Effects of the substratum on the migration of primordial germ cells

It is now clear from work on defined cell types on artificial substrates that various chemical and physical inhomogeneities in the substrates can guide cell locomotion. It is also becoming clear that less well defined inhomogeneities in living cell substrates can guide the normal locomotion of embryonic migratory cells in vivo. The primordial germ cells (p.g.cs) of early anuran amphibian embryos are proving a useful model for the study of cell migration. When isolated from the embryo and cultured on living cellular substrate, p.g.cs become oriented by the shapes of the underlying cells or by their stress fibre cytoskeleton, or both. A combination of scanning and transmission electron microscopy in vivo shows a clearly aligned cellular substrate for p.g.c. migration along part of their route. Furthermore, we find that the glycoprotein fibronectin is involved in p.g.c. adhesion, which suggests a link between orientation of the substrate cells and p.g.c. guidance.     

Mouse primordial germ cells use fibronectin as a substrate for migration

The behavior in vitro of mouse primordial germ cells (PGCs) on different substrates was studied by means of microcinematography. It was observed that for displacement, the PGCs required fibronectin in the substrate and that this glycoprotein was present in vivo in those routes along which the cells moved. It was concluded that fibronectin plays an important role in the migration of the PGCs to the genital crests.     

Guidance of primordial germ cell migration

Primordial germ cells (PGCs), the progenitors of the gametes, migrate from the position where they are specified towards the region where the gonad develops. To reach their target, the PGCs obtain directional cues from cells positioned along their migration path. One such cue, the chemokine SDF-1, has recently been found to be critical for proper PGC migration in zebrafish and in mice. In Drosophila, too, a molecule that is structurally related to chemokine receptors and is important for PGC migration has been identified. The ability to visualize chemokine-guided migration at a high resolution in vivo in these model organisms provides a unique opportunity to study this process, which is relevant for many events in normal development and disease.     

Migration and proliferation of primordial germ cells in the rat

Information about early primordial germ cell (PGC) formation and migration in rats is lacking. In utero developed and in vitro cultivated whole rat embryos were studied on days 10-13 postcoitum (p.c.). The development of the PGCs was investigated in serial sections stained for alkaline phosphatase activity. On postcoital day 10, PGCs were found in the invaginating visceral yolk sac endoderm and at the base of the allantois. At day 11 p.c. PGCs were mostly found in the ventral and lateral gut wall or in the mesenchyme between the gut and the future genital ridges. At day 12 p.c. most of the PGCs (94%) could be localised in the mesenchyme or in the future genital ridges. On postcoital day 13 almost all PGCs had reached the now-well-developed genital ridges. Quantitative measurements showed an increase in the number of PGCs from 84 at day 10 p.c. up to 2,768 at day 13 p.c. Only slight differences were found between in vivo and in vitro embryos with respect to the number of PGCs and their developmental pattern. The in vitro culture of whole rat embryos enables the discrimination between the effects of indirect (maternal) and direct action of PGC-toxic agents.     

Response to fibronectin of mouse primordial germ cells before, during and after migration.

The adhesive extracellular matrix glycoprotein fibronectin is thought to play a central role in cell migration during embryogenesis. In order to define this role, we have examined the response to fibronectin in cell culture of mouse primordial germ cells (PGCs) before, during and after their migration from the hindgut into their target tissue, the genital ridges. Using an explant culture system, we show that PGCs will emigrate from tissue fragments containing hindgut, and that fibronectin stimulates this migration. Adhesion assays show that the start of PGC migration is associated with a fall in adhesion to fibronectin. Double-labelling studies using in situ hybridization and histochemistry demonstrate that migrating PGCs do not contain detectable fibronectin mRNA, suggesting that they do not synthesize and secrete the fibronectin within their migratory substratum. Taken together, these findings are consistent with an important role for fibronectin in stimulating PGC migration. In addition, however, they suggest that the interaction between PGCs and fibronectin may be important in timing the start of migration, with the fall in adhesion allowing the PGCs to commence their migration towards the genital ridges.     

