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 BMP4 activates the Wnt-Lin28A-Blimp1-Wnt pathway to promote primordial germ cells formation via altering H3K4me2

2020 ◽  
pp. jcs.249375
Author(s):  
Qisheng Zuo ◽  
Kai Jin ◽  
Man Wang ◽  
Yani Zhang ◽  
Guohong Chen ◽  
...  
Keyword(s):  
Wnt Signaling ◽  
Formation Mechanism ◽  
Germ Cells ◽  
Positive Feedback ◽  
Gene Editing ◽  
Wnt Pathway ◽  
Primordial Germ Cells ◽  
Bird Species ◽  
Developmental Characteristics ◽  
Feedback Pathway

The unique developmental characteristics of Chicken primordial germ cells (PGCs) enable it to be used in recovery of endangered bird species, gene editing and the generation of transgenic birds, but the limited number of PGCs greatly limits its application. Studies have shown that the formation of mammalian PGCs is induced by BMP4 signal, but the formation mechanism of chicken PGCs has not been determined. Here, we confirmed that Wnt signaling activated via BMP4 activates transcription of Lin28A by inducing β-catenin to compete with LSD1 for binding to TCF7L2, causing LSD1 to dissociate from the Lin28A promoter and enhanced H3K4me2 methylation in this region. Lin28A promotes PGCs 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, that ensures PGCs formation. In summary, our data provide a new insights in the development of PGCs in chickens.

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.

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)

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.

Functional requirement of gp130-mediated signaling for growth and survival of mouse primordial germ cells in vitro and derivation of embryonic germ (EG) cells

Development ◽  
1996 ◽  
Vol 122 (4) ◽  
pp. 1235-1242 ◽  
Author(s):  
U. Koshimizu ◽  
T. Taga ◽  
M. Watanabe ◽  
M. Saito ◽  
Y. Shirayoshi ◽  
...  
Keyword(s):  
Germ Cells ◽  
Intracellular Signaling ◽  
Primordial Germ Cells ◽  
Cell Formation ◽  
Embryonic Stem ◽  
Oncostatin M ◽  
Receptor Complexes ◽  
Binding Subunit

Leukemia inhibitory factor (LIF) is a cytokine known to influence proliferation and/or survival of mouse primordial germ cells (PGC) in culture. The receptor complex for LIF comprises LIF-binding subunit and non-binding signal transducer, gp130. The gp130 was originally identified as a signal-transducing subunit of interleukin (IL)-6 and later also found to be a functional component of receptor complexes for other LIF-related cytokines (oncostatin M [OSM], ciliary neurotrophic factor [CNTF] and IL-11). In this study, we have analyzed the functional role of gp130-mediated signaling in PGC growth in vitro. OSM was able to fully substitute for LIF; both cytokines promoted the proliferation of migratory PGC (mPGC) and enhanced the viability of postmigratory (colonizing) PGC (cPGC) when cultured on SI/SI4-m220 cells. Interestingly, IL-11 stimulated mPGC growth comparable to LIF and OSM, but did not affect cPGC survival. IL-6 and CNTF did not affect PGC. In addition, a combination of IL-6 and soluble IL-6 binding subunit (sIL-6R), which is known to activate intracellular signaling via gp130, fully reproduced the LIF action of PGC. Both in the presence and absence of LIF, addition of neutralizing antibody against gp130 in culture remarkably blocked cPGC survival. These results suggest a pivotal role of gp130 in PGC development, especially that it is indispensable for cPGC survival as comparable to the c-KIT-mediated action. We have further demonstrated that a combination of LIF with forskolin or retinoic acid, a potent mitogen for PGC, supported the proliferation of PGC, leading to propagation of the embryonic stem cell-like cells, termed embryonic germ (EG) cells. Since EG cells were also obtained by using OSM or the IL-6/sIL-6R complex in place of LIF, a significant contribution of gp130-mediated signaling in EG cell formation was further suggested.

207 ESTABILSHMENT OF CYNOMOLGUS MONKEY EMBRYONIC STEM CELL LINES AND CONFIRMATION OF THE POSSIBILITY FOR GERMINAL COMPETENCY

2006 ◽  
Vol 18 (2) ◽  
pp. 211
Author(s):  
T. Teramura ◽  
N. Kawata ◽  
N. Fujinami ◽  
M. Takenoshita ◽  
N. Sagawa ◽  
...  
Keyword(s):  
Germ Cell ◽  
Cell Lines ◽  
Cynomolgus Monkey ◽  
Primordial Germ Cell ◽  
Cell Formation ◽  
Embryonic Stem ◽  
Alp Activity ◽  
Weak Expression ◽  
Germ Cell Formation

