The germline of multicellular animals is segregated from somatic tissues, which is an essential developmental process for the next generation. Although certain ecdysozoans and chordates segregate their germline during embryogenesis, animals from other taxa segregate their germline after embryogenesis from multipotent progenitor cells. An overlapping set of genes, including vasa, nanos and piwi, operate in both multipotent precursors and in the germline. As we propose here, this conservation implies the existence of an underlying germline multipotency program in these cell types that has a previously underappreciated and conserved function in maintaining multipotency.Key words: Germline, Stem cell, Vasa, Piwi, Nanos, Sea urchin Introduction Multicellular animals consist of specialized cells, most of which are differentiated and devoid of reproductive potential. However, some cells, such as stem cells, retain both the ability to self-renew and to create new differentiated cells. One such stem cell, the germline stem cell (GSC), gives rise to a continual supply of germ cells, which are specialized for the task of reproduction. Yet once egg and sperm fuse, the resulting zygote is totipotent, and thus can give rise to all cell types of the animal. Although GSCs normally give rise only to germ cells in vivo, and thus are unipotent, a significant body of evidence shows that these cells maintain the potential to acquire many cell fates. For example, mouse primordial germ cells (PGCs) acquire the morphological and cytological characteristics of embryonic stem (ES) cells when cultured in the presence of three growth factors: steel factor, leukemia inhibitory factor (LIF) and fibroblast growth factor (FGF) (Matsui et al., 1992). These cells create teratomas when injected into adult mice and chimeric pups when injected into blastocysts, thus demonstrating their pluripotency. Isolated human PGCs can also be converted to pluripotent stem cells without genetic manipulation (Shamblott et al., 1998). Furthermore, pluripotent cells spontaneously arise from isolated adult mouse spermatogonial stem cells under specific culture conditions (Guan et al., 2006). This phenomenon is not limited to vertebrates as mutations in the translational regulators mex-3 and gld-1 in Caenorhabditis elegans leads to transdifferentiation of germ cells into muscle, neurons and intestinal cells (Ciosk et al., 2006). Thus, although germ cells are highly specialized, they seemingly retain the capacity to give rise to all cell types. As such, it is possible that the developmental potential of GSCs in these organisms is not intrinsically limited but might instead be restricted by environment. What, then, is the underlying regulatory program that controls this highly potent cell type?The molecular regulation of the germline is best understood in a handful of animals, including in C. elegans, Drosophila melanogaster and the mouse. Here, we explore the diversity of germline origins more broadly by examining germline segregation mechanisms across diverse taxa f...

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A crucial event in animal development is the specification of primordial germ cells (PGCs), which become the stem cells that create sperm and eggs. How PGCs are created provides a valuable paradigm for understanding stem cells in general. We find that the PGCs of the sea urchin Strongylocentrotus purpuratus exhibit broad transcriptional repression, yet enrichment for a set of inherited mRNAs. Enrichment of several germline determinants in the PGCs requires the RNA-binding protein Nanos to target the transcript that encodes CNOT6, a deadenylase, for degradation in the PGCs, thereby creating a stable environment for RNA. Misexpression of CNOT6 in the PGCs results in their failure to retain Seawi transcripts and Vasa protein. Conversely, broad knockdown of CNOT6 expands the domain of Seawi RNA as well as exogenous reporters. Thus, Nanos-dependent spatially restricted CNOT6 differential expression is used to selectively localize germline RNAs to the PGCs. Our findings support a 'time capsule' model of germline determination, whereby the PGCs are insulated from differentiation by retaining the molecular characteristics of the totipotent egg and early embryo.

SUMMARYIndirect development, in which embryogenesis gives rise to a larval form, requires that some cells retain developmental potency until they contribute to the different tissues in the adult, including the germ line, in a later, post-embryonic phase. In sea urchins, the coelomic pouches are the major contributor to the adult, but how coelomic pouch cells (CPCs) are specified during embryogenesis is unknown. Here we identify the key signaling inputs into the CPC specification network and show that the forkhead factor foxY is the first transcription factor specifically expressed in CPC progenitors. Through dissection of its cis-regulatory apparatus we determine that the foxY expression pattern is the result of two signaling inputs: first, Delta/Notch signaling activates foxY in CPC progenitors; second, Nodal signaling restricts its expression to the left side, where the adult rudiment will form, through direct repression by the Nodal target pitx2. A third signal, Hedgehog, is required for coelomic pouch morphogenesis and institution of laterality, but does not directly affect foxY transcription. Knockdown of foxY results in a failure to form coelomic pouches and disrupts the expression of virtually all transcription factors known to be expressed in this cell type. Our experiments place foxY at the top of the regulatory hierarchy underlying the specification of a cell type that maintains developmental potency.

