Human primordial germ cells (hPGCs), the precursors of sperm and eggs, originate during week 2-3 of early postimplantation development1. Using in vitro models of hPGC induction2–4, recent studies suggest striking mechanistic differences in specification of human and mouse PGCs5. This may partly be due to the divergence in their pluripotency networks, and early postimplantation development6–8. Since early human embryos are inaccessible for direct studies, we considered alternatives, including porcine embryos that, as in humans, develop as bilaminar embryonic discs. Here we show that porcine PGCs (pPGCs) originate from the posterior pre-primitive streak competent epiblast by sequential upregulation of SOX17 and BLIMP1 in response to WNT and BMP signalling. Together with human and monkey in vitro models simulating peri-gastrulation development, we show conserved principles for epiblast development for competency for PGC fate, followed by initiation of the epigenetic programme9–11, regulated by a balanced SOX17–BLIMP1 gene dosage. Our combinatorial approach using human, porcine and monkey in vivo and in vitro models, provides synthetic insights on early human development.

Background: This work describes the first genome-wide analysis of the transcriptional landscape of the pig. A new porcine Affymetrix expression array was designed in order to provide comprehensive coverage of the known pig transcriptome. The new array was used to generate a genome-wide expression atlas of pig tissues derived from 62 tissue/cell types. These data were subjected to network correlation analysis and clustering.Results: The analysis presented here provides a detailed functional clustering of the pig transcriptome where transcripts are grouped according to their expression pattern, so one can infer the function of an uncharacterized gene from the company it keeps and the locations in which it is expressed. We describe the overall transcriptional signatures present in the tissue atlas, where possible assigning those signatures to specific cell populations or pathways. In particular, we discuss the expression signatures associated with the gastrointestinal tract, an organ that was sampled at 15 sites along its length and whose biology in the pig is similar to human. We identify sets of genes that define specialized cellular compartments and region-specific digestive functions. Finally, we performed a network analysis of the transcription factors expressed in the gastrointestinal tract and demonstrate how they subdivide into functional groups that may control cellular gastrointestinal development. Conclusions: As an important livestock animal with a physiology that is more similar than mouse to man, we provide a major new resource for understanding gene expression with respect to the known physiology of mammalian tissues and cells. The data and analyses are available on the websites http://biogps.org and http://www.macrophages.com/pig-atlas.

High-resolution molecular programmes delineating the cellular foundations of mammalian embryogenesis have emerged recently. Similar analysis of human embryos is limited to pre-implantation stages, since early post-implantation embryos are largely inaccessible. Notwithstanding, we previously suggested conserved principles of pig and human early development. For further insight on pluripotent states and lineage delineation, we analysed pig embryos at single cell resolution. Here we show progressive segregation of inner cell mass and trophectoderm in early blastocysts, and of epiblast and hypoblast in late blastocysts. We show that following an emergent short naive pluripotent signature in early embryos, there is a protracted appearance of a primed signature in advanced embryonic stages. Dosage compensation with respect to the X-chromosome in females is attained via X-inactivation in late epiblasts. Detailed human-pig comparison is a basis towards comprehending early human development and a foundation for further studies of human pluripotent stem cell differentiation in pig interspecies chimeras.

