Migratory mechanisms of chick primordial germ cells toward gonadal anlage.

After appearing at the germinal crescent region, chick primordial germ cells (PGCs) migrate toward the presumptive gonads (pG) till stage 19 (Hamburger and Hamilton, 1951). This study seeks to elucidate the roles of passive and active factors in the PGC-migration, physical trapping of circulating PGCs by the capillary network and PGC attraction by chemotactic factor from presumptive gonads. Firstly, latex beads/pollens (the same size or larger than PGCs) were injected into the embryonic bloodstream at stage 13-19 (when PGCs are in the migrating and settlement phase to the presumptive gonad) in ovo in order to determine whether the PGCs passively reach pG. Most of such particles accumulated in the head region (60%), whereas the remainder did the same in the gonadal region (23% at the peak) at stage 16 when both the head and gonadal regions are rich in capillary plexus. After 3 days, most particles in the gonadal region were located at the angles of dorsal mesentery near the developing gonads where many extra-gonadal PGCs had been located, and a few particles were detected close to the gonad. These results suggest that one of the mechanisms of PGC-migration to the developing gonads is an autonomous trapping of PGCs by the capillary network quite close to the germinal epithelium (GE) and passive translocation by morphogenetic movement. Secondly, the attraction for PGCs by the gonadal anlage proper was examined in ovo using chick and quail embryos. Grafts of quail gonadal anlage containing gonadal epithelium and neighbouring mesenchymal tissue were excised from the quail embryo at stages 12 to 16 (staging by Zacchei, 1961). With the aims of eliminating the influence of surrounding tissue, the quail graft was ectopically transplanted into the posterior to the optic vesicle of 8 to 17 somite chick embryo from the point of a posterior region to the auditory vesicle by a fine tungsten needle under the illumination by the method of Hara (1971). Then the region posterior to the level of presumptive vitelline arteries was surgically excised in ovo. After a 48 hrs.-incubation, the host PGCs which lost their own gonadal anlage as a target organ accumulated in the transplanted quail gonadal anlage originating from the embryo at PGC-migrating periods. This result strongly suggested the presence of some attractive factor that may be emitted from the gonadal anlage proper. Furthermore, it was demonstrated that the PGCs in vitro showed no contact inhibition in relation to other PGCs or fibroblasts in their moving pathway.     

Segregation of primordial germ cells: Their numbers and fate during early development of Barbus conchonius (Cyprinidae,Teleostei) as indicated by 3H-thymidine incorporation

³H-Thymidine incorporation experiments in Barbus conchonius showed that presumptive primordial germ cells (PGCs) terminated their mitotic activity between midepibolys, and late epiboly. At the ten-somite stage, shortly after labeling of PGCs by uptake of ³H-thymidine became arrested, they could be recognized by their relatively large size and large nucleus. They were located in two longitudinal rows of cells between mesoderm and periblast, always at the same distance to the left and right of the notochord. Contact with the endoderm was not observed before the 16- to 23-somite stage. The numbers of PGCs were small (mean number, 18–19) and remained small for nearly 3 weeks. Mitotic activity was not observed in PGCs during that period; thereafter, rapid proliferation began. There is no evidence for active migration of PGCs; it is assumed that they are merely translocated passively together with their surrounding tissues. No specific constituents were detected with histochemical methods for glycogen, alkaline phosphatase, and RNA. Electron microscopy revealed the presence of “nuage” around the nucleus of PGCs. This material corresponded with perinuclear dense bodies as seen with light microscopy from the 19-somite stage onward. It is concluded that presumptive PGCs segregate from the somatic cells between midepiboly and late epiboly, before the three germ layers have been formed, and that locations of PGCs in the endodermal or mesodermal layer may be merely transitory stages during their translocation toward the gonadal primordia.     

