dead end,a novel vertebrate germ plasm component,is required for zebrafish primordial germ cell migration and survival

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.     

Expression of axolotl DAZL RNA, a marker of germ plasm: widespread maternal RNA and onset of expression in germ cells approaching the gonad

How germ cell specification occurs remains a fundamental question in embryogenesis. The embryos of several model organisms contain germ cell determinants (germ plasm) that segregate to germ cell precursors. In other animals, including mice, germ cells form in response to regulative mechanisms during development. To investigate germ cell determination in urodeles, where germ plasm has never been conclusively identified, we cloned a DAZ-like sequence from axolotls, Axdazl. Axdazl is homologous to Xdazl, a component of Xenopus germ plasm found in the vegetal pole of oocytes and eggs. Axdazl RNA is not localized in axolotl oocytes, and, furthermore, these oocytes do not contain the mitochondrial cloud that localizes Xdazl and other germ plasm components in Xenopus. Maternal Axdazl RNA is inherited in the animal cap and equatorial region of early embryos. At gastrula, neurula, and tailbud stages, Axdazl RNA is widely distributed. Axdazl first shows cell-specific expression in primordial germ cells (PGCs) approaching the gonad at stage 40, when nuage (germ plasm) appears in PGCs. These results suggest that, in axolotls, germ plasm components are insufficient to specify germ cells.     

The protein encoded by the germ plasm RNA Germes associates with dynein light chains and functions in Xenopus germline development

Germ plasm plays a prominent role in germline formation in a large number of animal taxons. We previously identified a novel maternal RNA named Germes associated with Xenopus germ plasm. In the present work, we addressed possible involvement of Germes protein in germ plasm function. Expression in oocytes followed by confocal microscopy revealed that the EGFP fused to Germes, in contrast to the free EGFP, co-localized with the germ plasm. Overexpression of intact Germes and Germes lacking both leucine zipper motifs (GermesDeltaLZs) resulted in a statistically significant reduction of the number of primordial germ cells (PGCs). Furthermore, the GermesDeltaLZs mutant inhibited PGC migration and produced abnormalities in germ plasm intra-cellular distribution at tailbud stages. To begin unraveling biochemical interactions of Germes during embryogenesis, we searched for Germes partners using yeast two-hybrid (YTH) system. Two closely related sequences were identified, encoding Xenopus dynein light chains dlc8a and dlc8b. Tagged versions of Germes and dlc8s co-localize in VERO cells upon transient expression and can be co-immunoprecipitated after injection of the corresponding RNAs in Xenopus embryos, indicating that their interactions occur in vivo. We conclude that Germes is involved in organization and functioning of germ plasm in Xenopus, probably through interaction with motor complexes.     

Analysis of SDF-1/CXCR4 signaling in primordial germ cell migration and survival or differentiation in Xenopus laevis

Directional migration of primordial germ cells (PGCs) toward future gonads is a common feature in many animals. In zebrafish, mouse and chicken, SDF-1/CXCR4 chemokine signaling has been shown to have an important role in PGC migration. In Xenopus, SDF-1 is expressed in several regions in embryos including dorsal mesoderm, the target region that PGCs migrate to. CXCR4 is known to be expressed in PGCs. This relationship is consistent with that of more well-known animals. Here, we present experiments that examine whether chemokine signaling is involved in PGC migration of Xenopus. We investigate: (1) Whether injection of antisense morpholino oligos (MOs) for CXCR4 mRNA into vegetal blastomere containing the germ plasm or the precursor of PGCs disturbs the migration of PGCs? (2) Whether injection of exogenous CXCR4 mRNA together with MOs can restore the knockdown phenotype? (3) Whether the migratory behavior of PGCs is disturbed by the specific expression of mutant CXCR4 mRNA or SDF-1 mRNA in PGCs? We find that the knockdown of CXCR4 or the expression of mutant CXCR4 in PGCs leads to a decrease in the PGC number of the genital ridges, and that the ectopic expression of SDF-1 in PGCs leads to a decrease in the PGC number of the genital ridges and an increase in the ectopic PGC number. These results suggest that SDF-1/CXCR4 chemokine signaling is involved in the migration and survival or in the differentiation of PGCs in Xenopus.     

