Genital ridges exert long-range effects on mouse primordial germ cell numbers and direction of migration in culture

Development ◽  
1990 ◽  
Vol 108 (2) ◽  
pp. 357-363 ◽  
Author(s):  
I. Godin ◽  
C. Wylie ◽  
J. Heasman
Keyword(s):  
Long Range ◽  
Primordial Germ Cells ◽  
Primordial Germ Cell ◽  
Great Majority ◽  
Early Embryo ◽  
Extrinsic Factors ◽  
Alkaline Phosphatase Staining ◽  
Hind Gut ◽  
Feeder Cell Layers

The functional gametes of all vertebrates first arise in the early embryo as a migratory population of cells, the primordial germ cells (PGCs). These migrate to, and colonise, the genital ridges (GR) during the early organogenesis period, giving rise to the complete differentiating gonad. PGCs first become visible by alkaline phosphatase staining in the root of the developing allantois at 8.5 days post coitum (dpc). At 9.5 dpc they are found in the wall of the hind-gut and, during the following three days, they migrate along the hind-gut mesentery to the dorsal body wall, and then to the genital ridges. By 12.5 dpc, the great majority of PGCs have colonised the genital ridges. During this period the number of PGCs increases from less than 100 to approximately 4000. In a previous paper (Donovan et al. 1986), we showed that 10.5 dpc PGCs can be explanted from the hind-gut mesentery, and will spread and migrate on feeder cell layers. We showed also that the intrinsic ability of PGCs to spread and migrate changes as they colonise the genital ridges. In this paper, we examine extrinsic factors that control PGC behaviour in vitro. Using PGCs taken from 8.5 dpc embryos, at the beginning of their migratory phase, we show that culture medium conditioned by 10.5 dpc genital ridges causes an increase in the number of PGCs in these cultures. We also show that PGCs migrate towards 10.5 dpc genital ridges in preference to other explanted organs. These experiments show that genital ridges exert long-range effects on the migrating population of PGCs.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Association of culture of mouse urogenital complexes in media containing rodent sera with the appearance of primordial germ cell-like cells

Reproduction ◽  
2003 ◽  
pp. 519-526 ◽  
Author(s):  
T Mayanagi ◽  
K Ito ◽  
J Takahashi
Keyword(s):  
Germ Cell ◽  
Germ Cells ◽  
Primordial Germ Cells ◽  
Primordial Germ Cell ◽  
Calf Serum ◽  
Culture Method ◽  
Embryonic Germ Cells ◽  
Alkaline Phosphatase Staining

Primordial germ cells differentiate into germ cells and have the ability to reacquire totipotency. Mouse primordial germ cells are identified by alkaline phosphatase staining of the extraembryonic mesoderm, and they proliferate and migrate to reach the genital ridges. Mouse primordial germ cells have never been maintained in culture exclusively for longer than a week without differentiation or dedifferentiation. Moreover, primordial germ cells have not been proliferated with urogenital complexes in vitro, because gonad culture has never been successful. It was thought that primordial germ cells could proliferate in a culture of urogenital complex under modified medium conditions resembling those in vivo; however, organ culture of mouse gonad has been performed with fetal calf serum or equine serum, and those sera produce conditions different from those in vivo. Therefore, mouse urogenital complexes were cultured in media containing rodent sera. As a result, it was possible to proliferate primordial germ cell-like cells outside gonads, and these cells very closely resembled primordial germ cells. In addition, motile primordial germ cell-like cells could be obtained. The ability to maintain primordial germ cell-like cells in culture by this intra-species culture method is important in the study of gametogenesis. Furthermore, this method is useful as a source of stem cells such as embryonic germ cells.

Slow muscle induction by Hedgehog signalling in vitro

2000 ◽  
Vol 113 (15) ◽  
pp. 2695-2703 ◽  
Author(s):  
W. Norris ◽  
C. Neyt ◽  
P.W. Ingham ◽  
P.D. Currie
Keyword(s):  
Cell Fate ◽  
Sonic Hedgehog ◽  
Culture System ◽  
Early Embryo ◽  
Extrinsic Factors ◽  
Fast Myosin ◽  
Neural Input ◽  
Slow Twitch

