Hermes is a localized factor regulating cleavage of vegetal blastomeres in Xenopus laevis

We have identified the RNA-binding protein Hermes in a screen for vegetally localized RNAs in Xenopus oocytes. The RNA localizes to the vegetal cortex through both the message transport organizer (METRO) and late pathways. Hermes mRNA and protein are both detected at the vegetal cortex of the oocyte; however, the protein is degraded within a several hour period during oocyte maturation. Injection of antisense morpholino oligonucleotides (HE-MO) against Hermes caused a precocious reduction in Hermes protein present during maturation and resulted in a phenotype characterized by cleavage defects in vegetal blastomeres. The phenotype can be partially rescued by injecting Hermes mRNA. These results demonstrate that the localized RNA-binding protein Hermes functions during oocyte maturation to regulate the cleavage of specific vegetally derived cell lineages. Hermes most likely performs its function by regulating the translation or processing of one or more target RNAs. This is an important mechanism by which the embryo can generate unique cell lineages. The regulation of region-specific cell division is a novel function for a localized mRNA.     

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.     

Pix1 and Pix2 are novel WD40 microtubule-associated proteins that colocalize with mitochondria in Xenopus germ plasm and centrosomes in human cells

In many animals, the germ line develops from a distinct mitochondria-rich region of embryonic cytoplasm called the germ plasm. However, the protein composition of germ plasm and its formation remain poorly understood, except in Drosophila. Here, we show that Xpat, a recently identified protein component of Xenopus germ plasm, interacts via its C-terminal domain with a novel protein, xPix1. Xpat and xPix1 are co-expressed in ovaries, eggs and early embryos and colocalize to the mitochondrial cloud and germ plasm in stage I and stage VI oocytes, respectively. Although Xpat appears unique to Xenopus, Pix proteins, which contain an N-terminal WD40 domain and C-terminal coiled-coil, are widely conserved. In humans, two proteins, Pix1 and Pix2, are expressed at varying levels in different cancer cell lines. Importantly, as well as localizing to mitochondria, human Pix proteins localize to centrosomes and associate with microtubules in vitro and in vivo. Although, Pix proteins are stably expressed through the cell cycle, Pix2 concentrates on microtubule structures in mitosis and microinjection of Pix antibodies interferes with cell division. Based on these data, we propose that Pix1 and Pix2 are microtubule-associated adaptor proteins that likely contribute to a range of developmental and cell division processes.     

Ectopic formation of primordial germ cells by transplantation of the germ plasm: Direct evidence for germ cell determinant in Xenopus

In many animals, the germ line is specified by a distinct cytoplasmic structure called germ plasm (GP). GP is necessary for primordial germ cell (PGC) formation in anuran amphibians including Xenopus. However, it is unclear whether GP is a direct germ cell determinant in vertebrates. Here we demonstrate that GP acts autonomously for germ cell formation in Xenopus.EGFP-labeled GP from the vegetal pole was transplanted into animal hemisphere of recipient embryos. Cells carrying transplanted GP (T-GP) at the ectopic position showed characteristics similar to the endogenous normal PGCs in subcellular distribution of GP and presence of germ plasm specific molecules. However, T-GP-carrying-cells in the ectopic tissue did not migrate towards the genital ridge. T-GP-carrying cells from gastrula or tailbud embryos were transferred into the endoderm of wild-type hosts. From there, they migrated into the developing gonad. To clarify whether ectopic T-GP-carrying cells can produce functional germ cells, they were identified by changing the recipients, from the wild-type Xenopus to transgenic Xenopus expressing DsRed2. After transferring T-GP carrying cells labeled genetically with DsRed2 into wild-type hosts, we could find chimeric gonads in mature hosts. Furthermore, the spermatozoa and eggs derived from T-GP-carrying cells were fertile. Thus, we have demonstrated that Xenopus germ plasm is sufficient for germ cell determination.     

