scholarly journals Study on the Function and Mechanism of Lin28B in the Formation of Chicken Primordial Germ Cells

Animals ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 43
Author(s):  
Qisheng Zuo ◽  
Jing Zhou ◽  
Man Wang ◽  
Yani Zhang ◽  
Guohong Chen ◽  
...  
Keyword(s):  
Germ Cells ◽  
High Throughput Sequencing ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Periodic Acid ◽  
Primordial Germ Cells ◽  
Negative Regulator ◽  
Marker Genes ◽  
Periodic Acid Schiff

Lin28A and Lin28B are two homologues of the same family of RNA binding proteins (RBPs). The function and molecular mechanism of Lin28A in the formation of primordial germ cells (PGCs) are very clear, but the related research on Lin28B is rarely reported. Here, we found that the overexpression of Lin28B can promote the formation of PGC in vivo. Furthermore, the overexpression of Lin28B also resulted in the inhibition of totipotency gene expression and upregulated the PGCs marker genes, and a significant increase in the number of PGCs in genital ridge, as detected by Periodic Acid-Schiff(PAS) staining. However, the inhibited Lin28B expression showed completely opposite results, which were confirmed on the PGC induction model in vitro. Mechanistically, we found that the overexpression of Lin28B can inhibit the maturation of let-7a-3p, and the results of high-throughput sequencing indicated that let-7a-3p was a negative regulator of the formation process of PGCs. Therefore, we conclude that our results determine that Lin28B participates in the formation of PGCs through let-7a-3p, which set a theoretical foundation for improving the function and mechanism of Lin28 family in the formation of PGCs.

Isolation, culture and characterization of chicken primordial germ cells

2006 ◽  
Vol 3 (3) ◽  
pp. 183-188 ◽  
Author(s):  
Tang Xin-Yan ◽  
Zeng Wei-Dong ◽  
Mi Yu-Ling ◽  
Liu Hong-Yun ◽  
Zhang Cai-Qiao
Keyword(s):  
Germ Cells ◽  
Gradient Centrifugation ◽  
Periodic Acid ◽  
Primordial Germ Cells ◽  
Culture Conditions ◽  
Nuclear Antigen ◽  
Periodic Acid Schiff ◽  
Ficoll Density Gradient Centrifugation ◽  
Pas Staining ◽  
Proliferating Activity

AbstractPrimordial germ cells (PGCs) were isolated from the genital ridges of chicken (Gallus domesticus) embryos at the 19th stage and purified by Ficoll density-gradient centrifugation. PGCs were co-cultured with somatic cells in preliminary culture and subcultured. Identification of PGCs was carried out by histochemical methods, including alkaline phosphatase (AKP) and periodic acid–Schiff (PAS). The proliferating activity of PGCs in subculture was demonstrated by immunocytochemistry of proliferating cell nuclear antigen (PCNA). Meanwhile, proliferating PGCs were compared under different culture conditions of 5–20% fetal cattle serum (FCS), insulin–transferrin–selenite (ITS) medium, conditioned medium (CM), 15% FCS+ITS, 15% FCS+40% CM. The results showed that the cultured PGCs were positive for AKP and PAS staining and displayed intensive proliferating activity by PCNA. The PGCs without centrifugation grew better than those with centrifugation. The PGCs formed larger colonies in media with 5% FCS or ITS than other media, indicating that 5% FCS or ITS supplemented media could be an ideal culture system for PGC proliferation in the PGC-somatic cell co-culture, in addition to the embryonic fibroblast feeder layer.

scholarly journals Bayesian inference of transcriptional branching identifies regulators of early germ cell development in humans

2017 ◽  
Author(s):  
Christopher A. Penfold ◽  
Anastasiya Sybirna ◽  
John Reid ◽  
Aracely Castillo Venzor ◽  
Elena Drousioti ◽  
...  
Keyword(s):  
Cell Fate ◽  
Germ Cells ◽  
Temporal Dynamics ◽  
Primordial Germ Cells ◽  
Series Data ◽  
Human Embryos ◽  
Marker Genes ◽  
Cell Fate Decisions ◽  
Stage Of Development

