Molecular analyses of yolk syncytial layer formation and its role in zebrafish embryogenesis

鱼类胚胎由两个主要部分组成：动物极的囊胚和植物极的卵黄。囊胚由构成鱼身体的细胞组成，而卵黄是一种重要的结构，用于储存营养物质和信号分子。在这两个隔室之间，有一层薄薄的组织叫做卵黄合胞层（YSL）。YSL是具有许多细胞核的单个细胞层（具有多个细胞核的单个细胞的状态称为合胞体）。YSL细胞的前体是具有单个细胞核的正常细胞。然而，在500-1000个细胞阶段，当YSL形成时，这些细胞膜崩溃，细胞融合并成为具有多核的单细胞。YSL分离胚皮和蛋黄，YSL的作用是转移和代谢营养物质，以及为胚胎的模式发送信号。YSL作为模式的信号中心，向邻近的囊胚细胞发送信号，引导这些细胞成为中胚层（肌肉、肾脏、性腺、血液、消化系统等的前体），并获得区域特征（例如YSL引导细胞成为头、干或尾）。虽然哺乳动物中不存在YSL，但具有类似活动的等效细胞确实存在，因此对YSL的研究将引起包括研究人类发育的医生在内的所有脊椎动物研究人员的普遍兴趣。尽管YSL在胚胎发育中很重要，但YSL形成的分子机制尚未被研究。从我们之前的遗传筛选中，我们分离出一个在YSL中特异性表达的基因（我们将其命名为南斯拉夫-b）。当我们敲除南斯拉夫-b和相关基因南斯拉夫-a的基因功能时，我们发现YSL的形成被特别破坏。因此，这将是第一个被确定为YSL形成的关键调节因子的基因。以该基因为实验工具，探讨YSL细胞融合形成合胞体的分子机制。由于斑马鱼胚胎是透明的，并且YSL的形成在特定的时间以同步的方式发生，因此YSL是研究胚胎发育中发生的细胞融合事件的理想系统。通过利用斑马鱼系统中可用的许多工具（遗传学，基因组信息，使用morphlino反义寡核苷酸进行基因敲低，用荧光蛋白标记细胞结构，全载原位杂交等），我们的目标是获得一系列结果，从而深入了解YSL的形成过程以及所涉及的不同成分，结构，机制和分子。

Fish embryos consist of two major compartments: the blastoderm in the animal pole and yolk in the vegetal pole. The blastoderm consists of cells that make the fish body, while the yolk is a structure that is important for storage of nutrients and signalling molecules. Between these two compartments, there is a thin layer of tissue called the Yolk Syncytial Layer (YSL). The YSL is a single cell layer that has many nuclei (the status of a single cell with multiple nuclei is called a syncytium). The precursors of YSL cells are normal cells that have a single nucleus. However, at 500-1000 cell stage, when the YSL is forming, these cell membranes collapse, cells fuse and become single cells with multiple nuclei. The YSL separates the blastoderm and yolk, and the role of the YSL is to transfer and metabolise nutrients as well as to send signals for the patterning of the embryo. As the signalling centre for patterning, the YSL sends signals to neighbouring blastoderm cells and guides these cells to become mesendoderm (precursor of muscle, kidney, gonad, blood, digestive system and others) and also to obtain regional character (e.g. the YSL guides cells to become head, trunk or tail). Although the YSL does not exist in mammals, equivalent cells with similar activities do, therefore the study of the YSL would be of general interest to all vertebrate researchers including physicians studying human development. Despite the importance of the YSL in embryonic development, the molecular mechanisms under which the YSL is formed have not yet been investigated. From our previous genetic screening, we isolated a gene (we named it yugo-b) that is specifically expressed in the YSL. When we knocked down the gene function of yugo-b and related gene, yugo-a, we found that YSL formation is specifically disrupted. Therefore this would be the first gene that has been identified as a crucial regulator of YSL formation. By using this gene as an experimental tool, we aim to investigate the molecular mechanisms of syncytium formation by cell fusion occurring in the YSL. Since the zebrafish embryo is transparent, and YSL formation occurs at specific times in a synchronised manner, the YSL is an ideal system to study cell fusion events occurring in embryonic development. By utilising many tools available in the zebrafish system (genetics, genome information, gene-knockdown using morphlino antisense oligonucleotides, labelling cell structures with fluorescent proteins, whole mount in situ hybridisation, etc.) we aim to obtain a range of results providing insight into the processes of formation of the YSL and the different components, structures, mechanisms and molecules involved.