Spontaneous germ line virus infection and retroviral insertional mutagenesis in eighteen transgenic Srev lines of mice.

SWR/J-RF/J hybrid mice spontaneously acquire new germ line ecotropic proviruses at high frequency. In the studies described here, we used these hybrids to produce 18 transgenic mouse lines, each carrying a single newly acquired Srev locus (SWR/J-RF/J ecotropic proviral locus). All of the newly acquired proviruses identified in mosaic founder SWR/J-RF/J mice that could be transmitted through the germ line were also present in somatic tissues, demonstrating that viral integration occurred before the germ line was set aside from the somatic lineages. Quantitative analysis of proviral DNA copy numbers in somatic and germinal tissues of mosaic founder parents combined with structural analysis of Srev loci indicated that these proviruses are acquired after multiple rounds of somatic viral reinfection and that most of these viral integration events occurred after DNA replication in the zygote and before DNA replication in the four-cell embryo. The frequency of provirus acquisition in Srev lines that expressed the infectious ecotropic virus was similar to that in SWR.RF mice carrying Emv-16 and Emv-17, suggesting that the chromosomal integration site of the parental locus is not an important determinant for high-frequency provirus acquisition. The frequency of recessive lethal mutations induced by spontaneous viral integration was 5%, which was similar to that induced by preimplantation embryo infection. This approach represents a simple and viable strategy for inducing and studying mutations that affect mammalian development.     

Reversal of cellular polarity and early cell-cell interaction in the embryos of Caenorhabditis elegans

During early embryogenesis of Caenorhabditis elegans the serial stem cell-like cleavages of the germ line cells P0-P3 generate a number of somatic founder cells with different developmental potentials. Observations on partial embryos show that in the first two of these unequal divisions in the germ line the somatic daughter cell comes to lie anterior to the new germ line cell. In the following two, however, the somatic daughter cell comes to lie posterior to the new germ line cell, suggesting a reversal of polarity in the germ line. By the use of a laser microbeam, egg fragments can be extruded from young embryos; the fragments often cleave like partial twins. Depending on whether the fragment is derived from the posterior region of the uncleaved zygote P0 or its daughter P1, the mirror image duplications that are generated are joined at their larger soma-like cells or at their smaller germ line-like cells, respectively. This result is best explained as a reversal of polarity taking place in the germ line cell P2. This notion is strengthened by the finding that partial embryos derived from the posterior region of the P2 cell in late interphase do not undergo stem cell-like (i.e., unequal) cleavages in contrast to those derived from P0 or P1. Finally, an apparent early cell-cell interaction is described which is inconsistent with the classical notion of "mosaic" nematode development: removal of the germline cell P2 results in an altered developmental pattern of its somatic sister cell EMS. A working model is presented linking reversal of polarity and cell-cell interaction and offers an explanation for the unique behavior of the EMS cell in normal development.     

Distinct chromatin organization in the germ line founder cell of the Caenorhabditis elegans embryo

Cells belonging to the germ lineage segregate physically and molecularly from their somatic neighbors during embryogenesis. While germ line‐specific chromatin modifications have been identified at later stages in the Caenorhabditis elegans nematode, none have been found in the single P₄ germ line founder cell that arises at the beginning of gastrulation. Using light and electron microscopy, we now report that the chromatin organization in the germ line founder cell of the early C. elegans embryo is distinct from that in the neighboring somatic cells. This unique organization is characterized by a greater chromatin compaction and an expansion of the interchromatin compartment. The ultrastructure of individual chromatin domains does not differ between germ line and somatic cells, pointing to a specific organization mainly at the level of the whole nucleus. We show that this higher order reorganization of chromatin is not a consequence of the P₄ nucleus being smaller than somatic nuclei or having initiated mitosis. Imaging of living embryos expressing fluorescent markers for both chromatin and P granules revealed that the appearance of a distinct chromatin organization in the P₄ cell occurs approximately 10 min after its birth and coincides with the aggregation of P granules around the nucleus, suggesting a possible link between these two events. The higher order reorganization of chromatin that is reported here occurs during the establishment of definitive germ cell identity. The changes we have observed could therefore be a prerequisite for the programming of chromatin totipotency.     