Highlights d CENP-A nucleosomes are gradually incorporated in quiescent cells and oocytes d CENP-A deposition during quiescence is required for future chromosome segregation d RNA Polymerase transcription at centromeres promotes gradual CENP-A exchange d Terminally differentiated muscle cells fail to retain CENP-A nucleosomes

Members of the Vasa family of helicases are specifically localized to germ line lineages in embryos of many animal groups and, in some cases, have been shown to be required for germ line formation. Despite considerable attention to the embryology of gastropod molluscs, the germ line has not been identified in the early cleavage stages of these embryos. We have cloned a Vasa ortholog in the snail Ilyanassa and examined the distribution of IoVasa mRNA during early cleavage. Initially, the transcript is present in all cells and non-specifically localized to centrosomes in a subset of cells. The IoVasa mRNA becomes progressively more enriched in the dorsal quadrant of the embryo, and then becomes restricted to particular cells in the 4d lineage. At the 64-cell stage, IoVasa mRNA is detected in 4dL11, 4dL12, 4dR11, and 4dR12. Following another round of division in the 4d lineage, the mRNA is restricted to two cells: 4dL121 and 4dR121. By the 108-cell stage, IoVasa mRNA is no longer detectable. Because the germ line is thought to arise from the 4d lineage in spiralians, these data are consistent with the hypothesis that the Ilyanassa germ line is marked by inheritance of IoVasa and derived from the cells 4dL121 and 4dR121. Alternatively, IoVasa may be required in somatic lineages where it is expressed, and the germ line may be specified later in development.

Centromeres provide a robust model for epigenetic inheritance as they are specified by sequence-independent mechanisms involving the histone H3-variant CENP-A.Prevailing models indicate that the high intrinsic stability of CENP-A nucleosomes maintains centromere identity indefinitely. Here, we demonstrate that CENP-A is not stable at centromeres, but is instead gradually and continuously incorporated in quiescent cells including G0-arrested tissue culture cells and prophase I-arrested oocytes. Quiescent CENP-A incorporation involves the canonical CENP-A deposition machinery, but displays distinct requirements from cell cycle-dependent deposition. We demonstrate that Plk1 is required specifically for G1 CENP-A deposition, whereas transcription promotes CENP-A incorporation in quiescent oocytes. Preventing CENP-A deposition during quiescence results in significantly reduced CENP-A levels and perturbs chromosome segregation following the resumption of cell division. In contrast to quiescent cells, terminally differentiated cells fail to maintain CENP-A levels. Our work reveals that quiescent cells actively maintain centromere identity providing an indicator of proliferative potential. Bodor, D.L., Valente, L.P., Mata, J.F., Black, B.E., and Jansen, L.E. (2013). Assembly in G1 phase and long-term stability are unique intrinsic features of CENP-A nucleosomes. Mol Biol Cell 24, 923-932.

SUMMARY The formation of the germ line in an embryo marks a fresh round of reproductive potential. The developmental stage and location within the embryo where the primordial germ cells (PGCs) form, however, differs markedly among species. In many animals, the germ line is formed by an inherited mechanism, in which molecules made and selectively partitioned within the oocyte drive the early development of cells that acquire this material to a germ-line fate. In contrast, the germ line of other animals is fated by an inductive mechanism that involves signaling between cells that directs this specialized fate. In this review, we explore the mechanisms of germ-line determination in echinoderms, an early-branching sister group to the chordates. One member of the phylum, sea urchins, appears to use an inherited mechanism of germ-line formation, whereas their relatives, the sea stars, appear to use an inductive mechanism. We first integrate the experimental results currently available for germ line determination in the sea urchin, for which considerable new information is available, and then broaden the investigation to the lesser-known mechanisms in sea stars and other echinoderms. Even with this limited insight, it appears that sea stars, and perhaps the majority of the echinoderm taxon, rely on inductive mechanisms for germ-line fate determination. This enables a strongly contrasted picture for germ-line determination in this phylum, but one for which transitions between different modes of germ-line determination might now be experimentally addressed.