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

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.

(1) Whole mount immunofluorescent staining: To visualise the localisation of Yugo protein and other basic machineries that regulate membrane dynamics, we will stain the embryo with antibodies and fluorescent probes (Alexa488, Alexa564, Cy5). Besides commercially available antibodies, we will raise new polyclonal antibodies for N and C terminal peptides for some proteins (e.g. yugo-a and b). By staining, we will visualise these proteins under the confocal microscope, reconstruct a three-dimensional image and examine the relationship of these proteins with the cell membrane dynamics during YSL formation. (2) Raising nuclear-GFP (nGFP)/membrane-RFP (mRFP) transgenic fish: We observed the dynamics of membrane and nuclei by injecting nGFP and mRFP RNAs that are often not bright enough at early stages (Case for support Fig3). Therefore, we will produce transgenic fish that expresses nGFP and mRFP. I confirmed these nGFP and mRFP fish have high expression of GFP and RFP at 1000 cell stage and they are better tools for monitoring cell dynamics. We will cross them and raise double transgenic fish of nGFP and mRFP and monitor cell dynamics in the YSL formation. (3) Drug treatment of zebrafish embryos: Zebrafish embryos at 250-1000 cell stage will be treated with various drugs that can block the cell cycle, translation, transcription, polymerisation of cytoskeleton and small G protein activities. In some cases, injecting drugs into embryos might be more effective than to expose embryos for targeting particular part of embryo (e.g. prospective YSL cells). (4) Whole mount in situ hybridisation of yugo morphants: We have obtained many established and novel marker genes for early patterning (Kudoh 2001). Using these markers for in situ staining of yugo-morphants, we will examine what tissues are specifically affected by the knock down of yugo genes and discuss the role of yugos and the YSL in early patterning.