Dynamic intracellular localization of Dazl protein during <Emphasis Type="Italic">Xenopus</Emphasis> germline development

Xenopus dazl encoding an RNA-binding protein has been identified as a component of the germ plasm and is involved in the migration and differentiation of the primordial germ cells (PGCs). Here, we investigated the intracellular localization of Dazl in germline cells throughout the lifetime of Xenopus. In early embryogenesis, Dazl was detected initially in the germ plasm and then translocated to a perinuclear region. Then, it was detected within the nucleus in PGCs. Dazl was observed only in the cytoplasm in PGCs when sex differentiation began in the gonads. Dazl was distributed in both the nucleus and cytoplasm of the primary oogonium and spermatogonium, but only in the cytoplasm of the secondary oogonium and spermatogonium. In spermatocytes, Dazl was distributed throughout cytoplasm and localized at the spindles and cytoplasm during meiosis. Then, it was detected as speckles in the nucleus in the round spermatid. The dynamic intracellular localization suggests that Dazl is a multifunctional protein regulating RNA metabolism required for Xenopus germline development.     

Zebrafish Staufen1 and Staufen2 are required for the survival and migration of primordial germ cells

In sexually reproducing organisms, primordial germ cells (PGCs) give rise to the cells of the germ line, the gametes. In many animals, PGCs are set apart from somatic cells early during embryogenesis. Work in Drosophila, C. elegans, Xenopus, and zebrafish has shown that maternally provided localized cytoplasmic determinants specify the germ line in these organisms (Raz, E., 2003. Primordial germ-cell development: the zebrafish perspective. Nat. Rev., Genet. 4, 690--700; Santos, A.C., Lehmann, R., 2004. Germ cell specification and migration in Drosophila and beyond. Curr. Biol. 14, R578-R589). The Drosophila RNA-binding protein, Staufen is required for germ cell formation, and mutations in stau result in a maternal effect grandchild-less phenotype (Schupbach,T., Weischaus, E., 1989. Female sterile mutations on the second chromosome of Drosophila melanogaster:1. Maternal effect mutations. Genetics 121, 101-17). Here we describe the functions of two zebrafish Staufen-related proteins, Stau1 and Stau2. When Stau1 or Stau2 functions are compromised in embryos by injecting antisense morpholino modified oligonucleotides or dominant-negative Stau peptides, germ layer patterning is not affected. However, expression of the PGC marker vasa is not maintained. Furthermore, expression of a green fluorescent protein (GFP):nanos 3'UTR fusion protein in germ cells shows that PGC migration is aberrant, and the mis-migrating PGCs do not survive in Stau-compromised embryos. Stau2 is also required for survival of neurons in the central nervous system (CNS). These phenotypes are rescued by co-injection of Drosophila stau mRNA. Thus, staufen has an evolutionarily conserved function in germ cells. In addition, we have identified a function for Stau proteins in PGC migration.     

Drosophila pericentrin‐like protein promotes the formation of primordial germ cells

Primordial germ cells (PGCs) are the precursors to the adult germline stem cells that are set aside early during embryogenesis and specified through the inheritance of the germ plasm, which contains the mRNAs and proteins that function as the germline fate determinants. In Drosophila melanogaster, formation of the PGCs requires the microtubule and actin cytoskeletal networks to actively segregate the germ plasm from the soma and physically construct the pole buds (PBs) that protrude from the posterior cortex. Of emerging importance is the central role of centrosomes in the coordination of microtubule dynamics and actin organization to promote PGC development. We previously identified a requirement for the centrosome protein Centrosomin (Cnn) in PGC formation. Cnn interacts directly with Pericentrin‐like protein (PLP) to form a centrosome scaffold structure required for pericentriolar material recruitment and organization. In this study, we identify a role for PLP at several discrete steps during PGC development. We find PLP functions in segregating the germ plasm from the soma by regulating microtubule organization and centrosome separation. These activities further contribute to promoting PB protrusion and facilitating the distribution of germ plasm in proliferating PGCs.     

