Germ Cell Determination Research Articles

Localization of vasa, a component of Drosophila polar granules, in maternal-effect mutants that alter embryonic anteroposterior polarity

Cytoplasm at the posterior pole of the early Drosophila embryo, known as polar plasm, serves as a source of information necessary for germ cell determination and for specification of the abdominal region. Likely candidates for cytoplasmic elements important in one or both of these processes are polar granules, organelles concentrated in the cortical cytoplasm of the posterior pole. Females homozygous for any one of the maternal-effect mutations, tudor, oskar, staufen, vasa, or valois give rise to embryos that lack localized polar granules, fail to form the germ cell lineage and have abdominal segment deletions. Using antibodies against a polar granule component, the vasa protein, we find that vasa synthesis or localization is affected by these mutations. In vasa mutants, synthesis of vasa protein is absent or severely restricted. In oskar and staufen mutant females, vasa synthesis appears normal, but the vasa protein is not localized. In tudor and valois mutant females, vasa is localized to the posterior pole of oocytes, but this localization is lost following egg activation. In addition to the posterior localized vasa, there is a low level of vasa distributed throughout the embryo. A function for this distributed vasa is postulated based on the observation that embryos from Bicaudal-D mothers, in which abdominal determinants are incorrectly localized to the anterior pole, do not show any ectopic vasa localization, though abdomen development at the anterior end depends on the amount of vasa protein in the embryo.

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.

Primordial germ cells of Xenopus laevis are not irreversibly determined early in development

In anuran amphibians such as Rana pipiens and Xenopus laevis, the germ lineage is widely thought to be determined at the earlist stages in development, in response to a prelocalised cytoplasmic determinant (the germ plasm) present in the vegetal pole of the egg. However, the determined state of germ plasm-bearing cells has not been tested in anurans. In this work, we have labelled germ plasm-bearing cells from two stages of development with a cell-autonomous fluorescent label, and inserted them into unlabelled host embryos. In both cases the donor cells were pluripotent, and differentiated into a variety of cell types. This result may help resolve the paradoxical data on germ plasm and germ cell determination from different animal groups.

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.

Germ plasm and germ cell determination in Xenopus laevis as studied by cell transplantation analysis.

This paper will review separate but related groups of data on the ontogeny and possible roles of germ plasm in the establishment of the germ line in anuran amphibian embryos. We shall discuss the paradoxical situation that germ plasm is apparently necessary for the normal formation and/or the migration of primordial germ cells (PGCs), but that at the same time PGCs remain pluripotent, at least until they reach the gonad, and are not determined in the classical sense of being restricted in potential to form only germ line cells.

Photoreversible inhibition by ultraviolet light of germ line development in Smittia sp. (Chironomidae, Diptera)

Pole cell formation in embryos of the parthenogenetic midge, Smittia sp., can be delayed or inhibited by irradiation of the posterior egg pole with ultraviolet light (uv). This leaves the schedule of nuclear divisions and chromosome eliminations virtually unaffected. However, uv irradition delays the precocious migration to the posterior pole of one nucleus, which normally becomes included in the first pole cell. This effect is photoreversible, i.e., mitigated by application of blue light after uv. Photoreversibility indicates that a nucleic acid component is involved as an effective target. During normal development of Smittia a number of chromosomes are eliminated during mitosis V, not only from somatic nuclei but also in the germ line. In the latter, this mitosis takes place during the first gonial division in the larva. After uv irradiation, the first pole cell nucleus has undergone supernumerary mitoses before pole cell formation and, as a result, is driven into mitosis V precociously as the pole cell divides. This is frequently associated with chromosome elimination from pole cells, which in turn is correlated with subsequent disappearance of already formed pole cells. Adults derived from embryos without pole cells do not form ovaries. Pole cell formation, pole cell preservation, and ovary development are separately inhibited by uv, and inhibition of each step is photoreversible. The results are discussed in the context of germ cell determination, protection against chromosome elimination, and the role of chromosomes limited to the germ line.

