scholarly journals Sex differentiation in grayling (Salmonidae) goes through an all-male stage and is delayed in genetic males who instead grow faster

2017 ◽  
Author(s):  
Diane Maitre ◽  
Oliver M. Selmoni ◽  
Anshu Uppal ◽  
Lucas Marques da Cunha ◽  
Laetitia G. E. Wilkins ◽  
...  
Keyword(s):  
Gene Expression ◽  
Sex Determination ◽  
Sex Differentiation ◽  
Sex Ratios ◽  
Molecular Genetic ◽  
Specific Gene ◽  
Genetic Sexing ◽  
Experimental Conditions ◽  
Two Samples

AbstractFish can be threatened by distorted sex ratios that arise during sex differentiation. It is therefore important to understand sex determination and differentiation, especially in river-dwelling fish that are often exposed to environmental factors that may interfere with sex differentiation. However, sex differentiation is not sufficiently understood in keystone taxa such as the Thymallinae, one of the three salmonid subfamilies. Here we study a wild grayling (Thymallus thymallus) population that suffers from distorted sex ratios. We found sex determination in the wild and in captivity to be genetic and linked to the sdY locus. We therefore studied sex-specific gene expression in embryos and early larvae that were bred and raised under different experimental conditions, and we studied gonadal morphology in five monthly samples taken after hatching. Significant sex-specific changes in gene expression (affecting about 25,000 genes) started around hatching. Gonads were still undifferentiated three weeks after hatching, but about half of the fish showed immature testes around seven weeks after hatching. Over the next few months, this phenotype was mostly replaced by the “testis-to-ovary” or “ovaries” phenotypes. The gonads of the remaining fish, i.e. approximately half of the fish in each sampling period, remained undifferentiated until six months after fertilization. Genetic sexing of the last two samples revealed that fish with undifferentiated gonads were all males, who, by that time, were on average larger than the genetic females (verified in 8-months old juveniles raised in another experiment). Only 12% of the genetic males showed testicular tissue six months after fertilization. We conclude that sex differentiation starts around hatching, goes through an all-male stage for both sexes (which represents a rare case of “undifferentiated” gonochoristic species that usually go through an all-female stage), and is delayed in males who, instead of developing their gonads, grow faster than females during these juvenile stages.Author contributionMRR and CW initiated the project. DM, OS, AU, LMC, LW, and CW sampled the adult fish, did the experimental in vitro fertilizations, and prepared the embryos for experimental rearing in the laboratory. All further manipulations on the embryos and the larvae were done by DM, OS, AU, LMC, and LW. The RNA-seq data were analyzed by OS, JR, and MRR, the histological analyses were done by DM, supervised by SK, and the molecular genetic sexing was performed by DM, OS, AU, and KBM. DM, OS, and CW performed the remaining statistical analyses and wrote the first version of the manuscript that was then critically revised by all other authors.

scholarly journals Sex-specific changes in gene expression and delayed sex differentiation in response to estrogen pollution in grayling (Salmonidae)

2017 ◽  
Author(s):  
Oliver M. Selmoni ◽  
Diane Maitre ◽  
Julien Roux ◽  
Laetitia G. E. Wilkins ◽  
Lucas Marques da Cunha ◽  
...  
Keyword(s):  
Gene Expression ◽  
Sex Differentiation ◽  
Molecular Genetic ◽  
Gonadal Development ◽  
Yolk Sac ◽  
Specific Gene ◽  
Genetic Sexing ◽  
Early Stages ◽  
Study Gene Expression

