Differences in Fine Structures between Normal and RNA-induced Drosophila Pole Cells

Fine structures were compared between normal pole cells and those induced in embryos that had been uv-irradiated and then injected with intact polar plasm or with poly(A)⁺RNA extracted from cleavage embryos. Nuclei in nomal pole cells were spherical. In contrast, those in the induced pole cells were deformed to variable extents depending on materials injected with. Polar granules were smaller in pole cells induced by injection of poly(A)⁺RNA than in normal pole cells. The size of polar granules in polar-plasm-induced pole cells was intermediate between those in poly(A)⁺RNA-induced and normal pole cells. Small polar granules were observed in posterior cells of embryos uv-irradiated, nevertheless those cells were columnar and with identical morphology to somatic cells. Nuclear bodies showed a similar tendency in size differences as observed in polar granules in three types of pole cells observed.     

Delocalization of polar plasm components caused by grandchildless mutations, gs(1)N26 and gs(1)N441, in Drosophila melanogaster

Two maternal-effect grandchildless (gs) mutations of Drosophila melanogaster, gs(1)N26 and gs(1)N441, cause delay in nuclear arrival at the polar plasm. In mutant embryos, polar plasm loses its ability to induce pole cells during retarded nuclear migration to the posterior pole of embryos. In the present study, it was shown that in N26 and N441 embryos, mitochondrial large rRNA (mtlrRNA), an essential factor for pole cell formation, is delocalized during the delay in nuclear arrival. This suggests that the loss of mtlrRNA causes failure of the mutants to form pole cells. Furthermore, it was shown that all of the other polar plasm components examined, namely Vasa protein, Germ cell-less protein, nanos mRNA and Polar granule component RNA start to be delocalized during the delay in nuclear arrival. This suggests that polar plasm integrity is not maintained in mutant embryos. It was finally shown that Vas is also delocalized in embryos that are inhibited to form pole cells by reducing the amount of mtlrRNA. This indicates that the segregation of polar plasm into pole cells is required to maintain polar plasm integrity. The mechanism regulating polar plasm integrity in embryos is discussed.     

Isolation of a factor inducing pole cell formation from Drosophila embryos

Summary

An attempt to isolate an ooplasmic factor active in inducing pole cells in Drosophila embryos is described. With the help of a bioassay system, we demonstrated that RNA extracted from embryos was active in inducing pole cells. These RNA-induced pole cells were morphologically identical to the normal ones. In addition, a local application of cycloheximide suggests that translation in the posterior pole cytoplasm is a precondition for pole cell formation.     

Functions of maternal mRNA as a cytoplasmic factor responsible for pole cell formation in Drosophila embryos

Injection of mRNA extracted from Drosophila cleavage embryos or mature oocytes restored pole cell-forming ability to embryos that had been deprived of this ability by uv irradiation. However, mRNA extracted from blastoderms did not show the restoration activity. Pole cells thus formed in uv-irradiated embryos bear similarities to normal pole cells both in their morphology and their ability to migrate to the gonadal rudiments. But this mRNA does not appear to be capable of rescuing uv-induced sterility, or inducing pole cells in the anterior polar region.     

Analysis of the Drosophila maternal effect mutant MAT(3)1 by pole cell transplantation experiments

Summary Pole cell transplantations were used to construct germ line mosaics of the Drosophila melanogaster maternal effect mutant mat(3)1. The mutant is of particular interest since the development of embryos derived from homozygous mat(3)1 females is arrested at the pole cell stage. Such embryos form exclusively pole cells and no blastoderm cells. By means of germ line mosaics we could demonstrate the primary target tissue of mutant gene expression. For normal development the mat(3)1 ⁺gene has to be expressed in the germ line. Pole cells formed in defective embryos derived from homozygous mutant mothers were transplanted into normal recipient embryos to test their developmental potential. Heterozygous mat(3)1 pole cells were found to form fertile gametes in both sexes whereas homozygous mat(3)1 pole cells form fertile gametes only in males. The lack of progeny derived from homozygous mat(3)1 donor pole cells in recipient females further demonstrates the germ line autonomy of the mat(3)1 mutation. Pole cells from defective embryos that are transplanted into normal hosts colonize the gonads with the same frequency as donor pole cells derived from normal embryos. This indicates that mat(3)1 derived pole cells are normal with respect to their function as germ cells and that the mat(3)1 mutant might therefore offer a convenient source for the mass isolation of functional pole cells.     