Induction of pole cells in sterilized Drosophila embryos by injection of subcellular fraction from eggs

A subcellular fraction isolated from a homogenate of young Drosophila embryos was shown to be capable of inducing pole cells when injected into UV-sterilized Drosophila embryos. Most of the pole cell-inducing activity was recovered from the precipitate after centrifugation at 27,000 x g. The activity remained in this precipitate (called F-3 fraction hereafter) even after membranous structures were removed from it through centrifugation on a sucrose density gradient. Dialysis, lyophilization, and heating at 80 degrees C for 10 min did not inactivate the F-3 fraction. The pole cells, which were produced when the F-3 fraction was injected at the posterior pole of UV-sterilized embryos, did not develop into germ cells. Furthermore, the F-3 fraction was unable to induce pole cells when injected into the anterior region of the egg. These results can be explained by assuming that (i) pole cell formation and germ cell determination are controlled by different factors, (ii) pole cell formation requires at least two factors, which are normally localized in the posterior-pole cytoplasm, one of which is sensitive and one resistant to the UV dosage we used, and (iii) the subcellular fraction we obtained contains the UV-sensitive factor but not the UV-resistant factor.

Pole cell formation in Drosophila melanogaster

The development of the pole cells of Drosophila melanogaster has been investigated in a temperature-sensitive developmental mutant strain, shibire ts. Shibire ( shi ts) mutants have periods of temperature sensitivity in early embryogenesis; embryos shifted to 29°C during those periods subsequently lack pole cells. The temperature-sensitivity profiles of four independently isolated alleles indicate that the shibire mutation is responsible for this defect in development. Carefully timed heat pulses established that there is a broad range in times of sensitivity extending even to the stage when the pole cells have begun to form. Scanning electron microscopy of the heattreated embryos was compared to that of controls to illustrate the extent of the defects in mutants. Transmission electron microscopy of sectioned embryos lacking pole cells revealed normal stage-specific morphology of polar granules in the posterior end. Speculations are presented on the significance of pole cell formation as a means of segregating germ cell determinants.

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

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.

Occurrence of nuage in fetal human germ cells.

Germ cells of human fetuses aged 7-13 weeks were studied. Dense fibrous material called nuage was found in the cytoplasm of human fetal germ cells. The nuage increased in frequency in the germ cells during the interval studied. In oogonia the nuage occurred much more frequently than in spermatogonia and the frequency of nuage in oogonia increased earlier than in spermatogonia. The number of nuage was continuously at a higher level in oogonia than in spermatogonia. It is suggested that these findings support the theory that the nuage may act as a germ cell determinant.

Effects of γ-Ray Irradiation on Primordial Germ Cells in Embryos of Oryzias latipes

Effects of ..gamma.. rays on different stages of primordial germ cells were quantitatively studied in Oryzias latipes embryos. Stage 9, which is before the germ cell determination, exhibited the highest radiosensitivity to the elimination of primordial germ cells in embryos and hatching fry. Irradiation of embryos at this stage with 500-2000-rad ..gamma..-ray doses showed an exponential dose-effect relationship. A time-course study was also carried out on embryos and fry irradiated with 2000 rad at stage 9. A possibility was indicated that the onset of sex differentiation of germ cells may be independent of the population size of primordial germ cells at the genital ridges. Primordial germ cells became highly radioresistant from the stage 3 days after fertilization. In the time-course study of the embryos irradiated at 4 days after fertilization with 2000 rad, germ cells with enlarged nuclei were observed after 48 hr. Under the present experimental conditions, no ''immediate cell death'' was encountered.

