Piwi proteins specify an animal-specific subclass of the Argonaute family that, in vertebrates, is specifically expressed in germ cells. We demonstrate that zebrafish Piwi (Ziwi) is expressed in both the male and the female gonad and is a component of a germline-specifying structure called nuage. Loss of Ziwi function results in a progressive loss of germ cells due to apoptosis during larval development. In animals that have reduced Ziwi function, germ cells are maintained but display abnormal levels of apoptosis in adults. In mammals, Piwi proteins associate with approximately 29-nucleotide-long, testis-specific RNA molecules called piRNAs. Here we show that zebrafish piRNAs are present in both ovary and testis. Many of these are derived from transposons, implicating a role for piRNAs in the silencing of repetitive elements in vertebrates. Furthermore, we show that piRNAs are Dicer independent and that their 3' end likely carries a 2'O-Methyl modification.

MicroRNAs (miRNAs) are inhibitors of gene expression capable of controlling processes in normal development and cancer. In mammals, miRNAs use a seed sequence of 6-8 nucleotides (nt) to associate with 3' untranslated regions (3'UTRs) of mRNAs and inhibit their expression. Intriguingly, occasionally not only the miRNA-targeting site but also sequences in its vicinity are highly conserved throughout evolution. We therefore hypothesized that conserved regions in mRNAs may serve as docking platforms for modulators of miRNA activity. Here we demonstrate that the expression of dead end 1 (Dnd1), an evolutionary conserved RNA-binding protein (RBP), counteracts the function of several miRNAs in human cells and in primordial germ cells of zebrafish by binding mRNAs and prohibiting miRNAs from associating with their target sites. These effects of Dnd1 are mediated through uridine-rich regions present in the miRNA-targeted mRNAs. Thus, our data unravel a novel role of Dnd1 in protecting certain mRNAs from miRNA-mediated repression.

The signals directing primordial germ cell (PGC) migration in vertebrates are largely unknown. We demonstrate that sdf-1 mRNA is expressed in locations where PGCs are found and toward which they migrate in wild-type as well as in mutant embryos in which PGC migration is abnormal. Knocking down SDF-1 or its receptor CXCR4 results in severe defects in PGC migration. Specifically, PGCs that do not receive the SDF-1 signal exhibit lack of directional movement toward their target and arrive at ectopic positions within the embryo. Finally, we show that the PGCs can be attracted toward an ectopic source of the chemokine, strongly suggesting that this molecule provides a key directional cue for the PGCs.

Primordial germ cell (PGC) migration in zebrafish is directed by the chemokine SDF-1a that activates its receptor CXCR4b. Little is known about the molecular mechanisms controlling the distribution of this chemoattractant in vivo. We demonstrate that the activity of a second SDF-1/CXCL12 receptor, CXCR7, is crucial for proper migration of PGCs toward their targets. We show that CXCR7 functions primarily in the somatic environment rather than within the migrating cells. In CXCR7 knocked-down embryos, the PGCs exhibit a phenotype that signifies defects in SDF-1a gradient formation as the cells fail to polarize effectively and to migrate toward their targets. Indeed, somatic cells expressing CXCR7 show enhanced internalization of the chemokine suggesting that CXCR7 acts as a sink for SDF-1a, thus allowing the dynamic changes in the transcription of sdf-1a to be mirrored by similar dynamics at the protein level.

In most animals, primordial germ cell (PGC) specification and development depend on maternally provided cytoplasmic determinants that constitute the so-called germ plasm. Little is known about the role of germ plasm in vertebrate germ cell development, and its molecular mode of action remains elusive. While PGC specification in mammals occurs via different mechanisms, several germ plasm components required for early PGC development in lower organisms are expressed in mammalian germ cells after their migration to the gonad and are involved in gametogenesis. Here we show that the RNA of dead end, encoding a novel putative RNA binding protein, is a component of the germ plasm in zebrafish and is specifically expressed in PGCs throughout embryogenesis; Dead End protein is localized to perinuclear germ granules within PGCs. Knockdown of dead end blocks confinement of PGCs to the deep blastoderm shortly after their specification and results in failure of PGCs to exhibit motile behavior and to actively migrate thereafter. PGCs subsequently die, while somatic development is not effected. We have identified dead end orthologs in other vertebrates including Xenopus, mouse, and chick, where they are expressed in germ plasm and germ-line cells, suggesting a role in germ-line development in these organisms as well.

