Interspecific transplantation of polar plasm between Drosophila embryos

Posterior polar plasm of the Drosophila egg has been shown to function autonomously in germ cell determination after transplantation to either the anterior or mid-ventral region of the early embryo. By means of similar transplantations, we have tested the ability of polar plasm of Drosophila immigrans to induce the formation of pole cells in a Drosophila melanogaster embryo. After the transplantation of polar plasm, "hybrid" pole cells were found in which both pole cell-specific organelles, the polar granules and nuclear body, were structurally similar to those characteristic of the transplanted cytoplasm. In order to determine whether these hybrid cells can function as germ cell precursors, these cells were transplanted to the posterior tip of genetically marked embryos. Approximately 5% of the flies obtained from embryos receiving potential pole cells produce offspring derived from the induced pole cells. This result demonstrates that polar plasm can function in interspecific species combinations and indicates that the molecular mechanisms of germ cell determination are conservative in evolution. Finally, in order to test whether there is any evidence for cytoplasmic inheritance of polar granules, embryos derived from hybrid pole cells were examined for their polar granule morphology. The fine structure of the granules conformed to that of the nucleus. Thus, no evidence was found for the cytoplasmic inheritance of these particular organelles.     

The ontogeny of germ plasm during oogenesis in Drosophila

Primordial germ cells can be induced at both the anterior and ventral region of the Drosophila egg by transplanted posterior polar plasm. Two questions arise from these results: (1) Is fertilization required for germ plasm to be functional, and (2) at what stage during oogenesis does the posterior polar plasm become established as a germ-cell determinant?Polar plasm from unfertilized eggs and from oocytes at stage 10 to 14 of Drosophila melanogaster was implanted into the anterior region of cleavage embryos. Some injected embryos were analyzed at the ultrastructural level during blastoderm formation. Polar plasm from unfertilized eggs and from oocytes of stages 13 and 14 was found to be integrated into several anterior cells that resembled morphologically normal pole cells. The formation of such cells, however, could not be detected in embryos injected with polar plasm from oogenetic stages 10 to 12. Experimentally induced pole cells proved to be capable of differentiating into functional germ cells when cycled through the germ line of genetically different host embryos. About 5% of the flies developing from these embryos produced progeny that originated from the induced pole cells. Germ-line mosaicism in those flies also could be detected histochemically in their gonads. No germ cells were recovered with polar plasm transplants from oogenetic stages 10 to 12.The results show that posterior polar plasm of the unfertilized egg is functional in germ-cell determination, and that prior to egg maturation this cytoplasm has already acquired its determinative ability. This is the first demonstration that specific developmental information stored in the cytoplasm can be traced back to a particular region of the oocyte.     

The germ cell-less gene product: a posteriorly localized component necessary for germ cell development in Drosophila.

The first cell fate specification process in the Drosophila embryo, formation of the germline precursors, requires posteriorly localized germ plasm. We have cloned a gene, germ cell-less (gcl), required for germline formation. Posterior localization of the gcl messenger RNA (mRNA) requires the function of those genes essential for the localization of both nanos RNA, which specifies the abdomen, and the germ cell determinants. Mothers with reduced gcl function give rise to sterile adult progeny that lack germ cells. In embryos with reduced maternal gcl product, the germ cell precursors fail to form properly. Consistent with this phenotype, gcl protein specifically associates with those nuclei that later become the nuclei of the germ cell precursors. These observations suggest that gcl functions in the germ cell specification pathway.     

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.     

Oskar organizes the germ plasm and directs localization of the posterior determinant nanos

Oskar is one of seven Drosophila maternal-effect genes that are necessary for germline and abdomen formation. We have cloned oskar and show that oskar RNA is localized to the posterior pole of the oocyte when germ plasm forms. This polar distribution of oskar RNA is established during oogenesis in three phases: accumulation in the oocyte, transport toward the posterior, and finally maintenance at the posterior pole of the oocyte. The colocalization of oskar and nanos in wild-type and bicaudal embryos suggests that oskar directs localization of the posterior determinant nanos. We propose that the pole plasm is assembled stepwise and that continued interaction among its components is required for germ cell determination.     

