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.

A common feature of development in most vertebrate models is the early segregation of the germ line from the soma. For example, in Xenopus and zebrafish embryos primordial germ cells (PGCs) are specified by germ plasm that is inherited from the egg; in mice, Blimp1 expression in the epiblast mediates the commitment of cells to the germ line. How these disparate mechanisms of PGC specification evolved is unknown. Here, in order to identify the ancestral mechanism of PGC specification in vertebrates, we studied PGC specification in embryos from the axolotl (Mexican salamander), a model for the tetrapod ancestor. In the axolotl, PGCs develop within mesoderm, and classic studies have reported their induction from primitive ectoderm (animal cap). We used an axolotl animal cap system to demonstrate that signalling through FGF and BMP4 induces PGCs. The role of FGF was then confirmed in vivo. We also showed PGC induction by Brachyury, in the presence of BMP4. These conditions induced pluripotent mesodermal precursors that give rise to a variety of somatic cell types, in addition to PGCs. Irreversible restriction of the germ line did not occur until the mid-tailbud stage, days after the somatic germ layers are established. Before this, germline potential was maintained by MAP kinase signalling. We propose that this stochastic mechanism of PGC specification, from mesodermal precursors, is conserved in vertebrates.

In bilaterian animals, germ cells are specified by the inductive/regulative mode or the predetermined (germ plasm) mode. Among tetrapods, mammals and urodeles use the inductive mode, whereas birds and anurans use the predetermined mode. From histological data it has been predicted that some reptiles including turtles use the inductive mode. Examining turtle oocytes, we find that Dazl RNA, Vasa RNA, and Vasa protein are not localized, suggesting that germ plasm is not present. In turtle embryos at somite stages, primordial germ cells (PGCs) expressing Dazl lie on a path from the lateral posterior extraembryonic endoderm through the gut to the gonad as previously described. In gastrulating embryos, cells expressing Dazl are found in the blastoporal plate and subsequently below the blastoporal plate, indicating that PGCs are generated at the equivalent of the early posterior primitive streak of mammals. Vasa RNA is expressed in somatic cells of gastrula to early somite stages, and Vasa RNA and protein are expressed in PGCs of later embryos. Taken together the evidence strongly suggests that turtles, and other reptiles (lacertoid lizards) with the same location of PGCs in embryos, use the inductive mode of germ cell specification. Phylogenetic analysis of the available evidence supports the following hypotheses: (1) the inductive mode is basal among reptiles, indicating that this mode was maintained as basal tetrapods evolved to amniotes, (2) the predetermined mode arose twice within reptiles, and (3) the induced mode may be used in several lepidosaurs whose PGCs are located in an unusual pattern distributed around the embryo.

Pluripotency defines the unlimited potential of cells in the primitive ectoderm of vertebrate embryos, from which all adult somatic cells and germ cells are derived. Understanding how the programing of pluripotency evolved has been obscured by the study of early development in models from lower vertebrates in which pluripotency is not conserved. Here we investigated how the axolotl ortholog of the mammalian core pluripotency factor NANOG , programs pluripotency during axolotl development to model the tetrapod ancestor from which terrestrial vertebrates evolved. We show that in axolotl primitive ectoderm (animal caps; AC) NANOG synergizes with NODAL activity and the epigenetic modifying enzyme DPY30 to direct the deposition of H3K4me3 in chromatin prior to the waves of transcription required for lineage commitment and developmental progression. We show that the interaction of NANOG and NODAL with DPY30 is required to direct development downstream of pluripotency and this is conserved in axolotls and human. These data demonstrate that the interaction of NANOG and NODAL signaling represents the basal state of vertebrate pluripotency.

Pluripotency defines the unlimited potential of individual cells of vertebrate embryos, from which all adult somatic cells and germ cells are derived. Understanding how the programming of pluripotency evolved has been obscured in part by a lack of data from lower vertebrates; in model systems such as frogs and zebrafish, the function of the pluripotency genes NANOG and POU5F1 have diverged. Here, we investigated how the axolotl ortholog of NANOG programs pluripotency during development. Axolotl NANOG is absolutely required for gastrulation and germ-layer commitment. We show that in axolotl primitive ectoderm (animal caps; ACs) NANOG and NODAL activity, as well as the epigenetic modifying enzyme DPY30, are required for the mass deposition of H3K4me3 in pluripotent chromatin. We also demonstrate that all 3 protein activities are required for ACs to establish the competency to differentiate toward mesoderm. Our results suggest the ancient function of NANOG may be establishing the competence for lineage differentiation in early cells. These observations provide insights into embryonic development in the tetrapod ancestor from which terrestrial vertebrates evolved.