Activin/Nodal signaling is required for maintaining pluripotency and self-renewal of mouse epiblast stem cells and human embryonic stem cells (hESC). In this study, we investigated whether this signaling mechanism is also operative in cultured epiblasts derived from Days 10.5-12 pig embryos. Pig epiblast stem cell lines (pEpiSC) were established on mouse feeder layers and medium supplemented with basic fi broblast growth factor (bFGF). pEpiSC express the core pluripotency factors OCT4 (or POU5F1 ), NANOG , SOX2 , and NODAL , but they do not express REX1 or alkaline phosphatase activity. Blocking leukemia inhibitory factor (LIF)/JAK/STAT3 pathway by adding the specifi c JAK I inhibitor 420099 and an anti-LIF antibody over 3 passages did not affect pluripotency of pEpiSC. In contrast, cells grown with the Alk-5 inhibitor SB431542, which blocks Activin/Nodal pathway, differentiated readily toward the neural lineage. pEpiSC are pluripotent, as established by their differentiation potential to ectoderm, mesoderm, and endoderm. These cells can be induced to differentiate toward trophectoderm and to germ cell precursors in response to bone morphogenetic protein 4 (BMP-4). In conclusion, our study demonstrates that pig epiblasts express the core pluripotency genes and that the capacity for maintaining self-renewal in pEpiSC depends on Activin/Nodal signaling. This study provides further evidence that maintenance of pluripotency via Activin/Nodal signal is conserved in mammals. Introduction Mouse embryonic stem cells (mESC), conventionally derived from the inner cell mass (ICM) of preimplantation blastocysts, are pluripotent and can self-renew indefinitely in culture. mESC depend on the cytokines leukemia inhibitory factor (LIF) and bone morphogenetic protein 4 (BMP-4) to maintain the undifferentiated state [ 1 , 2 ]. Human embryonic stem cells (hESC) are also derived from blastocysts; however, these cells depend on basic fi broblast growth factor (bFGF) and Activin A for pluripotency and selfrenewal [ 3 , 4 ]. Interestingly, pluripotent cells derived from mouse epiblasts, a part of the ICM, also require bFGF and Activin A for pluripotency and self-renewal [ 5 , 6 ]. hESC share multiple features with mouse epiblast stem cells (mEpiSC), such as growing as fl at and compact colonies [5][6][7], they respond to BMP-4 by differentiating to trophectoderm (TE) [ 8 ] and germ cell progenitors [ 9 ], and they do not require LIF/ JAK/STAT3 signaling for self-renewal [ 4 , 10 ]. The functional and phenotypic similarities between these cell types suggest a developmental relationship [ 5 , 6 ], and demonstrate that there are at least 2 signaling mechanisms involved in capturing pluripotency in vitro. Recent reports show that bFGF and Activin A are also necessary for maintaining rabbit ESC [ 11 , 12 ], indicating that this signaling pathway may be a conserved mechanism of pluripotency in mammals.Here we tested this hypothesis in pigs, a representative of ungulates. Attempts to derive stem cells from pig embryos have traditionally reli...

SUMMARYCells in the pluripotent ground state can give rise to somatic cells and germ cells, and the acquisition of pluripotency is dependent on the expression of Nanog. Pluripotency is conserved in the primitive ectoderm of embryos from mammals and urodele amphibians, and here we report the isolation of a Nanog ortholog from axolotls (axNanog). axNanog does not contain a tryptophan repeat domain and is expressed as a monomer in the axolotl animal cap. The monomeric form is sufficient to regulate pluripotency in mouse embryonic stem cells, but axNanog dimers are required to rescue LIF-independent self-renewal. Our results show that protein interactions mediated by Nanog dimerization promote proliferation. More importantly, they demonstrate that the mechanisms governing pluripotency are conserved from urodele amphibians to mammals.

The establishment of the pluripotent ICM during early mammalian development is characterized by the differential expression of the transcription factors NANOG and GATA4/6, indicative of the epiblast and hypoblast, respectively. Differences in the mechanisms regulating the segregation of these lineages have been reported in many species, however little is known about this process in the porcine embryo. The aim of this study was to investigate the signalling pathways participating in the formation of the porcine ICM, and to establish whether their modulation can be used to increase the developmental potential of pluripotent cells. We show that blocking MEK signalling enhances the proportion of NANOG expressing cells in the ICM, but does not prevent the segregation of GATA-4 cells. Interestingly, inhibition of FGF signalling does not alter the segregation of NANOG and GATA-4 cells, but affects the number of ICM cells. This indicates that FGF signalling participates in the formation of the founders of the ICM. Inhibition of MEK signalling combined with GSK3β inhibition and LIF supplementation was used to modulate pluripotency in porcine iPS (piPS) cells. We demonstrate that under these stringent culture conditions piPS cells acquire features of naive pluripotency, characterized by the expression of STELLA and REX1, and increased in vitro germline differentiation capacity. We propose that small molecule inhibitors can be used to increase the homogeneity of induced pluripotent stem cell cultures. These improved culture conditions will pave the way for the generation of germline competent stem cells in this species.

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