Drosophila tudor is essential for polar granule assembly and pole cell specification, but not for posterior patterning

Pole cells and posterior segmentation in Drosophila are specified by maternally encoded genes whose products accumulate at the posterior pole of the oocyte. Among these genes is tudor (tud). Progeny of hypomorphic tud mothers lack pole cells and have variable posterior patterning defects. We have isolated a null allele to further investigate tud function. While no pole cells are ever observed in embryos from tud-null mothers, 15% of these embryos have normal posterior patterning. OSKAR (OSK) and VASA (VAS) proteins, and nanos (nos) RNA, all initially localize to the pole plasm of tud-null oocytes and embryos from tud-null mothers, while localization of germ cell-less (gcl) and polar granule component (pgc), is undetectable or severely reduced. In embryos from tud-null mothers, polar granules are greatly reduced in number, size, and electron density. Thus, tud is dispensable for somatic patterning, but essential for pole cell specification and polar granule formation.     

Studies on the expression of intracellular and surface polarity in animal pole cells of Xenopus embryos cultured on various substrata

The expression of intracellular and surface polarity in animal pole cells of Xenopus embryos (stage 10) cultured on various substrata was studied by electron microscopy. When animal pole cells of Xenopus embryos were cultured on type I collagen- or gelatin-coated dishes until control embryos reached stage 23, the cells in confluent layers expressed an apical-basal polarity so that the apical surface membrane domain faced the culture medium. However, the cells in confluent layers cultured on naked plastic dishes were suppressed to express intracellular and surface polarity. In addition, single attached cells which were formed by being sparsely plated neither spread on any substrata nor displayed the apical-basal polarity perpendicular to the dishes. These results indicate that the expression of intracellular and surface polarity in cultured animal pole cells of Xenopus embryos requires not only cell-cell contact but also an adhesive substrata such as type I collagen or gelatin.     

Cellular polarity in cultured animal pole cells of Xenopus embryos

The expression of intracellular and surface polarity in cultured animal pole cells of Xenopus embryos (stages 6, 8, and 10) was examined morphologically and immunocytochemically. When control embryos reached stage 23, daughter cells derived from a single or a few animal pole cells formed aggregates. Outer cells of the aggregates displayed intracellular and surface polarity and expressed an epidermis-specific antigen (XEPI-1) on the apical surface circumference, while these characteristics had not yet been established in the animal pole cells at the time of isolation. However, inner cells of the aggregates did not display the cellular polarity along an outer-inner axis of the aggregates and displayed the antigen randomly within the aggregates. These results indicate that the expression of cellular polarity in epidermal differentiation of Xenopus embryos in vitro depends on the position within the aggregates formed by daughter cells derived from isolated animal pole cells.     

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.     

Abdominal segmentation, pole cell formation, and embryonic polarity require the localized activity of oskar, a maternal gene in Drosophila

Embryos derived from oskar females lack pole cells and the specialized pole plasm including polar granules. In addition, the abdominal region remains unsegmented and eventually dies. Transplantation of cytoplasm from normal embryos into mutant embryos reveals that osk-dependent activity is strictly localized at the posterior pole and has three distinct functions. In mutant embryos the activity will normalize pole cell formation when transplanted into the posterior pole and abdominal segmentation after transplantation to a more anterior, the prospective abdominal, region. Furthermore, osk activity can provoke the formation of a second "posterior center" at the anterior. The participation of the osk product in the establishment of a source of morphogenetic activity in the posterior pole plasm is discussed.     

Elongated Microvilli on Vegetal Pole Cells in Sea Urchin Embryos

The ultrastructure of cells in the vegetal pole region of sea urchin embryos during early development to the mesenchyme blastula stage was examined by scanning electron microscopy. Vegetal pole cells in the ectoderm with longer microvilli than those of neighboring cells were first detectable at the early blastula stage just before hatching. These cells with elongated microvilli remained in the central region of the vegetal plate when most vegetal plate cells ingressed into the blastocoel to form primary mesenchyme. When first detectable in the sea urchin, Anthocidaris crassispina , four vegetal pole cells had elongated microvilli, but at the time of primary mesenchyme cell ingression, the number of cells with elongated microvilli had increased to eight, apparently by cell division. These vegetal pole cells were wedge-shaped with a broad surface adhering to the hyaline layer at the time of primary mesenchyme cell ingression. SEM observation of the outer surface of embryos showed that the microvilli extended into the hyaline layer. The reinforced attachment of vegetal pole cells to the hyaline layer through their elongated microvilli may explain why these cells could remain at the vegetal pole when the surrounding cells ingressed into the blastocoel as primary mesenchyme cells.     