Targeted disruption of exogenous EGFP gene in medaka using zinc-finger nucleases

Zinc-finger nucleases (ZFNs) are artificial enzymes that create site-specific double-strand breaks and thereby induce targeted genome editing. Here, we demonstrated successful gene disruption in somatic and germ cells of medaka (Oryzias latipes) using ZFN to target exogenous EGFP genes. Embryos that were injected with an RNA sequence pair coding for ZFNs showed mosaic loss of green fluorescent protein fluorescence in skeletal muscle. A number of mutations that included both deletions and insertions were identified within the ZFN target site in each embryo, whereas no mutations were found at the non-targeted sites. In addition, ZFN-induced mutations were introduced in germ cells and efficiently transmitted to the next generation. The mutation frequency varied (6-100%) in the germ cells from each founder, and a founder carried more than two types of mutation in germ cells. Our results have introduced the possibility of targeted gene disruption and reverse genetics in medaka.     

Cell lineage determination in the mouse

During the peri-implantation development of the mouse embryo from the blastocyst through gastrulation, Pou5f1 (OCT-4) down-regulation is closely linked to the initial step of lineage allocation to extraembryonic and embryonic somatic tissues. Subsequently, differentiation of the lineage precursors is subject to inductive tissue interactions and intercellular signalling that regulate cell proliferation and the acquisition of lineage-specific morphological and molecular characteristics. A notable variation of this process of lineage specification is the persistence of Pou5f1 activity throughout the differentiation of the primordial germ cells, which may underpin their ability to produce pluripotent progeny either as stem cells (embryonic germ cells) in vitro or as gametes in vivo. Nevertheless, intercellular signalling still plays a critical role in the specification of the primordial germ cells. The findings that primordial germ cells can be induced from any epiblast cells and that they share common progenitors with other somatic cells provide compelling evidence for the absence of a pre-determined germ line in the mouse embryo.     

Proviruses of mouse mammary tumor virus in normal and neoplastic tissues from GR and C3Hf mouse strains.

We analyzed two experimental situations to assess the role of endogenous mouse mammary tumor virus (MMTV) DNA in the genesis of mammary carcinomas. (i) GR mice carry in their germ line one or more proviruses indistinguishable by limited restriction mapping from the proviruses introduced into cells by experimental infection with the highly tumorigenic virus isolated from GR mouse milk, MMTV(GR). Most tumors arising in GR mice contain one or more proviruses at various sites in tumor DNA in addition to those present endogenously. Detection of these new proviruses is possible as a consequence of the clonal or quasiclonal character of the tumors. (ii) C3H/He mice carry three units of endogenous viral DNA, none of which resembles the DNA of the commonly encountered strains of milk-borne MMTV. Nevertheless, MMTV-associated tumors arise late in life when these animals are removed from the influence of milk-borne virus; the responsible agent, MMTV(C3Hf), can also produce tumors in BALB/c mice. We found that tumors arising in both C3Hf/He mice and BALB/c mice infected with MMTV(C3Hf) were clonal or quasiclonal and contained one or more new copies of proviral DNA at various sites in the host genome. These new proviruses were readily distinguished from the proviruses of the common milk-borne virus strains and closely resembled unit II of endogenous MMTV DNA (Cohen et al., J. Virol., 32:483-496). Thus, in both experimental systems, we found evidence for new proviruses in mammary tumors, despite the preexistence of similar or identical proviruses in the germ line. The results suggest that the repositioning of MMTV proviruses may be required for the full expression of the oncogenic potential of endogenous MMTV DNA.     

Recurrence of lethal osteogenesis imperfecta due to parental mosaicism for a dominant mutation in a human type I collagen gene (COL1A1).

We have determined that two infants with perinatal lethal osteogenesis imperfecta in one family had the same new dominant point mutation. Although not detected in his dermal fibroblast DNA, the mutation was detected in somatic DNA from the father's hair root bulbs and lymphocytes. The mutation was also detected in the father's sperm, demonstrating that mosaicism in the father's germ line explains recurrence. The presence of both germ-line and somatic mosaicism indicates that the mutation occurred prior to segregation of the germ-line and somatic cell progenitors. About one in eight sperm carry the mutation, which implies that at least four progenitor cells populate the germ line in human males. The observation that the mosaic individual is clinically normal suggests that genetic diseases can have both qualitative and quantitative components.     