Evidence for substrate guidance of primordial germ cells

Primordial germ cells (PGCs) have been removed from their normal migratory route in early embryos of Xenopus laevis, and their behaviour studied in vitro. They adhere to, and move over the upper surface of, layers of outgrowing cells from expiants of adult Xenopus mesentery. They move by the extrusion of single filopodia, elongation, forward streaming of the yolky cytoplasm and retraction of their trailing ends. When the underlying cells are polarized in one direction only, PGCs always elongate and move along the same direction. Furthermore, when PGCs elongate and move over less obviously polarized cells, they always do so in the direction of ‘stress fibres’ (actin bundles) in the underlying cells. A substrate-guidance hypothesis for PGC migration is only tenable if there is some orientation in their natural substrate in vivo. Using the scanning electron microscope, we demonstrate that the coelpmic lining cells, beneath which PGCs migrate up the dorsal mesentery of the gut, are orientated in the direction of travel. Furthermore, this orientation changes at the time of gonadal ridge formation. This raises the intriguing possibility that PGCs are guided for at least part of their migration in Xenopus laevis embryos by a substrate-guidance mechanism.     

Asymmetrical Development of the Gonads in the Embryos and Fry of the Fish, Oryzias celebensis

The development of the gonad in Oryzias celebensis was studied by light and electron microscopy. The path of PGC-migration, the time of the sex-differentiation, and the pattern of germ cell proliferation were almost identical to those in O. latipes. The most conspicuous difference was in the distribution of germ cells after migration. The gonadal anlage in O. celebensis developed only on the right side of the dorsal mesentery, although PGCs were stituated on the both sides of the embryos before migration. The testis retained its unilateral condition throughout development and acquired a unilobed shape. In the female, the right presumptive ovary developed over the mesentery, and the ovary became bilobed. Thereafter, right and left parts of the presumptive ovary fused to develop into a single ovary in the adult fish. The situation appears to be comparable to the gonadal asymmetry in birds, but the present observations suggest that the developmental processes of the asymmetrical gonads in this fish are quite different from those in birds.     

Changes in the morphology of the germinal dense bodies in primordial germ cells of the teleost,Oryzias latipes

Summary In many organisms, the germinal dense bodies (GDBs) are known to be organelles unique to the cells of germ-line. In the present study, GDBs in primordial germ cells (PGCs) of the teleost, Oryzias latipes, were examined by electron microscopy. An obvious change was noticed in the morphology of GDBs. In PGCs situated in the endoderm, GDBs consisted of a loosely woven strand-like structure, whereas, GDBs in PGCs in the gonadal anlage, which were amorphous bodies of various sizes and shapes, were composed of electron-dense fine fibrils. The changes in the morphology of GDBs proceeded gradually according to the progress of the stages in migration of the PGCs. GDBs of intermediate morphology were found. The change in the morphology of the GDBs began at the stage of movement of the PGCs from endoderm to mesoderm. It is suggested that the differentiation of PGCs proceeds during their migratory stages under the influence of surrounding somatic cells.     

Regulation of zebrafish primordial germ cell migration by attraction towards an intermediate target.

Migration of primordial germ cells (PGCs) from their site of specification towards the developing gonad is controlled by directional cues from somatic tissues. Although in several animals the PGCs are attracted by signals emanating from their final target, the gonadal mesoderm, little is known about the mechanisms that control earlier steps of migration. We provide evidence that a key step of zebrafish PGC migration, in which the PGCs become organized into bilateral clusters in the anterior trunk, is regulated by attraction of PGCs towards an intermediate target. Time-lapse observations of wild-type and mutant embryos reveal that bilateral clusters are formed at early somitogenesis, owing to migration of PGCs towards the clustering position from medial, posterior and anterior regions. Furthermore, PGCs migrate actively relative to their somatic neighbors and they do so as individual cells. Using mutants that exhibit defects in mesoderm development, we show that the ability to form PGC clusters depends on proper differentiation of the somatic cells present at the clustering position. Based on these findings, we propose that these somatic cells produce signals that attract PGCs. Interestingly, fate-mapping shows that these cells do not give rise to the somatic tissues of the gonad, but rather contribute to the formation of the pronephros. Thus, the putative PGC attraction center serves as an intermediate target for PGCs, which later actively migrate towards a more posterior position. This final step of PGC migration is defective in hands off mutants, where the intermediate mesoderm of the presumptive gonadal region is mispatterned. Our results indicate that zebrafish PGCs are guided by attraction towards two signaling centers, one of which may represent the somatic tissues of the gonad.     