The mRNA coding for Xenopus glutamate receptor interacting protein 2 (XGRIP2) is maternally transcribed, transported through the late pathway and localized to the germ plasm

Using a large-scale in situ hybridization screening, we found that the mRNA coding for Xenopus glutamate receptor interacting protein 2 (XGRIP2) was localized to the germ plasm of Xenopus laevis. The mRNA is maternally transcribed in oocytes and, during maturation, transported to the vegetal germ plasm through the late pathway where VegT and Vg1 mRNAs are transported. In the 3'-untranslated region (UTR) of the mRNA, there are clusters of E2 and VM1 localization motifs that were reported to exist in the mRNAs classified as the late pathway group. With in situ hybridization to the sections of embryos, the signal could be detected in the cytoplasm of migrating presumptive primordial germ cells (pPGCs) until stage 35. At stage 40, when the cells cease to migrate and reach the dorsal mesentery, the signal disappeared. A possible role of XGRIP2 in pPGCs of Xenopus will be discussed.     

Ultrastructural observations on the germ line of Xenopus laevis

Summary The development of the male germ line in Xenopus laevis has been examined by electron microscopy. Findings have been compared to the parallel process in the female. Three structures unique to the germ line were found in both male and female cells: a fibrillar nuclear region free of DNA; largely proteinaceous masses of nuage material; and a chromatoid body. Germ plasm bodies of the egg and early embryo appear to represent a form of nuage material. The finding of a structure which can be identified as a chromatoid body in the female germ line is unique, as is its presence in sexually undifferentiated primordial germ cells. The chromatoid body in Xenopus, unlike that in mammals, does not persist in the spermatozoon. Instead, it dissociates into a series of coated vesicles during spermatogenesis. The chromosomal ultrastructure of meiotic prophase stages in Xenopus is similar in both sexes until diplotene, when male bivalents condense and enter meiotic metaphase instead of entering the extended lampbrush stage characteristic of the female. The multiple nucleoli present in gonia are lost at the onset of meiotic prophase, but no obvious mechanism for this process was observed.The author would like to thank Drs. Joseph Gall and Bernard Tandler for their helpful suggestions during the course of this investigation. The author is a postdoctoral fellow of the National Institutes of Health, U.S.A. This research was supported by N.I.H. Grants 51823 and 12427.     

Xvelo1 uses a novel 75-nucleotide signal sequence that drives vegetal localization along the late pathway in Xenopus oocytes

Vegetally localized RNAs in Xenopus laevis oocytes are involved in the patterning of the early embryo as well as in cell fate specification. Here we report on the isolation and characterization of a novel, vegetally localized RNA in Xenopus oocytes termed Xvelo1. It encodes a protein of unknown biological function and it represents an antisense RNA for XPc1 over a length of more than 1.8 kb. Xvelo1 exhibits a localization pattern reminiscent of the late pathway RNAs Vg1 and VegT; it contains RNA localization elements (LE) which do not match with the consensus structural features as deduced from Vg1 and VegT LEs. Nevertheless, the protein binding pattern as observed for Xvelo1-LE in UV cross-linking experiments and coimmunoprecipitation assays is largely overlapping with the one obtained for Vg1-LE. These observations suggest that the structural features recognized by the protein machinery that drives localization of maternal mRNAs along the late pathway in Xenopus oocytes must be redefined.     

Bazooka regulates microtubule organization and spatial restriction of germ plasm assembly in the Drosophila oocyte

Localization of the germ plasm to the posterior of the Drosophila oocyte is required for anteroposterior patterning and germ cell development during embryogenesis. While mechanisms governing the localization of individual germ plasm components have been elucidated, the process by which germ plasm assembly is restricted to the posterior pole is poorly understood. In this study, we identify a novel allele of bazooka (baz), the Drosophila homolog of Par-3, which has allowed the analysis of baz function throughout oogenesis. We demonstrate that baz is required for spatial restriction of the germ plasm and axis patterning, and we uncover multiple requirements for baz in regulating the organization of the oocyte microtubule cytoskeleton. Our results suggest that distinct cortical domains established by Par proteins polarize the oocyte through differential effects on microtubule organization. We further show that microtubule plus-end enrichment is sufficient to drive germ plasm assembly even at a distance from the oocyte cortex, suggesting that control of microtubule organization is critical not only for the localization of germ plasm components to the posterior of the oocyte but also for the restriction of germ plasm assembly to the posterior pole.     

