The Oct4 homologue PouV and Nanog regulate pluripotency in chicken embryonic stem cells

International audienceEmbryonic stem cells ( ESC) have been isolated from pregastrulation mammalian embryos. The maintenance of their pluripotency and ability to self- renew has been shown to be governed by the transcription factors Oct4 ( Pou5f1) and Nanog. Oct4 appears to control cell- fate decisions of ESC in vitro and the choice between embryonic and trophectoderm cell fates in vivo. In nonmammalian vertebrates, the existence and functions of these factors are still under debate, although the identification of the zebrafish pou2 ( spg; pou5f1) and Xenopus Pou91 ( XlPou91) genes, which have important roles in maintaining uncommitted putative stem cell populations during early development, has suggested that these factors have common functions in all vertebrates. Using chicken ESC ( cESC), which display similar properties of pluripotency and long- term self- renewal to mammalian ESC, we demonstrated the existence of an avian homologue of Oct4 that we call chicken PouV ( cPouV). We established that cPouV and the chicken Nanog gene are required for the maintenance of pluripotency and self- renewal of cESC. These findings show that the mechanisms by which Oct4 and Nanog regulate pluripotency and self- renewal are not exclusive to mammal

Bmi1 facilitates primitive endoderm formation by stabilizing Gata6 during early mouse development

International audienceThe transcription factors Nanog and Gata6 are critical to specify the epiblast versus primitive endoderm (PrE) lineages. However, little is known about the mechanisms that regulate the protein stability and activity of these factors in the developing embryo. Here we uncover an early developmental function for the Polycomb group member Bmi1 in supporting PrE lineage formation through Gata6 protein stabilization. We show that Bmi1 is enriched in the extraembryonic (endoderm [XEN] and trophectodermal stem [TS]) compartment and repressed by Nanog in pluripotent embryonic stem (ES) cells. In vivo, Bmi1 overlaps with the nascent Gata6 and Nanog protein from the eight-cell stage onward before it preferentially cosegregates with Gata6 in PrE progenitors. Mechanistically, we demonstrate that Bmi1 interacts with Gata6 in a Ring finger-dependent manner to confer protection against Gata6 ubiquitination and proteasomal degradation. A direct role for Bmi1 in cell fate allocation is established by loss-of-function experiments in chimeric embryoid bodies. We thus propose a novel regulatory pathway by which Bmi1 action on Gata6 stability could alter the balance between Gata6 and Nanog protein levels to introduce a bias toward a PrE identity in a cell-autonomous manner

Chicken embryonic stem cells as a non-mammalian embryonic stem cell model

International audienceEmbryonic stem cells (ESCs) were isolated in the early 1980s from mouse and in the late 1990s from primate and human. These cells present the unique property of self-renewal and the ability to generate differentiated progeny in all embryonic lineages both in vitro and in vivo. The mESCs (mouse embryonic stem cells) can contribute to both somatic and germinal lineages once re-injected into a recipient embryo at the blastocyst stage. In avian species, chicken embryonic stem cells (cESCs) have been isolated from the in vitro culture of early chicken blastodermal cells (cBCs) taken from stage X embryo (EG&K) These cESCs can be maintained under specific culture conditions and have been characterized on the basis of their morphology, biochemical features, in vitro differentiation potentialities and in vivo morphogenetic properties. The relationship between these cESCs and some of the chicken germ cells identified and grown under specific culture conditions are still under debate, in particular with the identification of the Cvh gene as a key factor for germ cell determination. Moreover, by cloning the avian homologue of the Oct4 mammalian gene, we have demonstrated that this gene, as well as the chicken Nanog gene, was involved in the characterization and maintenance of the chicken pluripotency. These first steps toward the understanding of pluripotency control in a non-mammalian species opens the way for the development and characterization of putative new cell types such as chicken EpiSC and raises the question of the existence of reprogramming in avian species. These different points are discussed

Pluripotence et compétence germinale des cellules souches embryonnaires aviaires

LYON-ENS Sciences (693872304) / SudocSudocFranceF

Astacin-like metallo-endopeptidase is dynamically expressed in embryonic stem cells and embryonic epithelium during morphogenesis

