Investigating the mechanisms that restrict the extra-embryonic Yolk Syncytial Layer

The normal development of many animals depends on cells that make the animal ("embryonic cells") as well as supporting cells that do not form a part of the final animal form per se ("extra-embryonic" cells). For instance, the human placenta is a supporting "extra-embryonic" tissue which is essential for normal human embryo development. In many fish species, an extra-embryonic tissue called the yolk syncytial layer (YSL) develops within a shared yolk cytoplasm. The YSL does not become a part of the fully-formed animal, but is a crucial source of nutrition and signals for the developing fish. If the developing fish embryo is separated from the yolk and YSL at very early stages, the embryo develops abnormally. Although the YSL is crucial for normal development, how it forms and how the YSL is controlled is not well understood. Protein factors deposited by the mother into the egg are thought to control YSL formation, but to date, only a few molecules have been found and how they act in YSL development is not well understood. We found that Zebrafish embryos lacking the proteins Igf2bp3 and Ybx1 have an abnormally expanded YSL at the expense of the embryo proper, leading to disastrous consequences for the embryo. How Igf2bp3 and Ybx1 control YSL formation is not known. Zebrafish embryos are transparent at early stages, making it possible to see the YSL as it forms. We will film the YSL as it forms in normal zebrafish embryos and compare to embryos lacking these proteins. We will investigate how some key cell communication pathways are controlled by these proteins during YSL formation. We will identify the key molecules through which Igf2bp3 and Ybx1 control development of the YSL. Through these strategies, we will find out how Igf2bp3 and Ybx1 function in YSL formation in zebrafish. Both the proteins are present in many animals. The mechanisms that we identify might be involved in extra-embryonic tissue formation in other fish. Therefore, the work is likely to be of broad relevance to extra-embryonic development across animal species.

In this project, we aim to investigate how the RNA-binding proteins Ybx1 and Igf2bp3 control growth factor signalling and cytoskeletal re-modelling to establish the extra-embryonic yolk syncytial layer (YSL) in zebrafish. It is based on our recent work showing that Ybx1 and Igf2bp3 translationally repress Nodal growth factor signalling in early zebrafish embryos. We will make use of our unique ybx1 and igf2bp3 mutants that show a massively expanded YSL and fail to undergo gastrulation. The increased YSL in these mutants suggests that during normal embryonic development, an inhibitory mechanism prevents uncontrolled spreading of the YSL. We will use high resolution time-lapse imaging (by spinning disk confocal and multi photon imaging), to investigate if defects in cell divisions, the actin and microtubule cytoskeleton, or membranes underlie YSL expansion in the mutant embryos.

We will investigate how regulation of Nodal growth factor signalling by Ybx1 and Igf2bp3 affects cell remodelling pathways and the YSL by inhibiting Nodal signalling and altering Rock and Src signalling in the mutant embryos. The YSL transcriptome is not well represented in existing zebrafish datasets. Based on analysis of our available ybx1 and igf2bp3 mutant transcriptomic datasets, we will identify the key molecules through which Ybx1 and Igf2bp3 regulate YSL formation. We will collaborate with KH Chen of the Genome institute of Singapore to study the YSL distribution of selected Ybx1 and Igf2bp3 targets. We will analyse the distribution and functions of two selected direct Ybx1 and Igf2bp3 target genes during YSL development through genome editing mutants, knockdown and YSL spatial transcriptomics.

The proposal builds upon our previous work on maternal regulation of early embryonic development, and will utilise a recent BBSRC-supported imaging system. Insights from this project will provide better understanding of how extra-embryonic tissues and syncytia develop in animals.