AbstractThe synthetic 17α-ethinylestradiol (EE2) is an estrogenic compound of oral contraceptives and therefore a common pollutant that has been suspected to affect the demography of river-dwelling salmonids. We study a population of European grayling (Thymallus thymallus) that suffers from sex ratio distortions. Here we test how ecologically relevant concentrations of EE2 affect sex-specific gene expression around early stages of sex differentiation. We collected gametes from F1s of wild spawners, used them for in vitro fertilizations, and raised the resulting embryos singly under experimentally controlled conditions. Embryos were either exposed to 1ng/L EE2 or sham-exposed. RNA was collected from samples taken 10 days before hatching, at the day of hatching, and towards the end of the yolk-sac stage, to study gene expression and relate it to genetic sex (sdY genotype). We found that EE2 affects gene expression of a very large number of genes especially at the day of hatching. The effects of EE2 on gene expression is strongly sex-specific. At the day of hatching, EE2 affected about twice as many genes in females than in males, and towards the end of the yolk-sac larval stage, EE2 effects were nearly exclusively observed in females. Among the many effects was, for example, a surprising EE2-induced molecular masculinization in the females’ heads. Histological examination of gonadal development of EE2-treated or sham-exposed juveniles during the first 4.5 months after hatching revealed a delaying effect of EE2 on sex differentiation. Because grayling sex determination goes through an all-male stage (a rare case of undifferentiated gonochorism), the rate of EE2-induced sex reversal could not be unequivocally determined during the observational period. However, two EE2-treated genetic males had ovarian tissues at the end of the study. We conclude that common levels of EE2 pollution affect grayling from very early stages on by interfering with male and female gene expression around the onset of sex differentiation, by delaying sex differentiation, and by feminizing some males.Author contributionMRR and CW initiated the project. OS, DM, LW, LMC, and CW sampled the adult fish, did the experimental in vitro fertilizations, and prepared the embryos for experimental rearing in the laboratory. All further manipulations on the embryos and the larvae were done by OS, DM, LW, and LMC. The RNA-seq data were analyzed by OS, JR, and MRR, the histological analyses were done by DM, supervised by SK, the molecular genetic sexing was performed by OS and DM, and EV supervised the EE2 analytics. OS and CW performed the remaining statistical analyses and wrote the first version of the manuscript that was then critically revised by all other authors.

Serial analysis of gene expression (SAGE) during porcine embryo development

2004 ◽  
Vol 16 (2) ◽  
pp. 87 ◽  
Author(s):  
Le Ann Blomberg ◽  
Kurt A. Zuelke
Keyword(s):  
Gene Expression ◽  
Functional Genomics ◽  
Embryo Development ◽  
Molecular Mechanisms ◽  
Physiological Role ◽  
Transgenic Animals ◽  
Specific Gene ◽  
Research Strategies

Functional genomics provides a powerful means for delving into the molecular mechanisms involved in pre-implantation development of porcine embryos. High rates of embryonic mortality (30%), following either natural mating or artificial insemination, emphasise the need to improve the efficiency of reproduction in the pig. The poor success rate of live offspring from in vitro-manipulated pig embryos also hampers efforts to generate transgenic animals for biotechnology applications. Previous analysis of differential gene expression has demonstrated stage-specific gene expression for in vivo-derived embryos and altered gene expression for in vitro-derived embryos. However, the methods used to date examine relatively few genes simultaneously and, thus, provide an incomplete glimpse of the physiological role of these genes during embryogenesis. The present review will focus on two aspects of applying functional genomics research strategies for analysing the expression of genes during elongation of pig embryos between gestational day (D) 11 and D12. First, we compare and contrast current methodologies that are being used for gene discovery and expression analysis during pig embryo development. Second, we establish a paradigm for applying serial analysis of gene expression as a functional genomics tool to obtain preliminary information essential for discovering the physiological mechanisms by which distinct embryonic phenotypes are derived.

scholarly journals A Dual Role for Oncostatin M Signaling in the Differentiation and Death of Mammary Epithelial Cells in Vivo

2008 ◽  
Vol 22 (12) ◽  
pp. 2677-2688 ◽  
Author(s):  
Paul G. Tiffen ◽  
Nader Omidvar ◽  
Nuria Marquez-Almuina ◽  
Dawn Croston ◽  
Christine J. Watson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Mammary Epithelial Cells ◽  
Mammary Epithelial ◽  
Oncostatin M ◽  
Specific Gene ◽  
Mammary Involution

