Distribution Of Primordial Germ Cells Research Articles

Identification, expression and functional analysis of prmt7 in medaka Oryzias latipes.

Arginine methylation is an important posttranslational modification and catalyzed by a family of protein arginine methyltransferases (PRMTs). PRMT7 is the type III PRMT and produces solely monomethylarginine products. PRMT7 has been found to play important roles in multiple biological processes in mammals. However, the expression pattern and function of Prmt7 remain largely unknown in fish. In this study, we characterized the medaka prmt7 gene and determined its expression pattern and function during embryogenesis and germ cell development. The results showed that the chromosomal location and gene structure of medaka prmt7 were similar to its mammalian orthologs. Comparisons of deduced amino acid sequences indicated that medaka Prmt7 was a homolog of human PRMT7 with two methyltransferase domains. Reverse transcription-polymerase chain reaction (RT-PCR) and real time RT-PCR revealed that medaka prmt7 had maternal origin with continuous and dynamical expression during embryonic development. Whole-mount in situ hybridization analysis observed that the transcripts of prmt7 were ubiquitous at morula and gastrula stage, and were later riched in the brain and otic vesicles during embryogenesis. In the adult stage, prmt7 messenger RNA was detected in all examined tissues with the high levels in the ovary and testis. The expression of prmt7 in the gonads was restricted to oocytes of the ovary and spermatids/sperm of the testis. Functional analysis showed that knockdown of medaka prmt7 did not reduce the total number of primordial germ cells (PGCs) in vivo but significantly affected PGCs distribution during embryonic development. These results indicate that prmt7 may be involved in germ cell development in medaka.

Migration behavior of PGCs and asymmetrical gonad formation in pond smelt Hypomesus nipponensis.

In teleost fish, the gonad originates from primordial germ cells (PGCs) and somatic cells. However, it is not clear whether the final gonadal position is determined by anteroposterior and dextrosinistral differentiation of endodermal organs or by the distribution of PGCs. The pond smelt has a transparent body even after hatching, enabling clear observation of PGC distribution and endodermal differentiation. Here, we first examined normal embryonic development to define the spatio-temporal characteristics of our developmental model. Second, the origin of PGCs was investigated by in situ hybridization. Third, the migration route of PGCs was tracked by microinjection of GFP-nos3 3' UTR mRNA and visualization of PGCs by green fluorescent protein. Lastly, differentiation of gonadal and endodermal organs was examined histologically. Maternal vasa transcripts were detected at the ends of cleavage furrows, indicating that PGCs differentiated by inheritance of germplasm as in other teleosts. During gastrulation, PGCs migrated following somatic cell movement and lined both sides of the embryonic body. During the segmentation period, PGCs moved posteriorly and were distributed in a line among dorsal mesentery cells around the posterior part of the intestinal bulb in the 16th to 24th somite region at 3 days post hatching. At 1 month post hatching, the gonad was formed at the 20th somite region. PGC distribution was biased to the left side of the body cavity, while the pancreas was formed on the right side. These results indicate that PGCs accumulate at the gonadal region by dorsal mesentery cells, and gonadal position is determined by the digestive system.

Primordial germ cell development in the marmoset monkey as revealed by pluripotency factor expression: suggestion of a novel model of embryonic germ cell translocation

Primordial germ cells (PGCs) are the embryonic progenitors of sperm and egg cells. Mammalian PGCs are thought to actively migrate from the yolk sac endoderm over long distances across the embryo to reach the somatic genital ridges. The general principles of mammalian PGC development were discovered in mice. In contrast, little is known about PGC development in primates due to extremely limited access to primate embryos. Here, we analyzed 12 well preserved marmoset monkey (Callithrix jacchus) embryos covering the phase from PGC emergence in the endoderm to the formation of the sexually differentiated gonad (embryonic day (E) 50 to E95). We show using immunohistochemistry that the pluripotency factors OCT4A and NANOG specifically mark PGCs throughout the period studied. In contrast, SALL4 and LIN28 were first expressed ubiquitously and only later down-regulated in somatic tissues. We further show, for the first time, that PGCs are located in the endoderm in E50 embryos in close spatial proximity to the prospective genital ridge, making a long-range migration of PGCs dispensable. At E65, PGCs are already present in the primitive gonad, while significantly later embryonic stages still exhibit PGCs at their original endodermal site, revealing a wide spatio-temporal window of PGC distribution. Our findings challenge the ‘dogma’ of active long-range PGC migration from the endoderm to the gonads. We therefore favor an alternative model based primarily on passive translocation of PGCs from the mesenchyme that surrounds the gut to the prospective gonad through the intercalar expansion of mesenchymal tissue which contains the PGCs. In summary, we (i) show differential pluripotency factor expression during primate embryo development and (ii) provide a schematic model for embryonic PGC translocation.