Spatiotemporal localization of germ plasm RNAs during zebrafish oogenesis

In zebrafish, primordial germ cells (PGCs) are determined by a specialized maternal cytoplasm, the germ plasm, which forms at the distal ends of the cleavage furrows in 4-cell embryos. The germ plasm includes maternal mRNAs from the germline-specific genes such as vasa and nanos1, and vegetally localized dazl RNA is also incorporated into the germ plasm. However, little is known about the distributions and assembly mechanisms of germ plasm components, especially during oogenesis. Here we report that the germ plasm RNAs vasa, nanos1, and dazl co-localize with the mitochondrial cloud (MC) and are transported to the vegetal cortex during early oogenesis. We found that a mitochondrial cloud localization element (MCLE) previously identified in the 3' untranslated region (3'UTR) of Xenopus Xcat2 gene can direct RNA localization to the vegetal cortex via the MC in zebrafish oocytes. In addition, the RNA-binding protein Hermes is a component of the MC in zebrafish oocytes, as is the case in Xenopus. Moreover, we provide evidence that the dazl 3'UTR possesses at least three types of cis-acting elements that direct multiple steps in the localization process: MC localization, anchorage at the vegetal cortex, and localization at the cleavage furrows. Taken together, the data show that the MC functions as a conserved feature that participates in transport of the germ plasm RNAs in Xenopus and zebrafish oocytes. Furthermore, we propose that the germ plasm components are assembled in a stepwise and spatiotemporally-regulated manner during oogenesis and early embryogenesis in zebrafish.     

nos-1 and nos-2, two genes related to Drosophila nanos, regulate primordial germ cell development and survival in Caenorhabditis elegans.

In Drosophila, the posterior determinant nanos is required for embryonic patterning and for primordial germ cell (PGC) development. We have identified three genes in Caenorhabditis elegans that contain a putative zinc-binding domain similar to the one found in nanos, and show that two of these genes function during PGC development. Like Drosophila nanos, C. elegans nos-1 and nos-2 are not generally required for PGC fate specification, but instead regulate specific aspects of PGC development. nos-2 is expressed in PGCs around the time of gastrulation from a maternal RNA associated with P granules, and is required for the efficient incorporation of PGCs into the somatic gonad. nos-1 is expressed in PGCs after gastrulation, and is required redundantly with nos-2 to prevent PGCs from dividing in starved animals and to maintain germ cell viability during larval development. In the absence of nos-1 and nos-2, germ cells cease proliferation at the end of the second larval stage, and die in a manner that is partially dependent on the apoptosis gene ced-4. Our results also indicate that putative RNA-binding proteins related to Drosophila Pumilio are required for the same PGC processes as nos-1 and nos-2. These studies demonstrate that evolutionarily distant organisms utilize conserved factors to regulate early germ cell development and survival, and that these factors include members of the nanos and pumilio gene families.     

Evidence for crucial role of hindgut expansion in directing proper migration of primordial germ cells in mouse early embryogenesis

During mouse gastrulation, primordial germ cells (PGCs) become clustered at the base of the allantois and move caudally into the hindgut endoderm before entering the genital ridges. The precise roles of endoderm tissues in PGC migration, however, remain unclear. By using Sox17 mutants with a specific endoderm deficiency, we provide direct evidence for the crucial role of hindgut expansion in directing proper PGC migration. In Sox17-null embryos, PGCs normally colonize in the allantois and then a small front-row population of PGCs moves properly into the most posterior gut endoderm. Defective hindgut expansion, however, causes the failure of further lateral PGC movement, resulting in the immobilization of PGCs in the hindgut entrance at the later stages. In contrast, the majority of the remaining PGCs moves into the visceral endoderm layer, but relocate outside of the embryonic gut domain. This leads to a scattering of PGCs in the extraembryonic yolk sac endoderm. This aberrant migration of Sox17-null PGCs can be rescued by the supply of wildtype hindgut cells in chimeric embryos. Therefore, these data indicate that hindgut morphogenic movement is crucial for directing PGC movement toward the embryonic gut side, but not for their relocation from the mesoderm into the endoderm.     

Preformation and epigenesis converge to specify primordial germ cell fate in the early Drosophila embryo

A critical step in animal development is the specification of primordial germ cells (PGCs), the precursors of the germline. Two seemingly mutually exclusive mechanisms are implemented across the animal kingdom: epigenesis and preformation. In epigenesis, PGC specification is non-autonomous and depends on extrinsic signaling pathways. The BMP pathway provides the key PGC specification signals in mammals. Preformation is autonomous and mediated by determinants localized within PGCs. In Drosophila, a classic example of preformation, constituents of the germ plasm localized at the embryonic posterior are thought to be both necessary and sufficient for proper determination of PGCs. Contrary to this longstanding model, here we show that these localized determinants are insufficient by themselves to direct PGC specification in blastoderm stage embryos. Instead, we find that the BMP signaling pathway is required at multiple steps during the specification process and functions in conjunction with components of the germ plasm to orchestrate PGC fate.     

