NANOG at the exit of pluripotency: new roles in the gastrulating mouse embryo

Abstract

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 13-07-2018Esta tesis tiene embargado el acceso al texto completo hasta el 13-01-2020Developmental biology studies the processes by which a cell divides and differentiates to generate a full functional organism. Up to the onset of gastrulation, cells from the embryo remain pluripotent, but precisely at this stage they differentiate towards mesoderm, endoderm, ectoderm and primordial germ cells. Pluripotency factors maintain uncommitted cells of the blastocyst and embryonic stem cells in culture in the pluripotent state. However, little is known about the role played by these factors during later development, despite their being expressed in the postimplantation epiblast. At this stage, progenitors of the first hematopoietic cells in the mouse arise in the early mesodermal progenitors in the posterior-proximal region of the epiblast, but the mechanisms that specify primitive blood cells are still largely unknown. At the same time, the embryo polarizes and asymmetry arises with the formation of the anterior-posterior axis. Using a dual transgene system for controlled expression at postimplantation stages, we found that NANOG, a pluripotency factor crucial for pluripotency maintenance, blocks primitive hematopoiesis in the gastrulating embryo, resulting in a loss of red blood cells and downregulation of erythropoietic genes. Accordingly, Nanog deficient embryonic stem cells are prone to erythropoietic differentiation. Moreover, Nanog expression in adults prevents the maturation of erythroid cells. By analysis of available data for NANOG binding during stem cell differentiation and CRISPR/Cas9 genome editing, we found that Tal1 is a direct target of NANOG. We also found that Nanog represses anteriorization of the epiblast. By analyzing NanogKO ES cell RNAseq during naïve to primed transition and single cell RNAseq of gastrulating mouse embryos, we discovered that Nanog directly downregulates Pou3f1, that codes for a transcription factor important for anteriorization and neural development. Our results show that Nanog exerts crucial functions at the exit of pluripotency, and that it regulates primitive hematopoiesis and anteriorization of the embryo by directly repressing critical lineage specifiers