Primordial germ cell development in the marmoset monkey as revealed by pluripotency factor expression: suggestion of a novel model of embryonic germ cell translocation.

Primordial germ cells (PGCs) are the embryonic progenitors of sperm and egg cells. Mammalian PGCs are thought to actively migrate from the yolk sac endoderm over long distances across the embryo to reach the somatic genital ridges. The general principles of mammalian PGC development were discovered in mice. In contrast, little is known about PGC development in primates due to extremely limited access to primate embryos. Here, we analyzed 12 well preserved marmoset monkey (Callithrix jacchus) embryos covering the phase from PGC emergence in the endoderm to the formation of the sexually differentiated gonad (embryonic day (E) 50 to E95). We show using immunohistochemistry that the pluripotency factors OCT4A and NANOG specifically mark PGCs throughout the period studied. In contrast, SALL4 and LIN28 were first expressed ubiquitously and only later down-regulated in somatic tissues. We further show, for the first time, that PGCs are located in the endoderm in E50 embryos in close spatial proximity to the prospective genital ridge, making a long-range migration of PGCs dispensable. At E65, PGCs are already present in the primitive gonad, while significantly later embryonic stages still exhibit PGCs at their original endodermal site, revealing a wide spatio-temporal window of PGC distribution. Our findings challenge the ‘dogma’ of active long-range PGC migration from the endoderm to the gonads. We therefore favor an alternative model based primarily on passive translocation of PGCs from the mesenchyme that surrounds the gut to the prospective gonad through the intercalar expansion of mesenchymal tissue which contains the PGCs. In summary, we (i) show differential pluripotency factor expression during primate embryo development and (ii) provide a schematic model for embryonic PGC translocation.

Primordial germ cells in the dorsal mesentery of the chicken embryo demonstrate left-right asymmetry and polarized distribution of the EMA1 epitope.

Despite the importance of the chicken as a model system, our understanding of the development of chicken primordial germ cells (PGCs) is far from complete. Here we characterized the morphology of PGCs at different developmental stages, their migration pattern in the dorsal mesentery of the chicken embryo, and the distribution of the EMA1 epitope on PGCs. The spatial distribution of PGCs during their migration was characterized by immunofluorescence on whole-mounted chicken embryos and on paraffin sections, using EMA1 and chicken vasa homolog antibodies. While in the germinal crescent PGCs were rounded and only 25% of them were labeled by EMA1, often seen as a concentrated cluster on the cell surface, following extravasation and migration in the dorsal mesentery PGCs acquired an elongated morphology, and 90% exhibited EMA1 epitope, which was concentrated at the tip of the pseudopodia, at the contact sites between neighboring PGCs. Examination of PGC migration in the dorsal mesentery of Hamburger and Hamilton stage 20-22 embryos demonstrated a left-right asymmetry, as migration of cells toward the genital ridges was usually restricted to the right, rather than the left, side of the mesentery. Moreover, an examination of another group of cells that migrate through the dorsal mesentery, the enteric neural crest cells, revealed a similar preference for the right side of the mesentery, suggesting that the migratory pathway of PGCs is dictated by the mesentery itself. Our findings provide new insights into the migration pathway of PGCs in the dorsal mesentery, and suggest a link between EMA1, PGC migration and cell-cell interactions. These findings may contribute to a better understanding of the mechanism underlying migration of PGCs in avians.

