The novel secreted factor MIG-18 acts with MIG-17/ADAMTS to control cell migration in Caenorhabditis elegans.

The migration of Caenorhabditis elegans gonadal distal tip cells (DTCs) offers an excellent model to study the migration of epithelial tubes in organogenesis. mig-18 mutants cause meandering or wandering migration of DTCs during gonad formation, which is very similar to that observed in animals with mutations in mig-17, which encodes a secreted metalloprotease of the ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family. MIG-18 is a novel secreted protein that is conserved only among nematode species. The mig-17(null) and mig-18 double mutants exhibited phenotypes similar to those in mig-17(null) single mutants. In addition, the mutations in fbl-1/fibulin-1 and let-2/collagen IV that suppress mig-17 mutations also suppressed the mig-18 mutation, suggesting that mig-18 and mig-17 function in a common genetic pathway. The Venus-MIG-18 fusion protein was secreted from muscle cells and localized to the gonadal basement membrane, a tissue distribution reminiscent of that observed for MIG-17. Overexpression of MIG-18 in mig-17 mutants and vice versa partially rescued the relevant DTC migration defects, suggesting that MIG-18 and MIG-17 act cooperatively rather than sequentially. We propose that MIG-18 may be a cofactor of MIG-17/ADAMTS that functions in the regulation of the gonadal basement membrane to achieve proper direction of DTC migration during gonadogenesis.

Characterization of the 38 kDa protein lacking in gastrula-arrested mutant Xenopus embryos.

We have reported elsewhere that offspring from the No. 65 female of Xenopus laevis cleaved normally, but their development was arrested at the onset of gastrulation, like the Ambystoma ova-deficient (o) mutant, irrespective of mating with different wild-type males, and that an acidic, 38 kDa protein present in wild-type eggs was lacking in eggs of the female. In the current study, we first determined the partial amino acid sequence (VANLE) of one of the well-separated tryptic peptides from the protein, which was found in elongation factor 1 delta (Ef1delta) in Xenopus, and finally identified the protein as one of the Ef1delta isoforms, Ef1delta2, by peptide mass spectrometry. RT-PCR analyses for Ef1delta2 and its close homolog Ef1delta1 in wild-type oocytes and embryos demonstrated that both transcripts are maternal and Ef1delta1 is present more abundantly than Ef1delta2 throughout the stages examined. Importantly, the amount of the Ef1delta2 transcript per embryo decreased gradually after gastrulation, in accordance with the gradual decrease of the 38 kDa protein per embryo reported in our earlier study. Because pharmacological inhibition of translation induces gastrulation arrest in wild-type embryos, it is reasonable to conclude that the mutant embryos arrest in development due to the lack of Ef1delta2 that is indispensable for translation. Thus, the present study provides the first molecular information on the cause of the gastrulation-defective mutation in Amphibia.

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.

A novel method for microinjection into Xenopus eggs and embryos supported in methylcellulose solution.

We have developed a novel method for microinjection into Xenopus eggs and embryos. Microinjection was performed into eggs or embryos that were placed in wells (ca. 2.5 mm x 2.5 mm x 0.8 mm for each well) at the bottom of a commercially available hybridoma dish, which was filled with 1.5% methylcellulose solution. Eggs or embryos, rotated to a desired orientation in the viscous methylcellulose solution with a hair loop, could remain in the orientation for more than twenty minutes. Accordingly, samples such as mRNAs, DNAs, proteins and antisense morpholino oligonucleotides could be easily and efficiently microinjected into any part (animal, vegetal, dorsal, lateral or ventral) of more than five hundred eggs and embryos in one day. In addition, methylcellulose did not interfere with the development of the injected eggs and embryos.

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 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.

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.

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.

A novel gene, the protein product of which is mainly expressed in germline cells and in the dorsal structures of Xenopus.

A novel cDNA was isolated from a Xenopus cDNA library using an antibody recognizing the germ plasm-specific germinal granules. The protein product of the gene ( Xdsg) was detected on the germinal granules of the cleaving embryo as determined by immunoelectron microscopy. The spatio-temporal distribution of the RNA transcript and protein product of the gene was investigated in oocytes and embryos. The protein was detected in the germ plasm and the germline cells from the cleavage to the tadpole stages, while both the protein and RNA transcript were prominent in the dorsal structures from the tailbud stage onward. The present study has shown for the first time that the protein product of the Xdsg gene was present on the germinal granules in Xenopus.

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.

Possible role of the 38 kDa protein, lacking in the gastrula-arrested Xenopus mutant, in gastrulation.