Evidence for common machinery utilized by the early and late RNA localization pathways in Xenopus oocytes

In Xenopus, an early and a late pathway exist for the selective localization of RNAs to the vegetal cortex during oogenesis. Previous work has suggested that distinct cellular mechanisms mediate localization during these pathways. Here, we provide several independent lines of evidence supporting the existence of common machinery for RNA localization during the early and late pathways. Data from RNA microinjection assays show that early and late pathway RNAs compete for common localization factors in vivo, and that the same short RNA sequence motifs are required for localization during both pathways. In addition, quantitative filter binding assays demonstrate that the late localization factor Vg RBP/Vera binds specifically to several early pathway RNA localization elements. Finally, confocal imaging shows that early pathway RNAs associate with a perinuclear microtubule network that connects to the mitochondrial cloud of stage I oocytes suggesting that motor driven transport plays a role during the early pathway as it does during the late pathway. Taken together, our data indicate that common machinery functions during the early and late pathways. Thus, RNA localization to the vegetal cortex may be a regulated process such that differential interactions with basal factors determine when distinct RNAs are localized during oogenesis.     

Differential subcellular sequestration of proapoptotic and antiapoptotic proteins and colocalization of Bcl-x(L) with the germ plasm, in Xenopus laevis oocytes

Apoptosis is an important element of normal embryonic development and gametogenesis in invertebrate and vertebrate species. Although the components of apoptotic machinery are present in Xenopus laevis fully grown stage VI oocytes and eggs, apoptosis in the developing Xenopus ovary is limited to the somatic cells with no indication of apoptosis in the germ cells. Considering the possibility that Xenopus previtellogenic oocytes might lack the components of the apoptotic pathway, we analyzed Xenopus Stage I oocytes for the presence of the proapoptotic factors Bax and tumor suppressor p53, and antiapoptotic factors Bcl-x(L) and mitochondrial heat shock protein 60 (Hsp60). We found that pro- and antiapoptotic proteins are present in Xenopus oocytes but, surprisingly, they are located in distinct subcellular compartments with proapoptotic proteins Bax and p53 being sequestered in the oocyte nucleus and antiapoptotic protein Bcl-x(L) sequestered in the cytoplasm and highly enriched in the METRO region of the mitochondrial cloud, where it colocalized with the germ plasm, and Hsp60 colocalizing with all mitochondria. The absence of apoptosis in Xenopus early oogenesis is maybe due to differential sequestration of pro- and antiapoptotic molecules.     

Sm proteins, the constituents of the spliceosome, are components of nuage and mitochondrial cement in Xenopus oocytes

A conserved feature of germ cells in many animal species is the presence of perinuclear electron-dense material called the "nuage" that is believed to be a precursor of germinal (or polar or P) granules. In Xenopus oogenesis the nuage is first observed near the nuclear envelope and subsequently in close contact with mitochondria, at which stage it is called the mitochondrial cement. In this study, we found that, in Xenopus pre-stage I and stage I oocytes, nuage and mitochondrial cement contain the spliceosomal Sm proteins, Xcat2 mRNA, and DEAD-box RNA helicase XVLG1. Other components of Cajal bodies or splicing machinery such as coilin, SMN protein, and snRNAs are absent from the nuage and mitochondrial cement. We suggest that Xenopus Sm proteins have adapted to a role independent of pre-mRNA splicing and that instead of binding to their traditional spliceosomal partner such as snRNA, they bind mRNAs that are the components of germinal granules (i.e., Xcat2 mRNA) and facilitate the transport of these mRNAs from the nucleus to the nuage that is a precursor of germinal granules. In addition, the presence of Vasa-like DEAD-box helicase in Xenopus nuage suggests involvement of nuage in the microRNA and/or RNAi pathway, similar to the role of nuage in Drosophila.     