The Germ Plasm ofDrosophila: An Experimental System for the Analysis of Determination

The posterior polar plasm of Drosophila melanogaster may provide a model for understanding processes involved in cellular determination during early embryonic development. The presence of germ cell determinants in the posterior tip of the egg has been demonstrated by transplanting this material to new locations and showing that cells, induced at the site of transplantation, can function as germ cells. Furthermore, by utilizing similar procedures, the posterior polar plasm of late stage oocytes has been shown to be functional in inducing germ cells, thus demonstrating that the active components are formed during oogenesis. A number of maternal effect mutations affecting germ cell formation have been analyzed ultrastructurally and the mutant phenotype described. Ultrastructural studies have focused on polar granules, the unique organelle of the polar plasm, and these studies indicate that they are nonmembrane bound structures containing RNA and protein. Although ultrastructural observations have suggested that the protein components of polar granules may be continuous in the cytoplasm of germ cells during the whole life cycle, nucleo-cytoplasmic hybrid pole cells indicate that the structure of the polar granule is dependent upon the nucleus in each new generation of oocytes. Finally, attempts are being made to obtain a purified polar granule fraction in order to test their role in germ cell determination. Initial results indicate that clusters of ribosomes are attached to polar granules. Pole cells have recently been isolated as an enriched source of polar granules. These isolated cells will also provide an opportunity to analyze the unique traits of these cells at the time that they become segregated from the remaining cells of the embryo.

Differentiation of primordial germ cells in the embryonic development ofThermobia domestica. Pack.(Thysanura):an ultrastructural study

The primordial germ cells(PGCs) of Thermobia domestica undergo some morphological changes during the embryonic development. Most conspicuous are the changes in the ultrastructure of the nucleus, whose envelope shows a high degree of activity. Two types of vesicles bled off from the nucleus; the ones with the light interior are called the accessory nuclei, the others, with electron-opaque contents, have been termed the dense bodies. The nucleolus, initially clustered at the nucleus centre, undergoes dispersion and assembles again towards the end of embryonic development. At the same time, the sex differentiation of PGCs takes place. It is preceded by an increase in the activity of Golgi complexes and in the volume of lysosomes and lamellar bodies, the latter giving rise to lipid droplets. At the early stages of postembryonic development, preoogonia and prespermatogonia can readily be distinguished. Preoogonia have a wavy-surfaced nucleus and their cytoplasm contains dense bodies. In prespermatogonia, the nucleus is spherical with smooth envelope and there are no dense bodies in the cytoplasm. Throughout the period studied there occur nucleolus-like bodies and nuage material considered to be the germ-cell determinants in this species.

Existence of ultraviolet-labile germ cell determinant in unfertilized eggs of Xenopus laevis and its sensitivity

Ultraviolet light (uv) irradiation of the vegetal hemisphere of unfertilized eggs was performed in Xenopus laevis. The experiments resulted in the elimination of primoridial germ cells in the tadpoles, demonstrating the existence of uv-labile germ cell determinant in unfertilized eggs. Moreover, through its sensitivity analysis, it was found that the unfertilized eggs were equally as sensitive as (or slightly more sensitive than) the stage of eggs immediately after fertilization. The present experimental results support the idea that the germ cell determinant already exists in unfertilized eggs as “stored information,” which has been postulated by many authors.

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.

A mathematical model for germ cell determination process and effect of ultraviolet light on the process

The process of germ cell determination may be regarded as one of the best examples of cell determination process in general. The germ cells can be readily traced through its developmental stages by their possession of “germinal plasm”. During cleavage, the germinal plasm is distributed among cells of vegetal blastomeres, and the germinal plasm-bearing cells later become germ cells. Tadpoles lacking germ cells could be obtained by irradiating the vegetal hemisphere of egg with ultraviolet light (UV). The present experimental data revealed that there is a numerical relationship between the germ cells formed and irradiating UV dose. In this article, we present a mathematical model of the germ cell determination process. At a critical period, germinal plasm is supposed to exert some reaction to the nucleus of a cell, and the amount of this reaction will decide whether this cell differentiates into a germ cell or a somatic cell. Following the introduction of the concept of the distribution function, germinal plasm partition model is presented. Our basic assumption is that germinal plasm partition follows the probabilistic rule governed only by geometric relation between area of the cell surface and the germinal plasm contained. After obtaining the general shape of the distribution function from the partition model, we draw the mathematical equation which describes the relationship between survival of germ cells and UV dose. This equation shows a good agreement with the actual experimental data. Finally, we point out that it is possible to get a clue to what kind of molecule the germ cell determinant. is, through the analysis of actual data using this mathematical equation.

