CELSR3和FZD3依赖的介导细胞在人胚前脑神经网络形成的作用研究

The complex neural circuits in forebrain are critical for sensory processing, motor control and cognition. Using animal, mostly murine models, several genes are identified and involved in establishing neural forebrain networks. However, their role during human brain development remain elusive. Using knockout mouse models, we previously demonstrated that Celsr3 and Fzd3 play an indispensible role to regulate forming a pioneer "sling" via their expression in guidepost cells, which then steer forebrain axonal projections .These facts led us to hypothesize that CELSR3 and FZD3expressing guidepost cells are also important for establishing neural circuits in the human forebrain. To test this, we will collect WPC (post conceptual weeks) 6-9 abortion human fetuses to: i) Study the expression patterns of putative markers in intermediate targets critical for early axonal projections; ii) Analyze the distribution of CELSR3 and FZD3 mRNAs by using in situ hybridization and the sub-cellular distribution; iii) Assess their function in regulating axonal projections by knocking down their expression in guidepost cells; and iv) Compare transcriptional profiles of guidepost cells between human and mouse using FACS sorting and RNASeq. We predict that our results will shed light on the role of CELSR3 and FZD3 dependent guidepost cells in the formation of human forebrain networks and point to significant differences of gene expression between human and mouse guidepost cells. This should contribute to our basic understanding of molecular mechanisms that govern human forebrain development.

前脑正常神经网络是机体执行感觉处理、运动控制、认知等高级功能的关键，调控其形成的功能基因在动物模型中陆续被发现，但它们在人胚脑发育中的作用依然不明确。通过基因敲除，我们发现Celsr3和Fzd3通过影响前脑介导细胞形成先驱“轴突桥梁”，从而导向投射神经元轴突生长、建立正常的靶向联系。为此，我们提出：CELSR3和FZD3依赖的介导细胞在人胚前脑神经联系建立过程是必需的。我们将收集6-9周流产胚胎，比较分析前脑神经元轴突投射早期形成过程经历不同“过渡区”的细胞标记物图谱、CELSR3和FZD3时空表达模式和亚细胞定位、投射神经元轴突与介导细胞的关系、介导细胞敲减CELSR3和FZD3对神经元轴突生长导向的影响、人和小鼠介导细胞生物学特性异同等。预期结果将阐明CELSR3和FZD3依赖的介导细胞调控人前脑神经网络形成的作用及其与小鼠的异同，以期望填补人胚前脑神经联系形成分子调控机制方面的空白。

前脑正常神经网络是机体执行感觉处理、运动控制、认知等高级功能的关键，调控其形成的功能基因在动物模型中陆续被发现，但它们在人胚脑发育中的作用依然不明确。基于前期基因敲除小鼠模型的研究结果，本项目提出：CELSR3和FZD3依赖的介导细胞在人胚前脑神经联系建立过程是必需的。本项目我们通过收集妊娠5-8周（WPC5-8）的胚胎，研究人脑的早期发育。我们研究发现大脑早期的神经元产生于神经上皮层阶段，皮质的神经祖细胞起源于局部端脑背侧的神经上皮细胞和复侧的嗅基板。在皮质发育的原基板（preplate）期，前脑的结构形成在人和小鼠十分相似，包括脑部区域的形成、Cajal-Retzius细胞的分化、先驱神经元的发育和轴突方向性生长等。在人胚脑，来自于前丘脑（prethalamus）、腹侧端脑、皮质原基板的先驱神经元发出轴突跨越间脑-端脑交界处（diencephalon-telencephalon junction）和皮质-皮质下交界处（pallial-subpallial boundary）形成“轴突桥梁”导向丘脑和皮质投射神经元轴突的靶向生长，这一现象与我们前期小鼠研究Celsr3和Fzd3依赖的介导细胞调控前脑神经网络形成的机理相似。在人胚胎前脑皮质板（cortical plate）发育早期，皮质投射神经元生长轴突在腹侧端脑与Celsr3和Fzd3阳性的介导细胞紧密接触。应用RNAscope，我们发现在人和小鼠胚胎前脑Celsr3和Fzd3表达十分相似，调节介导细胞和先驱轴突的形成。总之，本研究提示人和小鼠胚胎前脑的发育具有保守性，Celsr3和Fzd3依赖的介导细胞作为一个共性机理调控前脑神经网络的形成。该研究为解析人脑形成的分子调控机制提供了理论依据。