Ror2 enhances polarity and directional migration of primordial germ cells

The trafficking of primordial germ cells (PGCs) across multiple embryonic structures to the nascent gonads ensures the transmission of genetic information to the next generation through the gametes, yet our understanding of the mechanisms underlying PGC migration remains incomplete. Here we identify a role for the receptor tyrosine kinase-like protein Ror2 in PGC development. In a Ror2 mouse mutant we isolated in a genetic screen, PGC migration and survival are dysregulated, resulting in a diminished number of PGCs in the embryonic gonad. A similar phenotype in Wnt5a mutants suggests that Wnt5a acts as a ligand to Ror2 in PGCs, although we do not find evidence that WNT5A functions as a PGC chemoattractant. We show that cultured PGCs undergo polarization, elongation, and reorientation in response to the chemotactic factor SCF (secreted KitL), whereas Ror2 PGCs are deficient in these SCF-induced responses. In the embryo, migratory PGCs exhibit a similar elongated geometry, whereas their counterparts in Ror2 mutants are round. The protein distribution of ROR2 within PGCs is asymmetric, both in vitro and in vivo; however, this asymmetry is lost in Ror2 mutants. Together these results indicate that Ror2 acts autonomously to permit the polarized response of PGCs to KitL. We propose a model by which Wnt5a potentiates PGC chemotaxis toward secreted KitL by redistribution of Ror2 within the cell.     

Inter-species transplantation and migration of primordial germ cells in cyprinid fish

Primordial germ cells (PGCs) are the only cells in developing embryos that can transmit genetic information to the next generation. PGCs therefore have considerable potential value for gene banking and cryopreservation, particularly via production of donor gametes using germ-line chimeras. In some animal species, including teleost fish, the feasibility of using PGC transplantation to obtain donor-derived offspring, within and between species, has been demonstrated. Successful use of PGC transplantation to produce germ-line chimeras is absolutely dependent on the migration of the transplanted cells from the site of transplantation to the host gonadal region. Here, we induced germ-line chimeras between teleost species using two different protocols: blastomere transplantation and single PGC transplantation. We evaluated the methods using the rate of successful migration of transplanted PGCs to the gonadal region of the host embryo. First, we transplanted blastomeres from zebrafish, pearl danio, goldfish, or loach into blastula-stage zebrafish embryos. Some somatic cells, derived from donor blastomeres, were co-transplanted with the PGCs and formed aggregates in the host embryos; a low efficiency of PGC transfer was achieved. Second, a single PGC from the donor species was transplanted into a zebrafish embryo. In all inter-species combinations, the donor PGC migrated toward the gonadal region of the host embryo at a comparatively high rate, regardless of the phylogenetic relationship of the donor and host species. These transplantation experiments showed that the mechanism of PGC migration is highly conserved beyond the family barrier in fish and that transplantation of a single PGC is an efficient method for producing inter-species germ-line chimeras.     

Collagen and tenascin-C expression along the migration pathway of mouse primordial germ cells

Primordial germ cells (PGCs) are the progenitor cells of the vertebrate germ line. These cells originate outside of the embryo and, through separation, migration, and colonization, arrive at the genital ridge, contributing to gonad development. Diverse extracellular matrix molecules are present along the PGC migratory pathway, permitting or inhibiting PGC displacement. Collagens and tenascin form the substratum for in vitro migration of neural crest cells and PGCs. However, little is known about the expression and distribution of these molecules during in situ PGC migration. Using immunohistochemistry, we identified tenascin-C and types I, III, and V collagen along the mouse PGC migration pathway. These molecules were spatiotemporally expressed in basement membranes of hindgut, coelomic epithelia, and mesonephric tubules and mesenchyme throughout the study. Our results complement previous data from our laboratory and contribute to building comprehension of the composition of the mouse PGC migratory pathway extracellular matrix, thereby enhancing understanding of the process.     