BackgroundCXCR7 (RDC1), the recently discovered second receptor for CXCL12, is phylogenetically closely related to chemokine receptors, but fails to couple to G-proteins and to induce typical chemokine receptor mediated cellular responses. The function of CXCR7 is controversial. Some studies suggest a signaling activity in mammalian cells and zebrafish embryos, while others indicate a decoy activity in fish. Here we investigated the two propositions in human tissues.Methodology/Principal FindingsWe provide evidence and mechanistic insight that CXCR7 acts as specific scavenger for CXCL12 and CXCL11 mediating effective ligand internalization and targeting of the chemokine cargo for degradation. Consistently, CXCR7 continuously cycles between the plasma membrane and intracellular compartments in the absence and presence of ligand, both in mammalian cells and in zebrafish. In accordance with the proposed activity as a scavenger receptor CXCR7-dependent chemokine degradation does not become saturated with increasing ligand concentrations. Active CXCL12 sequestration by CXCR7 is demonstrated in adult mouse heart valves and human umbilical vein endothelium.Conclusions/SignificanceThe finding that CXCR7 specifically scavenges CXCL12 suggests a critical function of the receptor in modulating the activity of the ubiquitously expressed CXCR4 in development and tumor formation. Scavenger activity of CXCR7 might also be important for the fine tuning of the mobility of hematopoietic cells in the bone marrow and lymphoid organs.

The molecular and cellular mechanisms governing cell motility and directed migration in response to the chemokine SDF-1 are largely unknown. Here, we demonstrate that zebrafish primordial germ cells whose migration is guided by SDF-1 generate bleb-like protrusions that are powered by cytoplasmic flow. Protrusions are formed at sites of higher levels of free calcium where activation of myosin contraction occurs. Separation of the acto-myosin cortex from the plasma membrane at these sites is followed by a flow of cytoplasm into the forming bleb. We propose that polarized activation of the receptor CXCR4 leads to a rise in free calcium that in turn activates myosin contraction in the part of the cell responding to higher levels of the ligand SDF-1. The biased formation of new protrusions in a particular region of the cell in response to SDF-1 defines the leading edge and the direction of cell migration.

In mouse embryos, germ cells arise during gastrulation and migrate to the early gonad. First, they emerge from the primitive streak into the region of the endoderm that forms the hindgut. Later in development, a second phase of migration takes place in which they migrate out of the gut to the genital ridges. There, they co-assemble with somatic cells to form the gonad. In vitro studies in the mouse, and genetic studies in other organisms, suggest that at least part of this process is in response to secreted signals from other tissues. Recent genetic evidence in zebrafish has shown that the interaction between stromal cell-derived factor 1 (SDF1) and its G-protein-coupled receptor CXCR4, already known to control many types of normal and pathological cell migrations, is also required for the normal migration of primordial germ cells. We show that in the mouse, germ cell migration and survival requires the SDF1/CXCR4 interaction. First, migrating germ cells express CXCR4, whilst the body wall mesenchyme and genital ridges express the ligand SDF1. Second,the addition of exogenous SDF1 to living embryo cultures causes aberrant germ cell migration from the gut. Third, germ cells in embryos carrying targeted mutations in CXCR4 do not colonize the gonad normally. However, at earlier stages in the hindgut, germ cells are unaffected in CXCR4-/-embryos. Germ cell counts at different stages suggest that SDF1/CXCR4 interaction also mediates germ cell survival. These results show that the SDF1/CXCR4 interaction is specifically required for the colonization of the gonads by primordial germ cells, but not for earlier stages in germ cell migration. This demonstrates a high degree of evolutionary conservation of part of the mechanism, but also an area of evolutionary divergence.