Precise self-renewal of the germ cell lineage is fundamental to fertility and reproductive success. The early precursors for the germ lineage, primordial germ cells (PGCs), survive and proliferate in several embryonic locations during their migration to the embryonic gonad. By elucidating the active signaling pathways in migratory PGCs in vivo, we were able to create culture conditions that recapitulate this embryonic germ cell environment. In defined medium conditions without feeder cells, the growth factors FGF2, insulin, and Activin A, signaling through their cognate-signaling pathways, were sufficient for self-renewal of germline-competent PGCs. Forced expression of constitutively active MEK1, AKT, and SMAD3 proteins could replace their respective upstream growth factors. Unexpectedly, we found that BMP4 could replace Activin A in non-clonal growth conditions. These defined medium conditions identify the key molecular pathways required for PGC self-renewal and will facilitate efforts in biobanking of chicken genetic resources and genome editing.

https://www.pure.ed.ac.uk/ws/files/22168227/PIIS2213671115003057.pdf

FGF, Insulin, and SMAD Signaling Cooperate for Avian Primordial Germ Cell Self-Renewal

Background 
Long-term maintenance of avian primordial germ cells (PGCs) in vitro has tremendous potential because it can be used to deepen our understanding of the biology of PGCs. A transgenic bioreactor based on the unique migration of PGCs toward the recipients' sex cord via the bloodstream and thereby creating a germline chimeric bird has many potential applications. However, the growth factors and the signaling pathway essential for inducing proliferation of chicken PGCs are unknown.


Methodology/Principal Findings 
Therefore, we conducted this study to investigate the effects of various combinations of growth factors on the survival and proliferation of PGCs under feeder-free conditions. We observed proliferation of PGCs in media containing bFGF. Subsequent characterization confirmed that the cultured PGCs maintained expression of PGC-specific markers, telomerase activity, normal migrational activity, and germline transmission. We also found that bFGF activates the mitogen-activated protein kinase kinase/extracellular-signal regulated kinase (MEK/ERK) signaling. Also, the expression of 133 transcripts was reversibly altered by bFGF withdrawal.


Conclusions/Significance 
Our results demonstrate that chicken PGCs can be maintained in vitro without any differentiation or dedifferentiation in feeder free culture conditions, and subsequent analysis revealed that bFGF is one of the key factors that enable proliferation of chicken PGCs via MEK/ERK signaling regulating downstream genes that may be important for PGC proliferation and survival.

/pdf/basic-fibroblast-growth-factor-activates-mek-erk-cell-1v70nlcrds.pdf

Basic Fibroblast Growth Factor Activates MEK/ERK Cell Signaling Pathway and Stimulates the Proliferation of Chicken Primordial Germ Cells

Production of transgenic chickens from purified primordial germ cells infected with a lentiviral vector

During gastrulation, chicken primordial germ cells (PGCs) are present in an extraembryonic region of the embryo from where they migrate towards the genital ridges. This is also observed in mammals, but in chicken the vehicle used by the migratory PGCs is the vascular system. We have analysed the migratory pathway of chicken PGCs, focusing on the period of transition from the extraembryonic region to the intraembryonic vascular system.Our findings show that at Hamburger and Hamilton developmental stage HH12-HH14 the majority of PGCs concentrate axially in the sinus terminalis and favour transport axially via the anterior vitelline veins into the embryonic circulation. Moreover, directly blocking the blood flow through the anterior vitelline veins resulted in an accumulation of PGCs in the anterior region and a decreased number of PGCs in the genital ridges. We further confirmed the key role for the anterior vitelline veins in the correct migration of PGCs using an ex ovo culture method that resulted in defective morphogenetic development of the anterior vitelline veins.We propose a novel model for the migratory pathway of chicken PGCs whereby the anterior vitelline veins play a central role at the extraembryonic and embryonic interface. The chicken model of PGC migration through the vasculature may be a powerful tool to study the process of homing (inflammation and metastasis) due to the striking similarities in regulatory signaling pathways (SDF1-CXCR4) and the transient role of the vasculature.

Chicken primordial germ cells use the anterior vitelline veins to enter the embryonic circulation.

Embryonic stem (ES) cells were first isolated in 1981 in the mouse from the in vitro proliferation of the inner cell mass of a 3.5 days post-coitum (dpc) blastocyst. Later on, epiblast stem cells (EpiSC) were identified from in vitro culture of the epiblast of a 6.5 dpc mouse embryo, leading to the concept of naive and primed stem cells. Among non-mammalian species, ES cells have been characterized both in birds and fish; here, we focus on cells derived from chicken and the pluripotent associated markers such as OCT4, SOX2, NANOG, and KLF, previously identified in mammalian cells. In this review, we present both published and original data regarding the involvement of those pluripotent associated genes in the ES cells and early embryo of chicken.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/dgd.12021

Pluripotent genes in avian stem cells

Primordial germ cells (PGCs) are precursors of germline cells. Although avian PGCs have been used to produce transgenic birds, their characteristics largely remain unknown. In this study, we isolated PGCs from chicken embryos at various developmental stages and analyzed the gene expression. Using the expression of stage-specific embryonic antigen-1 (SSEA-1) as a marker of chicken PGCs, we purified PGCs from embryos by fluorescence-activated cell sorting after incubation for 2.5–8.5 days. The number of SSEA-1+ cells was almost unchanged during days 2.5–8.5 of incubation in females but continuously increased in male. Expression of several genes, including Blimp1, SOX2, and CXCR4, was observed in SSEA-1+ cells but not in SSEA-1− cells in both female and male embryos. Quantitative reverse-transcription PCR analysis revealed that the expression of CXCR4, a chemokine receptor essential for migration of PGCs from the bloodstream to the gonads, was reduced after the circulating PGC stage (day 2.5).

Takuya Ohashi

Papers

Analysis of chicken primordial germ cells.