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.     

DEADSouth is a germ plasm specific DEAD-box RNA helicase in Xenopus related to eIF4A

DEADSouth was selected in a screen for localized RNAs in Xenopus oocytes. In situ hybridization analysis shows that DEADSouth localizes to the vegetal cortex via the mitochondrial cloud early in oogenesis and segregates with germ plasm during early embryogenesis. These results lend further support for the general concept that the role of the early RNA localization pathway in Xenopus is to localize germ cell components (reviewed in King, M.L., Zhou, Y., Bubunenko, M. , 1999. BioEssays 21, 546-557). Further analysis shows that DEADSouth is a germline specific RNA, found exclusively within the germ plasm of oocytes and PGCs, as well as in male germ cells. Sequence comparisons with DEADSouth show it to be a member of a small sub-family of the DEAD-box RNA-dependent helicases related to eIF4A.     

A late phase of germ plasm accumulation during Drosophila oogenesis requires lost and rumpelstiltskin

Asymmetric mRNA localization is an effective mechanism for establishing cellular and developmental polarity. Posterior localization of oskar in the Drosophila oocyte targets the synthesis of Oskar to the posterior, where Oskar initiates the assembly of the germ plasm. In addition to harboring germline determinants, the germ plasm is required for localization and translation of the abdominal determinant nanos. Consequently, failure of oskar localization during oogenesis results in embryos lacking germ cells and abdominal segments. oskar accumulates at the oocyte posterior during mid-oogenesis through a well-studied process involving kinesin-mediated transport. Through live imaging of oskar mRNA, we have uncovered a second, mechanistically distinct phase of oskar localization that occurs during late oogenesis and results in amplification of the germ plasm. Analysis of two newly identified oskar localization factors, Rumpelstiltskin and Lost, that are required specifically for this late phase of oskar localization shows that germ plasm amplification ensures robust abdomen and germ cell formation during embryogenesis. In addition, our results indicate the importance of mechanisms for adapting mRNAs to utilize multiple localization pathways as necessitated by the dramatic changes in ovarian physiology that occur during oogenesis.     

Fat facets interacts with vasa in the Drosophila pole plasm and protects it from degradation

Anterior-posterior patterning and germ cell specification in Drosophila requires the establishment, during oogenesis, of a specialized cytoplasmic region termed the pole plasm. Numerous RNAs and proteins accumulate to the pole plasm and assemble in polar granules. Translation of some of these RNAs is generally repressed and active only in pole plasm. Vasa (VAS) protein, an RNA helicase and a component of polar granules, is essential maternally for posterior patterning and germ cell specification, and VAS is a candidate translational activator in the pole plasm. VAS is stabilized within the pole plasm in that it is initially present throughout the entire embryo but strictly limited to the pole cells by the cellular blastoderm stage. hsp83 mRNA, which accumulates in the pole plasm through a stabilization-degradation mechanism, is another example. Here, we used a biochemical approach to identify proteins that copurify with VAS in crosslinked extracts. Prominent among these proteins was the ubiquitin-specific protease Fat facets (FAF), a pole plasm component [7], but one whose roles in posterior patterning and germ line specification have remained unclear. We present evidence that FAF interacts with VAS physically and reverses VAS ubiquitination, thereby stabilizing VAS in the pole plasm.     

Localization of RNAs to the mitochondrial cloud in Xenopus oocytes through entrapment and association with endoplasmic reticulum