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.

UV effect on germ cell determinant and a proposed scheme for germ cell formation process in embryos of Xenopus laevis.

Ultraviolet light (UV) irradiation of vegetal hemispheres of many anuran eggs results in an elimination of primordial germ cells in the tadpoles. Quantitative studies were performed on this phenomenon. In this report, UV irradiation experiments were performed on the eggs just before the first cleavage division, and a dose-response curve was obtained at the stage. The results supported the probabilistic model for germ cell determination process, previously presented by the authors. Cell diameter analysis suggested that the germinal plasm-containing cells (i. e. presumptive primordial germ cells) divide at the same rate as ordinary endodermal cells during stage 10-33. This led to a calculation of the doubling time of presumptive primordial germ cells, odtaining its value as about 30 hrs at 20°C. Finally, a scheme for the proliferation kinetics of germ cells in Xenopus laevis embryos was presented. Several UV experiments on the vegetal hemisphere of anuran eggs have presented favorable evidence that histologically identifiable germinal plasm contains the UV-labile germ cell determinant. Their significance was also discussed.

Differential responses to ultraviolet irradiation of the polar cytoplasm ofDrosophila eggs: II. Response to dose.

The posterior pole cytoplasm of early embryos ofDrosophila melanogaster was irradiated with increasing doses of U.V. light at a wave length specific for nucleic acids 35 and 75 min after ovoposition, when nuclei were still inside the yolk mass. Probit analysis showed that:Sterility was higher in irradiations performed at 35 min.Mortality was higher in the experiment performed at 75 min.After treatment at 35 min, sterility and mortality were independent phenomena.The numbers of targets involved in germ cell determination were of the order of tens and hundreds at 35 and 75 min respectively.The numbers of targets involved in viability were of several orders of magnitude in both developmental stages.The irradiation, even at very low doses, caused a background effect which was probably due to "old" embryos. The sex ratio was distorted in favour of females at high doses and of males at low doses.

The autonomous function of germ plasm in a somatic region of the Drosophila egg.

Abstract Posterior polar plasm from Binsc/B 6 Y cleavage embryos of Drosophila melanogaster was transferred to the mid-ventral region of mwh e 4 embryos of the same age. Between 15 min and 5 h after cytoplasmic injections, embryos were analyzed at the electron microscopic level for the formation of cells with implanted cytoplasm. Characteristic subcellular organelles, the polar granules, which are exclusively located at the posterior pole of the egg, served as a morphological marker to identify the transferred cytoplasm. The appearance of round nuclear bodies, which are only present in nuclei of cells containing posterior cytoplasm, the so-called pole cells, was used as a further criterion for successful transfers. Polar granules and nuclear bodies could be found in eight and ten cells in two of the seven embryos and analyzed at the blastoderm stage. Experimentally induced pole cells were inserted among pole cells of y sn 3 mal blastoderm hosts in order to determine if they could function normally and give rise to offspring. About 3% of the flies, which were derived from these embryos and mated to mwh e 4 partners, had y sn 3 mal germ cells as well as mwh e 4 ones. In addition, germ-line mosaicism in these flies could be “visualized” by testing histochemically for aldehyde oxidase activity. Mid-ventral blastoderm cells when transferred from non-injected mwh e 4 embryos to the posterior pole of y sn 3 mal blastoderm embryos did not differentiate into germ cells but instead participated in the development of somatic tissues of the hosts. To find out if a pole cell experimentally induced at the mid-ventral region of the egg can be incorporated into the germ line, normal mwh e 4 pole cells were implanted into this area of y sn 3 mal blastoderm hosts. About 5% of the fertile flies were found to have a mosaic germ line. Our results show (1) that cytoplasm of the posterior region of the Drosophila egg contains germ-cell determinants which function autonomously after transplantation to a somatic, i.e. mid-ventral region of the egg; (2) that pole cells either formed in or transferred to this ectopic region can still reach the gonads and contribute to the germ line.