Evolutionary origin of vertebrate OCT4/POU5 functions in supporting pluripotency.

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.

Polycomb enables primitive endoderm lineage priming in embryonic stem cells.

Mouse embryonic stem cells (ESCs), like the blastocyst from which they are derived, contain precursors of the epiblast (Epi) and primitive endoderm (PrEn) lineages. While transient in vivo, these precursor populations readily interconvert in vitro. We show that altered transcription is the driver of these coordinated changes, known as lineage priming, in a process that exploits novel polycomb activities. We find that intragenic levels of the polycomb mark H3K27me3 anti-correlate with changes in transcription, irrespective of the gene's developmental trajectory or identity as a polycomb target. In contrast, promoter proximal H3K27me3 is markedly higher for PrEn priming genes. Consequently, depletion of this modification stimulates the degree to which ESCs are primed towards PrEn when challenged to differentiate, but has little effect on gene expression in self-renewing ESC culture. These observations link polycomb with dynamic changes in transcription and stalled lineage commitment, allowing cells to explore alternative choices prior to a definitive decision.

Differentiation of Mouse Embryonic Stem Cells into Ventral Foregut Precursors.

Anterior definitive endoderm (ADE), the ventral foregut precursor, is both an important embryonic signaling center and a unique multipotent precursor of liver, pancreas, and other organs. Here, a method is described for the differentiation of mouse embryonic stem cells (mESCs) to definitive endoderm with pronounced anterior character. ADE-containing cultures can be produced in vitro by suspension (embryoid body) culture or in a serum-free adherent monolayer culture. ESC-derived ADE cells are committed to endodermal fates and can undergo further differentiation in vitro towards ventral foregut derivatives.

Resolving Heterogeneity: Fluorescence-Activated Cell Sorting of Dynamic Cell Populations from Feeder-Free Mouse Embryonic Stem Cell Culture.

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).

Clathrin is essential for meiotic spindle function in oocytes.

In the mammalian ovary, oocytes are arrested at prophase of meiosis I until a hormonal stimulus triggers resumption of meiosis. During the subsequent meiotic maturation process, which includes completion of the first meiotic division and formation of the second metaphase spindle, oocytes acquire competence for fertilization. Recently, it was shown that clathrin, a cytosolic protein complex originally defined for its role in intracellular membrane traffic, is also involved in the stabilization of kinetochore fibers in mitotic spindles of dividing somatic cells. However, whether clathrin has a similar function in meiotic spindles in oocytes has not been investigated previously. Our results show that endogenous clathrin associates with the meiotic spindles in oocytes. To study the function of clathrin during meiotic maturation, we microinjected green fluorescent protein-tagged C-terminal and N-terminal dominant-negative clathrin protein constructs into isolated porcine oocytes prior to in vitro maturation. Both protein constructs associated with meiotic spindles similar to endogenous clathrin, but induced misalignment and clumping of chromosomes, occurrence of cytoplasmic chromatin and failure of polar body extrusion. These data demonstrate that clathrin plays a crucial role in meiotic spindle function in maturing oocytes, possibly through spindle stabilization.

CDC2/SPDY transiently associates with endoplasmic reticulum exit sites during oocyte maturation.

BACKGROUND: Mammalian oocytes acquire competence to be fertilized during meiotic maturation. The protein kinase CDC2 plays a pivotal role in several key maturation events, in part through controlled changes in CDC2 localization. Although CDC2 is involved in initiation of maturation, a detailed analysis of CDC2 localization at the onset of maturation is lacking. In this study, the subcellular distribution of CDC2 and its regulatory proteins cyclin B and SPDY in combination with several organelle markers at the onset of pig oocyte maturation has been investigated. RESULTS: Our results demonstrate that CDC2 transiently associates with a single domain, identified as a cluster of endoplasmic reticulum (ER) exit sites (ERES) by the presence of SEC23, in the cortex of maturing porcine oocytes prior to germinal vesicle break down. Inhibition of meiosis resumption by forskolin treatment prevented translocation of CDC2 to this ERES cluster. Phosphorylated GM130 (P-GM130), which is a marker for fragmented Golgi, localized to ERES in almost all immature oocytes and was not affected by forskolin treatment. After removal of forskolin from the culture media, the transient translocation of CDC2 to ERES was accompanied by a transient dispersion of P-GM130 into the ER suggesting a role for CDC2 in redistributing Golgi components that have collapsed into ERES further into the ER during meiosis. Finally, we show that SPDY, rather than cyclin B, colocalizes with CDC2 at ERES, suggesting a role for the CDC2/SPDY complex in regulating the secretory pathway during oocyte maturation. CONCLUSION: Our data demonstrate the presence of a novel structure in the cortex of porcine oocytes that comprises ERES and transiently accumulates CDC2 prior to germinal vesicle breakdown. In addition, we show that SPDY, but not cyclin B, localizes to this ERES cluster together with CDC2.