The isolation of functional pole cells from theDrosophila melanogaster maternal effect mutantmat(3)1

Summary A procedure for pole cell isolation has been developed that takes advantage of theDrosophila melanogaster maternal effect mutantmat(3) 1. Embryos derived from homozygousmat(3)1 mothers form exclusively pole cells. By outcrossing we could substantially increase the expressivity of the original mutant stock. We further introduced theTM8 balancer chromosome, which carries the dominant temperature sensitive mutationDTS-4. This allows the accumulation of large homozygousmat(3) 1 fly populations by eliminating the heterozygous flies at the restrictive temperature.Early embryos were mechanically fragmented and the cells were isolated by means of metrizamide step gradients. The isolated cells were demonstrated to exhibit the various ultrastructural and histochemical characteristics of pole cells. The isolated cells were transplanted into genetically marked host embryos. The germ line mosaics that were obtained indicate that the isolated cells represent functional pole cells.Proteins synthesized by the isolated pole cells during short term in vitro labelling with³⁵S-methionine were compared to the proteins synthesized by blastoderm cells fromOregon-R embryos. At least one protein could be demonstrated in the pole cell samples that is not synthesized byOregon-R blastoderm cells.The method allows a fast and gentle isolation of highly enriched pole cell populations which are a prerequisite for the biochemical analysis of germ cell determination and differentiation.     

Maternal Pumilio acts together with Nanos in germline development in Drosophila embryos

The maternal RNA-binding proteins Pumilio (Pum) and Nanos (Nos) act together to specify the abdomen in Drosophila embryos. Both proteins later accumulate in pole cells, the germline progenitors. Nos is required for pole cells to differentiate into functional germline. Here we show that Pum is also essential for germline development in embryos. First, a mutation in pum causes a defect in pole-cell migration into the gonads. Second, in such pole cells, the expression of a germline-specific marker (PZ198) is initiated prematurely. Finally, pum mutation causes premature mitosis in the migrating pole cells. We show that Pum inhibits pole-cell division by repressing translation of cyclin B messenger RNA. As these phenotypes are indistinguishable from those produced by nos mutation, we conclude that Pum acts together with Nos to regulate these germline-specific events.     

Spatial and developmental changes in the respiratory activity of mitochondria in early Drosophila embryos.

Mitochondria of early Drosophila embryos were observed with a transmission electron microscope and a fluorescent microscope after vital staining with rhodamine 123, which accumulates only in active mitochondria. Rhodamine 123 accumulated particularly in the posterior pole region in early cleavage embryos, whereas the spatial distribution of mitochondria in an embryo was uniform throughout cleavage stages. In late cleavage stages, the dye showed very weak and uniform accumulation in all regions of periplasm. Polar plasm, sequestered in pole cells, restored the ability to accumulate the dye. Therefore, it is concluded that the respiratory activity of mitochondria is higher in the polar plasm than in the other regions of periplasm in early embryos, and this changes during development. The temporal changes in rhodamine 123-staining of polar plasm were not affected by u.v. irradiation at the posterior of early cleavage embryos at a sufficient dosage to prevent pole cell formation. This suggests that the inhibition of pole cell formation by u.v. irradiation is not due to the inactivation of the respiratory activities of mitochondria. In addition, we found that the anterior of Bicaudal-D mutant embryos at cleavage stage was stained with rhodamine 123 with the same intensity as the posterior of wild-type embryos. No pole cells form in the anterior of Bic-D embryos, where no restoration of mitochondrial activity occurs in the blastoderm stage. The posterior group mutations that we tested (staufen, oskar, tudor, nanos) and the terminal mutation (torso) did not alter staining pattern of the posterior with rhodamine 123.     

