On the Origin of Primordial Germ Cells in the Chick Embryo

An attempt was made to re-examine the location of the primordial germ cells (PGCs) in very young chick embryos. Freshly laid blastoderms, prior to hypoblast formation, of a known anterio-posterior axis, were transversely bisected and each half was separately grown in vitro. Both anterior and posterior halves were shown to be fertile and each was shown to contain roughly the same amount of PGCs as a normal control embryo. It has been concluded that in the chick as well as in the duck there is no concentration of cells containing germinal plasm in the posterior part of the blastoderm.
Two other possibilities should be investigated:
1. A concentric arrangement of cells containing germinal plasm. 2. The absence of a germinal plasm and a relatively late appearance of PGCs as a result of induction.     

Interactions between Germ Cells and Extracellular Matrix Glycoproteins during Migration and Gonad Assembly in the Mouse Embryo

Cells are known to bind to individual extracellular matrix glycoproteins in a complex and poorly understood way. Overall strength of adhesion is thought to be mediated by a combinatorial mechanism, involving adhesion of a cell to a variety of binding sites on the target glycoproteins. During migration in embryos, cells must alter their overall adhesiveness to the substrate to allow locomotion. The mechanism by which this is accomplished is not well understood. During early development, the cells destined to form the gametes, the primordial germ cells (PGCs), migrate from the developing hind gut to the site where the gonad will form. We have used whole-mount immunocytochemistry to study the changing distribution of three extracellular matrix glycoproteins, collagen IV, fibronectin, and laminin, during PGC migration and correlated this with quantitative assays of adhesiveness of PGCs to each of these. We show that PGCs change their strength of adhesion to each glycoprotein differentially during these stages. Furthermore, we show that PGCs interact with a discrete tract of laminin at the end of migration. Closer analysis of the adhesion of PGCs to laminin revealed that PGCs adhere particularly strongly to the E3 domain of laminin, and blocking experiments in vitro suggest that they adhere to this domain using a cell surface proteoglycan.     

A Possibility of an in Vitro Differentiation of Primordial Germ Cells (PGCs) from Blastomeres Containing Germinal Plasm of Early Cleavage Stage in Xenopus laevis

The blastomeres containing the germinal plasm were isolated from 32-cell stage Xenopus embryos and cultured in vitro for various periods of time till the control embryos developed to stage 28, 33/34, 40 and 45, respectively. The cells containing the plasm in the 'stage-28', '33/34' and '40' explants were similar in external shape, and in distribution in the spherical endodermal cell mass to the presumptive primordial germ cells (pPGCs) in normal embryos of the corresponding stages. In addition, the cells in explants as well as the pPGCs were separated by a large intercellular space from the surrounding endodermal cells. The change in proportion of the compact or the loosely structured germinal granules and the irregularly shaped-stringlike bodies (ISBs) occurred in the cells of the explants with the prolongation of the culture period. In the cells of the 'stage-45' explant as well as in the PGCs of normal stage-45 tadpoles the ISBs and granular materials replace those germinal granules. These facts lead to the conclusion that the change of the germinal granules through the ISBs, to the granular materials, noticed in the normal course of differentiation of pPGCs into PGCs (see (1)), also takes place in the cells of the explants during the culture. Therefore, it is likely that the cells in the explants are genuine pPGCs or PGCs. This is the first demonstration of a possibility of the in vitro differentiation of PGCs from the blastomeres containing the germinal plasm of early cleavage stage.     

Appearance of Primordial Germ Cells in Young Chick Blastoderms Cultured in vitro

Appearance of primordial germ cells (PGCs) in young chick blastoderms was investigated by the cultivation of only the epiblast or hypoblast. Presumptive PGCs exist in the epiblast before primitive-streak formation. They translocate gradually to the lower layer during early stages of primitive-streak formation, though substantial number of presumptive PGCs remain in the upper layer. The existing primary hypoblast under the epiblast is dispensable for the further differentiation of the PGCs.     

Dullard/Ctdnep1 Modulates WNT Signalling Activity for the Formation of Primordial Germ Cells in the Mouse Embryo

Dullard/Ctdnep1 is a member of the serine/threonine phosphatase family of the C-terminal domain of eukaryotic RNA polymerase II. Embryos lacking Dullard activity fail to form primordial germ cells (PGCs). In the mouse, the formation of PGCs is influenced by BMP4 and WNT3 activity. Although Dullard is reputed to negatively regulate BMP receptor function, in this study we found mutations in Dullard had no detectable effect on BMP4 and p-Smad activity. Furthermore Dullard mutations did not influence the dosage-dependent inductive effect of Bmp4 in PGC formation. However, Dullard may function as a positive regulator of WNT signalling. Combined loss of one copy each of Dullard and Wnt3 had a synergistic effect on the reduction of PGC numbers in the compound heterozygous embryo. In addition, loss of Dullard function was accompanied by down-regulation of WNT/β-catenin signalling activity and a reduction in the level of Dishevelled 2 (Dvl2). Therefore, Dullard may play a role in the fine-tuning of WNT signalling activity by modulating the expression of ligands/antagonists and the availability of Dvl2 protein during specification of the germ cell lineage.     