Molecular cloning and characterization of an adaptor protein Shc isoform from Xenopus laevis oocytes

In order to gain further insight into IGF-1 receptor signaling in Xenopus laevis oocytes and embryos, we have undertaken the characterization of the adapter protein Shc and studied its implication in oocyte maturation induced after IGF-1 receptor activation, especially since expression of this molecule has been indirectly evidenced in Xenopus oocytes, eggs and embryos. We report herein the cloning from Xenopus postvitellogenic oocytes of a complementary DNA encoding a protein of 470 amino acids which shows the higher identity with the mammalian adaptor protein p52(ShcA). Western blot analysis using homologous antibodies evidenced a 60-kDa protein, p60(Xl)(Shc), that is predominantly expressed in oocytes and in early embryos. We also demonstrate that, like p60(Xl)(Shc), Grb2 and the guanine nucleotide exchange factor Sos are expressed in oocytes throughout vitellogenesis and in early embryos and that overexpression of a dominant-negative form of Grb2 specifically inhibits insulin-induced resumption of meiosis. We finally show that Grb2 binds to p60(Shc) in oocytes specifically upon insulin treatment. Altogether, these results suggest that Shc and Grb2-Sos are implicated in ras-dependent Xenopus oocyte maturation induced by insulin/IGF-1; they also indicate that inability of insulin/IGF-1 to activate the Ras-MAPK cascade in vitellogenic oocytes does not result from an insufficient expression level of Shc, Grb2 and Sos.     

Acrosome reaction in sperm of the frog, Xenopus laevis: its detection and induction by oviductal pars recta secretion

Previous electron microscopic observations have shown that the acrosome of the sperm of the frog, Xenopus laevis, comprises a membrane-bounded vesicle covering the anterior-most position of the head. We obtained a sperm suspension from the testes and stained it with LysoSensor Green for observation under a confocal laser scanning microscope and found a bright fluorescence reflecting the presence of the acrosomes at the top of the sperm head in about 64% of the sperm, with no deterioration of their capacity to fertilize. About 40% of the sperm with an acrosome underwent an acrosome reaction in response to Ca(2+) ionophore A23187, as evidenced by a loss of LysoSensor Green stainability, accompanied by breakdown of the acrosomal vesicle. About 53% of the sperm bound to isolated vitelline envelopes underwent an acrosome reaction, whereas both jelly water and solubilized vitelline envelopes weakly induced an acrosome reaction. When the sperm were treated with an oviductal extract obtained from the pars recta, but not the pars convoluta region, about 40% of the sperm with acrosomes underwent an acrosome reaction. The substance containing acrosome reaction-inducing activity in the pars recta extract seemed to be a heat-unstable substance with a molecular weight of greater than 10 kDa. The activity was not inhibited by protease inhibitors but required extracellular Ca(2+) ions. These results indicate that the acrosome reaction occurs on the vitelline envelopes in response to the substance deposited from the pars recta during the passage of the oocytes through the oviduct.     

Cold-inducible RNA binding protein is required for the expression of adhesion molecules and embryonic cell movement in Xenopus laevis

We have previously shown that the Xenopus homologue of cold-inducible RNA binding protein, XCIRP-1, is required for the morphogenetic migration of the pronephros during embryonic development. However, the underlying molecular mechanisms remain elusive. Here, we report that XCIRP is essential for embryonic cell movement, as suppression of XCIRP by microinjection of anti-sense mRNA and morpholino antisense oligonucleotides (MOs) significantly reduced protein expression, inhibited the cell migration rate, and inhibited eFGF and activin-induced animal cap elongation. By immunoprecipitation and RT-PCR, we further showed that the mRNA of a panel of adhesion molecules, including alphaE- and beta-catenin, C- and E-cadherin, and paraxial proto-cadherin, are the targets of XCIRP. Consistently, in animal cap explant studies, suppression of XCIRP by MOs inhibited the expression of these adhesion molecules, while over-expression of sense XCIRP-1 mRNA fully rescued this inhibition. Taken together, these results suggest for the first time that XCIRP is required to maintain the expression of adhesion molecules and cell movement during embryonic development.