Embryonic stem cells (ESCs) of nonhuman primate are important tools for human gametogenesis research. Generally, ESCs, embryos, and fetuses of nonhuman primates are similar to these of human. Recently, germ cell formation of mouse ESCs in vitro has been reported. In this study, we established new cynomolgus monkey ES (cyES) lines and determined germinal competency by assessing expression of mRNA markers. CyES lines were established using blastocysts produced by intracytoplasmic sperm injection (ICSI). For inducing super-ovulation, females were treated with 25 IU/kg pregnant mare serum gonadotropin (PMSG) once a day for 9 days, followed by 400 IU/kg hCG. Oocytes were collected 40 h after injection of hCG. After sperm injection, embryos were cultured in mCMRL medium to the blastocyst stage. For ES line establishment, inner cell masses (ICMs) were isolated by immunosurgery. ESC colonies emerged at about 10 days after ICM plating; three cyES cell lines were successfully obtained (3/11; 27.3%). We characterized these lines by immunocytochemistry for Oct-3/4, SSEA-3, and SSEA-4, which are diagnostic markers for primate ESCs, and by assay for alkaline phosphatase (ALP) activity. All cell lines expressed Oct-3/4, SSEA-4 and ALP activity. The previously reported SSEA-3 weak expression in cyES cells was not observed. These lines differentiated spontaneously when they were replaced in non-adherent culture (embryoid body: EB) or injected into SCID mice subcutaneously. To assess germ cell competency in vitro, we analyzed for the presence of vasa mRNA which shows a restricted expression pattern to germ cell formation, and DMC1 and SYCP1 which show specific existence on synaptonema complex in meiosis. Detection of these germ cell markers was performed by RT-PCR with total cDNA from ESCs and EBs. Nanog mRNA was detected only in ESCs. Oct-4 was detected in gonadal tissue of both sexes, ESCs, and EBs. Vasa was expressed in testis, but not in ESCs or somatic cells. Interestingly, we recognized weak expression of Vasa in Day 12-16 EBs. DMC1 and SYCP1 as meiosis markers were not detected. Because Oct-4 and Vasa mRNA are transcribed simultaneously, similar to that in the early part of gametogenesis such as the latter period of primordial germ cell (PGC) migration, PGC formation in cynomolgus EBs could occurr as in some cases of mouse or human EBs previously reported. Although detailed properties such as the functions of these Vasa-positive cells have not been confirmed, these results demonstrate that cyES cells obtained in the current study might contribute to putative germ cells in vitro by differentiating to EBs. This study was supported by a Grant-in-Aid for the 21st Century COE Program of the Japan Mext and by a grant for the Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technology Excellence of the JST.

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 Direct repression of Nanog and Oct4 by OTX2 modulates contribution of epiblast-derived cells to germline and somatic lineage

Development ◽  
2021 ◽  
Author(s):  
Luca Giovanni Di Giovannantonio ◽  
Dario Acampora ◽  
Daniela Omodei ◽  
Vincenzo Nigro ◽  
Pasquale Barba ◽  
...  
Keyword(s):  
Wnt Signaling ◽  
Regulatory Network ◽  
Primordial Germ Cells ◽  
Culture Conditions ◽  
Specific Cell ◽  
Temporal Window ◽  
Pluripotency Gene ◽  
Gene Regulatory ◽  
Cell Culture Conditions

In mammals the pre-gastrula proximal epiblast gives rise to Primordial Germ Cells (PGCs) or somatic precursors in response to BMP4 and WNT signaling. Entry into the germline requires activation of a naïve-like pluripotency gene regulatory network (GRN). Recent work showed that suppression of OTX2 expression in the epiblast by BMP4 allows cells to develop a PGC fate in a precise temporal window. However, the mechanisms by which OTX2 suppresses PGC fate are unknown. Here we show that OTX2 prevents epiblast cells from activating the pluripotency GRN by direct repression of Oct4 and Nanog. Loss of this control during PGC differentiation in vitro causes widespread activation of the pluripotency GRN and deregulated response to LIF, BMP4 and WNT signaling. These abnormalities, in specific cell culture conditions, result in massive germline entry at the expense of somatic mesoderm differentiation. Increased generation of PGCs occurs also in mutant embryos. We propose that the OTX2 repressive control of Oct4 and Nanog is at the basis of the mechanism determining epiblast contribution to germline and somatic lineage.

scholarly journals Long Noncoding RNA LncPGCR Mediated by TCF7L2 Regulates Primordial Germ Cell Formation in Chickens

Animals ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 292
Author(s):  
Jingyi Jiang ◽  
Chen Chen ◽  
Shaoze Cheng ◽  
Xia Yuan ◽  
Jing Jin ◽  
...  
Keyword(s):  
Stem Cells ◽  
Noncoding Rna ◽  
Marker Gene ◽  
Primordial Germ Cell ◽  
Cell Formation ◽  
Embryonic Stem ◽  
Positive Role ◽  
Dependent Inhibition

Although lncRNAs have been identified as playing critical roles in the development of germ cells, their potential involvement in the development of PGCs in chickens remains poorly understood. Differentially expressed lncRNAs (DELs) from previous RNA-seq of embryonic stem cells (ESCs), PGCs, and spermatogonial stem cells (SSCs) were analyzed by K-means clustering, from which a key candidate, lncRNA (lncRNA PGC regulator, LncPGCR) was obtained. We confirmed that LncPGCR plays a positive role in the development of PGCs by increasing the expression of the PGC marker gene (Cvh and C-kit), while downregulating the pluripotency-associated gene (Nanog) in vitro and in vivo. The activation and expression of LncPGCR are regulated by histone acetylation, and transcription factor TCF7L2. Mechanistically, a rescue assay was performed to further confirm that LncPGCR contributed to the development of PGCs by regulating the gga-miR-6577-5p/Btrc signaling pathway. Adsorption of gga-miR-6577-5p activated the WNT signaling cascade by relieving the gga-miR-6577-5p-dependent inhibition of Btrc expression. Taken together, our study discovered the growth-expedited role of LncPGCR in PGCs development, showing the potential LncPGCR/miR-6577-5p/Btrc pathway. The results and findings provide a novel insight into the development of PGCs.

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.

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