Abstract Recent studies in breast cancer cell lines have shown that oncostatin M (OSM) not only inhibits proliferation but also promotes cell detachment and enhances cell motility. In this study, we have looked at the role of OSM signaling in nontransformed mouse mammary epithelial cells in vitro using the KIM-2 mammary epithelial cell line and in vivo using OSM receptor (OSMR)-deficient mice. OSM and its receptor were up-regulated approximately 2 d after the onset of postlactational mammary regression, in response to leukemia inhibitory factor (LIF)-induced signal transducer and activator of transcription-3 (STAT3). This resulted in sustained STAT3 activity, increased epithelial apoptosis, and enhanced clearance of epithelial structures during the remodeling phase of mammary involution. Concurrently, OSM signaling precipitated the dephosphorylation of STAT5 and repressed expression of the milk protein genes β-casein and whey acidic protein (WAP). Similarly, during pregnancy, OSM signaling suppressed β-casein and WAP gene expression. In vitro, OSM but not LIF persistently down-regulated phosphorylated (p)-STAT5, even in the continued presence of prolactin. OSM also promoted the expression of metalloproteinases MMP3, MMP12, and MMP14, which, in vitro, were responsible for OSM-specific apoptosis. Thus, the sequential activation of IL-6-related cytokines during mammary involution culminates in an OSM-dependent repression of epithelial-specific gene expression and the potentiation of epithelial cell extinction mediated, at least in part, by the reciprocal regulation of p-STAT5 and p-STAT3.

scholarly journals A molecular genetic approach for sex determination on helmeted hornbill (Rhinoplax vigil) casque: a forensic casework

2020 ◽  
Vol 19 ◽  
pp. 00020
Author(s):  
Yuli S. Fitriana ◽  
Mohammad Irham ◽  
Hari Sutrisno ◽  
Abinawanto
Keyword(s):  
Sex Determination ◽  
Molecular Genetic ◽  
Independent Sets ◽  
Black Market ◽  
Critically Endangered ◽  
Genetic Approach ◽  
Genetic Sexing ◽  
Male And Female ◽  
Forensic Casework ◽  
Males And Females

Helmeted Hornbill (Rhinolax vigil) is the only hornbill that equipped with solid casque made from keratin for both males and females. The demand for casque in the black market was huge and resulted in IUCN status leaped up from vulnerable to critically endangered. We received a total of 68 confiscated helmeted hornbill casques. As part of the casework and the objectives of the study, we determined to reveal the sex status of those casques and the best methods to work with keratinous material. Molecular methods to determining sex in birds rely on the CHD gene located on male and female chromosomes ZZ and ZW, respectively. We optimized laboratory protocols for genetic sexing using three independent sets of primers P2/P8, 2550F/2718R, and CHD1F/CHD1R to amplify regions of the sexlinked CHD-Z and CHD-W genes. The CHD1F/CHD1R determined sex 80.88% of samples. The 2550F/2718R were quite successful, sexing 51.47% of samples. In contrast, the P2/P8 only identified the sex around 20.58% of samples. These results showed that CHD1F/CHD1R works the most effective for sexing the casques with 52.9% females, 27.9% males, and 19.1% unidentified. Therefore, the most accurate and suitable primers are CHD1F/CHD1R, 2550F/2718R, and P2/P8, respectively for keratinous samples.

scholarly journals In vitro-selected RNA cleaving DNA enzymes from a combinatorial library are potent inhibitors of HIV-1 gene expression

2000 ◽  
Vol 352 (3) ◽  
pp. 667-673 ◽  
Author(s):  
Bandi SRIRAM ◽  
Akhil C. BANERJEA
Keyword(s):  
Gene Expression ◽  
Mammalian Cell ◽  
Specific Gene ◽  
Cleavage Sites ◽  
Dna Enzymes ◽  
Biological Tool ◽  
Catalytic Motif ◽  
Target Rna ◽  
Hiv 1