OBSERVATIONS OF PRIMORDIAL GERM CELLS IN THE TURTLE EMBRYO (CARETTA CARETTA): LIGHT AND ELECTRON MICROSCOPIC STUDIES

Primordial germ cells (PGCs) in the turtle embryo ( Caretta caretta ) were observed with light and transmission electron microscopes. Identification of the PGCs for light microscopy was made by the periodic acid-Schiff (PAS) technique. PGCs were first found in the yolk-sac endoderm through the 5th to 6th day of development. PGCs freed from the endoderm then migrated to the root area of the dorsal mesentery and the coelomic angle between the 7th and the 11th day of development, and finally settled down in the gonadal anlage by the 14th day. Turtle PGCs were characterized by a large size (16 μm in diameter) and large nuclei with distinct nucleoli, and by the presence of large numbers of lipid droplets, yolk platelets and glycogen particles in the cytoplasm. Cell organelles were well-developed in PGCs at later stages. Amoeboid features of the PGCs were observed in the mesenchyme, indicating active locomotion. PGCs were usually surrounded or encircled by neighboring somatic cells. No intravascular PGCs were detected at any stage of development examined.     

Mesodermal cell migration during Xenopus gastrulation

The adhesive glycoprotein fibronectin (FN), which is a component of the network of extracellular matrix fibrils on the inner surface of the blastocoel roof (BCR), has been proposed to play a major role in directing mesodermal cell migration during amphibian gastrulation. In the first part of this paper, the adhesion of Xenopus mesodermal cells to FN in vitro is examined. Cells from several mesoderm regions, which differ in developmental fate and morphogenetic activity, are able to bind specifically to the RGD cell-binding site of FN. Dorsal mesodermal cell adhesion to FN varies along the anterior-posterior (a-p) axis: adhesion is strongest in the anterior head mesoderm, and gradually decreases posteriorly. This a-p gradient of mesodermal adhesiveness to FN does not change during mesodermal involution, and is reflected in the morphology of mesodermal explants on FN. An a-p strip of mesoderm develops a spreading, leading anterior margin and a compact, retracting posterior end, thus moving slowly and directionally over the FN substrate at about 0.8 micron/min. Although dissociated cells from all levels of the dorsal mesodermal axis adhere to FN, only the anterior, leading prospective head mesoderm cells migrate as single cells on a FN substrate in vitro. Locomotion by means of lamelliform protrusions occurs at an average rate of about 1.5 micron/min. Cells of the more posterior axial mesoderm merely shift position at random without substantial net translocation, and preinvolution mesoderm cells are completely stationary. On the BCR, the in vivo substrate for mesodermal cell migration, dissociated prospective head mesoderm cells spread and migrate as on FN in vitro, at 2.2 microns/min. In the presence of an RGD peptide which inhibits cell-FN interaction, cells remain globular and do not spread. They are still motile, but change direction frequently, which leads to less efficient net translocation. Apparently, interaction with the RGD cell-binding site of FN and concomitant spreading of head mesoderm cells is required for the stabilization of cell locomotion. In contrast to the directional migration of the mesoderm cell population toward the animal pole in the embryo, the pathways of dissociated cells on the BCR are randomly oriented. Coherent explants of migratory mesoderm do not move at all on the BCR, although they translocate on FN in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)     

Activin and TGFbeta limit murine primordial germ cell proliferation

Mammalian primordial germ cells (PGCs) proliferate as they migrate from their initial location in the extraembryonic mesoderm to the genital ridge, the gonadal anlage. Once in the genital ridge, PGCs cease dividing and differentiate according to their gender. To identify ligands that might limit PGC proliferation, we analyzed growth factor receptors encoded in RNA obtained from purified germ cells shortly after their arrival in the genital ridge. Receptors for two members of the TGFbeta superfamily were found, TGFbeta1 and activin. As the signal-transducing domains of both receptor systems are highly conserved, the effects of both TGFbeta1 and activin on PGCs would be expected to be similar. We found that both ligands limited the accumulation of germ cells in primary PGC cultures. BrdU incorporation assays demonstrated that either ligand inhibits PGC proliferation. These results suggest that these signal transduction pathways are important elements of the mechanism that determines germ cell endowment.     