Muscles are composed of several fibre types, the precise combination of which determines muscle function. Whereas neonatal and adult fibre type is influenced by a number of extrinsic factors, such as neural input and muscle load, there is little knowledge of how muscle cells are initially determined in the early embryo. In the zebrafish, fibres of the slow twitch class arise from precociously specified myoblasts that lie close to the midline whereas the remainder of the myotome differentiates as fast myosin expressing muscle. In vivo evidence has suggested the Sonic Hedgehog glycoprotein, secreted from the notochord, controls the formation of slow twitch and fast twitch muscle fates. Here we describe an in vitro culture system that we have developed to test directly the ability of zebrafish myoblasts to respond to exogenous Sonic Hedgehog peptide. We find that Sonic Hedgehog peptide can control the binary cell fate choice of embryonic zebrafish myoblasts in vitro. We have also used this culture system to assay the relative activities of different Hedgehog-family proteins and to investigate the possible involvement of heterotrimeric G-proteins in Hedgehog signal transduction.

The active migration of Drosophila primordial germ cells

Development ◽  
1995 ◽  
Vol 121 (11) ◽  
pp. 3495-3503 ◽  
Author(s):  
M.K. Jaglarz ◽  
K.R. Howard
Keyword(s):  
Primary Culture ◽  
Germ Cells ◽  
Actin Polymerization ◽  
Primordial Germ Cells ◽  
Primordial Germ Cell ◽  
Cell Polarization ◽  
Germ Cell Migration ◽  
Oriented Movement

We describe our analysis of primordial germ cell migration in Drosophila wild-type and mutant embryos using high resolution microscopy and primary culture in vitro. During migratory events the germ cells form transient interactions with each other and surrounding somatic cells. Both in vivo and in vitro they extend pseudopodia and the accompanying changes in the cytoskeleton suggest that actin polymerization drives these movements. These cellular events occur from the end of the blastoderm stage and are regulated by environmental cues. We show that the vital transepithelial migration allowing exit from the gut primordium and passage into the interior of the embryo is facilitated by changes in the structure of this epithelium. Migrating germ cells extend processes in different directions. This phenomenon also occurs in primary culture where the cells move in an unoriented fashion at substratum concentration-dependent rates. In vivo this migration is oriented leading germ cells to the gonadal mesoderm. We suggest that this guidance involves stabilization of states of an intrinsic cellular oscillator resulting in cell polarization and oriented movement.

154 MOLECULAR CHARACTERIZATION OF CAT PRIMORDIAL GERM CELLS

2016 ◽  
Vol 28 (2) ◽  
pp. 207
Author(s):  
J. Galiguis ◽  
C. E. Pope ◽  
C. Dumas ◽  
G. Wang ◽  
R. A. MacLean ◽  
...  
Keyword(s):  
Stem Cells ◽  
Germ Cell ◽  
Germ Cells ◽  
Primordial Germ Cells ◽  
Primordial Germ Cell ◽  
Embryonic Stem ◽  
Transcript Abundance ◽  
Domestic Cat ◽  
Sample Type

As precursors to germline stem cells and gametes, there are many potential applications for primordial germ cells (PGC). Primordial germ cell-like cells have been generated from mouse embryonic stem cells and induced pluripotent stem cells, which subsequently were used to produce functional spermatozoa, oocytes, and healthy offspring (Hayashi et al. 2012 Science 338(6109), 971–975). Applying this approach to generate sperm and oocytes of endangered species is an appealing prospect. Detection of molecular markers associated with PGC is essential to optimizing the process of PGC induction. In the current study, in vitro-derived domestic cat embryos were assessed at various developmental stages to characterise the expression of markers related to the specification process of cat PGC. In vivo-matured, IVF oocytes were cultured until Days 7, 9, and 12 post-insemination. Then, embryos were assessed by RT-qPCR to determine relative transcript abundance of the pluripotency markers NANOG, POU5F1, and SOX2; the epiblast marker DNMT3B; the primitive endoderm marker GATA4; the PGC marker PRDM14; and the germ cell marker VASA; RPS19 was used as the internal reference gene. To validate the qPCR results, fibroblasts served as the negative control cells, whereas spermatogonial stem cells (SSC) served as the positive control cells for GATA4, PRDM14, and VASA. Total mRNA were isolated using the Cells-to-cDNA™ II Kit (Ambion/Thermo Fisher Scientific, Waltham, MA, USA) from either pools of 2 to 6 embryos or ~25 000 fibroblasts/SSC. A minimum of 2 biological replicates for each sample type was analysed, with transcript abundance detected in 2 technical replicates by SYBR Green chemistry. Student’s t-tests were performed on the ΔCts for statistical analysis. PRDM14, specific to the germ cell lineage, was detected as early as Day 7, suggesting the presence of PGC precursor cells. Compared with their levels at Day 7, PRDM14 expression was 0.34-fold lower in SSC (P < 0.05), whereas expression of VASA and GATA4 were 1964-fold and 144-fold higher, respectively (P < 0.05). This seems to emphasise the relative importance of PRDM14 in pre-germ cell stages. In general, all genes analysed were up-regulated from Day 7 to Day 9. This up-regulation was statistically significant for SOX2 and GATA4 (P < 0.05). Relative to that at Day 9, all transcripts were relatively less abundant at Day 12 (P < 0.05 for NANOG, POU5F1, SOX2, DNMT3B, and PRDM14). The data suggest that PGC specification takes place near Day 9, with peak specification activity concluding by Day 12. Although much needs be explored about PGC specification in the cat before applying induction and in vitro germ cell production techniques, these findings represent the first step towards a new potential strategy for preserving endangered and threatened felids.