Selective inactivation of a target gene by antisense mechanisms is an important biological tool to delineate specific functions of the gene product. Approaches mediated by ribozymes and RNA-cleaving DNA enzymes (DNA enzymes) are more attractive because of their ability to catalytically cleave the target RNA. DNA enzymes have recently gained a lot of importance because they are short DNA molecules with simple structures that are expected to be stable to the nucleases present inside a mammalian cell. We have designed a strategy to identify accessible cleavage sites in HIV-1 gag RNA from a pool of random DNA enzymes, and for isolation of DNA enzymes. A pool of random sequences (all 29 nucleotides long) that contained the earlier-identified 10Ő23 catalytic motif were tested for their ability to cleave the target RNA. When the pool of random DNA enzymes was targeted to cleave between any A and U nucleotides, DNA enzyme 1836 was identified. Although several DNA enzymes were identified using a pool of DNA enzymes that was completely randomized with respect to its substrate-binding properties, DNA enzyme-1810 was selected for further characterization. Both DNA enzymes showed target-specific cleavage activities in the presence of Mg2+ only. When introduced into a mammalian cell, they showed interference with HIV-1-specific gene expression. This strategy could be applied for the selection of desired target sites in any target RNA.

scholarly journals Epigenetic regulation of neural cell differentiation plasticity in the adult mammalian brain

2008 ◽  
Vol 105 (46) ◽  
pp. 18012-18017 ◽  
Author(s):  
Jun Kohyama ◽  
Takuro Kojima ◽  
Eriko Takatsuka ◽  
Toru Yamashita ◽  
Jun Namiki ◽  
...  
Keyword(s):  
Gene Expression ◽  
Target Genes ◽  
Epigenetic Modification ◽  
Mammalian Brain ◽  
Cytokine Signaling ◽  
Specific Gene ◽  
Neural Cells ◽  
Specific Gene Expression

Neural stem/progenitor cells (NSCs/NPCs) give rise to neurons, astrocytes, and oligodendrocytes. It has become apparent that intracellular epigenetic modification including DNA methylation, in concert with extracellular cues such as cytokine signaling, is deeply involved in fate specification of NSCs/NPCs by defining cell-type specific gene expression. However, it is still unclear how differentiated neural cells retain their specific attributes by repressing cellular properties characteristic of other lineages. In previous work we have shown that methyl-CpG binding protein transcriptional repressors (MBDs), which are expressed predominantly in neurons in the central nervous system, inhibit astrocyte-specific gene expression by binding to highly methylated regions of their target genes. Here we report that oligodendrocytes, which do not express MBDs, can transdifferentiate into astrocytes both in vitro (cytokine stimulation) and in vivo (ischemic injury) through the activation of the JAK/STAT signaling pathway. These findings suggest that differentiation plasticity in neural cells is regulated by cell-intrinsic epigenetic mechanisms in collaboration with ambient cell-extrinsic cues.

scholarly journals Gene expression analysis by non-radioactive RNA-RNA in situ hybridization techniques

2004 ◽  
Vol 23 (2) ◽  
pp. 127-133 ◽  
Author(s):  
Danijela Drakulic ◽  
Milena Stevanovic ◽  
Gordana Nikcevic
Keyword(s):  
Gene Expression ◽  
In Situ Hybridization ◽  
Cultured Cells ◽  
Specific Gene ◽  
Rna In Situ Hybridization ◽  
Sox Gene ◽  
Labeled Rna ◽  
Set Up

RNA-RNA in situ hybridization is a reliable method for studying tissue and cell specific gene expression, which enables visualization of labeled antisense RNA probe hybridized to specific mRNA. In this study we employed non-radioactive RNA-RNA in situ hybridization using biotin- or digoxigenin-labeled RNA probes in order to detect SOX gene expression in carcinoma cell lines. By this approach we confirmed results obtained by Northern blot analysis, where the presence of SOX2 mRNA in NT2/D1 and SOX14 mRNA in HepG2 cells has been established. Our aim was to set up RNA-RNA in situ hybridization method in in vitro cultured cells in order to perform further analyses of SOX gene expression on various normal and cancer tissues.

scholarly journals OncogenicGata1causes stage-specific megakaryocyte differentiation delay

2019 ◽  
Author(s):  
Gaëtan Juban ◽  
Nathalie Sakakini ◽  
Hedia Chagraoui ◽  
Qian Cheng ◽  
Kelly Soady ◽  
...  
Keyword(s):  
Gene Expression ◽  
Es Cells ◽  
Erythroid Differentiation ◽  
Detailed Examination ◽  
S Phase ◽  
Myeloproliferative Disorder ◽  
Specific Gene ◽  
Specific Stage

