Involvement of the protein of Xenopus vasa homolog (Xenopus vasa-like gene 1, XVLG1) in the differentiation of primordial germ cells

In order to understand the role of the protein of Xenopus vasa homolog ( Xenopus vasa -like gene 1, XVLG1 ) in germ line cells, an attempt was made to perturb the function of the protein with the anti-vasa antibody 2L-13. The 2L-13 or the control antibody was microinjected with a lineage tracer (FITC-dextran-lysine, FDL) into single vegetal blastomeres containing the germ plasm of Xenopus 32-cell embryos, the descendants of which were destined to differentiate into a small number of primordial germ cells (PGC) and a large number of somatic cells, mostly of endodermal tissues at the tadpole stage. No significant effect of the injection of the antibodies on FDL-labeled, presumptive PGC (pPGC) was observed in embryos until stage 37/38. However, FDL-labeled PGC were not observed in almost all the 2L-13 antibody-injected tadpoles, although a similar number of labeled somatic cells were always present. As 2L-13 antibody specifically reacts with XVLG1 protein in the embryos by immunoblotting, the present results suggest that the antibody perturbed the function of XVLG1 protein in the pPGC, resulting in failure of PGC differentiation at the tadpole stage.     

A trial for induction of supernumerary primordial germ cells in Xenopus tadpoles by injecting RNA of Xenopus vasa homologue into germline cells of 32-cell embryos

Whether overexpression of Xenopus vasa homologue or Xenopus vasa-like gene 1 (XVLG1) in germline cells of Xenopus embryos can induce supernumerary primordial germ cells (PGC) at tadpole stage was investigated. XVLG1 RNA (0.1-2.0 ng) and beta-gal RNA (0.5 ng) were injected into one of, usually, four germ plasm-bearing cells (GPBC) of 32-cell embryos, with the beta-gal RNA (2.0 ng) serving as both lineage tracer and control for XVLG1 RNA. The total number of PGC, including X-gal-stained and unstained PGC of injected and uninjected GPBC origins respectively, was examined in the experimental tadpoles developed from the injected embryos. The injected RNA, XVLG1 and beta-gal RNA, were translated, resulting in a large amount of corresponding proteins in presumptive PGC (pPGC) as well as in somatic cells derived from the injected GPBC. Nevertheless, the average number of total PGC per tadpole found in the experimental tadpoles from the XVLG1 RNA-injected embryos was not significantly different from that of beta-gal RNA-injected ones, irrespective of the injected dose of XVLG1 RNA. This indicates that the extra XVLG1 protein in pPGC is not sufficient to increase the number of PGC in the tadpoles.     

Immuno-Localization of DEAD Family Proteins in Germ Line Cells of Xenopus Embryos.

In order to investigate whether a vasa -like protein is present in germ line cells of Xenopus , antibodies were produced which react specifically with synthetic oligopeptides of sequences from near the N- or C-termini or with one including the DEAD box of the Drosophila vasa protein.
Only the antibody against the oligopeptide including the DEAD box reacted strongly with germ plasm (GP) or with cytoplasm of germ line cells of Xenopus embryos by immunofluorescence microscopy. By immunoelectron microscopy, the antibody was demonstrated to react with the GP-specific structure, germinal granules, in cleaving embryos, and with their derivatives in the germ line cells of embryos at stages extending from gastrula to feeding tadpole. It also reacted with mitochondria not only in the GP and the germ line cells but also in somatic cells, and with myofibrils in muscle cells. By Western blotting, the antibody was shown to react with several bands of Mr 42–69 ± 10³ in protein samples from Xenopus embryos. In samples from Drosophila ovaries, it reacted with a Mr 71 ± 10³ band which was probably the vasa protein. This indicates the possibility that Xenopus embryos contain several DEAD family proteins. One of these is present on germinal granules, resembling the vasa protein on polar granules of Drosophila .     

Cause of the decreased number of PGC in albino Xenopus: analysis of the number and position of pPGC in albino embryos during and after cleavage

In order to understand the cause for the decreased number of primordial germ cells (PGC) in Xenopus albino (a(p)/a(p)) tadpoles, the number of presumptive PGC (pPGC) in the albino and wild-type embryos at Nieuwkoop and Faber's stages 6-37/38 were examined using the antibody specific to germ plasm. The positions of pPGC in the endodermal cell mass in embryos of both types at stages 28 and 33/34 were also observed to learn the migratory behavior of pPGC. The number of pPGC in the albino increased up to stage 28 with development, but decreased thereafter. In contrast, the number in the wild-type increased to stage 33/34 as development proceeded, and the number of pPGC in stage 33/34 embryos reached nearly that of PGC of the feeding tadpoles in the same batches. Judging from the positions of pPGC, the migration of pPGC from the median part through the lateral to the dorsal part of the endodermal cell mass in the albino was suspected to be somewhat later than that in the wild-type. These results, together with the results in previous studies, suggest that the decreased number of PGC in the albino would be closely related to the sudden decrease in number of pPGC at stage 33/34, as well as to the ectopic position of pPGC in endodermal cell mass, the latter of which had already been demonstrated to be responsible for the differentiation into PGC.     