AbstractDuring embryonic development, cells undertake a series of fate decisions to form a complete organism comprised of various cell types, epitomising a branching process. A striking example of branching occurs in humans around the time of implantation, when primordial germ cells (PGCs), precursors of sperm and eggs, and somatic lineages are specified. Due to inaccessibility of human embryos at this stage of development, understanding the mechanisms of PGC specification remains difficult. The integrative modelling of single cell transcriptomics data from embryos and appropriate in vitro models should prove to be a useful resource for investigating this system, provided that the cells can be suitably ordered over a developmental axis. Unfortunately, most methods for inferring cell ordering were not designed with structured (time series) data in mind. Although some probabilistic approaches address these limitations by incorporating prior information about the developmental stage (capture time) of the cell, they do not allow the ordering of cells over processes with more than one terminal cell fate. To investigate the mechanisms of PGC specification, we develop a probabilistic pseudotime approach, branch-recombinant Gaussian process latent variable models (B-RGPLVMs), that use an explicit model of transcriptional branching in individual marker genes, allowing the ordering of cells over developmental trajectories with arbitrary numbers of branches. We use first demonstrate the advantage of our approach over existing pseudotime algorithms and subsequently use it to investigate early human development, as primordial germ cells (PGCs) and somatic cells diverge. We identify known master regulators of human PGCs, and predict roles for a variety of signalling pathways, transcription factors, and epigenetic modifiers. By concentrating on the earliest branched signalling events, we identified an antagonistic role for FGF receptor (FGFR) signalling pathway in the acquisition of competence for human PGC fate, and identify putative roles for PRC1 and PRC2 in PGC specification. We experimentally validate our predictions using pharmacological blocking of FGFR or its downstream effectors (MEK, PI3K and JAK), and demonstrate enhanced competency for PGC fate in vitro, whilst small molecule inhibition of the enzymatic component of PRC1/PRC2 reveals reduced capacity of cells to form PGCs in vitro. Thus, B-RGPLVMs represent a powerful and flexible data-driven approach for dissecting the temporal dynamics of cell fate decisions, providing unique insights into the mechanisms of early embryogenesis. Scripts relating to this analysis are available from: https://github.com/cap76/PGCPseudotime

scholarly journals Functional reconstruction of NANOS3 expression in the germ cell lineage by a novel transgenic reporter reveals distinct subcellular localizations of NANOS3

Reproduction ◽  
2010 ◽  
Vol 139 (2) ◽  
pp. 381-393 ◽  
Author(s):  
Masashi Yamaji ◽  
Takashi Tanaka ◽  
Mayo Shigeta ◽  
Shinichiro Chuma ◽  
Yumiko Saga ◽  
...  
Keyword(s):  
Germ Cell ◽  
Germ Cells ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Primordial Germ Cells ◽  
Cell Lineage ◽  
Regulatory Elements ◽  
Initiation Factor ◽  
Processing Bodies

Mutations of RNA-binding proteins such as NANOS3, TIAL1, and DND1 in mice have been known to result in the failure of survival and/or proliferation of primordial germ cells (PGCs) soon after their fate is specified (around embryonic day (E) 8.0), leading to the infertility of these animals. However, the mechanisms of actions of these RNA-binding proteins remain largely unresolved. As a foundation to explore the role of these RNA-binding proteins in germ cells, we established a novel transgenic reporter strain that expresses NANOS3 fused with EGFP under the control of Nanos3 regulatory elements. NANOS3–EGFP exhibited exclusive expression in PGCs as early as E7.25, and continued to be expressed in female germ cells until around E14.5 and in male germ cells throughout the fetal period with declining expression levels after E16.5. NANOS3–EGFP resumed strong expression in postnatal spermatogonia and continued to be expressed in undifferentiated spermatogonial cells in adults. Importantly, the Nanos3–EGFP transgene rescued the sterile phenotype of Nanos3 homozygous mutants, demonstrating the functional equivalency of NANOS3–EGFP with endogenous NANOS3. We found that throughout germ cell development, a predominant amount of  NANOS3–EGFP co-localized with TIAL1 (also known as TIAR) and phosphorylated eukaryotic initiation factor 2α, markers for the stress granules, whereas a fraction of it showed co-localization with DCP1A, a marker for the processing bodies. On the other hand, NANOS3–EGFP did not co-localize with Tudor domain-containing protein 1, a marker for the intermitochondrial cements, in spermatogenic cells. These findings unveil the presence of distinct posttranscriptional regulations in PGCs soon after their specification, for which RNA-binding proteins such as NANOS3 and TIAL1 would play critical functions.