The germ cell lineage in Xenopus is specified by the inheritance of germ plasm, which originates within a distinct "mitochondrial cloud" (MC) in previtellogenic oocytes. Germ plasm contains localized RNAs implicated in germ cell development, including Xcat2 and Xdazl. To understand the mechanism of the early pathway through which RNAs localize to the MC, we applied live confocal imaging and photobleaching analysis to oocytes microinjected with fluorescent Xcat2 and Xdazl RNA constructs. These RNAs dispersed evenly throughout the cytoplasm through diffusion and then became progressively immobilized and formed aggregates in the MC. Entrapment in the MC was not prevented by microtubule disruption and did not require localization to germinal granules. Immobilized RNA constructs codistributed and showed coordinated movement with densely packed endoplasmic reticulum (ER) concentrated in the MC, as revealed with Dil16(3) labeling and immunofluorescence analysis. Vg1RBP/Vera protein, which has been implicated in linking late pathway RNAs to vegetal ER, was shown to bind specifically both wild-type Xcat2 3' untranslated region and localization-defective constructs. We found endogenous Vg1RBP/Vera and Vg1RBP/Vera-green fluorescent protein to be largely excluded from the MC but subsequently to codistribute with Xcat2 and ER at the vegetal cortex. We conclude that germ line RNAs localize into the MC through a diffusion/entrapment mechanism involving Vg1RBP/Vera-independent association with ER.     

Live imaging of endogenous RNA reveals a diffusion and entrapment mechanism for nanos mRNA localization in Drosophila

BACKGROUND: Localization of nanos mRNA to the posterior pole of the Drosophila embryo directs local synthesis of Nanos protein that is essential for patterning of the anterior-posterior body axis and germ cell function. While nanos RNA is synthesized by the ovarian nurse cells and appears at the posterior pole of the ooctye late in oogenesis, the mechanism by which this RNA is translocated to and anchored at the oocyte posterior is unknown. RESULTS: By labeling endogenous nanos RNA with GFP, we have been able to follow the dynamic pathway of nanos localization in living oocytes. We demonstrate that nanos localization initiates immediately upon nurse cell dumping, whereby diffusion, enhanced by microtubule-dependent cytoplasmic movements, translocates nanos RNA from the nurse cells to the ooctye posterior. At the posterior, nanos is trapped by association, in particles, with the posteriorly localized germ plasm. Actin-dependent anchoring of nanos RNA complexed to the germ plasm at the posterior maintains localization in the face of rapid cytoplasmic movements. CONCLUSIONS: These results reveal a diffusion-based, late-acting posterior localization mechanism for long-range transport of nanos mRNA. This mechanism differs from directed transport-based localization mechanisms in its reliance on bulk movement of RNA.     

Intermolecular interactions of homologs of germ plasm components in mammalian germ cells

In some species such as flies, worms, frogs and fish, the key to forming and maintaining early germ cell populations is the assembly of germ plasm, microscopically distinct egg cytoplasm that is rich in RNAs, RNA-binding proteins and ribosomes. Cells which inherit germ plasm are destined for the germ cell lineage. In contrast, in mammals, germ cells are formed and maintained later in development as a result of inductive signaling from one embryonic cell type to another. Research advances, using complementary approaches, including identification of key signaling factors that act during the initial stages of germ cell development, differentiation of germ cells in vitro from mouse and human embryonic stem cells and the demonstration that homologs of germ plasm components are conserved in mammals, have shed light on key elements in the early development of mammalian germ cells. Here, we use FRET (Fluorescence Resonance Energy Transfer) to demonstrate that living mammalian germ cells possess specific RNA/protein complexes that contain germ plasm homologs, beginning in the earliest stages of development examined. Moreover, we demonstrate that, although both human and mouse germ cells and embryonic stem cells express the same proteins, germ cell-specific protein/protein interactions distinguish germ cells from precursor embryonic stem cells in vitro; interactions also determine sub-cellular localization of complex components. Finally, we suggest that assembly of similar protein complexes may be central to differentiation of diverse cell lineages and provide useful diagnostic tools for isolation of specific cell types from the assorted types differentiated from embryonic stem cells.     

