。研究神经外胚层向神经管-神经嵴细胞分化命运的调控，有助于理解神经嵴病的发病机制。我们建立了人ES细胞向神经管和神经嵴细胞分化的体系，并通过miRNA芯片发现miR-29b在神经管上皮细胞中特异性上调。生物信息学分析提示miR-29b可能通过下调DNA甲基转移酶3a(DNMT3a)从而调控人ES细胞神经分化中的细胞命运决定。在此基础上，我们拟对miR-29b在人ES神经分化中神经管-神经嵴命运决定的确切调控作用进行验证，并运用报告基因、信号通路芯片和DNA甲基化芯片，探索在神经分化中调控miR-29b上游信号通路和DNMT3a的下游靶基因。本项目从miRNA视角探讨神经分化中细胞分化命运决定的调控机制，为神经嵴病的发病机制研究和治疗提供新的思路。

神经系统在结构和功能上都是一个高度复杂的系统，其起源于原肠胚时期由外胚层形成的神经板。在神经发生阶段，两侧神经板隆起，相互接近并逐渐融合，形成神经管，而在神经管形成过程中，位于神经板的两侧缘的一群神经上皮细胞从神经管背壁分离出来，形成与神经管及覆盖它的表皮细胞都有明显区别的纵向长条细胞带，即神经嵴。在发育过程中，最初的神经外胚层细胞分化成为性质相异的神经管与神经嵴，研究在这一发育过程中，神经外胚层细胞向神经管-神经嵴分化命运决定的调控机制，不仅有助于阐明这一神经发育中的基础性问题，也为今后神经嵴发育缺陷相关疾病的研究与治疗提供了研究基础。在本项研究中，我们将胚胎干细胞高效地定向分化为神经管上皮细胞和神经嵴细胞，并发现相比较于分化起始点，miR-29b在神经管上皮细胞中上调表达，在神经嵴细胞中下调表达。继而发现在胚胎干细胞向神经管上皮细胞分化过程中需要miR-29b的表达，外源过表达miR-29b会促进神经管上皮细胞的分化，并抑制神经嵴细胞的分化。机制研究显示，miR-29b受神经分化过程中重要转录因子Pou3f1调控，并且靶向Dnmt3a而发挥其调控神经分化的作用。本工作目前已发表在“Stem Cell Reports”杂志上。