Developmental regulation of locomotive activity in Xenopus primordial germ cells

Primordial germ cells (PGCs) arise in the early embryo and migrate toward the future gonad through species-specific pathways. They are assumed to change their migration properties dependent on their own genetic program and/or environmental cues, though information concerning the developmental change in PGC motility is limited. First, we re-examined the distribution of PGCs in the endodermal region of Xenopus embryos at various stages by using an antibody against Xenopus Daz-like protein, and found four stages of migration, namely clustering, dispersing, directionally migrating and re-aggregating. Next, we isolated living PGCs at each stage and directly examined their morphology and locomotive activity in cell cultures. PGCs at the clustering stage were round in shape with small blebs and showed little motility. PGCs in both the dispersing and the directionally migrating stages alternated between the locomotive phase with an elongated morphology and the pausing phase with a rugged morphology. The locomotive activity of the elongated PGCs was accompanied by the persistent formation of a large bleb at the leading front. The duration of the locomotive phase was shortened gradually with the transition from the dispersing stage to the directionally migrating stage. At the re-aggregating stage, PGCs became round in shape and showed no motility. Thus, we directly showed that the locomotive activity of PGCs changes dynamically depending upon the migrating stage. We also showed that the locomotion and blebbing of the PGCs required F-actin, myosin II activity and RhoA/Rho-associated protein kinase (ROCK) signaling.

Medaka piwi is Essential for Primordial Germ Cell Migration

Piwi controls the number of primordial germ cells (PGCs) via protecting maternal mRNA from decay and adult germ stem cell division in Drosophila. In mouse and zebrafish, piwi controls maintenance and differentiation of adult germ stem cell during gametogenesis. Whether piwi plays a role in PGC development of vertebrates remains unsolved. We addressed this issue by using medaka (Oryzias latipes) as a vertebrate model. Molecular cloning, sequence comparison and analyses of genomic organization and chromosome synteny led to the identification in this fish of a single piwi gene, called Opiwi. By RT-PCR analyses and in situ hybridization, the Opiwi transcript is maternally supplied and becomes restricted to PGCs and the central nervous system (CNS). Opiwi knockdown did not prevent PGC formation even in the absence of any somatic structures but did significantly reduce the number of PGCs in vivo and in vitro and affect the distribution of PGCs in developing embryos. Surprisingly, depletion of zygotic Opiwi severely and specifically affected PGC migration. We conclude that Opiwi is required not only for determining the PGC number but also for controlling PGC migration. Our results demonstrate that piwi plays a generally conserved role in germ cell development from Drosophila to vertebrate and a specific role in PGC migration.

The proliferation and migration of immature germ cells in the mussel, Mytilus galloprovincialis: observation of the expression pattern in the M. galloprovincialis vasa-like gene (Myvlg) by in situ hybridization

In bivalve, the distribution of primordial germ cells can be traced from early embryogenesis to the veliger larva by the expression of the vasa ortholog. However, the distribution of germ cells from metamorphosis to maturation in bivalves has not been examined extensively. In this study, we used in situ hybridization to observe expression of the Mytilus galloprovincialis vasa-like gene (Myvlg). The distribution of germ cells was clarified in immature mussels. We observed germ cells in adult mussels during the non-reproductive and reproductive seasons. Myvlg was specifically expressed in germ cells. Gametogenesis occurs in acini surrounded by connective tissue. Myvlg expression was detected in spermatogonia, spermatocytes, oogonia, and oocytes. In the non-reproductive season, gametes were not observed in the acini, but Myvlg was expressed in germinal stem cells along the acini. The expression intensity in the non-reproductive season, however, was much weaker than that in the reproductive season. Myvlg-positive cells proliferated during the non-reproductive season. In immature mussels, a pair of germ cell clumps was distributed laterally in the connective tissue between the nephric tubules and posterior byssal retractor muscle. Germ cells were also observed along pericardium. When immature mussels grew, a pair of germ cell clumps migrated anteriorly in the connective tissue along the outer epithelium at the dorsal region of the mantle base between the mantle and gill. The number of germ cells increased significantly as the mussels grew. This is the first report to observe the proliferation and migration of germ cells in immature mussels.

