Cellular and molecular aspects of mouse primordial germ cell migration and proliferation in culture.

The development of mouse primordial germ cells is followed from their first appearance in the extraembryonic mesoderm of the posterior amniotic fold (7 dpc embryo) to their settlement in the genital ridges (12.5 dpc embryo). The role of fibronectin as adhesive substrate and/or in stimulating cell motility during PGC migration is discussed. Recent papers showing how PGCs migrate when cultured in vitro on cellular monolayers are reviewed. The process of PGC homing is proposed to be controlled by chemotaxis as well by developmentally regulated cell-to-cell interactions. Finally, evidence that survival and proliferation of PGCs is strictly dependent on growth factors such as LIF and MGF, and possibly on a cAMP-dependent mechanism is reported.     

Growth factors in mouse primordial germ cell migration and proliferation

Information obtained mainly from in vitro culture studies and genetic analysis of mouse mutants White spotting and Steel indicate a pivotal role of growth factors in the development of mouse primordial germ cells (PGCs). While stem cell factor (SCF) and TGFβ1 seem to have a role in PGC migration (as an adhesion factor and a chemoattractant, respectively), the former is certainly required for PGC survival in vitro and probably in vivo as well. Recent findings suggest that the mechanism by which SCF supports PGC survival is by preventing PGC apoptosis. A similar action appears to be exerted by leukemia inhibitory factor (LIF), a further growth factor influencing PGC growth in culture.PGC proliferation seems to be mainly induced by cAMP dependent mechanisms, but futther investigations are needed to clarify the interrelationships among the different molecular pathways activated by SCF, LIF, cAMP and other putative PGC growth factors (i.e. bFGF). Stimulation of long-term proliferation of PGCs, leading to derivation of ES-like cells (embryonal germ cells) obtained by using a combination of growth factors (bFGF, SCF and LIF), opens new intriguing perspectives for such studies and transgenic technology.     

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.     

In vitro culture of mouse primordial germ cells

Germ cells were isolated from mouse fetal gonads 11 1/2-16 1/2 days post coitum (dpc), and exposed to various methods of in vitro culture. From 13 1/2 dpc onwards, both male and female germ cells survived well at 37 degrees C for several days. During the culture period the proportion of female germ cells in meiosis increased and later stages of meiotic prophase were seen. The gonadal environment is therefore not essential for the progress of meiosis. Male germ cells in vitro did not enter meiosis. Germ cells isolated from gonads 11 1/2 or 12 1/2 dpc did not survive at 37 degrees C in any of the three culture systems used (Petri dishes, microtest plate wells, drops under oil); cell density, substrate and culture medium were varied, and several additives tested, but no improvement in viability was detected. Below 30 degrees C, on the other hand, 11 1/2 and 12 1/2 day germ cells survived in vitro for at least a week. They did not enter meiosis in culture, but continued to undergo mitotic proliferation.     

小鼠原生殖细胞体外培养及其应用研究

原生殖细胞（ｐｒｉｍｏｒｄｉａｌｇｅｒｍｃｅｌｌ,ＰＧＣ）是胚胎生殖谱系最原始形式的细胞,在体胚胎迁移期ＰＧＣ增殖极为旺盛。体外培养的小鼠迁移期ＰＧＣ在饲养层细胞和三种生长因子（干细胞生长因子、碱性成纤维细胞生长因子及白血病抑制因子）的共同作用下,可发展为长期增殖并维持不分化状态的胚胎性干细胞,即胚胎生殖细胞（ｅｍｂｒｙｏｎｉｃｇｅｒｍｃｅｌｌ,ＥＧ）,具全能性发育潜能。ＥＧ建系成功对于研究生殖细胞发育以及寻找新的转基因动物操作的有效载体具有重要价值。     

Autonomous modes of behavior in primordial germ cell migration

Zebrafish primordial germ cells (PGCs) are guided toward their targets by the chemokine SDF-1a. PGCs were followed during three phases of their migration: when migrating as individual cells, while remaining in a clustered configuration, and when moving as a cell cluster within the embryo. We found that individually migrating PGCs alternate between migratory and pausing modes. Pausing intervals are characterized by loss of cell polarity and correlate with subsequent changes in the direction of migration. These properties constitute an intrinsic behavior of PGCs, enabling erasure of prior polarity and re-sampling of the environment. Following migration arrest at a site of high SDF-1a levels, PGCs resume migration as a cluster. The seemingly coordinated cluster migration is a result of single-cell movement in response to local variations in SDF-1a distribution. Together, these behavioral modes allow the cells to arrive at specific destinations with high fidelity and remain at their target site.     