Centroid, a novel putative DEAD-box RNA helicase maternal mRNA, is localized in the mitochondrial cloud in Xenopus laevis oocytes

In Xenopus species, the early stages of oogenesis take place in the developing tadpole ovary when the oocytes are in a period critical for the organization of the germ plasm (believed to be a determinant of germ-cell fate) and the initial stages of localization of RNAs involved in germ plasm functions. We constructed a cDNA library from the ovaries of stage 64 Xenopus tadpoles with the idea that it will be enriched for oogonia and pre-stage I and stage I oocytes and thus, RNAs involved in oocyte development and germ plasm formation and function. From this cDNA library, we cloned a new maternal localized mRNA which we named centroid. This RNA codes for the protein belonging to the DEAD-box RNA helicase family. Some of the members of this protein family are components of the messenger ribonucleoprotein (mRNP) particles stored in the germ plasm in oocytes of Xenopus, Drosophila and Caenorhabditis species and are believed to play a role in translational activation of stored mRNPs and sorting of mRNPs into the germ plasm. We found that centroid mRNA is localized in Xenopus oocytes by a combination of early and late pathways, a pattern of localization that is very similar to the intermediate pathway localization of fatvg mRNA, another germ-plasm-localized RNA in Xenopus oocytes. Also, centroid mRNA is present in the mitochondrial cloud and in the germ plasm at the surface of germinal granules. This suggests that centroid is involved in the regulation of germ plasm-stored mRNPs and/or germ plasm function.     

Polarizing genetic information in the egg: RNA localization in the frog oocyte

RNA localization is a powerful strategy used by cells to localize proteins to subcellular domains and to control protein synthesis regionally. In germ cells, RNA targeting has profound implications for development, setting up polarities in genetic information that drive cell fate during embryogenesis. The frog oocyte offers a useful system for studying the mechanism of RNA localization. Here, we discuss critically the process of RNA localization during frog oogenesis. Three major pathways have been identified that are temporally and spatially separated in oogenesis. Each pathway uses a different mechanism to effect RNA localization. In some cases, localization elements within the 3' untranslated region have been identified and have provided unique insights into the localization process. This important field is still in its infancy, however, and much remains to be learned. BioEssays 21:546–557, 1999. © 1999 John Wiley & Sons, Inc.     

Arginine methyltransferase Capsuleen is essential for methylation of spliceosomal Sm proteins and germ cell formation in Drosophila

Although arginine modification has been implicated in a number of cellular processes, the in vivo requirement of protein arginine methyltransferases (PRMTs) in specific biological processes remain to be clarified. In this study we characterize the Drosophila PRMT Capsuléen, homologous to human PRMT5. During Drosophila oogenesis, catalytic activity of Capsuléen is necessary for both the assembly of the nuage surrounding nurse cell nuclei and the formation of the pole plasm at the posterior end of the oocyte. In particular, we show that the nuage and pole plasm localization of Tudor, an essential component for germ cell formation, are abolished in csul mutant germ cells. We identify the spliceosomal Sm proteins as in vivo substrates of Capsuléen and demonstrate that Capsuléen, together with its associated protein Valois, is essential for the synthesis of symmetric di-methylated arginyl residues in Sm proteins. Finally, we show that Tudor can be targeted to the nuage in the absence of Sm methylation by Capsuléen, indicating that Tudor localization and Sm methylation are separate processes. Our results thus reveal the role of a PRMT in protein localization in germ cells.     