An acidic, 38 kDa protein that is present in Xenopus wild-type embryos has been previously shown to be lacking in gastrula-arrested mutant embryos. To gain understanding of the role of this protein, its spatio-temporal distribution and involvement in gastrulation was investigated using the monoclonal antibody (9D10) against it. The protein was prominent in the cortical cytoplasm of cells facing the outside in the animal hemisphere of embryos until the gastrula stage, and in ciliated epithelial cells of embryos at stages later than the late neurula. When the 9D10 antibody was injected into fertilized wild-type eggs, they cleaved normally, but most of them had arrested development, always at the early stage of gastrulation, as in the mutant embryos. In contrast, the majority of the control antibody-injected eggs gastrulated normally and developed further. Cytoskeletal F-actin, which was mainly observed in the area beneath the plasma membrane facing the outside of the epithelial layer of not only the dorsal involuting marginal zone but also the dorsal, vegetal cell mass of the control antibody-injected embryos at the early gastrula stage, was scarcely recognized in the corresponding area of the 9D10 antibody-injected embryos. It is likely that the paucity of the F-actin caused by the 9D10 antibody inhibition of the 38 kDa protein might lead to a failure of cell movement in gastrulation, resulting in developmental arrest.

Spatio-temporal distribution of the protein of Xenopus vasa homologue (Xenopus vasa-like gene 1, XVLG1) in embryos.

In order to know when the protein of Xenopus vasa homolog (Xenopus vasa-like gene 1, XVLG1) first appears in germ line cells and whether the protein is also present in somatic cells as is vasa protein in Drosophila, the spatio-temporal distribution of the protein in Xenopus embryos was carefully investigated by fluorescent microscopy. Part of the observation was performed by whole-mount immunocytochemistry and immunoblotting. A distinct fluorescence of XVLG1 protein was first recognized in a juxta-nuclear location of germ line cells or presumptive primordial germ cells (pPGC) at stage 12 (late gastrula) and remained associated with the pPGC or primordial germ cells (PGC) throughout the following stages until stage 46 (feeding tadpole). In contrast, weak fluorescence was seen in the animal hemisphere rather than in the vegetal hemisphere of cleaving embryos and in the perinuclear region of somatic cells at stages 10-42 (early gastrula to young tadpole), respectively. Nearly the same pattern as revealed by fluorescence was seen by whole-mount immunocytochemistry, except that a small amount of XVLG1 protein seemed to be present in the germ plasm and pPGC of embryos earlier than stage 12. The presence of the protein in the somatic cells and the PGC was also shown by immunoblotting.

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.

Presumptive Primordial Germ Cells (pPGCs) and PGCs in Tadpoles from UV-irradiated embryos of Xenopus: (UV-irradiation/primordial germ cell/monoclonal antibody/complete sterility/Xenopus laevis).

In order to determine whether or not tadpoles that once lacked primordial germ cells (PGCs) in the genital ridges and dorsal mesentery as a result of ultraviolet (UV) irradiation subsequently contained germ cells at more advanced stages of larval development, the numbers of presumptive PGCs or PGCs were carefully examined in Xenopus tadpoles at Nieuwkoop and Faber's stage 35/36-52 that developed normally from UV-irradiated eggs. No late-appearing germ cells were observed in almost all the UV-irradiated tadpoles examined at stages 49-52. This same population had completely lacked PGCs at about stage 46. Moreover, presumptive PGCs (pPGCs) or cells with granular cytoplasm that reacted with a monoclonal antibody specific for the germ plasm of cleaving Xenopus eggs stayed in the central part of the endoderm cell mass in the irradiated tadpoles at stage 35/36, when the majority of those cells were located in the dorsal part of the endoderm in unirradiated controls. Furthermore, in the irradiated embryos pPGCs were demonstrated to decrease in number with development and eventually to disappear in tadpoles at about stage 40. The results strongly suggest that UV irradiation under the conditions used here totally eliminated germline cells from the irradiated animals.

Xenopus Heterochronic Presumptive Primordial Germ Cells (pPGCs) Implanted in the Correct Position in Host Neurula Embryos can Differentiate into PGCs: (Xenopus laevis, PGCs/heterochronic presumptive PGCs/explant/germ plasm-bearing cells).

Presumptive primordial germ cells (pPGCs) in explants, derived from single germ plasm-bearing cells of Xenopus 32-cell embryos, at the equivalent of neurula stage (stage 20) in control embryos (designated as 'stage-20' explants) were demonstrated to be able to differentiate into PGCs, when implanted into a prospective place of pPGCs in host embryos (stage 20) (Ikenishi & Tsuzaki, 1988). According to a recent proposal that individual early embryonic cells in Xenopus, at both in vivo and in vitro, are able to measure elapsed time since fertilization (Cooke and Smith, 1990), the result means that the implanted pPGCs having the same elapsed time as the host embryos (isochronic pPGCs) could differentiate into PGCs. In the present study, in order to know whether the compatibility in elapsed times of implanted pPGCs and host embryos is necessary for the differentiation of PGCs, labelled, heterochronic pPGCs in 'stages 12-33/34' explants were implanted into unlabelled, host neurulae (stage 19). Those heterochronic pPGCs could differentiate into PGCs like isochronic pPGCs in 'stage-19' explants as the control. By comparing the average diameters and yolk contents of labelled PGCs with those of unlabelled, host ones in experimental tadpoles, the possibility that a certain mechanism modulating the elapsed time of heterochronic pPGCs to that of host pPGCs is present in host embryos was also suggested.

