不同Bmp配体差异调节斑马鱼外植体延伸和胚胎集中延伸运动的机制研究

Convergence and extension is one of the most important events during early embryonic development. Transforming Growth Factor-β (TGF-β) member BMP, are a group of morphogens that play important roles in early embryonic development, organogenesis and regeneration. Although the convergence and extension defect caused by abnormal BMP signaling has long been reported, the mechanism is not very clear so far, meanwhile the differences of three Bmp ligands in early embryonic development especially the CE cell movements are poorly understood. Our preliminary data in zebrafish animal pole explant showed that Bmp4 induced extension of the explant while Bmp2b can not, suggesting obvious differences between BMP ligands. Zebrafish explant extension mimics the convergence and extension movements during early embryonic development, thus the mechanism by which Bmp4 driving explant extension could apply to convergence and extension movements in vivo. Based on these observations, firstly we plan to find Bmp4 specific target genes using RNA-seq and single cell sequencing in the explant, then in these candidates, we will screen for cell movement related genes using knockdown, knockout and overexpression methods. We plan to figure out how cell movements are activated by these genes using high resolution live imaging, and its related molecular mechanisms. This project is expected to discover new mechanisms of convergence and extension, aiming to find out the distinct roles of different Bmp ligands in regulating cell movements and cell fate decision during embryonic development.

集中延伸运动是胚胎发育早期的重要事件，它的异常会导致神经管闭合不全等严重的出生缺陷。BMP信号通路对于集中延伸运动是必需的，但机制尚不明确，尤其是斑马鱼三个Bmp配体对集中延伸过程的调控作用更是了解甚少。我们前期的研究发现，Bmp4可以诱导外植体延伸而Bmp2b不可以，显示了BMP配体之间明显的差异性。我们认为外植体的延伸是集中延伸运动在体外的表现，Bmp4诱导外植体延伸的机制应该同样也适用于体内的集中延伸运动。我们拟利用RNA-seq和单细胞测序的方法，寻找Bmp4诱导外植体差异表达的基因。在外植体和胚胎中，利用敲低、敲除或高表达的方法筛选Bmp4影响细胞运动的靶基因。利用活体荧光成像技术观察靶基因的异常导致的细胞运动的变化并研究其生化机制。本项目利用斑马鱼体外外植体和体内两种系统，有望阐明BMP不同配体调节细胞运动的异同，发现集中延伸这一重要发育事件新的调控机制。

BMP信号通路在早期胚胎发育中发挥重要作用，目前被广泛用于体外诱导类胚胎和类器官。.在本研究中，我们利用斑马鱼外植体的系统，发现bmp4在体外有中胚层诱导能力而bmp2b没有。对比bmp2b和bmp4的氨基酸序列，发现只有若干碱基不同。然后对bmp4进行单碱基的突变分析，我们发现C端的3个碱基决定了不同配体诱导胚胎干细胞进行胚层分化的能力的不同。然后我们针对bmp4诱导产生的类胚胎的细胞命运决定和形态发生进行了详细的分析。我们发现原肠期bmp4主要诱导了轴旁中胚层和侧部中胚层，在体节期体节的依次形成十分明显并存在血管，心脏，原肾管等的细胞类型的特化。通过长时间荧光成像分析，我们发现bmp4外植体的形态是通过原肠期的内卷运动和体节期的尾部延伸运动形成的。本研究首次报导了BMP信号通路配体bmp2b和bmp4存在功能差异，并揭示了bmp4自身就足以诱导尾部结构的形成。