Hematopoietic development of primordial germ cell-derived mouse embryonic germ cells in culture.

Induction of hematopoiesis in an embryonic germ (EG) cell line derived from mouse primordial germ cells (PGCs) was examined. When single undifferentiated EG-1 cells were inoculated directly into the methylcellulose medium, both primitive and definitive erythropoiesis were seen in embryoid bodies derived from the EG cells as observed in ES cells, and production of myeloid cell lineages was stimulated by IL-3. These results indicate that EG cells acquired in vitro potency to differentiate toward hematopoietic cells, although they were derived from PGC and are distinct from inner cell mass-derived ES cells with regard to gene expression and patterns of DNA methylation corresponding to genomic imprinting. It turns out that they are useful for study of cell differentiation in the animals whose ES cells are not available.     

Migratory and postmigratory mouse primordial germ cells behave differently in culture

In all vertebrate groups, the progenitors of the germ line, the primordial germ cells (PGCs) arise extragonadally and move to the developing gonad early in embryonic development. We have examined the behavior of isolated pregonadal and gonadal PGCs in vitro on feeder layers of an embryo-derived cell line. Histochemically and serologically identified pregonadal germ cells are found to be actively motile in vitro and, furthermore, show behavior characteristic of invasive cells. PGCs isolated from the developing gonad, however, show little locomotory activity and are not invasive on the same cellular substrate. These observations suggest that PGCs undergo a major change in phenotype at the time of their entry into the gonad anlagen.     

The role of cadherins during primordial germ cell migration and early gonad formation in the mouse

Primordial germ cells (PGCs) are the founder cells of the gametes. In mammals, PGCs migrate from the hindgut to the genital ridges, where they coalesce with each other and with somatic cells to form the primary sex cords. We show here that, in both sexes, PGCs express P- and E-cadherins during and after migration, and N-cadherin at post-migratory stages. E-Cadherin is not expressed by PGCs whilst in the hindgut, but is upregulated as they leave. Blocking antibodies against E-, but not P-cadherin cause defective PGC-PGC coalescence, and in some cases, ectopic PGCs.     

Regulation of primordial germ cell development in the mouse

Primordial germ cells (PGCs) are the founders of the gametes. They arise at the earliest stages of embryonic development and migrate to the gonadal ridges, where they differentiate into oogonia/oocytes in the ovary, and prospermatogonia in the testis. The present article is a review of the main studies undertaken by the author with the aim of clarifying the mechanisms underlying the development of primordial germ cells. Methods for the isolation and purification of migratory and post-migratory mouse PGCs devised in the author's laboratory are first briefly reviewed. Such methods, together with the primary culture of PGCs onto suitable cell feeder layers, have allowed the analysis of important aspects of the control of their development, concerning in particular survival, proliferation and migration of mouse PGCs. Compounds and growth factors affecting PGC numbers in culture have been identified. These include survival anti-apoptotic factors (SCF, LIF) and positive regulators of proliferation (cAMP, PACAPs, RA). Evidence has been provided that the motility of migrating PGCs relies on integrated signals from extracellular matrix molecules and the surrounding somatic cells. Moreover, homotypic PGC-PGC interaction has been evidenced that might play a role in PGC migration and in regulating their development. Several molecules (i.e. integrins, specific types of oligosaccharides, E-cadherin, the tyrosine kinase receptor c-kit) have been found to be expressed on the surface of PGCs and to mediate adhesive interactions of PGCs with the extracellular matrix, somatic cells and neighbouring PGCs.     

A role for E-cadherin in mouse primordial germ cell development

In this study we show that mouse primordial germ cells and fetal germ cells at certain stages of differentiation express E-cadherin and alpha and beta catenins. Moreover, we demonstrate that the formation of germ cell aggregates that rapidly occurs when monodispersed germ cell populations are released from embryonic gonads in culture is E-cadherin mediated, developmentally regulated, and dependent on the sex of the germ cells. Immunoblotting analyses indicate that the lower ability to form aggregates of primordial germ cells in comparison to fetal germ cells is not due to gross changes in E-cadherin expression, altered association with beta catenin, or changes in beta catenin phosphorylation. Investigating possible functions of E-cadherin-mediated adhesion in primordial germ cell development, we found that E-cadherin-mediated adhesion may stimulate the motility of primordial germ cells. Moreover, treatment of primordial germ cells cultured on STO cell monolayers with an anti-E-cadherin antibody caused a significant decrease in their number and markedly reduced their ability to form colonies in vitro. The same in vitro treatment of explanted undifferentiated gonadal ridges cultured for 4 days results in decreased numbers and altered localization of the germ cell inside the gonads. Taken together these results suggest that E-cadherin plays an important role in primordial germ cell migration and homing and may act as a modulator of primordial germ cell development.     