AbstractThe megakaryocyte/erythroid Transient Myeloproliferative Disorder (TMD) in newborns with Down Syndrome (DS) occurs when N-terminal truncating mutations of the hemopoietic transcription factor GATA1, that produce GATA1short protein (GATA1s), are acquired early in development. Prior work has shown that murine GATA1s, by itself, causes a transient yolk sac myeloproliferative disorder. However, it is unclear where in the hemopoietic cellular hierarchy GATA1s exerts its effects to produce this myeloproliferative state. Here, through a detailed examination of hemopoiesis from murine GATA1s ES cells and GATA1s embryos we define defects in erythroid and megakaryocytic differentiation that occur relatively in hemopoiesis. GATA1s causes an arrest late in erythroid differentiationin vivo, and even more profoundly in ES-cell derived cultures, with a marked reduction of Ter-119 cells and reduced erythroid gene expression. In megakaryopoiesis, GATA1s causes a differentiation delay at a specific stage, with accumulation of immature, kit-expressing CD41himegakaryocytic cells. In this specific megakaryocytic compartment, there are increased numbers of GATA1s cells in S-phase of cell cycle and reduced number of apoptotic cells compared to GATA1 cells in the same cell compartment. There is also a delay in maturation of these immature GATA1s megakaryocytic lineage cells compared to GATA1 cells at the same stage of differentiation. Finally, even when GATA1s megakaryocytic cells mature, they mature aberrantly with altered megakaryocyte-specific gene expression and activity of the mature megakaryocyte enzyme, acetylcholinesterase. These studies pinpoint the hemopoietic compartment where GATA1s megakaryocyte myeloproliferation occurs, defining where molecular studies should now be focussed to understand the oncogenic action of GATA1s.Scientific CategoryHaematopoiesis and Stem CellsKey PointsGATA1s-induced stage-specific differentiation delay increases immature megakaryocytesin vivoandin vitro, during development.Differentiation delay is associated with increased numbers of cells in S-phase and reduced apoptosis.

scholarly journals TMOD-13. MODELING THE GENETIC, TRANSCRIPTOMIC, AND CELLULAR HETEROGENEITY OF GLIOBLASTOMA USING TUMOR ORGANOIDS

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi265-vi265
Author(s):  
Daniel Zhang ◽  
Fadi Jacob ◽  
Ryan Salinas ◽  
Phuong Nguyen ◽  
Guo-li Ming ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Cellular Heterogeneity ◽  
Definitive Treatment ◽  
Functional Study ◽  
Specific Gene ◽  
Fresh Tissue ◽  
Mechanistic Investigation

Abstract Glioblastoma exhibits enormous genetic, transcriptional, and cellular heterogeneity at the macroscopic level across regions of the tumor as well as at the microscopic level between neighboring cells, all of which present significant challenges towards creating a definitive treatment for this devastating disease. We have developed a method of generating glioblastoma organoids (GBOs) from fresh tissue obtained directly from surgical resection and maintaining them in a defined medium without bFGF/EGF. Whole exome sequencing revealed that GBOs maintain the genomic landscape of their parent tumors. Somatic and copy number variants are present in the GBOs at similar allele frequencies or copy ratios as in the parent tumor, suggesting that the relative proportions of clonal populations are largely maintained in the organoids. Bulk transcriptomic analysis demonstrated strong gene expression correlations between the parent tumor and corresponding GBOs through 12 weeks of culture. Some tumors were sampled at multiple different anatomic regions, and the corresponding GBOs maintained region-specific gene expression signatures and genomic variants. EGFRvIII, a tumor-specific variant targeted in a number of emerging therapies, also remains present in the GBOs at similar transcript frequencies, reflecting the native heterogeneity of the parent tumor. Finally, we used single cell transcriptomics to examine cellular heterogeneity and find that GBOs contain many different cell types that exhibit similar gene expression profiles as the matching cell type in the corresponding parent tumor. Notably, these GBOs retain neoplastic as well as non-neoplastic cells, such as tumor associated macrophages / microglia, T-cells, endothelial cells, stromal cells, and oligodendrocytes. These GBOs preserve complex tumor heterogeneity an in vitro environment, creating opportunities for extended manipulation, characterization, and functional study for mechanistic investigation and therapeutic testing.

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