scholarly journals Generation of Primordial Germ Cell-like Cells from iPSCs Derived from Turner Syndrome Patients

Cells ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 3099
Author(s):  
Aline Fernanda de Souza ◽  
Fabiana Fernandes Bressan ◽  
Naira Caroline Godoy Pieri ◽  
Ramon Cesar Botigelli ◽  
Tamas Revay ◽  
...  
Keyword(s):  
Germ Cell ◽  
Turner Syndrome ◽  
Genetic Disorder ◽  
Primordial Germ Cells ◽  
Primordial Germ Cell ◽  
Cell Formation ◽  
In Vitro Models ◽  
Germline Development ◽  
Germ Cell Specification

Turner syndrome (TS) is a genetic disorder in females with X Chromosome monosomy associated with highly variable clinical features, including premature primary gonadal failure leading to ovarian dysfunction and infertility. The mechanism of development of primordial germ cells (PGCs) and their connection with ovarian failure in TS is poorly understood. An in vitro model of PGCs from TS would be beneficial for investigating genetic and epigenetic factors that influence germ cell specification. Here we investigated the potential of reprogramming peripheral mononuclear blood cells from TS women (PBMCs-TS) into iPSCs following in vitro differentiation in hPGCLCs. All hiPSCs-TS lines demonstrated pluripotency state and were capable of differentiation into three embryonic layers (ectoderm, endoderm, and mesoderm). The PGCLCs-TS recapitulated the initial germline development period regarding transcripts and protein marks, including the epigenetic profile. Overall, our results highlighted the feasibility of producing in vitro models to help the understanding of the mechanisms associated with germ cell formation in TS.

scholarly journals Enhancement of chicken primordial germ cell in vitro maintenance using an automated cell image analyser

2018 ◽  
Vol 66 (4) ◽  
pp. 518-529 ◽  
Author(s):  
Mahek Anand ◽  
Bence Lázár ◽  
Roland Tóth ◽  
Emőke Páll ◽  
Eszter Patakiné Várkonyi ◽  
...  
Keyword(s):  
Growth Rate ◽  
Doubling Time ◽  
Primordial Germ Cells ◽  
Genotypic Variation ◽  
Primordial Germ Cell ◽  
Significant Difference ◽  
The Difference ◽  
In Vitro Maintenance

Primordial germ cells (PGCs) were isolated from blood samples of chicken embryos. We established four PGC lines: two males (FS-ZZ-101, GFP-ZZ-4ZP) and two females (FS-ZW-111, GFP-ZW-5ZP). We could not detect a significant difference in the marker expression profile, but there was a remarkable difference between the proliferation rates of these PGC lines. We monitored the number of PGCs throughout a three-day period using a high-content screening cell imaging and analysing system (HCS). We compared three different initial cell concentrations in the wells: ~1000 cells (1×, ~4000 (4× and ~8000 (8×. For the GFPZW- 5ZP, FS-ZZ-101 and FS-ZW-111 PGC lines the lowest doubling time was observed at 4× concentration, while for GFP-ZZ-4ZP we found the lowest doubling time at 1× concentration. At 8× initial concentration, the growth rate was high during the first two days for all cell lines, but this was followed by the appearance of cell aggregates decreasing the cell growth rate. We could conclude that the difference in proliferation rate could mainly be attributed to genotypic variation in the established PGC lines, but external factors such as cell concentration and quality of the culture medium also affect the growth rate of PGCs.