The unique responses of the primordial germ cells in the fish Oryzias latipes to gamma-rays

The effects of gamma-radiation on the development of the primordial germ cells (PGCs) of medaka embryos (Oryzias latipes) during the early stages of development were quantitatively examined and compared to the effects on the intestinal cells. The PGCs develop in three stages: an extra-gonadal proliferative stage (1-2.5 days after fertilization), a mitotically inactive stage after the termination of the migration into the gonad (2.5-4.5 days), and an extensive proliferative stage (between 4.5 days and hatching). A dose-rate effect was absent in the PGCs, regardless of their mitotic activity, when dose rates were 2.5 and 0.14 Gy/min. The radiation effect on the PGCs was not reduced by hypoxia and was not enhanced by heat treatment during the proliferating stages. Conversely, radiation resistance was induced in the PCGs during the mitotocally inactive stage by hypoxia and, unexpectedly, by heat treatment. From the present data, we conclude that the PGCs have a small repair ability, and we discuss the radiation resistance induced in the PGCs by hypoxia and heat treatment.     

Ontogenesis of primordial germ cells

In sexually reproducing animals all gametes of either sex arise from primordial germ cells (PGC). PGC represent a small cell population, appearing early during embryo development. They represent a key cell population responsible for the survival and the evolution of a species. Indeed, the production of gametes will assure fertilisation and therefore the establishment of the next generation. Until recently only few laboratories were working on PGC biology. A new interest emerged since these cells have the ability to function as pluripotent stem cells when established as cell lines. Indeed, like embryonic stem cells (ESC), embryonic germ cells (EGC) are able to differentiate in a wide variety of tissues. In vivo, EGC are able, after injection into a host blastocyst cavity to colonise the inner cell mass and to participate in embryonic development. In vitro studies in human and mouse have also shown their capacity to differentiate into a large variety of cell types allowing the study of processes involved in cardiomyocyte, haematopoietic, neuronal and myogenic differentiation pathways. We present here the last updates of PGC ontogeny focusing mainly on the murine model.     

In vitro differentiation of germ cells from stem cells: a comparison between primordial germ cells and in vitro derived primordial germ cell-like cells

Stem cells are unique cell types capable to proliferate, some of them indefinitely, while maintaining the ability to differentiate into a few or any cell lineages. In 2003, a group headed by Hans R. Schöler reported that oocyte-like cells could be produced from mouse embryonic stem (ES) cells in vitro. After more than 10 years, where have these researches reached? Which are the major successes achieved and the problems still remaining to be solved? Although during the last years, many reviews have been published about these topics, in the present work, we will focus on an aspect that has been little considered so far, namely a strict comparison between the in vitro and in vivo developmental capabilities of the primordial germ cells (PGCs) isolated from the embryo and the PGC-like cells (PGC-LCs) produced in vitro from different types of stem cells in the mouse, the species in which most investigation has been carried out. Actually, the formation and differentiation of PGCs are crucial for both male and female gametogenesis, and the faithful production of PGCs in vitro represents the basis for obtaining functional germ cells.     

Heparan sulfate glycosaminoglycans modulate migration and survival in zebrafish primordial germ cells

Early in embryonic development, primordial germ cells (PGCs) are specified and migrate from the site of their origin to where the gonad develops, following a specific route. Heparan sulfate glycosaminoglycans (HS-GAGs) are ubiquitous in extracellular matrix and the cell surface and have long been speculated to play a role during the migration of PGCs. In line with this speculation, whole-mount immunohistochemistry revealed the existence of HS-GAGs in the vicinity of migrating PGCs in early zebrafish embryos. To examine the roles of HS-GAGs during PGC migration, zebrafish heparanase 1 (hpse1), which degrades HS-GAGs, was cloned and overexpressed specifically in PGCs. The guidance signal for the migration of PGCs was disrupted with the overexpression of hpse1, as cluster formation and marginal localization at the blastoderm were significantly perturbed at 6 hours postfertilization. Furthermore, the number of PGCs was significantly decreased with the lack of vicinal HS-GAGs, as observed in the whole-mount in situ hybridization and quantitative PCR of the PGC marker gene vasa. Terminal deoxynucleotidyl transferase dUTP nick-end labeling indicated significantly increased apoptosis in PGCs overexpressing hpse1, suggesting that HS-GAGs contribute to the maintenance of PGC survival. In conclusion, HS-GAGs play multifaceted roles in PGCs during migration and are required both for guidance signals and multiplication of PGCs.     