Molecular cloning and expression of dead end homologue in chicken primordial germ cells

Chicken primordial germ cells (PGCs) dynamically migrate towards the prospective gonadal area through the germinal crescent region and bloodstream at early embryonic stages. To date, chicken PGCs have been mainly identified by histochemical and immunohistochemical methods or by their morphological characteristics. However, their origin, migration and differentiation are not fully understood because of the lack of specific PGC molecular markers. Here, we have isolated the chicken dead end homologue (CDH) in order to clone its full-length cDNA with an open reading frame of 329 amino acids. The RNA-binding motif present in CDH at amino acids 54-133 was highly homologous to those in the dead end proteins of human, mouse and Xenopus. The temporal and spatial distribution of PGCs was also investigated by in situ hybridization (ISH) on whole-mount embryos with CDH cRNA as a probe. Chicken embryos from stage X to stage 20 were subjected to ISH. The hybridized samples were then sectioned to analyse the translocation of PGCs. CDH-positive cells could be counted from stage X to stage 4, with minimally 30 cells at the blastderm and approximately 260 cells at the germinal crescent. Thus, specific expression of CDH mRNA has been established in chicken PGCs located at the blastderm, germinal crescent and prospective gonadal area by ISH and reverse transcription/polymerase chain reaction. We conclude that isolated CDH is specifically expressed in chicken PGCs during embryogenesis.

Meiosis Onset Is Postponed to Postnatal Stages during Ovotestis Development in Female Moles

In mammals, germ cells are important both during development and for the function of female gonads, whereas male gonads may develop in the absence of germ cells. The gonads of female moles (genus Talpa) develop according to a testis-like pattern which results in the formation of ovotestes. In this paper, we studied the expression pattern of several pre-meiotic and meiotic germ cell markers, in order to establish the precise time of meiosis onset in the mole species T. occidentalis, and to investigate the location and possible role of germ cells in ovotestis organogenesis. Our results evidenced that: (1) the asymmetrical distribution of primordial germ cells, which concentrate in the cortex of the XX gonad, is brought about by germ cell depletion from the medulla between the s5a and s5b stages, (2) XX germ cells enter meiosis postnatally, which is quite exceptional among eutherian mammals, and (3) XX but not XY germ cells of moles express DMRT1 during premeiotic stages of development, an expression pattern not described previously in vertebrates.

Mutations affecting early distribution of primordial germ cells in Medaka ( Oryzias latipes) embryo

The development of germ cells has been intensively studied in Medaka ( Oryzias latipes). We have undertaken a large-scale screen to identify mutations affecting the development of primordial germ cells (PGCs) in Medaka. Embryos derived from mutagenized founder fish were screened for an abnormal distribution or number of PGCs at embryonic stage 27 by RNA in situ hybridization for the Medaka vasa homologue ( olvas). At this stage, PGCs coalesce into two bilateral vasa-expressing foci in the ventrolateral regions of the trunk after their migration and group organization. Nineteen mutations were identified from a screen corresponding to 450 mutagenized haploid genomes. Eleven of the mutations caused altered PGC distribution. Most of these alterations were associated with morphological abnormalities and could be grouped into four phenotypic classes: Class 1, PGCs dispersed into bilateral lines; Class 2, PGCs dispersed in a region more medial than that in Class 1; Class 3, PGCs scattered laterally and over the yolk sac area; and Class 4, PGCs clustered in a single median focus. Eight mutations caused a decrease in the number of PGCs. This decrease was observed in the offspring of heterozygous mothers, indicating the contribution of a maternal factor in determining PGC abundance. Taken together, these mutations should prove useful in identifying molecular mechanisms underlying the early PGC development and migration.

Mutations affecting the formation of posterior lateral line system in Medaka, Oryzias latipes