scholarly journals Genetic and structural analysis of the in vivo functional redundancy between murine NANOS2 and NANOS3

Development ◽  
2020 ◽  
pp. dev.191916
Author(s):  
Danelle Wright ◽  
Makoto Kiso ◽  
Yumiko Saga
Keyword(s):  
Germ Cell ◽  
Germ Cells ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Double Knockout ◽  
Germ Cell Differentiation ◽  
Evolutionarily Conserved ◽  
Male Specific

NANOS2 and NANOS3 are evolutionarily conserved RNA-binding proteins involved in murine germ cell development. NANOS3 is required for protection from apoptosis during migration and gonadal colonization in both sexes, whereas NANOS2 is male-specific and required for the male-type differentiation of germ cells. Ectopic NANOS2 rescues the functions of NANOS3, but NANOS3 cannot rescue NANOS2 function even though its expression is up-regulated in Nanos2-null conditions. It is unknown why NANOS3 cannot rescue NANOS2 function and it is unclear whether NANOS3 plays any role in male germ cell differentiation. To address these questions, we made conditional Nanos3/Nanos2 knockout mice and chimeric mice expressing chimeric NANOS proteins. Conditional double knockout of Nanos2 and 3 led to the rapid loss of germ cells, and in vivo and in vitro experiments revealed that DND1 and NANOS2 binding is dependent on the specific NANOS2 zinc finger structure. Moreover, murine NANOS3 failed to bind CNOT1, an interactor of NANOS2 at its N-terminal. Collectively, our study suggests that the inability of NANOS3 to rescue NANOS2 function is due to poor DND1 recruitment and CNOT1 binding.

scholarly journals Regulation of Klf16 by Double Strand RNA-binding Protein STAU1 in Adipocyte Differentiation in vitro

2020 ◽  
Author(s):  
Ya Qun Guan ◽  
Xuan Yu Meng ◽  
Xiao Di Liang ◽  
Ting Ting Hu ◽  
Nurbierye Nuermamati ◽  
...  
Keyword(s):  
Rna Binding ◽  
Adipocyte Differentiation ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
Adipogenic Differentiation ◽  
Regulation Of Gene Expression ◽  
Rna Binding Protein ◽  
Negative Regulator ◽  
Transcriptional Level

Abstract Background: Adipogenesis is an essential process in organismal development and plays a significant role in adipose tissue homeostasis. Post-transcriptional regulation of gene expression plays a key role in adipogenesis and involves many RNA-binding proteins (RBPs). In mammals, Staufen1 (STAU1) is a conserved RBP(RNA Binding Protein )consisting of several dsRBP (double strand RNA). STAU1 plays an important role in the Stau1-mediated mRNA decay (SMD) pathway, which is related to adipocyte formation, myocyte development, and neural differentiation. Klf16 (Kruppel like transcription factor 16) is a negative regulator that inhibits adipocyte differentiation. AIM:This study was conducted to determine the role of Klf16 in adipocyte differentiation in the context of the SMD pathway.Methods: 3T3-L1 cells were induced and cultured in vitro by cocktail method, Knockdown and Overexpression of STAU1 and KLF16. Then, adipocyte differentiation andexpression of adipogenic-related genes (STAU1, KLF16, PPARγ, and Lipin1) were measured by RT-qPCR and Western blot.RNA immunoprecipitation (RIP) method verified that STAU1 protein can bind to KLF16.Results: The results revealed that STAU1 regulates Klf16 expression at the post-transcriptional level during the adipogenic differentiation of 3T3-L1 cells.STAU1 candirectly bind the 3′UTR of Klf16 mRNA. Klf16 mRNA was found to be degraded through the SMD pathway, thus promoting adipocyte differentiation.Conclusions: In this study, the mechanism of adipocyte differentiation regulation at the post-transcriptional level is demonstrated, and Klf16 is shown as a substrate of the SMD pathway, thus providing new insights into adipogenesis.