On the Segregation of the Germ and Somatic Cell Lines in the Embryo

Abstract. In this paper we argue that the mechanisms underlying the segregation of the somatic and germ cell lines are basically similar. The interaction of the genome with specific cytoplasmic factors is responsible for the restriction of their developmental potencies in the somatic cell lines, and for the prevention of such a restriction in the germ cell line. In particular, it is suggested that meiosis is part of the differentiation program of the germ cells.     

Extracellular Matrix in Platyhelminths,with Special Reference to the Presence of Fibronectin

The extracellular matrix (ECM) was studied in the turbellarians Polycelis nigra and Microstomum lineare, the cestode Diphyllobothrium dendriticum and the trematode Dicrocoelium dendriticum. Routine LM staining methods for connective tissue gave positive results only in P. nigra. Positive staining reactions were observed in the subepithelial basal lamina, around the pharynx and as strings in the tissues. Peroxidase-anti-peroxidase and immunofluorescence methods were used to identify fibronectin. Positive results were obtained in all species. Positive reactivity to anti-fibronectin was observed in the subepithelial basal lamina and as a thin network between the cells of the tissues. Some intracellular reactivity occurred, but the cell type was not identified. In M. lineare a strong positive reactivity was observed in the regenerating area.     

Direct Evidence for in vitro Binding of a Lectin from Arachis hypogea to Endoblastic Primordial Germ Cells of Amphibian Embryos

Peanut agglutinin was previously shown to have a specific affinity for primordial germ cells (PGCs) from anuran amphibian embryos. For separation of these cells from endoblastic ones, suspensions of dissociated cells from the endoblastic masses of Xenopus laevis and Rana dalmatina embryos were treated with peanut agglutinin. This treatment resulted in agglutination of a small number of cells, and these aggregates were separated from unaggregated single cells by gravity in 50% calf serum medium. Histological and ultrastructural analysis of numerous sections of the aggregated cells showed that they contained the germinal plasm characteristic of PGCs. The specificity of the PGCs agglutination was confirmed by disocciation of the aggregates with 0, 2 M D-galactose solution.
This embryonic cellular population of PGCs should be useful in further in vitro experiments.     

Xenopus Heterochronic Presumptive Primordial Germ Cells (pPGCs) Implanted in the Correct Position in Host Neurula Embryos can Differentiate into PGCs

Presumptive primordial germ cells (pPGCs) in explants, derived from single germ plasm-bearing cells of Xenopus 32-cell embryos, at the equivalent of neurula stage (stage 20) in control embryos (designated as 'stage-20' explants) were demonstrated to be able to differentiate into PGCs, when implanted into a prospective place of pPGCs in host embryos (stage 20) (Ikenishi & Tsuzaki, 1988). According to a recent proposal that individual early embryonic cells in Xenopus , at both in vivo and in vitro , are able to measure elapsed time since fertilization (Cooke and Smith, 1990), the result means that the implanted pPGCs having the same elapsed time as the host embryos (isochronic pPGCs) could differentiate into PGCs. In the present study, in order to know whether the compatibility in elapsed times of implanted pPGCs and host embryos is necessary for the differentiation of PGCs, labelled, heterochronic pPGCs in 'stages 12–33/34' explants were implanted into unlabelled, host neurulae (stage 19).
Those heterochronic pPGCs could differentiate into PGCs like isochronic pPGCs in 'stage-19' explants as the control. By comparing the average diameters and yolk contents of labelled PGCs with those of unlabelled, host ones in experimental tadpoles, the possibility that a certain mechanism modulating the elapsed time of heterochronic pPGCs to that of host pPGCs is present in host embryos was also suggested.     

Improvement of Transfection Efficiency in Cultured Chicken Primordial Germ Cells by Percoll Density Gradient Centrifugation

Chicken primordial germ cells (PGCs) differentiate into germ cells in gonads. Because PGCs can be cloned and cultured maintaining germline competency, they are a good means of modifing the chicken genome, but the efficiency of plasmid transfection into PGCs is very low. In this study, I attempted to improve the efficiency of PGC transfection. Cultured PGCs were purified by Percoll density gradient centrifugation, and were then transfected with plasmid DNA. For transient transfection, the transfection efficiency increased more than 7-fold by the Percoll method. The efficiency of stable transfection of PGCs also increased significantly. The stable transfectants that were isolated by this method accumulated in the developing gonads after microinjection into bloodstream of chick embryos, indicating that gene transfection by Percoll purification did not alter the function of PGCs in vivo.     