Immunocytological and FISH analysis of pole cell formation and soma elimination of germ line-limited chromosomes in the chironomid Acricotopus lucidus

In the chironomid Acricotopus lucidus, germ line-soma differentiation becomes evident with the formation of the pole cells and the elimination of the germ line-limited chromosomes (Ks) from the future somatic nuclei of the embryo. Unlike in Drosophila, the early nuclear divisions do not proceed synchronously in A. lucidus. Usually, only one nucleus, the future pole nucleus, penetrates into the pole plasm, always at a telophase stage in the course of a regular mitosis. This happens by chance, depending on the orientation of the mitotic spindles of the early syncytial nuclei. Consequently, the time and the cell cycle at which a nucleus reaches the pole plasm, and pole cells arise, vary between embryos of the same oviposition. When entering the first germ line mitosis, while polar plasm and syncytial plasm are still not separated, some future somatic nuclei begin to eliminate their Ks. While the soma chromosomes (Ss) undergo normal anaphasic migration to the opposite poles, the K chromatids do not separate and remain in the equatorial plane, as demonstrated by fluorescence in situ hybridization using germ line-specific DNA probes. The elimination of the Ks does not occur at the same time in all future somatic nuclei. Nondisjunction of Ks was observed in the first mitosis of the pole nucleus, leading to primordial germ cells with different compositions of their K complements. The pattern and timing of elimination mitoses in the embryos indicate that each of the future somatic nuclei seems to regulate the elimination of the Ks autonomously.     

tudor, a gene required for assembly of the germ plasm in Drosophila melanogaster

Developmental analysis of a newly isolated maternal effect grandchildless mutant, tudor (tud), in Drosophila melanogaster indicates that tud+ activity is required during oogenesis for the determination and/or formation of primordial germ cells (pole cells) and for normal embryonic abdominal segmentation. Regardless of their genotype, progeny of females homozygous for strong alleles (tud1 and tud3) never form pole cells, apparently lack polar granules in the germ plasm, and approximately 40% of them die during late embryogenesis exhibiting severe abdominal segmentation pattern defects. Females carrying weak allele, tud4, produce progeny with some functional pole cells and form polar granules approximately one-third the size of those observed in wild-type oocytes and embryos. No segmentation abnormalities are observed in the inviable embryos derived from tud4/tud4 females.     

Drosophila pericentrin‐like protein promotes the formation of primordial germ cells

Primordial germ cells (PGCs) are the precursors to the adult germline stem cells that are set aside early during embryogenesis and specified through the inheritance of the germ plasm, which contains the mRNAs and proteins that function as the germline fate determinants. In Drosophila melanogaster, formation of the PGCs requires the microtubule and actin cytoskeletal networks to actively segregate the germ plasm from the soma and physically construct the pole buds (PBs) that protrude from the posterior cortex. Of emerging importance is the central role of centrosomes in the coordination of microtubule dynamics and actin organization to promote PGC development. We previously identified a requirement for the centrosome protein Centrosomin (Cnn) in PGC formation. Cnn interacts directly with Pericentrin‐like protein (PLP) to form a centrosome scaffold structure required for pericentriolar material recruitment and organization. In this study, we identify a role for PLP at several discrete steps during PGC development. We find PLP functions in segregating the germ plasm from the soma by regulating microtubule organization and centrosome separation. These activities further contribute to promoting PB protrusion and facilitating the distribution of germ plasm in proliferating PGCs.     

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.     

Evolution of predetermined germ cells in vertebrate embryos: implications for macroevolution

The germ line is established in animal embryos with the formation of primordial germ cells (PGCs), which give rise to gametes. Therefore, the need to form PGCs can act as a developmental constraint by inhibiting the evolution of embryonic patterning mechanisms that compromise their development. Conversely, events that stabilize the PGCs may liberate these constraints. Two modes of germ cell determination exist in animal embryos: (a) either PGCs are predetermined by the inheritance of germ cell determinants (germ plasm) or (b) PGCs are formed by inducing signals secreted by embryonic tissues (i.e., regulative determination). Surprisingly, among the major extant amphibian lineages, one mechanism is found in urodeles and the other in anurans. In anuran amphibians PGCs are predetermined by germ plasm; in urodele amphibians PGCs are formed by inducing signals. To determine which mechanism is ancestral to the tetrapod lineage and to understand the pattern of inheritance in higher vertebrates, we used a phylogenetic approach to analyze basic morphological processes in both groups and correlated these with mechanisms of germ cell determination. Our results indicate that regulative germ cell determination is a property of embryos retaining ancestral embryological processes, whereas predetermined germ cells are found in embryos with derived morphological traits. These correlations suggest that regulative germ cell formation is an important developmental constraint in vertebrate embryos, acting before the highly conserved pharyngula stage. Moreover, our analysis suggests that germ plasm has evolved independently in several lineages of vertebrate embryos.     