Protein Interactions in Xenopus Germ Plasm RNP Particles

Hermes is an RNA-binding protein that we have previously reported to be found in the ribonucleoprotein (RNP) particles of Xenopus germ plasm, where it is associated with various RNAs, including that encoding the germ line determinant Nanos1. To further define the composition of these RNPs, we performed a screen for Hermes-binding partners using the yeast two-hybrid system. We have identified and validated four proteins that interact with Hermes in germ plasm: two isoforms of Xvelo1 (a homologue of zebrafish Bucky ball) and Rbm24b and Rbm42b, both RNA-binding proteins containing the RRM motif. GFP-Xvelo fusion proteins and their endogenous counterparts, identified with antisera, were found to localize with Hermes in the germ plasm particles of large oocytes and eggs. Only the larger Xvelo isoform was naturally found in the Balbiani body of previtellogenic oocytes. Bimolecular fluorescence complementation (BiFC) experiments confirmed that Hermes and the Xvelo variants interact in germ plasm, as do Rbm24b and 42b. Depletion of the shorter Xvelo variant with antisense oligonucleotides caused a decrease in the size of germ plasm aggregates and loosening of associated mitochondria from these structures. This suggests that the short Xvelo variant, or less likely its RNA, has a role in organizing and maintaining the integrity of germ plasm in Xenopus oocytes. While GFP fusion proteins for Rbm24b and 42b did not localize into germ plasm as specifically as Hermes or Xvelo, BiFC analysis indicated that both interact with Hermes in germ plasm RNPs. They are very stable in the face of RNA depletion, but additive effects of combinations of antisense oligos suggest they may have a role in germ plasm structure and may influence the ability of Hermes protein to effectively enter RNP particles.     

C-terminal moiety of Tudor contains its in vivo activity in Drosophila

Background

In early Drosophila embryos, the germ plasm is localized to the posterior pole region and is partitioned into the germline progenitors, known as pole cells. Germ plasm, or pole plasm, contains the polar granules which form during oogenesis and are required for germline development. Components of these granules are also present in the perinuclear region of the nurse cells, the nuage. One such component is Tudor (Tud) which is a large protein containing multiple Tudor domains. It was previously reported that specific Tudor domains are required for germ cell formation and Tud localization.

Methodology/Principal Findings

In order to better understand the function of Tud the distribution and functional activity of fragments of Tud were analyzed. These fragments were fused to GFP and the fusion proteins were synthesized during oogenesis. Non-overlapping fragments of Tud were found to be able to localize to both the nuage and pole plasm. By introducing these fragments into a tud mutant background and testing their ability to rescue the tud phenotype, I determined that the C-terminal moiety contains the functional activity of Tud. Dividing this fragment into two parts reduces its localization in pole plasm and abolishes its activity.

Conclusions/Significance

I conclude that the C-terminal moiety of Tud contains all the information necessary for its localization in the nuage and pole plasm and its pole cell-forming activity. The present results challenge published data and may help refining the functional features of Tud.     

Oskar organizes the germ plasm and directs localization of the posterior determinant nanos

Oskar is one of seven Drosophila maternal-effect genes that are necessary for germline and abdomen formation. We have cloned oskar and show that oskar RNA is localized to the posterior pole of the oocyte when germ plasm forms. This polar distribution of oskar RNA is established during oogenesis in three phases: accumulation in the oocyte, transport toward the posterior, and finally maintenance at the posterior pole of the oocyte. The colocalization of oskar and nanos in wild-type and bicaudal embryos suggests that oskar directs localization of the posterior determinant nanos. We propose that the pole plasm is assembled stepwise and that continued interaction among its components is required for germ cell determination.     

The fat facets gene is required for Drosophila eye and embryo development.

In a screen for mutations affecting Drosophila eye development, we have identified a gene called fat facets (faf) which is required for cell interactions that prevent particular cells in the developing eye from becoming photoreceptors. Analysis of eyes mosaic for faf+ and faf- cells shows that faf is required in cells near to, but outside, normal developing photoreceptors and also outside of the ectopic photoreceptors in mutant facets. faf is also essential during oogenesis, and we show that a faf-lacZ hybrid protein is localized via the first 392 amino acids of faf to the posterior pole of oocytes. Posterior localization of faf-lacZ depends on oskar. oskar encodes a key organizer of the pole plasm, a specialized cytoplasm at the posterior pole of embryos. The pole plasm is required for germ cell formation and contains the determinant of posterior polarity, encoded by nanos. Although other pole plasm components are required for localization of nanos RNA or for nanos protein function, faf is not. We have cloned the faf gene, and have shown that it encodes two similar large (approximately 300 x 10(3) M(r)) proteins that are unique with respect to other known proteins.