Endoderm patterning by the notochord: development of the hypochord in Xenopus

The patterning and differentiation of the vertebrate endoderm requires signaling from adjacent tissues. In this report, we demonstrate that signals from the notochord are critical for the development of the hypochord, which is a transient, endodermally derived structure that lies immediately ventral to the notochord in the amphibian and fish embryo. It appears likely that the hypochord is required for the formation of the dorsal aorta in these organisms. We show that removal of the notochord during early neurulation leads to the complete failure of hypochord development and to the elimination of expression of the hypochord marker, VEGF. Removal of the notochord during late neurulation, however, does not interfere with hypochord formation. These results suggest that signals arising in the notochord instruct cells in the underlying endoderm to take on a hypochord fate during early neural stages, and that the hypochord does not depend on further notochord signals for maintenance. In reciprocal experiments, when the endoderm receives excess notochord signaling, a significantly enlarged hypochord develops. Overall, these results demonstrate that, in addition to patterning neural and mesodermal tissues, the notochord plays an important role in patterning of the endoderm.     

维甲酸直接诱导鸡原始生殖细胞启动并完成减数分裂

维甲酸（RA）在胚胎期生殖细胞启动减数分裂过程中发挥重要的调控作用,但RA与性腺细胞的作用机制及其能否诱导生殖细胞完成整个减数分裂生成配子的问题尚不清楚.本文以鸡原始生殖细胞体外无饲养层培养体系为模型,避开性腺体细胞的影响,研究RA诱导PGC进入减数分裂的作用机理.研究发现,在无体细胞的情况下,RA显著上调鸡胚PGC中STRA8,SYCP3和DMC1的mRNA和蛋白表达水平,从而促进其进入减数分裂;同时,流式细胞分析和吉姆萨染色结果表明,RA能使鸡胚PGC经历各个减数分裂时期,最终生成36.5%～58.4%单倍体生殖细胞;此外,本实验还对雌性和雄性PGC对RA的应答能力进行了研究,发现两者对RA的敏感程度相似.综上所述,RA能直接诱导PGC启动并完成整个减数分裂过程,生成单倍体生殖细胞,无需体细胞或其他因子的介导.这为临床上治疗不孕不育及配子形成的机理研究提供了基础.     

Chronological Changes in the Accumulation of Poly(A)+ RNA in Developing Cells of Xenopus laevis, with Special Reference to Primordial Germ Cells

Chronological changes in the accumulation of poly(A)⁺RNA in developing cells of Xenopus laevis were traced by in situ hybridization with ³H-poly(U) as a probe. Almost all the somatic cells acquired ³H-poly(U) binding sites by 3.5 days after fertilization (stage 44). At this stage, the primordial germ cells (PGCs), which had just moved to the genital ridges from the deep endodermal cell mass showed moderate ³H-poly(U) binding activity. The silver grains disappeared from the PGCs after stage 47 (6 days after fertilization), while the somatic cells, which constituted the neural tube, genital ridges, kidney and intestine exhibited strong ³H-poly(U) binding activity. Activity for ³H-poly(U) binding reappeared in the FGCs after stage 52 (21 days after fertilization) when sexual differentiation of gonads became detectable morphologically. The chronological accumulation and disappearance of ³H-poly(U) binding activity in the PGCs in contrast to the constant activity in somatic cells is discussed.     

Autonomous Regulation of Proliferation and Growth Arrest in Mouse Primordial Germ Cells Studied by Mixed and Clonal Cultures

In culture, mouse primordial germ cells (PGCs) proliferate and undergo growth arrest with a time course similar to thatin vivo.It is unclear whether this behavior is regulated autonomously or by coexisting somatic cells. We performed mixed culture experiments using PGCs from 8.5- and 11.5-d.p.c. embryos and found no interaction between the PGCs and somatic cells at the two stages. Next, we carried out clonal culture of PGCs and examined the proliferation of and morphological change in individual clones. Such clonal culture did not reveal any subpopulation of PGCs with an increased growth rate or less differentiated characteristics, which might have been suggested by formation of the embryonic germ cell lines. Our results suggest that there is an autonomous regulation of growth and cell shape change in PGCs which occur as stochastical events but are not strictly timed by the number of cell divisions.     