Topology of the germ plasm and development of primordial germ cells in inverted amphibian eggs

Abstract. Inverted Xenopus eggs have reduced numbers of primordial germ cells (PGCs). The extent of the reduction varies from spawning to spawning. Histologic examination revealed that PGC counts were lowest in inverted eggs which displayed the greatest amount of shift in the vegetal mass of large yolk platelets, although the germ plasm itself always remained localized in the egg's original vegetal hemi-sphere. Even at blastulation the germ plasm continued to be localized in the egg's original vegetal hemisphere. In many cases, however, it was confined to the periphery of the embryo, which probably accounts for the reduced PGC number in some tadpoles. In other cases it may have been dispersed and therefore not detectable in histologic analyses.
Although the altered site of involution in inverted embryos did not influence PGC development, subsequent cell movement patterns apparently did. Those embryos which displayed the largest degree of pattern reversal at the tail-bud stage also exhibited the most extreme reduction in PGC numbers. A brief cold shock (4° C, 10 min) prior to first cleavage leads to a further reduction in PGC numbers in inverted embryos, probably as a result of the displace-ment of the germ plasm away from its original vegetal pole location.     

Topology of the germ plasm and development of primordial germ cells in inverted amphibian eggs

Inverted Xenopus eggs have reduced numbers of primordial germ cells (PGCs). The extent of the reduction varies from spawning to spawning. Histologic examination revealed that PGC counts were lowest in inverted eggs which displayed the greatest amount of shift in the vegetal mass of large yolk platelets, although the germ plasm itself always remained localized in the egg's original vegetal hemisphere. Even at blastulation the germ plasm continued to be localized in the egg's original vegetal hemisphere. In many cases, however, it was confined to the periphery of the embryo, which probably accounts for the reduced PGC number in some tadpoles. In other cases it may have been dispersed and therefore not detectable in histologic analyses. Although the altered site of involution in inverted embryos did not influence PGC development, subsequent cell movement patterns apparently did. Those embryos which displayed the largest degree of pattern reversal at the tail-bud stage also exhibited the most extreme reduction in PGC numbers. A brief cold shock (4 degrees C, 10 min) prior to first cleavage leads to a further reduction in PGC numbers in inverted embryos, probably as a result of the displacement of the germ plasm away from its original vegetal pole location.     

Receptor tyrosine kinase signaling and primordial germ cell development

Primordial germ cells (PGCs) give rise to sperms and eggs. Their development is crucial to species propagation and has to be precisely controlled. Studies in several model organisms have identified many genes involved in the specification and guided migration of PGCs. However, the mechanisms governing the behaviors of these unique cells remain to be investigated. Interestingly, PGCs share certain cellular properties with metastasizing cancer cells including proliferation, invasion of other tissues, survival and migration. Recently we have shown that in Drosophila the receptor tyrosine kinase Torso activates both STAT and Ras during the early phase of PGC development. In later stages, activation of both STAT and Ras, likely by other molecules, is required continuously for PGC migration. The requirement for RTK suggests molecular conservation between flies and mice in PGC development and also suggests that germ cells and cancer cells share certain intracellular signaling strategies.     

Mitochondrial trafficking through Rhot1 is involved in the aggregation of germinal granule components during primordial germ cell formation in Xenopus embryos

In many animals, the germ plasm is sufficient and necessary for primordial germ cell (PGC) formation. It contains germinal granules and abundant mitochondria (germline‐Mt). However, the role of germline‐Mt in germ cell formation remains poorly understood. In Xenopus, the germ plasm is distributed as many small islands at the vegetal pole, which gradually aggregates to form a single large mass in each of the four vegetal pole cells at the early blastula stage. Polymerized microtubules and the adapter protein kinesin are required for the aggregation of germ plasm. However, it remains unknown whether germline‐Mt trafficking is important for the cytoplasmic transport of germinal granules during germ plasm aggregation. In this study, we focused on the mitochondrial small GTPase protein Rhot1 to inhibit mitochondrial trafficking during the germ plasm aggregation. Expression of Rhot1ΔC, which lacks the C‐terminal mitochondrial transmembrane domain, inhibited the aggregation of germline‐Mt during early development. In Rhot1‐inhibited embryos, germinal granule components did not aggregate during cleavage stages, which reduced the number of PGCs on the genital ridge at tail‐bud stage. These results suggest that mitochondrial trafficking is involved in the aggregation of germinal granule components, which are essential for the formation of PGCs.