Mammalian and human primordial germ cells: Differentiation,identification, migration

In recent years, a large amount of data on gene expression at different stages of primordial germ cell (PGC) development has been acquired. The process of germ line segregation in various species is realized differently, i.e., as preformation or epigenesis. The review surveys the mechanisms of the initial lineage specification of mammalian and human germ cells. The data on PGC identification from their initial detection in the epiblast to gonadal anlagen where they migrate has been analyzed. Information on the PGC markers of the different development, the mechanisms of PGC migration towards genital ridges and the chemokines that direct migration are discussed.     

Medaka vasa is required for migration but not survival of primordial germ cells

vasa is essential for germline development. However, the precise processes in which vasa involves vary considerably in diverse animal phyla. Here we show that vasa is required for primordial germ cell (PGC) migration in the medakafish. vasa knockdown by two morpholinos led to the PGC migration defect that was rescued by coinjection of vasa RNA. Interestingly, vasa knockdown did not alter the PGC number, identity, proliferation and motility even at ectopic locations. We established a cell culture system for tracing PGCs at the single cell level in vitro. In this culture system, control and morpholino-injected gastrulae produced the same PGC number and the same time course of PGC survival. Importantly, vasa-depleted PGCs in culture had similar motility and locomotion to normal PGCs. Expression patterns of wt1a, sdf1b and cxcr4b in migratory tissues remained unchanged by vasa knockdown. By chimera formation we show that PGCs from vasa-depleted blastulae failed to migrate properly in the normal environment, whereas control PGCs migrated normally in vasa-disrupted embryos. Furthermore, ectopic PGCs in vasa-depleted embryos also retained all the PGC properties examined. Taken together, medaka vasa is cell-autonomously required for PGC migration, but dispensable to PGC proliferation, motility, identity and survival.     

Isolation of mouse primordial germ cells

Primordial germ cells (PGCs) were obtained from fetal mouse gonads of both sexes days post coitum (dpc), either by collagenase treatment, or by mechanical procedures with or without prior EDTA treatment. With mechanical procedures alone, yield was relatively low and many of the cells released were dead. After EDTA treatment, both yield and viability were significantly improved. Collagenase treatment gave the best yield of cells, since the entire gonad was disaggregated, but contamination with somatic cells was substantial, and the adhesive properties of the germ cells were altered by the treatment. When cells released following EDTA treatment were fractionated on a simple Percoll gradient, several thousand viable PGCs per gonad could be obtained in 2–3 h, with not more than 10–20% somatic cell contamination.     

禽类原始生殖细胞的迁移能力

Blood samples were collected from chicken embryos at stage 11-15,and labeled with fluorescent dye PKH26.Primordial germ cells (PGCs)were then isolated from blood samples by nycodenz density gradient centrifugation.After PGCs were labeled and isolated,about 200 PGCs in one microliter were injected into the subgerminal cavity of quail blastoderm at stageX.After 48 hours incubation,chicken PGCs were identified by fluorescent microscopy.Red fluorescence emitted from PKH26 labeled chicken PGCs was observed in the head, the heart and the developing gonadal anlage of quail embryos.The result suggests that chicken PGCs still keep migration ability after 56 hours[Acta Zoologica Sinica 49(6):868-872,2003].