The roles of pluripotency factors in the germ layer specification in vertebrate embryo

• Applicant: Dr. Daria Onichtchouk
• Subject Area: Developmental Biology
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 497658974

Zygotic Genome Activation (ZGA) in zebrafish embryos relies on Pou5f3, Sox19b and Nanog, homologs of mammalian pluripotency transcription factors. Binding of these factors to thousands of the regulatory regions of early zygotic genes increases chromatin accessibility and primes the genes for activation. It remains poorly understood, how Pou5f3, Sox19b and Nanog interact with each other and with the signals patterning the embryo to three germ layers: ectoderm, endoderm and mesoderm. The purpose of the current proposal is to resolve the regulatory roles of pluripotency factors in the differentiating tissues of the early embryo in time and space. We will first simultaneously profile gene expression (single nuclear transcriptome) and open chromatin (single nuclear ATAC-seq) of the wild-type embryos, to add the regulatory layer to the existing single cell transcriptomics data. We will use the embryos in several stages between ZGA and beginning of organogenesis, encompassing the time period from pluripotent cells to pluripotency exit. The stages will be matched to the bulk ATAC-seq data and the large-scale single-cell transcriptome developmental reference tree for zebrafish embryogenesis which is publicly available. According to this tree, somatic cells select between three specification routes (axial mesoderm, other mesoderm and ectoderm) shortly before the gastrulation onset. We expect therefore, that combination of ATAC-seq and nuclear transcription data will allow us to cluster differentiating cell populations even earlier, as the formation of nucleosome-free regions at gene regulatory sites is thought to precede gene expression. Using the wild-type developmental regulatory atlas obtained at the first step, we will select the time point, where the cell populations can be robustly resolved. Then we will perform similar single nuclear ATAC-seq and transcriptome analysis on the single, double and triple mutants by the pluripotency factors at this time point. The data analysis will address the question, if the different cell populations can be resolved at all in the mutants, and, if yes, which of the embryonic tissues are missing or changed compared to the wild-type. We also aim to identify potential tissue-specific interaction partners of the pluripotency genes and shed the light into the causes of premature expression of the late regulatory factors in the pluripotency mutants. The results will provide new insights into the connection of pluripotency, differentiation and developmental plasticity.

• DFG Programme: Research Grants