A critical role for Xdazl, a germ plasm-localized RNA, in the differentiation of primordial germ cells in Xenopus

Xdazl is an RNA component of Xenopus germ plasm and encodes an RNA-binding protein that can act as a functional homologue of Drosophila boule. boule is required for entry into meiotic cell division during fly spermatogenesis. Both Xdazl and boule are related to the human DAZ and DAZL, and murine Dazl genes, which are also involved in gamete differentiation. As suggested from its germ plasm localization, we show here that Xdazl is critically involved in PGC development in Xenopus. Xdazl protein is expressed in the cytoplasm, specifically in the germ plasm, from blastula to early tailbud stages. Specific depletion of maternal Xdazl RNA results in tadpoles lacking, or severely deficient in, primordial germ cells (PGCs). In the absence of Xdazl, PGCs do not successfully migrate from the ventral to the dorsal endoderm and do not reach the dorsal mesentery. Germ plasm aggregation and intracellular movements are normal indicating that the defect occurs after PGC formation. We propose that Xdazl is required for early PGC differentiation and is indirectly necessary for the migration of PGCs through the endoderm. As an RNA-binding protein, Xdazl may regulate translation or expression of factors that mediate migration of PGCs.     

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.     

Three-dimensional ultrastructural analysis of RNA distribution within germinal granules of Xenopus.

The germ plasm is a specialized region of oocyte cytoplasm that contains determinants of germ cell fate. In Xenopus oocytes, the germ plasm is a part of the METRO region of mitochondrial cloud. It contains the germinal granules and a variety of coding and noncoding RNAs that include Xcat2, Xlsirts, Xdazl, DEADSouth, Xpat, Xwnt11, fatVg, B7/Fingers, C10/XFACS, and mitochondrial large and small rRNA. We analyzed the distribution of these 11 different RNAs within the various compartments of germ plasm during Xenopus oogenesis and development by using whole-mount electron microscopy in situ hybridization. Serial EM sections were used to reconstruct a three-dimensional image of germinal granule distribution within the METRO region of the cloud and the distribution of RNAs on the granules in oocytes and embryos. We found that, in the oocytes, the majority of RNAs were associated either with the precursor of germinal granules or with the germ plasm matrix. Only Xcat2, Xpat, and DEADSouth RNAs were associated with the mature germinal granules in oocytes, while only Xcat2 and Xpat were associated with germinal granules in embryos. However, Xcat2 was the only RNA that was consistently sequestered inside the germinal granules, while the others were located on the periphery. Xdazl, which functions in germ cell migration/formation, was detected on the matrix between granules. Later in development, Xcat2 mRNA was released from the germinal granules. This coincides with the timing of its translational derepression. These results demonstrate that there is a dynamic three-dimensional architecture to the germinal granules that changes during oogenesis and development. They also indicate that association of specific RNAs with the germinal granules is not a prerequisite for their serving a germ cell function; however, it may be related to their state of translational repression.     

Visualization of the Xenopus primordial germ cells using a green fluorescent protein controlled by cis elements of the 3' untranslated region of the DEADSouth gene

We succeeded in visualization of the primordial germ cells (PGCs) in a living Xenopus embryo. The mRNA of the reporter Venus protein, fused to the 3' untranslated region (UTR) of DEADSouth, which is a component of the germ plasm in Xenopus eggs, was microinjected into the vegetal pole of fertilized eggs and then the cells with Venus fluorescence were monitored during development. The behavior of the cells was identical to that previously described for PGCs. Almost all Venus-expressing cells were Xdazl-positive in the stage 48 tadpoles, indicating that they were PGCs. In addition, we found three sub-regions (A, B and C) in the 3' UTR, which were involved in the PGC-specific expression of the reporter protein. Sub-region A, which was identified previously as a localization signal for the germ plasm during oogenesis, participated in anchoring of the mRNA at the germ plasm and the degradation of the mRNA in the somatic cells. Sub-regions B and C were also involved in anchoring of the mRNA at the germ plasm. Sub-region B participated in the enhancement of translation.     

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.     

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.     

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.     

Distribution of XTdrd6/Xtr protein during oogenesis and early development in Xenopus laevis: Zygotic translation begins only in germ cells that have entered the genital ridge

We previously identified Xenopus tudor domain containing 6/Xenopus tudor repeat (Xtdrd6/Xtr), which was exclusively expressed in the germ cells of adult Xenopus laevis. Western blot analysis showed that the XTdrd6/Xtr protein was translated in St. I/II oocytes and persisted as a maternal factor until the tailbud stage. XTdrd6/Xtr has been reported to be essential for the translation of maternal mRNA involved in oocyte meiosis. In the present study, we examined the distribution of the XTdrd6/Xtr protein during oogenesis and early development, to predict the time point of its action during development. First, we showed that XTdrd6/Xtr is localized to germinal granules in the germplasm by electron microscopy. XTdrd6/Xtr was found to be localized to the origin of the germplasm, the mitochondrial cloud of St. I oocytes, during oogenesis. Notably, XTdrd6/Xtr was also found to be localized around the nuclear membrane of St. I oocytes. This suggests that XTdrd6/Xtr may immediately interact with some mRNAs that emerge from the nucleus and translocate to the mitochondrial cloud. XTdrd6/Xtr was also detected in primordial germ cells and germ cells throughout development. Using transgenic Xenopus expressing XTdrd6/Xtr with a C-terminal FLAG tag produced by homology-directed repair, we found that the zygotic translation of the XTdrd6/Xtr protein began at St. 47/48. As germ cells are surrounded by gonadal somatic cells and are considered to enter a new differentiation stage at this phase, the newly synthesized XTdrd6/Xtr protein may regulate the translation of mRNAs involved in the new steps of germ cell differentiation.     

