Search

The support of pluripotent cells over time is an essential feature of development. In eutherian embryos, pluripotency is maintained from naïve states in peri-implantation to primed pluripotency at gastrulation. To understand how these states emerged, we reconstruct the evolutionary trajectory of the Pou5 gene family, which contains the central pluripotency factor OCT4. By coupling evolutionary sequence analysis with functional studies in mouse embryonic stem cells, we find that the ability of POU5 proteins to support pluripotency originated in the gnathostome lineage, prior to the generation of two paralogues, Pou5f1 and Pou5f3 via gene duplication. In osteichthyans, retaining both genes, the paralogues differ in their support of naïve and primed pluripotency. The specialization of these duplicates enables the diversification of function in self-renewal and differentiation. By integrating sequence evolution, cell phenotypes, developmental contexts and structural modelling, we pinpoint OCT4 regions sufficient for naïve pluripotency and describe their adaptation over evolutionary time., (© 2022. The Author(s).)

Embryonic stem cell (ESC) culture comprises a mixture of cells that are primed to differentiate into different lineages. In conditions where ESCs self-renew, these primed populations continuously interconvert and consequently show highly dynamic coordinated changes in their expression of different sets of pluripotency and differentiation markers. It has become increasingly apparent that this transcriptional heterogeneity is an important characteristic of ESC culture. By sorting for specific populations of ESCs it is possible to enrich for cells with a capacity to colonize the embryo proper or the extra-embryonic lineages such as the descendents of the primitive endoderm or trophoblast. Here, we describe a method of isolating specific sub-sets of ESCs from the pluripotent cells present in in vitro ESC culture using SSEA1 antibody staining in combination with reporter lines and fluorescence activated cell sorting (FACS).

Manipulation of mammalian oocytes at the molecular level is hampered by low transcriptional activity and the presence of large stores of mRNA and protein. Microinjection of interfering macromolecules has become an important tool in studying oocyte maturation, although injection success, final concentrations of injected substances and viability after injection remain difficult to assess with current techniques. To address these problems, we developed an epifluorescence microscopy based technique to evaluate oocytes directly after (co-)injection of green fluorescent protein (GFP)., (Copyright © 2011 Elsevier Inc. All rights reserved.)