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

1997 ◽  
Vol 138 (2) ◽  
pp. 471-480 ◽  
Author(s):  
Martín I. García-Castro ◽  
Robert Anderson ◽  
Janet Heasman ◽  
Christopher Wylie
Keyword(s):  
Extracellular Matrix ◽  
Germ Cells ◽  
Binding Sites ◽  
Primordial Germ Cells ◽  
A Cell ◽  
Hind Gut ◽  
Combinatorial Mechanism ◽  
Cell Surface Proteoglycan ◽  
Strength Of Adhesion

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.

scholarly journals BMP4 Signaling Activates the Wnt-Lin28A-Blimp1-Wnt Positive Feedback Pathway to Promote Primordial Germ Cell Formation via Altering H3K4me2

2020 ◽  
Author(s):  
Qisheng Zuo ◽  
Yani Zhang ◽  
Guohong Chen ◽  
Bichun Li
Keyword(s):  
Wnt Signaling ◽  
Positive Feedback ◽  
Primordial Germ Cells ◽  
Primordial Germ Cell ◽  
Cell Formation ◽  
Feedback System ◽  
Developmental Characteristics ◽  
Technical Basis ◽  
Feedback Pathway

Abstract (Background) The unique developmental characteristics of bird primordial germ cells (PGC) have enabled genetic engineering–based breeding and restoration of endangered birds via transplantation in vitro. However, the limited number of PGC has limited their application. Thus, there is an urgent need to elucidate the mechanism of PGC formation in vitro to enhance its efficiency. (Results) Here, we confirmed that activation of BMP4 and Wnt signaling (Wnt5A/β-catenin/TCF7L2) is critical for PGC formation via RNA-seq (ESCs, PGC and SSCs) and in vitro induction models. Wnt signaling activated via BMP4 in turn activates transcription of Lin28A by inducing β-catenin to compete with LSD1 for binding to the transcription factor TCF7L2, causing LSD1 to dissociate from the Lin28A promoter and enhanced H3K4me2 methylation in this region. Lin28A promotes PGC formation by inhibiting gga-let7a-3p maturation to initiate Blimp1 expression. Interestingly, expression of Blimp1 helped sustain Wnt5A expression by preventing LSD1 binding to the Wnt5A promoter. We thus elucidated a positive feedback pathway involving Wnt-Lin28-Blimp1-Wnt, with BMP4 functioning as an activator that ensures PGC formation. (Conclusion) In summary, our study clarified the molecular mechanism by which BMP4 and Wnt signaling regulate PGC formation via a positive feedback system. Our data provide both a theoretical and technical basis for studies aimed at enhancing the generation of PGC in vitro.

scholarly journals Epigenetics and the germline

Reproduction ◽  
2005 ◽  
Vol 129 (2) ◽  
pp. 137-149 ◽  
Author(s):  
Cinzia Allegrucci ◽  
Alexandra Thurston ◽  
Emma Lucas ◽  
Lorraine Young
Keyword(s):  
Current Knowledge ◽  
Primordial Germ Cells ◽  
Primordial Germ Cell ◽  
Cell Lineage ◽  
Epigenetic Modifications ◽  
Germline Development ◽  
Genome Wide ◽  
Three Stages ◽  
Epigenetic Processes

Epigenetic processes affect three stages of germline development, namely (1) specification and formation of primordial germ cells and their germline derivatives through lineage-specific epigenetic modifications, in the same manner as other embryonic lineages are formed, (2) a largely genome-wide erasure and re-establishment of germline-specific epigenetic modifications that only occurs in the embryonic primordial germ cell lineage, followed by re-establishment of sex-specific patterns during gametogenesis, and (3) differential epigenetic modifications to the mature male and female gamete genomes shortly after fertilisation. This review will detail current knowledge of these three processes both at the genome-wide level and at specific imprinted loci. The consequences of epigenetic perturbation are discussed and newin vitromodels which may allow further understanding of a difficult developmental period to study, especially in the human, are highlighted.

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