The vegetally localized mRNA fatvg is associated with the germ plasm in the early embryo and is later expressed in the fat body

Vegetally localized RNAs in Xenopus oocytes have been implicated in the establishment of the primary germ layers and the formation and development of the primordial germ cells. fatvg mRNA is localized through the late pathway to the vegetal cortex. Like Vg1 mRNA fatvg is distributed throughout the entire cortex; however, unlike Vg1 there is a small fraction of the fatvg mRNA that is associated with the mitochondrial cloud. In early cleavage stage embryos, fatvg mRNA is associated with the germ plasm located at the tips of the vegetal blastomeres of the embryo. While several localized RNAs that follow the Message Transport Organizer (METRO) pathway have been found in the germ plasm in embryos, fatvg is a late pathway RNA that is associated with the germ plasm. In tadpoles, fatvg mRNA shows a novel pattern of expression which is distinct from the germ cell lineage and is detected at the dorso-anterior margin of the endodermal mass along the midline in two clusters of cells. fatvg mRNA expression is also detected later in the developing fat bodies, the major adipose tissues of the frog.     

Status of RNAs, localized in Xenopus laevis oocytes, in the frogs Rana pipiens and Eleutherodactylus coqui

Early development in the frog model, Xenopus laevis, is governed by RNAs, localized to the vegetal cortex of the oocyte. These RNAs include Xdazl RNA, which is involved in primordial germ cell formation, and VegT RNA, which specifies the mesoderm and endoderm. In order to determine whether orthologues of these RNAs are localized and have similar functions in other frogs, we cloned RpDazl and RpVegT from Rana pipiens, a frog that is phylogenetically distant from X. laevis. RNAs from both genes are localized to the vegetal cortex of the R. pipiens oocyte, indicating that the vegetal localization is likely the basal state. The animal location of EcVegT RNA in Eleutherodactylus coqui that we found previously (Beckham et al., 2003) is then a derived state, probably due to the great increase in egg size required for direct development of this species. To answer the question of function, we injected RpVegT or EcVegT RNAs into X. laevis embryos, and assayed animal caps for gene expression. Both of these RNAs induced the expression of endodermal, mesodermal, and organizer genes, showing that the function of RpVegT and EcVegT as meso-endodermal determinants is conserved in frogs. The RNA localizations and the function of VegT orthologues in germ layer specification may be synapomorphies for anuran amphibians.     

孤儿受体TR3在小鼠睾丸中的定位和表达

本文采用原位杂交和免疫组织化学技术,观察孤儿受体ＴＲ３及其ｍＲＮＡ在小鼠睾丸中的表达及细胞定位。结果表明,在小鼠睾丸中有显著量的孤儿受体ＴＲ３ ｍＲＮＡ和蛋白表达,其表达量在不同曲细精管有明显的差异;孤儿受体ＴＲ３蛋白主要定位于生精细胞,其ｍＲＮＡ在生精细胞特异表达,主要在精原细胞和发育早期的初级精母细胞表达,提示孤儿受体ＴＲ３在小鼠曲细精管精子发生的早期阶段中起着调控作用。     

Examination of the expression of the heat shock protein gene, hsp110, in Xenopus laevis cultured cells and embryos

Eukaryotic organisms respond to various stresses with the synthesis of heat shock proteins (HSPs). HSP110 is a large molecular mass HSP that is part of the HSP70/DnaK superfamily. In this study, we have examined, for the first time, the expression of the hsp110 gene in Xenopus laevis cultured cells and embryos. Sequence analysis revealed that the protein encoded by the hsp110 cDNA exhibited 74% identity with its counterparts in mammals and only 27-29% with members of the Xenopus HSP70 family. Hsp110 mRNA and/or protein was detected constitutively in A6 kidney epithelial cells and was inducible by heat shock, sodium arsenite, and cadmium chloride. However, treatment with ethanol or copper sulfate had no detectable effect on hsp110 mRNA levels. Similar results were obtained for hsp70 mRNA except that it was inducible with ethanol. In Xenopus embryos, hsp110 mRNA was present constitutively during development. Heat shock-inducible accumulation of hsp110 mRNA occurred only after the midblastula stage. Whole mount in situ hybridization analysis revealed that hsp110 mRNA accumulation in control and heat shocked embryos was enriched in selected tissues. These studies demonstrate that Xenopus hsp110 gene expression is constitutive and stress inducible in cultured cells and developmentally- and tissue specifically-regulated during early embryogenesis.