Functional gametes derived from explants of single blastomeres containing the "germ plasm" in Xenopus laevis: a genetic marker study.

Single blastomeres containing the "germ plasm" were isolated from 32-cell embryos of Xenopus albino (ap/ap) or wild type and cultured in vitro until the corresponding normal control embryos reached the neurula stage. The resulting explants from albinos were implanted into wild-type host neurulae and vice versa. The formation of functional gametes, eggs or sperm, of donor type was tested when the operated host embryos had reached sexual maturity. The color of the eggs laid by the experimental females and the presence or absence of melanophores in the epidermis and of pigment granules in the eyes of hatched larvae from matings of the experimental males with albino females made possible the identification of donor-type gametes. Twelve males and 12 females of the wild-type hosts, and 16 males and 14 females of the albino hosts survived. Six animals produced donor-type eggs or sperm, most of them being germ line chimeras. This shows that functional gametes can develop from explants derived from single blastomeres containing the "germ plasm."

Germ plasm in Caenorhabditis elegans, Drosophila and Xenopus.

Special cytoplasm, called germ plasm, that is essential for the differentiation of germ cells is localized in a particular region of Caenorhabditis elegans, Drosophila and Xenopus eggs. The mode of founder cell formation of germline, the origin and behavior of the germline granules, and the molecules localized in germline cells are compared in these organisms. The common characteristics of the organisms are mainly as follows. First, the founder cells of germline are established before the initiation of gastrulation. Second, the germline granules or their derivatives are always present in germline cells or germ cells throughout the life cycle in embryos, larvae, and adults. Lastly, among the proteins localized in the germ plasm, only Vasa protein or its homolog is detected in the germline cells or germ cells throughout the life cycle. As the protein of vasa homolog has been reported to be also localized in the germline-specific structure or nuage in some of the organisms without the germ plasm, the possibility that the mechanism for differentiation of primordial germ cells is basically common in all organisms with or without the germ plasm is discussed.

A possible maternal-effect mutant of Xenopus laevis I. Cytological and biochemical analyses of the unfertilized eggs and embryos.

In an effort to define the cause of the developmental arrest of offspring from a certain Xenopus female (designated as No. 65), we have examined eggs and embryos from the female both cytologically and biochemically. Light and electron microscopic observations revealed that all of the blastomeres from embryos of female No. 65 had multiple small spherical nuclei, while wild-type counterparts had a single lobulated nucleus. Two-dimensional gel electrophoretic analyses demonstrated that a major acidic protein, whose molecular weight was 38 kDa, was always found in samples from wild-type unfertilized eggs and embryos, but was not recognizable in those from female No. 65. These facts, coupled with the death of the offspring at gastrulation, suggest the possibility that female No. 65 carries a mutation of the ova-deficient type.

Monoclonal antibody production against a subcellular fraction of vegetal pole cytoplasm containing the germ plasm of Xenopus 2-cell eggs.

In attempts to understand the molecular nature of substances localized in the germ plasm (GP), monoclonal antibodies against a subcellular fraction of vegetal pole cytoplasm containing the GP of Xenopus 2-cell eggs were produced. The monoclonal antibody produced by the hybridoma cell line (designated as no 48) reacted specifically with the GP of embryos at the cleavage stages. Beyond the gastrula stage, the antibody reacted with not only the GP of germ line cells but also the cytoplasm of somatic cells. By immunoblotting analysis with two-dimensional (2-D) gel the antibody was shown to react with two protein spots of approx. 40 kDa, with a pI of 6.0-6.5.

Differentiation of presumptive primordial germ cell (pPGC)-like cells in explants into PGCs in experimental tadpoles.

A single blastomere containing the "germ plasm" of 32-cell stage Xenopus embryos was cultured with [3H]thymidine until the control embryos developed to the neurula stage. The explants, showing a spherical mass in which the nuclei of all cells were labeled, were implanted into the prospective place of presumptive primordial germ cells (pPGCs) in the endodermal cell mass of unlabeled host embryos of the neurula stage. Labeled PGCs as well as unlabeled, host PGCs were found in the genital ridges of experimental tadpoles. This indicates that the precursor of germ cells, corresponding to pPGCs in normal embryos of the neurula stage, in the explants migrated to genital ridges just at the right moment to become PGCs, and suggests that the developmental process progressed normally, even in the explants, as far as the differentiation of pPGCs is concerned.

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 or 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.

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.

Spatio-temporal expression of Xenopus vasa homolog, XVLG1, in oocytes and embryos: the presence of XVLG1 RNA in somatic cells as well as germline cells.