Influence of simulated microgravity on avian primordial germ cell migration and reproductive capacity

Fertilized eggs of chicken and quail were incubated under the simulated microgravity condition provided by a clinostat. The number of Primordial Germ Cells (PGCs) was counted in early embryogenesis, and the reproductive capacity of quail hatched following the simulated microgravity was investigated.Simulated microgravity caused significant decline of PGCs in the blood of early chicken embryos and in the gonads. The numbers of spermatogonia in the hatchling testis were also fewer than those in the control groups. Therefore, simulated microgravity may retard gonadial development and reduce the reproductive capacity.     

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.     

Ultraviolet irradiation of Rana pipiens embryos delays the migration of primordial germ cells into the genital ridges

Ultraviolet (UV) irradiation of the vegetal pole of anuran embryos at the two-cell stage has been reported to cause aberrant cleavage as well as a subsequent reduction in germ cell numbers. In this study, we find no correlation between UV-induced cleavage abnormalities and the absence of primordial germ cells in Rana pipiens tadpoles examined at stage 25. On the other hand, some tadpoles from a population which was lacking primordial germ cells at stage 25 subsequently contained germ cells. These late-appearing germs cells exhibited damaged mitochondria, autophagosomes, and secondary lysosomes, while surrounding somatic cells were morphologically normal. We suggest that these cytoplasmic abnormalities resulted from an effect of the initial UV irradiation of germ plasm. We conclude that one effect of UV irradiation of germ plasm is to delay or inhibit the migration of primordial germ cells into the genital ridges.     

Guidance of primordial germ cell migration by the chemokine SDF-1

The signals directing primordial germ cell (PGC) migration in vertebrates are largely unknown. We demonstrate that sdf-1 mRNA is expressed in locations where PGCs are found and toward which they migrate in wild-type as well as in mutant embryos in which PGC migration is abnormal. Knocking down SDF-1 or its receptor CXCR4 results in severe defects in PGC migration. Specifically, PGCs that do not receive the SDF-1 signal exhibit lack of directional movement toward their target and arrive at ectopic positions within the embryo. Finally, we show that the PGCs can be attracted toward an ectopic source of the chemokine, strongly suggesting that this molecule provides a key directional cue for the PGCs.     

Drosophila primordial germ cell migration requires epithelial remodeling of the endoderm

Trans-epithelial migration describes the ability of migrating cells to cross epithelial tissues and occurs during development, infection, inflammation, immune surveillance, wound healing and cancer metastasis. Here we investigate Drosophila primordial germ cells (PGCs), which migrate through the endodermal epithelium. Through live imaging and genetic experimentation we demonstrate that PGCs take advantage of endodermal tissue remodeling to gain access to the gonadal mesoderm and are unable to migrate through intact epithelial tissues. These results are in contrast to the behavior of leukocytes, which actively loosen epithelial junctions to migrate, and raise the possibility that in other contexts in which migrating cells appear to breach tissue barriers, they are actually exploiting existing tissue permeability. Therefore, the use of active invasive programs is not the sole mechanism to infiltrate tissues.     

Serum-free culture of murine primordial germ cells and embryonic germ cells

Fetal calf serum (FCS) has usually been used for culture of embryonic stem (ES) cell as a component of the culture medium. However, FCS contains undefined factors, which promote cell proliferation and occasionally stimulate differentiation of ES cells. Recently, a chemically-defined serum replacement, Knockout Serum Replacement (KSR), was developed to maintain ES cells in an undifferentiated state. In this experiment, we examined the effects of KSR on the growth and differentiation of primordial germ cells (PGCs) and embryonic germ (EG) cells. PGCs were collected 8.5 days postcoitum (dpc) from B6D2F1 (C57BL/6JxDBA/2J) female mice mated with B6D2F1 males. Most of the PGCs that were cultured in FCS-supplemented medium (FCS medium) had alkaline phosphatase (AP) activity and acquired a fibroblast cell shape. In contrast, PGCs in KSR-supplemented medium (KSR medium) proliferated, maintaining round and stem cell-like morphology. In addition, EG cells were established more easily from PGCs cultured in KSR medium than from PGCs cultured in FCS medium. The percentage of undifferentiated colonies of EG cells was significantly higher in KSR medium than in FCS medium. The germ line chimera was also produced from EG cells established in KSR medium. These results suggest that KSR can be used for sustaining an undifferentiated state of PGCs and EG cells in vitro.