Reprogramming of somatic cells to pluripotent cells promises to transform regenerative medicine. Recently many groups have achieved direct reprogramming of somatic cells by forced expression of defined factors using multiple viral vectors. However, such induced pluripotent stem (iPS) cells contain a number of viral vector integrations, any one of which could cause unpredictable genetic dysfunction. Moreover, viral vectors silenced in iPS cells can be re-activated when the cells differentiate. Here we show non-viral transfection with a single multiprotein expression vector can generate iPS cells from non-genetically modified mouse embryonic fibroblasts. This one vector system can achieve reprogramming from one integration site, enabling subsequent elimination of the reprogramming cassette by Cre-mediated excision. These non-viral iPS cells show robust expression of pluripotent markers and genuine pluripotency was confirmed by invitro differentiation assays and formation of adult chimeric mice. When the single vector reprogramming system was combined with a piggyBac transposon we succeeded in establishing reprogrammed human cell lines from embryonic fibroblasts with robust expression of pluripotency markers. This non-viral single vector system minimizes genome modification and eliminates the unpredictable reactivation of reprogramming factors, providing iPS cells more applicable to regenerative medicine, reliable drug screening and establishment of trustworthy disease models. The transcription factor Sox2 (SRY-related HMG-box gene 2), in synergy with Oct4, plays a pivotal role in maintaining embryonic stem (ES) cells self-renewing and pluripotent. After knock-down of Sox2 in mouse ES cells with three different shRNA constructs, alterations in transcript and protein expression of genes associated with pluripotency, commitment to trophectoderm, mesendoderm, neuroectoderm and cell-cycle regulation were detected. Absence of Sox2 resulted in loss of self-renewal (with down-regulation of Oct4, Nanog, Rex1 and Utf1), severe compromise of ES cell growth rate and cell-cycle arrest (as indicated by increased levels of p21 and p27, as well as decreased levels of Nucleostemin and Cyclin D2). However, apoptosis in Sox2 shRNA ES cells was not significantly different to mock-transfected control ES cells. In a culture system designed to promote neurogenesis, cells did not form neural precursors after Sox2 shRNA but acquired a more glial (astrocytic) phenotype, as demonstrated by increased levels of Musashi1 and Gfap by day 14, with parallel decreased levels of Sox1, Beta III tubulin and Pax6, compared to mock-transfected control ES cells. The most striking impact of knocking down Sox2 in mouse ES cells was observed within the first 3 days of the neural differentiation protocol with significant up-regulation of the trophectoderm markers Cdx2, Eomes and Fgfr2, as well as of the mesendoderm markers Mixl1, Goosecoid and Brachyury. Concomitant up-regulation of b-catenin and down-regulation of Lef1 in the absence of Sox2, suggests that ...

lineages. Because Nanog is required for the development of primordial germ cells (PGCs) in mice and axolotls, we propose that the deletion of Nanog in species often employed as experimental models, such as frogs and teleosts (Zebrafish; Danio rerio), has been compensated by the evolution of predetermined germ cells in these organisms. Our data highlights Nanog as a major component of the gene regulatory network that governs early development in the major trunk of chordate evolution. Maintenance of multipotency is a central question in stem cell biology. In the mouse embryo, mesodermal cells of the dorsal somite, the dermomyotome, give rise to different lineages such as vascular smooth and skeletal muscle. These multipotent stem cells express the Pax3 and Pax7 transcription factors, essential for the myogenic lineage. Here we identify a novel negative feedback loop between Pax3/7 and Foxc2. Using both genetic approaches and manipulation of external signals in somite explants, we demonstrate that the Pax3/7:Foxc2 ratio modulates myogenic versus vascular cell fates of dermomyotomal cells. This exemplifies the importance of such a mechanism of reciprocal repression in the maintenance and lineage commitment of multipotent stem cells. Fgf10 has been implicated in maintenance of stem cells in various embryonic and non-neural organs such as teeth and stomach. We have previously used Fgf10-LacZ reporter mice to show that the localization and fate of Fgf10-expressing cells in the adult mouse brain points to an involvement of Fgf10 in the control of neurogenesis in quiescent neurogenic niches (Hajihosseini etal., 2008. MCN 37: 857-868). In the adult hypothalamus, expression of Fgf10 is restricted to tanycytes which reside in the median eminence, at the floor of the third ventricle. Here, we have used a set of immunolabelling and neurosphere culture studies to study whether these Fgf10-expressing cells in the adult hypothalamus are neural stem cells or lineage-restricted neural progenitors.We find that in addition to Fgf10, tanycytes express BLBP, Nestin and Musashi. Fgf10 is downregulated as descendants of tanycytes migrate into the surrounding hypothalamic nuclei and express DARPP32 or the mature neuronal marker, NeuN. In invitro assays, we found that cells derived from the median eminence can generate neurospheres as efficiently as those derived from the lateral subventricular zones. Moreover, in differentiation assays, these neurospheres give rise to all three neural lineages: neurons (TuJ1+), astrocytes (GFAP+) and oligodendrocytes (olig2+). These data suggest that the adult hypothalamus contains a quiescent population of neural stem cells and, as in non-neural tissues, Fgf10 may play a critical role in the maintenance and/or differentiation of subpopulations of neural cells. Further studies are underway to verify and dissect the role of Fgf10 in this system. Pluripotency defines the ability of cells to produce all somatic cells and germ line cells. In mammals, primordial germ cells (PGCs) are induced from pluripotent c...