The expression of Xenopus vasa homolog or XVLG1 was examined in oocytes and embryos by whole-mount in situ hybridization and reverse transcription polymerase chain reaction (RT-PCR). To confirm the results in embryos, both methods were also applied to explants of germ plasm-bearing cells (GPBC) from 32-cell embryos and to those of partial embryos deprived of GPBC. By hybridization, XVLG1 ribonucleic acid (RNA) was shown to be present throughout the cytoplasm in oocytes at stages I-III, except for the mitochondrial cloud. It was barely recognizable in a portion of germline cells of embryos at specific stages, notwithstanding that XVLG1 protein was present in those cells almost throughout their life-span. A weak signal for the RNA was detectable in some of the presumptive primordial germ cells (pPGC, descendants of GPBC from the gastrula stage onward) from the late gastrula (stage 12) to the hatching tadpole stage (stage 33/34), and in some of the PGC at stages 49-50. The results for pPGC were confirmed by the hybridization of explants of GPBC at equivalent stages in control embryos. In contrast, XVLG1 RNA was detected in certain somatic cells of embryos until stage 46. These observations were supported in part by the results of RT-PCR for embryos and explants. The possible role of the product of XVLG1 was reconsidered given its presence in both germline and somatic cells.

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.

Isolation and characterization of a novel gene of the DEAD box protein family which is specifically expressed in germ cells of Xenopus laevis.

We have isolated a cDNA of the DEAD protein family from a Xenopus ovary cDNA library. Northern hybridization revealed that the mRNA corresponding to the cDNA [XVLG1 (Xenopus vasa-like gene)] was specifically expressed in adult testis and ovary. The deduced amino acid sequence of XVLG1 was relatively homologous to that of Drosophila vasa. XVLG1 protein was found to be expressed exclusively in adult testis and ovary by immunoblotting with a monoclonal antibody against XVLG1 protein. Immunocytological study showed that XVLG1 protein was expressed in oogonia, oocytes, spermatogonia, spermatocytes, and also in primordial germ cells in stage 46 tadpoles. The monoclonal antibody used in this study specifically stains germ line cells and is useful both as a germ cell lineage marker and in biochemical studies of germ cells.

Observations on the migration and proliferation of gonocytes in Xenopus laevis.

The process of primordial germ cell formation in the normal course of development of Xenopus laevis was examined with a light microscope on paraffin and Epon sections of embryos or tadpoles, extending over the period from the gastrula to the feeding tadpole stage. Positional changes of gonocytes with development were nearly the same as those reported on the same species by Blackler (1958) and Whitington & Dixon (1975). The following points were newly demonstrated. Gonocytes which have been located in a deep endodermal position till mid tail-bud stage come to be located in a rather peripheral region of the endoderm cell mass at stage 31 (late tail-bud), suggesting that the initial step of migration of the gonocytes towards the future genital ridge has already begun at this stage. Gonocytes at stages 33/34 and 35/36 were observed in a more dorsal part of the endoderm than at stage 31. Gonocytes which seem to have begun their migration are roundish in external shape and have a large intercellular space around them. At stage 40 gonocytes were located in the dorsal endodermal crest, and at stage 41 gonocytes were found with cell bodies extending over both the dorsal endoderm crest and the dorsal mesentery, indicating that the separation of the gonocytes from the endoderm was in progress at this stage. The present results seem to indicate that gonocytes migrate not passively but actively from the deep endodermal position to the genital ridge, passing through the dorsal mesentery. Counting the number of gonocytes at successive stages of development revealed that gonocytes proliferated exponentially throughout the developmental stages from gastrula to tadpole.

A decreased number of primordial germ cells and the small numbers and reduced sizes of germinal granules in the periodic albino mutant of Xenopus laevis.

Light and electron microscopy were used to examine the cause of the small number of primordial germ cells (PGCs) that were unexpectedly found in periodic albino (a(p)/a(p)) tadpoles of Xenopus laevis. The observations revealed that the volumes of germ plasm were not significantly different between a(p)/a(p) and wild-type eggs and that the germ plasm of ap/ap eggs contained a smaller number of germinal granules than are present in wild-type eggs and the granules were also smaller than in the wild-type eggs. It was concluded that the number of PGCs might be primarily determined by the number and size of germinal granules.

A possible maternal-effect mutant of Xenopus laevis: II. Studies of RNA synthesis in dissociated embryonic cells.

Embryos from a female of Xenopus laevis (designated as no. 65) arrest development at gastrulation and are assumed to be ova-deficient mutant. We dissociated these embryos and studied RNA synthesis at different stages. The cells from the ova-deficient embryos reaggregated quite actively as wild-type embryo cells until the late gastrula stage. RNA synthesis was normal at the early blastula stage but greatly inhibited by the late blastula (stage 9.5) stage, when the synthesis of DNA and protein was still not inhibited appreciably. Thus, inhibition in RNA synthesis appears to be the first manifestation of the maternal defect that occurs before the gastrulation arrest.