革胡子鲇卵巢在第一次性周期内分化与发育的研究

用光镜和电镜研究了革胡子鲇（Ｃｌａｒｉａｓｌａｚｅｒａ）原生殖细胞的起源迁移,卵巢在第１次性周期内的分化与发育以及各发育时期卵母细胞的超微结构。原生殖细胞起源于内胚层,有固定的迁移路线,进入生殖嵴后,生殖嵴进一步分化,出现卵巢腔分化成卵巢,卵母细胞在第１次性周期内的发育可分成６个时相,描述了各时相卵母细胞的显微结构与超微结构。同时,叙述了卵母细胞中卵黄发生的形态形成。     

Generation of germ-line chimera zebrafish using primordial germ cells isolated from cultured blastomeres and cryopreserved embryoids

Primordial germ cells (PGCs) are the only cells in developing embryos with the potential to transmit genetic information to the next generation. In our previous study, a single PGC transplanted into a host differentiated into fertile gametes and produced germ-line chimeras of cyprinid fish, including zebrafish. In this study, we aimed to induce germ-line chimeras by transplanting donor PGCs from various sources (normal embryos at different stages, dissociated blastomeres, embryoids, or embryoids cryopreserved by vitrification) into host blastulae, and compare the migration rates of the PGCs towards the gonadal ridge. Isolated, cultured blastomeres not subject to mesodermal induction were able to differentiate into PGCs that retained their motility. Moreover, these PGCs successfully migrated towards the gonadal ridge of the host and formed viable gametes. Motility depended on developmental stage and culture duration: PGCs obtained at earlier developmental stages and with shorter cultivation periods showed an increased rate of migration to the gonadal ridge. Offspring were obtained from natural spawning between normal females and chimeric males. These results provide the basis for new methods of gene preservation in zebrafish.     

Altered primordial germ cell migration in the absence of transforming growth factor beta signaling via ALK5

Transforming growth factor beta (TGFbeta) inhibits proliferation and promotes the migration of primordial germ cells (PGCs) towards explants of gonadal ridges in vitro. However, its effects in vivo are still unclear. Here, we analyzed the behavior of PGCs in embryos lacking TGFbeta signaling via the type I receptor ALK5. TGFbeta in vivo was neither a chemoattractant for PGCs, nor did it affect their proliferation during migration towards the gonadal ridges up to embryonic day (E)10. Unexpectedly, the absence of TGFbeta signaling in fact resulted in significant facilitation of PGC migration out of the hindgut, due to the reduced deposition of collagen type I surrounding the gut of Alk5-deficient mutant embryos. Migratory PGCs adhere strongly to collagen; therefore, reduced collagen type I along the gut may result in reduced adhesion, facilitating migration into the dorsal mesenterium and gonadal ridges. Our results provide new evidence for the role of TGFbeta signaling in migration of PGCs in vivo distinct from that described previously.     

Dual functions of the heartless fibroblast growth factor receptor in development of the Drosophila embryonic mesoderm

The Drosophila embryonic mesoderm forms by invagination of the ventral-most blastoderm cells. Subsequent development of this germ layer involves the dorsolateral migration of the internalized cells and expansion by cell division, followed by the specification of particular cell fates through the coordinate actions of both intrinsic and extrinsic regulatory mechanisms. The latter include several intercellular signals that function across germ layers. These processes combine to generate a diversity of mesodermal subtypes, including the cardial and pericardial cells of the heart or dorsal vessel, a complete set of somatic muscle founders each with its unique identity, a population of cells that form the visceral musculature, and other cells that develop into hemocytes and the fat body. Here, we review recent evidence for the involvement of a fibroblast growth factor receptor (FGFR) encoded by the heartless (htl) gene in early directional migration of the Drosophila mesoderm. In addition, we provide new data that 1) demonstrate a second role for Htl in promoting the specification of the precursors to certain cardiac and somatic muscle cells in the Drosophila embryo, independent of its cell migration function, 2) suggest that Ras and at least one other signal transduction pathway act downstream of Htl, and 3) establish a functional relationship between the Ras pathway and Tinman (Tin), a homeodomain factor that is essential for specifying some of the same dorsal mesodermal cells that are dependent on Htl. Finally, parallels between requirements for FGFR signaling in Drosophila and vertebrate mesoderm development are considered. Dev. Genet. 22:212–229, 1998. © 1998 Wiley-Liss, Inc.     