Mitochondrial small ribosomal RNA is present on polar granules in early cleavage embryos of Drosophila melanogaster

In Drosophila, formation of the germline progenitors, the pole cells, is induced by polar plasm localized in the posterior pole region of early embryos. The polar plasm contains polar granules, which act as a repository for the factors required for pole cell formation. It has been postulated that the factors are stored as mRNA and are later translated on polysomes attached to the surface of polar granules. Here, the identification of mitochondrial small ribosomal RNA (mtsrRNA) as a new component of polar granules is described. The mtsrRNA was enriched in the polar plasm of the embryos immediately after oviposition and remained in the polar plasm throughout the cleavage stage until pole cell formation. In situ hybridization at an ultrastructural level revealed that mtsrRNA was enriched on the surface of polar granules in cleavage embryos. Furthermore, the localization of mtsrRNA in the polar plasm depended on the normal function of oskar, vasa and tudor genes, which are all required for pole cell formation. The temporal and spatial distribution of mtsrRNA is essentially identical to that of mitochondrial large ribosomal RNA (mtlrRNA), which has been shown to be required for pole cell formation. Taken together, it is speculated that mtsrRNA and mtlrRNA are part of the translation machinery localized to polar granules, which is essential for pole cell formation.     

Tudor protein is essential for the localization of mitochondrial RNAs in polar granules of Drosophila embryos

In Drosophila, polar plasm contains polar granules, which deposit the factors required for the formation of pole cells, germ line progenitors. Polar granules are tightly associated with mitochondria in early embryos, suggesting that mitochondria could contribute to pole cell formation. We have previously reported that mitochondrial large and small rRNAs (mtrRNAs) are transported from mitochondria to polar granules prior to pole cell formation and the large rRNA is essential for pole cell formation. Here we show that the localization of mtrRNAs is diminished in embryos laid by tudor mutant females, although the polar granules are maintained. We also found that Tud protein was colocalized with mtrRNAs at the boundaries between mitochondria and polar granules when the transport of mtrRNAs takes place. These observations suggest that Tud mediates the transport of mtrRNAs from mitochondria to polar granules.     

A late phase of germ plasm accumulation during Drosophila oogenesis requires lost and rumpelstiltskin

Asymmetric mRNA localization is an effective mechanism for establishing cellular and developmental polarity. Posterior localization of oskar in the Drosophila oocyte targets the synthesis of Oskar to the posterior, where Oskar initiates the assembly of the germ plasm. In addition to harboring germline determinants, the germ plasm is required for localization and translation of the abdominal determinant nanos. Consequently, failure of oskar localization during oogenesis results in embryos lacking germ cells and abdominal segments. oskar accumulates at the oocyte posterior during mid-oogenesis through a well-studied process involving kinesin-mediated transport. Through live imaging of oskar mRNA, we have uncovered a second, mechanistically distinct phase of oskar localization that occurs during late oogenesis and results in amplification of the germ plasm. Analysis of two newly identified oskar localization factors, Rumpelstiltskin and Lost, that are required specifically for this late phase of oskar localization shows that germ plasm amplification ensures robust abdomen and germ cell formation during embryogenesis. In addition, our results indicate the importance of mechanisms for adapting mRNAs to utilize multiple localization pathways as necessitated by the dramatic changes in ovarian physiology that occur during oogenesis.     

Mitochondrial trafficking through Rhot1 is involved in the aggregation of germinal granule components during primordial germ cell formation in Xenopus embryos

In many animals, the germ plasm is sufficient and necessary for primordial germ cell (PGC) formation. It contains germinal granules and abundant mitochondria (germline‐Mt). However, the role of germline‐Mt in germ cell formation remains poorly understood. In Xenopus, the germ plasm is distributed as many small islands at the vegetal pole, which gradually aggregates to form a single large mass in each of the four vegetal pole cells at the early blastula stage. Polymerized microtubules and the adapter protein kinesin are required for the aggregation of germ plasm. However, it remains unknown whether germline‐Mt trafficking is important for the cytoplasmic transport of germinal granules during germ plasm aggregation. In this study, we focused on the mitochondrial small GTPase protein Rhot1 to inhibit mitochondrial trafficking during the germ plasm aggregation. Expression of Rhot1ΔC, which lacks the C‐terminal mitochondrial transmembrane domain, inhibited the aggregation of germline‐Mt during early development. In Rhot1‐inhibited embryos, germinal granule components did not aggregate during cleavage stages, which reduced the number of PGCs on the genital ridge at tail‐bud stage. These results suggest that mitochondrial trafficking is involved in the aggregation of germinal granule components, which are essential for the formation of PGCs.