scholarly journals Divergent RNA-binding Proteins, DAZL and VASA, Induce Meiotic Progression in Human Germ Cells Derived in Vitro

Stem Cells ◽  
2012 ◽  
Vol 30 (3) ◽  
pp. 441-451 ◽  
Author(s):  
Jose V. Medrano ◽  
Cyril Ramathal ◽  
Ha N. Nguyen ◽  
Carlos Simon ◽  
Renee A. Reijo Pera
Keyword(s):  
Germ Cells ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Meiotic Progression

scholarly journals Formative pluripotent stem cells show features of epiblast cells poised for gastrulation

Cell Research ◽  
2021 ◽  
Author(s):  
Xiaoxiao Wang ◽  
Yunlong Xiang ◽  
Yang Yu ◽  
Ran Wang ◽  
Yu Zhang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Germ Cells ◽  
Pluripotent Stem Cells ◽  
Primordial Germ Cells ◽  
Embryonic Stem ◽  
Large Set ◽  
Mouse Escs ◽  
Epiblast Stem Cells ◽  
Early Gastrula

AbstractThe pluripotency of mammalian early and late epiblast could be recapitulated by naïve embryonic stem cells (ESCs) and primed epiblast stem cells (EpiSCs), respectively. However, these two states of pluripotency may not be sufficient to reflect the full complexity and developmental potency of the epiblast during mammalian early development. Here we report the establishment of self-renewing formative pluripotent stem cells (fPSCs) which manifest features of epiblast cells poised for gastrulation. fPSCs can be established from different mouse ESCs, pre-/early-gastrula epiblasts and induced PSCs. Similar to pre-/early-gastrula epiblasts, fPSCs show the transcriptomic features of formative pluripotency, which are distinct from naïve ESCs and primed EpiSCs. fPSCs show the unique epigenetic states of E6.5 epiblast, including the super-bivalency of a large set of developmental genes. Just like epiblast cells immediately before gastrulation, fPSCs can efficiently differentiate into three germ layers and primordial germ cells (PGCs) in vitro. Thus, fPSCs highlight the feasibility of using PSCs to explore the development of mammalian epiblast.

scholarly journals The RNA-binding protein ELAVL1/HuR is essential for mouse spermatogenesis, acting both at meiotic and postmeiotic stages

2011 ◽  
Vol 22 (16) ◽  
pp. 2875-2885 ◽  
Author(s):  
Mai Nguyen Chi ◽  
Jacques Auriol ◽  
Bernard Jégou ◽  
Dimitris L. Kontoyiannis ◽  
James M.A. Turner ◽  
...  
Keyword(s):  
Germ Cells ◽  
Posttranscriptional Regulation ◽  
Target Gene ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Female Sterility ◽  
Targeted Deletion

Posttranscriptional mechanisms are crucial to regulate spermatogenesis. Accurate protein synthesis during germ cell development relies on RNA binding proteins that control the storage, stability, and translation of mRNAs in a tightly and temporally regulated manner. Here, we focused on the RNA binding protein Embryonic Lethal Abnormal Vision (ELAV) L1/Human antigen R (HuR) known to be a key regulator of posttranscriptional regulation in somatic cells but the function of which during gametogenesis has never been investigated. In this study, we have used conditional loss- and gain-of-function approaches to address this issue in mice. We show that targeted deletion of HuR specifically in germ cells leads to male but not female sterility. Mutant males are azoospermic because of the extensive death of spermatocytes at meiotic divisions and failure of spermatid elongation. The latter defect is also observed upon HuR overexpression. To elucidate further the molecular mechanisms underlying spermatogenesis defects in HuR-deleted and -overexpressing testes, we undertook a target gene approach and discovered that heat shock protein (HSP)A2/HSP70-2, a crucial regulator of spermatogenesis, was down-regulated in both situations. HuR specifically binds hspa2 mRNA and controls its expression at the translational level in germ cells. Our study provides the first genetic evidence of HuR involvement during spermatogenesis and reveals Hspa2 as a target for HuR.

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