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.

Direct Evidence for the Presence of Germ Cell Determinant in Vegetal Pole Cytoplasm of Xenopus laevis and in a Subcellular Fraction of It: (Xenopus laevis/germ cell determinant/germ plasm/PGC induction).

To test for the presence of germ cell determinant in Xenopus embryos, vegetal pole cytoplasm containing the "germ plasm", or a subcellular fraction of it, was microinjected into single somatic blastomeres isolated from 32-cell embryos. Injected or non-injected (control) blastomeres were cultured in 3 H-thymidine until normal control embryos reached the neurula stage. The labeled explants were then implanted into unlabeled host neurulae, which were allowed to develop to the tadpole stage. Labeled PGCs of explant origin in the genital ridges of the experimental tadpoles were examined by autoradiography. Isolated blastomeres were injected with vegetal pole cytoplasm of 32-cell embryos or with a 20,000 g pellet made from vegetal pole cytoplasm of 2-cell embryos. Labeled PGCs were found in 7.6% and 2.3% of the experimental tadpoles, respectively. No labeled PGCs were found in the control tadpoles, except for one tadpole in the first experiment. These results strongly suggest that the vegetal pole cytoplasm and its subcellular fractions act as germ cell determinant.

A Possibility of an in Vitro Differentiation of Primordial Germ Cells (PGCs) from Blastomeres Containing "Germinal Plasm" of Early Cleavage Stage in Xenopus laevis: (xenopus laevis/"germinal plasm"/germinal granule/PGCs).

The blastomeres containing the "germinal plasm" were isolated from 32-cell stage Xenopus embryos and cultured in vitro for various periods of time till the control embryos developed to stage 28, 33/34, 40 and 45, respectively. The cells containing the plasm in the 'stage-28', '33/34' and '40' explants were similar in external shape, and in distribution in the spherical endodermal cell mass to the presumptive primordial germ cells (pPGCs) in normal embryos of the corresponding stages. In addition, the cells in explants as well as the pPGCs were separated by a large intercellular space from the surrounding endodermal cells. The change in proportion of the compact or the loosely structured germinal granules and the irregularly shaped-stringlike bodies (ISBs) occurred in the cells of the explants with the prolongation of the culture period. In the cells of the 'stage-45' explant as well as in the PGCs of normal stage-45 tadpoles the ISBs and "granular materials" replace those germinal granules. These facts lead to the conclusion that the change of the germinal granules through the ISBs, to the "granular materials", noticed in the normal course of differentiation of pPGCs into PGCs (see (1)), also takes place in the cells of the explants during the culture. Therefore, it is likely that the cells in the explants are genuine pPGCs or PGCs. This is the first demonstration of a possibility of the in vitro differentiation of PGCs from the blastomeres containing the "germinal plasm" of early cleavage stage.

CORTICAL GRANULES PERSISTING IN GERM CELLS OF XENOPUS LAEVIS AFTER THE CLEAVAGE STAGES.

Cortical granules were demonstrated, in two successive Epon sections (0.7 µm thick) stained with PAS reagent and the triple staining method respectively, to persist beyond the cleavage stages of development to the tadpole stages in Xenopus laevis. They were also examined by electron microscope. The granules which are similar both cytochemically and ultrastructurally to the cortical granules of the unfertilized eggs were observed not only in germ cells, pPGCs and PGCs, but also in somatic cells at all the stages examined. An ultrastructural similarity between the granules found in the PGCs at the tadpole stages and chromatoid body was discussed.

LOCATION AND ULTRASTRUCTURE OF PRIMORDIAL GERM CELLS (PGCs) IN AMBYSTOMA MEXICANUM.

The location and ultrastructure of the primordial germ cells (PGCs) were studied in Ambystoma mexicanum larvae of stages 23 to 47. PGCs were found in the spaces between the endodermal cell mass and the lateral plate mesoderm at stages 23 to 35. Some of the PGCs at stage 35, and most of them at stages 40 and 42, were located near the Wolffian duct. At stages 46 and 47 all the PGCs were situated in the genital ridges. Cilia, which have hitherto never been reported in PGCs, were occasionally seen in PGCs of Ambystoma from stage 23 till stage 46. No "germinal plasm" was found in the PGCs prior to stage 40. Specific structures or "nuage material", corresponding to the germinal granules or their derivatives in Xenopus, were first recognized in the vicinity of the nucleus at stage 40. Between stages 40 and 46, the amount of "nuage material" markedly increased. It was finally localized mainly in "intermitochondrial spaces". A possible transfer of material from the nucleus to the cytoplasm or vice versa through nuclear pores was first noticed at stage 40, the material concerned being quite similar in ultrastructure to the "nuage material".