The mode and molecular mechanisms of the migration of presumptive PGC in the endoderm cell mass of Xenopus embryos

We investigated the mode of migration of presumptive primordial germ cells (pPGC) in the endoderm cell mass of Xenopus embryos at stages 7-40. The molecules underlying the migration were also studied cytochemically and immunocytologically. By examining the relative positions of pPGC and somatic cells derived from the single, fluorescein-dextran lysine (FDL)-injected, germ plasm-bearing cells of stage 6 embryos, pPGC in embryos at stages 7-23 and those at stages later than 24 were assumed to passively and actively migrate in the endoderm cell mass, respectively. This assumption was supported by the observation that F-actin, essential for active cell migration, was recognized on pPGC of the latter stages, but never on those of the former ones. In addition, the molecule like CXC chemokine receptor 4 (CXCR4) found on directionally migrating PGC in mouse and zebrafish, probably Xenopus CXCR4 (xCXCR4), was detected on pPGC only at latter stages. Accordingly, F-actin and xCXCR4, and probably beta1-integrin and collagen type IV, which are indispensable for the formation of F-actin, are thought to be involved in the active migration of pPGC in the endoderm cell mass.     

Ectopic germline cells in embryos of Xenopus laevis

Whether all descendants of germline founder cells inheriting the germ plasm can migrate correctly to the genital ridges and differentiate into primordial germ cells (PGCs) at tadpole stage has not been elucidated in Xenopus. We investigated precisely the location of descendant cells, presumptive primordial germ cells (pPGCs) and PGCs, in embryos at stages 23-48 by whole-mount in situ hybridization with the antisense probe for Xpat RNA specific to pPGCs and whole-mount immunostaining with the 2L-13 antibody specific to Xenopus Vasa protein in PGCs. Small numbers of pPGCs and PGCs, which were positively stained with the probe and the antibody, respectively, were observed in ectopic locations in a significant number of embryos at those stages. A few of the ectopic PGCs in tadpoles at stages 44-47 were positive in TdT-mediated dUTP digoxigenin nick end labeling (TUNEL) staining. By contrast, pPGCs in the embryos until stage 40, irrespective of their location and PGCs in the genital ridges of the tadpoles at stages 43-48 were negative in TUNEL staining. Therefore, it is evident that a portion of the descendants of germline founder cells cannot migrate correctly to the genital ridges, and that a few ectopic PGCs are eliminated by apoptosis or necrosis at tadpole stages.     

The cause of the decreased number of primordial germ cells in albino Xenopus resides not in the micro-environment but in the presumptive PGC

The number of primordial germ cells (PGC) in albino tadpoles of Xenopus is significantly decreased as compared with that of the wild-type. Whether the decreased number of PGC is caused by the presumptive PGC (pPGC) themselves or the micro-environment surrounding those cells in the albino, or both was investigated in the present study. [³H]thymidine-labeled pPGC of wild-type and albino were implanted into unlabeled, host neurulae of wild-type br albino and wild-type, respectively. Labeled PGC in the genital ridges of experimental tadpoles were examined by autoradiography. There were no significant differences in the proportion of tadpoles with labeled PGC and in the average number of those PGC between the albino and wild-type tadpoles, into which wild-type pPGC had been implanted. The proportion in wild-type tadpoles with albino pPGC was much lower than that in wild-type tadpoles with wild-type pPGC. These results suggest that the pPGC of the albino and not the micro-environment are responsible for the decreased number of PGC.     

The Xdsg protein in presumptive primordial germ cells (pPGCs) is essential to their differentiation into PGCs in Xenopus