Background: Astacin-like metallo-proteases are zinc endopeptidases conserved among vertebrates and invertebrates. First described as hatching gland enzymes, many members of the family possess other functions during embryonic development. In the chick, however, functions of Astacin-like proteins remain elusive. Results: We report here that Astacin-like (ASTL) is strongly expressed in mouse and chicken embryonic stem (ES) cells and exhibits a very dynamic expression pattern during embryogenesis and organogenesis, mostly in remodeled epithelia. Consistent with its expression in ES cells, chick ASTL is detected in vivo in the pluripotent cells of the epiblast and then disappears from the newly induced neural plate. ASTL expression remains at the junction of non-neural and neural ectoderm, just before neural tube closure. At later stages, chick ASTL is detected in the ventral epidermis before ventral closure, in the intermediate mesoderm, in the gonads and in the forming nephric duct and tubules of the mesonephros and metanephros. Conclusions: ASTL is dynamically expressed in the embryonic epithelium and in embryonic stem cells, suggesting an important function for the control of epithelial cell behavior during early development. © 2012 Wiley Periodicals, Inc.Grant sponsor: GenoStem project; Grant number: ANR-06 GANI-007; Grant sponsor: Epibird project; Grant number: ANR-09-GENM-004-03. F.L. and H.A. were supported by grants and fellowships from INRA and B.P. was funded by ANR (GenoStem project and Epibird project). H.A. was also supported by grants from the Spanish Ministry of Science and Innovation (BFU2008-01042 and CONSOLIDER INGENIO 2010 CSD2007-00017 and CSD2007-00023 attributed to M. Angela Nieto.Peer Reviewe

Chicken Stem Cells as a Model to Generate Transgenic Chicken: Present and Perspectives

Over the past 3 decades, different strategies were launched to obtain transgenic animals and chicken in particular. Among various technical approaches, the direct modification of the embryo with DNA vectors or viruses is one of the ways of choice which led to the generation of transgenic chicken. In parallel, cellular approaches were also actively developed and were mainly based either on the use of embryonic stem cells (ESC) or of primordial germ cells (PGC). Both cellular types present advantages and disadvantages. ESC can be easily amplified in vitro and genetically modified but until now failed to contribute efficienctly to the germ line. In contrast, PGC colonize the germ line but present a limited proliferative potential and are hardly modified in vitro. The exact developmental and molecular relationships between these two cell types are not well documented. Finally, recent development and demonstration of the lentiviruses efficiency revivifies the interest for transgenic chicken as a tool for developmental studies as well as potential biotechnological and agronomic purposes

Ectopic expression of Cvh (Chicken Vasa homologue) mediates the reprogramming of chicken embryonic stem cells to a germ cell fate

10 pages, 6 figures.-- PMID: 19324033 [PubMed].-- Printed version published Jun 1, 2009.-- Supporting information available at: http://dx.doi.org/10.1016/j.ydbio.2009.03.012When they are derived from blastodermal cells of the pre-primitive streak in vitro, the pluripotency of Chicken Embryonic Stem Cells (cESC) can be controlled by the cPouV and Nanog genes. These cESC can differentiate into derivatives of the three germ layers both in vitro and in vivo, but they only weakly colonize the gonads of host embryos. By contrast, non-cultured blastodermal cells and long-term cultured chicken primordial germ cells maintain full germline competence. This restriction in the germline potential of the cESC may result from either early germline determination in the donor embryos or it may occur as a result of in vitro culture. We are interested in understanding the genetic determinants of germline programming. The RNA binding protein Cvh (Chicken Vasa Homologue) is considered as one such determinant, although its role in germ cell physiology is still unclear. Here we show that the exogenous expression of Cvh, combined with appropriate culture conditions, induces cESC reprogramming towards a germ cell fate. Indeed, these cells express the Dazl, Tudor and Sycp3 germline markers, and they display improved germline colonization and adopt a germ cell fate when injected into recipient embryos. Thus, our results demonstrate that Vasa can drive ES cell differentiation towards the germ cell lineage, both in vitro and in vivo.This work was supported by grants from INRA and the GenAnimal program (GenStem FNS-2004; ANR-06 GANI-007 Genostem) to B.P. and from the Spanish Ministry of Science and Innovation (BFU2008-01042/BMC and CONSOLIDER-INGENIO 2010 CSD2007-00017 and CSD2007-00023) to M.A.N.Peer reviewe

Pluripotent genes in avian stem cells

International audienceEmbryonic 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

Molecular control of pluripotency and germ line competency in chicken embryonic stem cells

International audienc