We performed a systematic screen for mutations affecting the trajectory of axons visualized by immunohistochemical staining of Medaka embryos with anti-acetylated tubulin antibody. Among the mutations identified, yanagi ( yan) and kazura ( kaz) mutations caused specific defects in projection of the posterior lateral line (PLL) nerve. In yan and kaz mutant embryos, the PLL nerve main bundle was misrouted ventrally and dorsally or anteriorly. Medaka semaphorin3A, sdf1, and cxcr4 cDNA fragments were cloned to allow analysis of these mutants. There were no changes in semaphorin3A or sdf1 expression in mutant embryos, suggesting that the tissues expressing semaphorin3A or sdf1 that are involved in PLL nerve guidance are present in these mutant embryos. Double staining revealed that the mislocated PLL primordium and growth cone of the ectopically projected PLL nerve were always colocalized in both yan and kaz mutant embryos, suggesting that migration of PLL primordia and PLL nerve growth cones are not uncoupled in these mutants. Although homozygous yan larvae showed incomplete migration of the PLL primordium along the anteroposterior axis, ventral proneuromast migration was complete, suggesting that ventral migration of the proneuromast does not require the signaling affected in yan mutants. In addition to the PLL system, the distribution of primordial germ cells (PGCs) was also affected in both yan and kaz mutant embryos, indicating that yan and kaz genes are required for the migration of both PLL primordia and PGCs. Genetic linkage analysis indicated that kaz is linked to cxcr4, but yan is not linked to sdf1 or cxcr4. These mutations will provide genetic clues to investigate the molecular mechanism underlying formation of the PLL system.

Effects of endosulfan and nonylphenol on the primordial germ cell population in pre-larval zebrafish embryos

A variety of chemicals released into the aquatic environment are capable of targeting the reproductive system in fish and other vertebrates. Some of the effects observed in exposed adults may arise by permanent organizational changes that occur during embryogenesis, including changes in gonad structure and function. Little work has addressed the effects of pesticides and industrial chemicals, many of which are recognized as endocrine disrupting chemicals, on early embryos. The recent cloning of the vasa gene in zebrafish, the mRNA of which is found in fertilized eggs and is later segregated into the primordial germ cells (PGCs), has provided a unique opportunity to examine PGC migration and positioning in early embryos. We utilized antisense RNA probes to vasa mRNA in whole mount in situ hybridization analysis in order to examine the early migration and distribution of PGCs in embryos exposed to endosulfan and nonylphenol. The data reveal that these chemicals cause alterations in the distribution of PGCs along the anterior–posterior axis in 24-h-old embryos. This suggests that the previously reported alterations in juvenile and adult gonad structure of various aquatic vertebrates following exposure to pesticides and industrial chemicals could be related in part to alterations in early PGC distribution.

Localization of primordial germ cells or their precursors in stage X blastoderm of chickens and their ability to differentiate into functional gametes in opposite-sex recipient gonads

This study was performed to determine the distribution of primordial germ cells and their precursors in stage X blastoderm of chickens. The blastoderm (Barred Plymouth Rock chickens) isolated from the yolk was separated into three portions: the central disc, the marginal zone and the area opaca. The dissociated blastodermal cells derived from the central disc, marginal zone and area opaca were transferred into a recipient blastoderm (White Leghorn chicken) from which a cell cluster was removed from the centre of the central disc. The manipulated embryos were cultured in host eggshells until hatching. The chicks were raised until sexual maturity and test mated with Barred Plymouth Rock chickens to assess the donor cell contribution to the recipient germline. Germline chimaeric chickens were produced efficiently (46.7%, 7/15) when the blastodermal cells derived from the central disc were transferred into recipient embryos of the same sex, whereas no germline chimaeric chickens were produced when the blastodermal cells derived from the marginal zone or area opaca were transferred into recipient embryos of the same sex (0/12). Germline chimaeric chickens were also produced by transfer of blastodermal cells derived from the central disc (6.7%, 1/15), marginal zone (10.0%, 1/10) or area opaca (11.1%, 1/9) into recipient embryos of the opposite sex. It is concluded that primordial germ cells are induced during or shortly after stage X and that the cells derived from the central disc have the highest potential to give rise to germ cells. Cells derived from the marginal zone and area opaca can also give rise to germ cells, although the frequency is low.

Developmental origin of avian primordial germ cells and its unique differentiation in the gonads of mixed-sex chimeras.

Developmental origin of avian primordial germ cells and its unique differentiation in the gonads of mixed-sex chimeras.

Identical origin of adrenal cortex and gonad revealed by expression profiles of Ad4BP/SF-1.