In order to know the role of the Xdsg gene in presumptive PGCs (pPGCs) of Xenopus, we attempted to inhibit the translation of Xdsg mRNA in pPGCs by injecting antisense morpholino oligo (asMO), together with Fluorescein Dextran-Lysine (FDL), into single germ plasm-bearing cells of 32-cell embryos. Among three types of asMOs complementary to different parts of the 5'-untranslated region of Xdsg mRNA tested, only one asMO, designated as Xdsg-3, inhibited the translation of the mRNA in FDL-labeled pPGCs, resulting in the absence of labeled PGCs in experimental tadpoles. On the other hand, two other asMOs, Xdsg-1 and -2, did not inhibit the translation, so that a similar number of labeled PGCs found in FDL-injected but asMO-uninjected control tadpoles were observed in experimental tadpoles derived from asMO-injected embryos. Surprisingly, use of Xdsg-3 asMO resulted in the disappearance of the protein of Xenopus vasa homolog (Xenopus vasa-like gene 1, XVLG1) from FDL-labeled pPGCs by inhibiting the translation of XVLG1 mRNA. However, the effect of Xdsg-3 asMO on the translation of Xdsg and XVLG1 mRNAs and PGC formation could be canceled by the coinjection with Xdsg mRNA. Consequently, the Xdsg protein in pPGCs may play an important role in the formation of PGCs by regulating the production of XVLG1 protein.     

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.     

TGFβ相关蛋白在爪蟾早期胚胎中的免疫组织化学的研究

By the method of immunocytochemistry, using the polyclonal antibodies raised against the 1-29 N-terminal residues of TGF beta-1, we found that the protein could bind to the antibodies was present in the early embryos of Xenopus. The protein was named TGF beta-related protein. It was distributed mainly in the endoderm from blastula (stg. 7) to late neurula. In the blastula (stg. 8), the protein was localized in the vegetal hemisphere near the floor of the blastocoel [Plate I, Fig. 1]. In the early gastrula (stg. 10.5) [Plate I, Fig. 2], it was localized in the central part of the vegetal hemisphere. In late gastrula (stg. 12), it was mainly distributed around the gastrocoel [Plate I, Fig. 3], but the fluorescence in endoderm cells (ventral part beneath the gastrocoel) was stronger than in the mesoderm cells (dorsal part of the gastrocoel). In the early neurula (stg. 14), the whole endoderm displayed strong fluorescence and the part of dorsal mesoderm (presumptive somite & notochord) close to endoderm was also found to be positively stained [Plate I, Fig. 4,5], but the part close to neural plate was negative. In The late neurula (stg. 20) [Plate I, Fig. 6], it was found in the central area of yolk mass (endoderm cells). No positive stain was detected in the unfertilized egg, embryos earlier than stage and later than stage 20/21.2+ protein in early development.     

Characterization and localization of tropomyosin proteins in Xenopus embryos with specific antibodies

In the process of monoclonal antibody (mAb) production against the 38kDa protein which is lacking in the gastrula arrested mutant embryos in Xenopus we incidentally obtained two kinds of mAb (designated as B11 and 2D10 antibodies, respectively) recognizing tropomyosin (TM) proteins in Xenopus embryos. The characterization of the corresponding antigens to those mAb was performed by immunoblotting and silver staining for two-dimensional (2-D) gels in the present study. The localization of the antigens in Xenopus embryos was also investigated by fluorescent microscopy.
By 2-D immunoblots with those mAb, three distinct protein spots or TM isoforms were recognized in Xenopus embryos; a 38 kDa spot with a pl of approximately 4.8 reacted with both antibodies in embryos at stages later than the mid-tailbud (stage 28) and two 30 kDa spots, which are probably isomers, with a pl of approximately 4.8 were detected with 2D10 antibody in embryos at stages extending from the fertilized to the mid-neurula (stage 20). By immunofluorescent microscopy, B11 antibody was shown to react mainly with muscle cells and their precursor cells. In contrast, 2D10 antibody stained the cytoplasm of almost all cells in embryos at stages from the fertilized to the tadpole.
Judging from the results obtained with immunoblotting and fluorescent microscopy, it is likely that the 38 kDa spot is a skeletal muscle TM isoform and the two 30 kDa spots are non-muscle TM isoforms.     

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.     

Refinement of gene expression patterns in the early Xenopus embryo

During blastula and gastrula stages of Xenopus development, cells become progressively and asynchronously committed to a particular germ layer. We have analysed the expression of genes normally expressed in ectoderm, mesoderm or endoderm in individual cells from early and late gastrula embryos, by both in situ hybridization and single-cell RT-PCR. We show that at early gastrula stages, individual cells in the same region may express markers of two or more germ layers, and 'rogue' cells that express a marker outside its canonical domain are also observed at these stages. However, by the late gastrula stage, individual cells express markers that are more characteristic of their position in the embryo, and 'rogue' cells are seen less frequently. These observations exemplify at the gene expression level the observation that cells of the early gastrula are less committed to one germ layer than are cells of the late gastrula embryo. Ectodermal cells induced to form mesendoderm by the addition of Activin respond by activating expression of different mesodermal and endodermal markers in the same cell, recapitulating the response of marginal zone cells in the embryo.