Evidence for crucial role of hindgut expansion in directing proper migration of primordial germ cells in mouse early embryogenesis

During mouse gastrulation, primordial germ cells (PGCs) become clustered at the base of the allantois and move caudally into the hindgut endoderm before entering the genital ridges. The precise roles of endoderm tissues in PGC migration, however, remain unclear. By using Sox17 mutants with a specific endoderm deficiency, we provide direct evidence for the crucial role of hindgut expansion in directing proper PGC migration. In Sox17-null embryos, PGCs normally colonize in the allantois and then a small front-row population of PGCs moves properly into the most posterior gut endoderm. Defective hindgut expansion, however, causes the failure of further lateral PGC movement, resulting in the immobilization of PGCs in the hindgut entrance at the later stages. In contrast, the majority of the remaining PGCs moves into the visceral endoderm layer, but relocate outside of the embryonic gut domain. This leads to a scattering of PGCs in the extraembryonic yolk sac endoderm. This aberrant migration of Sox17-null PGCs can be rescued by the supply of wildtype hindgut cells in chimeric embryos. Therefore, these data indicate that hindgut morphogenic movement is crucial for directing PGC movement toward the embryonic gut side, but not for their relocation from the mesoderm into the endoderm.     

The hedgehog Pathway Gene shifted Functions together with the hmgcr-Dependent Isoprenoid Biosynthetic Pathway to Orchestrate Germ Cell Migration

The Drosophila embryonic gonad is assembled from two distinct cell types, the Primordial Germ Cells (PGCs) and the Somatic Gonadal Precursor cells (SGPs). The PGCs form at the posterior of blastoderm stage embryos and are subsequently carried inside the embryo during gastrulation. To reach the SGPs, the PGCs must traverse the midgut wall and then migrate through the mesoderm. A combination of local repulsive cues and attractive signals emanating from the SGPs guide migration. We have investigated the role of the hedgehog (hh) pathway gene shifted (shf) in directing PGC migration. shf encodes a secreted protein that facilitates the long distance transmission of Hh through the proteoglycan matrix after it is released from basolateral membranes of Hh expressing cells in the wing imaginal disc. shf is expressed in the gonadal mesoderm, and loss- and gain-of-function experiments demonstrate that it is required for PGC migration. Previous studies have established that the hmgcr-dependent isoprenoid biosynthetic pathway plays a pivotal role in generating the PGC attractant both by the SGPs and by other tissues when hmgcr is ectopically expressed. We show that production of this PGC attractant depends upon shf as well as a second hh pathway gene gγ1. Further linking the PGC attractant to Hh, we present evidence indicating that ectopic expression of hmgcr in the nervous system promotes the release/transmission of the Hh ligand from these cells into and through the underlying mesodermal cell layer, where Hh can contact migrating PGCs. Finally, potentiation of Hh by hmgcr appears to depend upon cholesterol modification.     

FGF8-like1 and FGF8-like2 encode putative ligands of the FGF receptor Htl and are required for mesoderm migration in the Drosophila gastrula

BACKGROUND: Mesoderm migration in the Drosophila gastrula depends on the fibroblast growth factor (FGF) receptor Heartless (Htl). During gastrulation Htl is required for adhesive interactions of the mesoderm with the ectoderm and for the generation of protrusive activity of the mesoderm cells during migration. After gastrulation Htl is essential for the differentiation of dorsal mesodermal derivatives. It is not known how Htl is activated, because its ligand has not yet been identified. RESULTS: We performed a genome-wide genetic screen for early zygotic genes and identified seven genomic regions that are required for normal migration of the mesoderm cells during gastrulation. One of these genomic intervals produces upon its deletion a phenocopy of the htl cell migration phenotype. Here we present the genetic and molecular mapping of this genomic region. We identified two genes, FGF8-like1 and FGF8-like2, that encode novel FGF homologs and were only partially annotated in the Drosophila genome. We show that FGF8-like1 and FGF8-like2 are expressed in the neuroectoderm during gastrulation and present evidence that both act in concert to direct cell shape changes during mesodermal cell migration and are required for the activation of the Htl signaling cascade during gastrulation. CONCLUSIONS: We conclude that FGF8-like1 and FGF8-like2 encode two novel Drosophila FGF homologs, which are required for mesodermal cell migration during gastrulation. Our results suggest that FGF8-like1 and FGF8-like2 represent ligands of the Htl FGF receptor.