Glp-3 Is Required for Mitosis and Meiosis in the Caenorhabditis Elegans Germ Line

The germ line is the only tissue in Caenorhabditis elegans in which a stem cell population continues to divide mitotically throughout life; hence the cell cycles of the germ line and the soma are regulated differently. Here we report the genetic and phenotypic characterization of the glp-3 gene. In animals homozygous for each of five recessive loss-of-function alleles, germ cells in both hermaphrodites and males fail to progress through mitosis and meiosis, but somatic cells appear to divide normally. Germ cells in animals grown at 15° appear by DAPI staining to be uniformly arrested at the G2/M transition with <20 germ cells per gonad on average, suggesting a checkpoint-mediated arrest. In contrast, germ cells in mutant animals grown at 25° frequently proliferate slowly during adulthood, eventually forming small germ lines with several hundred germ cells. Nevertheless, cells in these small germ lines never undergo meiosis. Double mutant analysis with mutations in other genes affecting germ cell proliferation supports the idea that glp-3 may encode a gene product that is required for the mitotic and meiotic cell cycles in the C. elegans germ line.     

Transfer of a mutant dihydrofolate reductase gene into pre- and postimplantation mouse embryos by a replication-competent retrovirus vector.

In order to explore the potential of retrovirus vectors for efficiently transferring foreign genes into mouse embryos, a replication-competent recombinant Moloney murine leukemia virus (Mo-MLV) vector carrying a mutant dihydrofolate reductase (DHFR) cDNA insert in the U3 region of the viral long terminal repeat was used to infect pre- and postimplantation embryos. When preimplantation mouse embryos were infected with the vector, as expected, the provirus integrated into the embryos and the germ line with the same efficiency as that observed with wild-type Mo-MLV, leading to inactivation of the recombinant virus. In contrast, when postimplantation mouse embryos were microinjected with virus-producing cells, between 90 to 100% of the surviving animals proved to be infected with the virus. The recombinant virus spread as efficiently as wild-type Mo-MLV in the infected embryos, resulting in up to three to five proviral copies per genome in heart, thymus, and brain tissues. Substantial expression of mutant DHFR*-coding viral message was found in all somatic tissues analyzed, the amounts correlating with the proviral copy number in the respective organ. These results suggest that replication-competent vectors are useful for efficient transfer and expression of foreign genes into tissues or whole animals when virus spread is needed.     

The symbiotically essential cbb(3)-type oxidase of Bradyrhizobium japonicum is a proton pump

The male germ line stem cell is the only cell type in the adult that can contribute genes to the next generation and is characterized by postnatal proliferation. It has not been determined whether this cell population can be used to deliberately introduce genetic modification into the germ line to generate transgenic animals or whether human somatic cell gene therapy has the potential to accidentally introduce permanent genetic changes into a patient's germ line. Here we report that several techniques can be used to achieve both in vitro and in vivo gene transfer into mouse male germ line stem cells using a retroviral vector. Expression of a retrovirally delivered reporter lacZ transgene in male germ line stem cells and differentiated germ cells persisted in the testis for more than 6 months. At least one in 300 stem cells could be infected. The experiments demonstrate a system to introduce genes directly into the male germ line and also provide a method to address the potential of human somatic cell gene therapy DNA constructs to enter a patient's germ line.     

No extension of lifespan by ablation of germ line in Drosophila

Increased reproduction is frequently associated with a reduction in longevity in a variety of organisms. Traditional explanations of this 'cost of reproduction' suggest that trade-offs between reproduction and longevity should be obligate. However, it is possible to uncouple the two traits in model organisms. Recently, it has been suggested that reproduction and longevity are linked by molecular signals produced by specific reproductive tissues. For example, in Caenorhabditis elegans, lifespan is extended in worms that lack a proliferating germ line, but which possess somatic gonad tissue, suggesting that these tissues are the sources of signals that mediate lifespan. In this study, we tested for evidence of such gonadal signals in Drosophila melanogaster. We ablated the germ line using two maternal effect mutations: germ cell-less and tudor. Both mutations result in flies that lack a proliferating germ line but that possess a somatic gonad. In contrast to the findings from C. elegans, we found that germ line ablated females had reduced longevity relative to controls and that the removal of the germ line led to an over-proliferation of the somatic stem cells in the germarium. Our results contrast with the widely held view that it is downstream reproductive processes such as the production and/or laying of eggs that are costly to females. In males, germ line ablation caused either no difference, or a slight extension, in longevity relative to controls. Our results indicate that early acting, upstream reproductive enabling processes are likely to be important in determining reproductive costs. In addition, we suggest that the specific roles and putative patterns of molecular signalling in the germ line and somatic tissues are not conserved between flies and worms.     