Incorporation of Tenascin-C into the Extracellular Matrix by Periostin Underlies an Extracellular Meshwork Architecture

Extracellular matrix (ECM) underlies a complicated multicellular architecture that is subjected to significant forces from mechanical environment. Although various components of the ECM have been enumerated, mechanisms that evolve the sophisticated ECM architecture remain to be addressed. Here we show that periostin, a matricellular protein, promotes incorporation of tenascin-C into the ECM and organizes a meshwork architecture of the ECM. We found that both periostin null mice and tenascin-C null mice exhibited a similar phenotype, confined tibial periostitis, which possibly corresponds to medial tibial stress syndrome in human sports injuries. Periostin possessed adjacent domains that bind to tenascin-C and the other ECM protein: fibronectin and type I collagen, respectively. These adjacent domains functioned as a bridge between tenascin-C and the ECM, which increased deposition of tenascin-C on the ECM. The deposition of hexabrachions of tenascin-C may stabilize bifurcations of the ECM fibrils, which is integrated into the extracellular meshwork architecture. This study suggests a role for periostin in adaptation of the ECM architecture in the mechanical environment.     

Origin of Germ Cells and Early Differentiation of Gonads in the Starfish, Asterina pectinifera

The origin of the germ cells and the development of the genital system in the annually spawning starfish, Asterina pectinifera , were studied by light and electron microscopy. Characteristic germ cells were first characterized in gonads after spawning: the gonia are larger than somatic cells, have large nuclei (with electron-lucent nucleoplasm), and show mitochondrial aggregation associated with nuage (electron-dense bodies). In young starfish without gonads similar cells were detected in the haemal sinus, where they were termed primordial germ cells (PGCs). Brachiolariae and metamorphosed juveniles had a cellular cluster in the coelomic epithelium, near the hydroporic canal. The cluster was comprised of cells endowed with the above-mentioned characteristics of the germ cells. The germ cell counts indicated that PGCs migrate from the aboral haemal sinus near the hydroporic canal, through the haemal sinus to the gonads, where they settle, proliferate, and differentiate into gonia.     

The Tetraspanin CD9 Influences the Adhesion, Spreading, and Pericellular Fibronectin Matrix Assembly of Chinese Hamster Ovary Cells on Human Plasma Fibronectin

The role of CD9 in cell adhesion and spreading on adhesive proteins was investigated using a transfected Chinese hamster ovary (CHO) cell system. CD9 cell surface expression resulted in reduced adhesion and increased spreading on fibronectin (Fn). Whereas mock-transfected (mock CHO) and na?ve CHO cells assumed a typical fibroblast spindle shape morphology, CD9-transfected (CD9-CHO) cells were polygonal with many filipodial projections and exhibited a twofold greater surface area. The spread morphology of CD9-CHO cells, but not mock CHO cells, was inhibited by PB1 mAb blockade of alpha(5)beta(1), suggesting that the coexpression of alpha(5)beta(1) and CD9 influenced cell activity on Fn. The second extracellular loop of CD9 was implicated in regulation of adhesion since reduced CD9-CHO cell adhesion on Fn was reversed by either anti-CD9 antibody ligation to the second extracellular loop or with cells expressing a CD9 mutant lacking the second extracellular loop domain. Using cell adhesion assays and ELISA, we demonstrated CD9 binding to the HEP2/IIICS region of Fn. Finally, CD9 expression resulted in a twofold reduction in Fn-rich pericellular matrix assembly. Our observations show that CD9 dramatically influences CHO cell interactions with Fn and suggest that CD9 has an important role in modulating cell-extracellular matrix interactions.     

Appearance and Distribution of RNA-Rich Cytoplasms in the Embryo of Xenopus laevis during Early Development

The occurrence of the dorsal yolk-free cytoplasm in the fertilized egg of Xenopus was re-examined, and the appearance and the distribution of RNA-rich cytoplasms in Xenopus embryos during early development were examined with their paraffin sections. The results show that the dorsal yolk-free cytoplasm does not occur solely in the dorsal part of the embryo but is continuous to similar cytoplasmic mass in the central and the ventral part. The whole mass of this continuous cytoplasm is denoted here as the mesoplasm. The locations of the mesoplasm in the embryo can be traced by its high RNA content during cleavage and blastulation. The cells endowed with the mesoplasm constitute a broad band about the equator of the blastula. At the lower edge of this band, the blastopore lip is formed during gastrulation. Another mass of yolk-poor and RNA-rich cytoplasm becomes distinct around every nucleus in the stage 4 embryo and is denoted here as the nucleophilic plasm. This plasm is diminished at every nuclear division and disappears in the stage 10 embryo. Origins and roles of the mesoplasm and the nucleophilic plasm were discussed and a mechanism of blastulation was suggested.