An Attempt to Hybridize Drosophila Species Using Pole Cell Transplantation

We have made hybrid embryos in Drosophila by pole cell transplants, by transfering pole cells from two species, D. rajasekari and D. eugracilis, into sterile D. melanogaster hosts. These females were then mated to melanogaster males and the older these females were, the further their hybrid offspring developed. In the case of the rajasekari/melanogaster hybrids, the embryos form cuticle but had defective heads, while the eugracilis/melanogaster hatched as larvae that grew but did not moult to the second instar. Hybrid pole cells could be transferred to melanogaster hosts but they failed to make eggs.     

Developmental analysis of grandchildless (gs(1)N441) mutation in Drosophila melanogaster; abnormal formation of pole cells

A detailed examination of the developmental features of abnormal formation of pole cells and a functional analysis of the germ plasma of gs(1)N441 embryos were carried out. The germ plasma is morphologically normal. Embryos in which cleavage nuclei show retarded migration to the posterior pole do not form pole cells. Pole cells, following formation, are abnormally segregated and then intermingled between the blastoderm cell layer but retaining normal morphology and differentiating into functional germ cells. The results of cytoplasmic transplantation experiments indicate the autonomous segregation ability of the mutant polar plasma to form pole cells to possibly be affected.     

Lineage analysis of transplanted individual cells in embryos of Drosophila melanogaster

Summary In this paper experiments concerning some aspects of the development of pole cells and midgut progenitors in Drosophila are reported. Cells were labelled by injecting horseradish-peroxidase (HRP) in embryos before pole bud formation and transplanted at different stages into unlabelled embryos, where the transplanted cells developed together with the unlabelled cells of the host. The hosts were then fixed and stained at different ages in order to demonstrate the presence of HRP in the progenies of transplanted cells. The main conlusions of the study are as follows. The gonads are the only organ to the formation of which pole cells normally contribute; those pole cells which do not participate in the formation of the gonads are finally eliminated or degenerate. Since the number of primordial germ cells in the gonads is the same irrespective of the number of pole cells present in the embryo, an (unknown) mechanism must exist regulating the final number of pole cells in each of the gonads. After their formation and before reaching the gonads, pole cells have been found to divide only up to two times. With respect to the midgut progenitors, the cells of both anlagen have been found to be committed to develop into midgut, although they behave as equivalent in that they do not apparently distinguish between the anterior and posterior anlage. Midgut progenitors have been found to divide a maximum of three times and to produce two different types of cells, epithelial cells of the midgut wall and spindle-like cells located internally in the gut.     

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.     

Ultraviolet irradiation of eggs and blastomere isolation experiments suggest that gastrulation in the direct developing ascidian, Molgula pacifica, requires localized cytoplasmic determinants in the egg and cell signaling beginning at the two-cell stage

Gastrulation in the maximum direct developing ascidian Molgula pacifica is highly modified compared with commonly studied "model" ascidians in that endoderm cells situated in the vegetal pole region do not undergo typical invagination and due to the absence of a typical blastopore the involution of mesoderm cells is highly modified. At the gastrula stage, embryos are comprised of a central cluster of large yolky cells that are surrounded by a single layer of ectoderm cells in which there is only a slight indication of an inward movement of cells at the vegetal pole. As a consequence, these embryos do not form an archenteron. In the present study, ultraviolet (UV) irradiation of fertilized eggs tested the possibility that cortical cytoplasmic factors are required for gastrulation, and blastomere isolation experiments tested the possibility that cell signaling beginning at the two-cell stage may be required for the development of the gastrula. Irradiation of unoriented fertilized eggs with UV light resulted in late cleavage stage embryos that failed to undergo gastrulation. When blastomeres were isolated from two-cell embryos, they developed into late cleavage stage embryos; however, they did not undergo gastrulation and subsequently develop into juveniles. These results suggest that cytoplasmic factors required for gastrulation are localized in the egg cortex, but in contrast to previously studied indirect developers, these factors are not exclusively localized in the vegetal pole region at the first stage of ooplasmic segregation. Furthermore, the inability of embryos derived from blastomeres isolated at the two-cell stage to undergo gastrulation and develop into juveniles suggests that important cell signaling begins as early as the two-cell stage in M. pacifica. These results are discussed in terms of the evolution of maximum direct development in ascidians.