Stem cells of Hydra magnipapillata can differentiate into somatic cells and germ line cells

We investigated whether all stem cells of Hydra can differentiate both somatic cells and gametes or if a separate germ line exists in these phylogenetically old organisms. The differentiation potential of single stem cells was analyzed by applying a statistical cloning procedure. All stem cell clones were found to differentiate somatic cells. No clone was found to contain stem cells which do not differentiate. Most of the clones could be induced to form gametes. No clone was found that produced gametes only. The results indicate that stem cells are multipotent in the sense that individual stem cells can differentiate into somatic cells as well as germ line cells.     

Germinal and Somatic Trisomy 21 Mosaicism: How Common is it,What are the Implications for Individual Carriers and How Does it Come About?

It is well known that varying degrees of mosaicism for Trisomy 21, primarily a combination of normal and Trisomy 21 cells within individual tissues, may exist in the human population. This involves both Trisomy 21 mosaicism occurring in the germ line and Trisomy 21 mosaicism documented in different somatic tissues, or indeed a combination of both in the same subjects. Information on the incidence of Trisomy 21 mosaicism in different tissue samples from people with clinical features of Down syndrome as well as in the general population is, however, still limited. One of the main reasons for this lack of detailed knowledge is the technological problem of its identification, where in particular low grade/cryptic Trisomy 21 mosaicism, i.e. occurring in less than 3-5% of the respective tissues, can only be ascertained by fluorescence in situ hybridization (FISH) methods on large cell populations from the different tissue samples.In this review we summarize current knowledge in this field with special reference to the question on the likely incidence of germinal and somatic Trisomy 21 mosaicism in the general population and its mechanisms of origin. We also highlight the reproductive and clinical implications of this type of aneuploidy mosaicism for individual carriers. We conclude that the risk of begetting a child with Trisomy 21 Down syndrome most likely is related to the incidence of Trisomy 21 cells in the germ line of any carrier parent. The clinical implications for individual carriers may likewise be dependent on the incidence of Trisomy 21 in the relevant somatic tissues. Remarkably, for example, there are indications that Trisomy 21 mosaicism will predispose carriers to conditions such as childhood leukemia and Alzheimer's Disease but there is on the other hand a possibility that the risk of solid cancers may be substantially reduced.     

Vasa and the germ line lineage in a colonial urochordate

Germ cell sequestering in Animalia is enlightened by either, launching true germ line along epigenetic or preformistic modes of development, or by somatic embryogenesis, where no true germ line is set aside. The research on germ line-somatic tissue segregation is of special relevancy to colonial organisms like botryllid ascidians that reconstruct, on a weekly basis, completely new sets of male and female gonads in newly formed somatic tissues. By sequencing and evaluating expression patterns of BS-Vasa, the Botryllus schlosseri orthologue of Vasa, in sexually mature and asexual colonies during blastogenesis, we have demonstrated that the BS-Vasa mRNA and protein are not expressed exclusively in germ cell lineages, but appeared in cells repeatedly emerging de novo in the colony, independently of its sexual state. In addition, we recorded an immediate Vasa response to cellular stress (UV irradiation) indicating additional functions to its germ line assignments. To confirm germ lineage exclusivity, we examined the expression of three more stem cell markers (BS-Pl10, Bl-piwi and Oct4). Vasa co-expression with Pl10 and Oct4 was detected in germ line derivatives and with Bl-piwi in somatic tissues. Presumptive primordial germ cells (PGC-like cells), that are Vasa⁺/Pl10⁺/Oct4⁺ and 6-12 μm in diameter, were first detected in wrapped-tail embryos, in oozooids, in sexual/asexual colonies, within a newly identified PGC niche termed as ‘budlet niche’, and in circulating blood borne cells, indicating epigenetic embryogenesis. Alternatively, BS-Vasa co-expression with piwi orthologue, an omnipresent bona fide stemness flag, in non germ line cell populations, may indicate germ cell neogenesis (somatic embryogenesis) in B. schlosseri. Both alternatives are not necessarily mutually exclusive.