Early Cell Fate Determination in Zebrafish

ESC/iPSC-derive somatic cells may be ideal for treating disorders caused by cellular deficiency or dysfunction. To form a lineage-specific cell population, ESCs/iPSCs undergo a multi-step process that recapitulates embryonic development. ESC/iPSC differentiation protocols are hampered by the limitation of our understanding in development. Zebrafish embryos are fertilized and developed externally, a feature facilitates the observation and manipulation of embryonic development. To explore the zebrafish as a system to study cell lineage determination, in this thesis, I 1) identified an ortholog of the key pluripotency regulator Nanog in zebrafish and examined its role in early cell fate determination; 2) developed a high-throughput image-based chemical screening system in zebrafish blastomere cell culture that is very similar to, but much faster than, ESC/iPSC differentiation screens. Specifically, in an effort to examine the role of Nanog in vivo, I identified a zebrafish Nanog ortholog, and found that its knockdown impaired endoderm formation. Genome-wide transcription analysis revealed that nanog-like morphants fail to develop the extra-embryonic yolk syncytial layer (YSL), which produces Nodal required for endoderm induction. I examined the genes that were regulated by Nanog-like, and identified the homeobox gene mxtx2, which is both necessary and sufficient for YSL induction. Chromatin immunoprecipitation assays and genetic studies indicated that Nanog-like directly activates mxtx2, which in turn specifies the YSL lineage by directly activating YSL genes. The study identifies a Nanog-like-Mxtx2-Nodal pathway and establishes a role for Nanog-like in regulating the formation of the extra-embryonic tissue required for endoderm induction.