Ad4BP/SF-1 was originally identified as a steroidogenic tissue-specific transcription factor. Recent gene disruption studies with the mammalian Ftz-F1 gene encoding Ad4BP/SF-1 clearly revealed the essential function of the factor for adrenal and gonadal differentiation. In this study, we examined the early development of these tissues using Ad4BP/SF-1 as the marker. In rat foetuses of 11.5 days post-coitum (d.p.c.), a cell population designated adrenogenital primordium was firstly observed on symmetrical lines extending from the dorsal aorta to the dorsal coelomic epithelia of the primitive urogenital ridges. From 12.5 d.p.c., the rostral half of the adreno-genital primordium started to separate into two distinct cell populations. Judging from the distribution of primordial germ cells, the cell population on the dorsal aortal side is a primordium for the adrenal cortex whereas that on the coelomic epithelial side is for the gonads. At 13.5 d.p.c., these two primordia have separated completely. These observations clearly identified a novel adreno-genital primordium from which both the adrenal cortex and the gonads originate. An RT-PCR study conducted to detect adrenal- and gonad-specific mRNAs supported the above observations.

Proliferation and migration of primordial germ cells in We/We mouse embryos.

We have examined the numbers and distribution of primordial germ cells in We/We, We/+, and +/+ mouse embryos using Southern blotting to determine embryo genotypes. At early somite stages (5-7 somites: approximately 8 1/2 days post coitum [dpc]) there are 50 to 100 germ cells in embryos of all genotypes. The number of germ cells in We/+ and +/+ embryos then begins to increase: at later somite stages (17-19 somites: approximately 9 1/2 dpc) they number about 200, and by 10 1/2 dpc there are approximately 725 We/+ and 850 +/+ germ cells. During this time, however, the number of germ cells in We/We embryos remains less than 100. At 8 1/2 dpc, the distribution of germ cells in the hindgut endoderm is the same in all genotypes. By 9 1/2 dpc, 30% of We/We germ cells are found in ectopic sites (allantois and vitelline artery); germ cell distribution along the length of the hindgut appears normal, but germ cells remain confined to the floor of the gut in We/We embryos, rather than being distributed around its circumference as in the other two genotypes. By 10 1/2 days, the migration of We/We germ cells through the dorsal mesentery lags behind that of the other genotypes, and a larger proportion remains in the gut wall.

Ectopic colonization of primordial germ cells in the chick embryo lacking the gonads

Chick primordial germ cells (PGCs) first appear in the extraembryonic region in the early embryo, then temporarily circulate via the blood vascular system and finally migrate into the gonadal anlagen. In the present study, we examined the trend of ectopic distribution of PGCs in the chick embryo when its future gonadal region had been removed at an early stage. Embryos at stage 10, from which the caudal third region was excised, were incubated until they reached stages 14 to 20. In embryos at stage 14, about 80% of the total PGCs were found in the capillaries of the yolk sac, whereas others were observed in the head, mainly in the mesenchyme and small vessels close to the neural tube. From stage 18 onward, many PGCs accumulated in the embryo proper; about 90% of them colonized in the head region around the neural tube. These ectopic PGCs in the head were found in the capillaries, sometimes as thrombi or emerging from them into the adjacent mesenchyme. These results show that, when the chick embryo lacked gonads, the PGCs could be concentrated in the head region and migrated from the capillaries into the mesenchyme.

Aspects quantitatifs du peuplement des gonades par les cellules germinales chez l’embryon de Caille (Coturnix coturnix japonica)

ABSTRACT Quantitative aspects of the colonization of the gonads by germ cells in the quail embryo (Coturnix coturnix japonica) A quantitative analysis made on quail embryos coming from 13 isolated parent-couples reveals some significative variations of a genetic origin, between some of the offspring studied : the differences observed concern both the quantitative importance of the colonization of gonads by germ cells and the asymmetrical distribution of PGCs (primordial germ cells) between the two genital ridges. The chronological study of the colonization in the quail shows, as in both the duck and the chick, two periods of rapid and regular increase of the number of gonadic PGC, at stages from 13 to 18 and from 24 to 30 of Hamburger & Hamilton. The distribution of germ cells between the two genital ridges is, at the beginning of the colonization, not very asymmetrical. Between stages 18 and 24 the asymmetry increases and remains stable so that the mean value of D % (percentage of the number of PGC contained in the right gonad) from that moment on is equal to 29 ·34 %. This value is specific for the quail embryo.