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1
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Soliman HK, Coughlan JM. United by conflict: Convergent signatures of parental conflict in angiosperms and placental mammals. J Hered 2024; 115:625-642. [PMID: 38366852 PMCID: PMC11498613 DOI: 10.1093/jhered/esae009] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2023] [Accepted: 02/13/2024] [Indexed: 02/18/2024] Open
Abstract
Endosperm in angiosperms and placenta in eutherians are convergent innovations for efficient embryonic nutrient transfer. Despite advantages, this reproductive strategy incurs metabolic costs that maternal parents disproportionately shoulder, leading to potential inter-parental conflict over optimal offspring investment. Genomic imprinting-parent-of-origin-biased gene expression-is fundamental for endosperm and placenta development and has convergently evolved in angiosperms and mammals, in part, to resolve parental conflict. Here, we review the mechanisms of genomic imprinting in these taxa. Despite differences in the timing and spatial extent of imprinting, these taxa exhibit remarkable convergence in the molecular machinery and genes governing imprinting. We then assess the role of parental conflict in shaping evolution within angiosperms and eutherians using four criteria: 1) Do differences in the extent of sibling relatedness cause differences in the inferred strength of parental conflict? 2) Do reciprocal crosses between taxa with different inferred histories of parental conflict exhibit parent-of-origin growth effects? 3) Are these parent-of-origin growth effects caused by dosage-sensitive mechanisms and do these loci exhibit signals of positive selection? 4) Can normal development be restored by genomic perturbations that restore stoichiometric balance in the endosperm/placenta? Although we find evidence for all criteria in angiosperms and eutherians, suggesting that parental conflict may help shape their evolution, many questions remain. Additionally, myriad differences between the two taxa suggest that their respective biologies may shape how/when/where/to what extent parental conflict manifests. Lastly, we discuss outstanding questions, highlighting the power of comparative work in quantifying the role of parental conflict in evolution.
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Affiliation(s)
- Hagar K Soliman
- Department of Ecology & Evolutionary Biology, Yale University, New Haven, CT 06511, United States
- Department of Biotechnology, Faculty of Science, Cairo University, Giza 12613, Egypt
| | - Jenn M Coughlan
- Department of Ecology & Evolutionary Biology, Yale University, New Haven, CT 06511, United States
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2
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Lee J, Cai L, Kim M, Choi H, Oh D, Jawad A, Lee E, Hyun SH. Tetraploid embryo aggregation produces high-quality blastocysts with an increased trophectoderm in pigs. Front Cell Dev Biol 2023; 11:1239448. [PMID: 38033873 PMCID: PMC10687364 DOI: 10.3389/fcell.2023.1239448] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2023] [Accepted: 11/01/2023] [Indexed: 12/02/2023] Open
Abstract
Tetraploid complementation is an ideal method for demonstrating the differentiation potential of pluripotent stem cells. In this study, we selected the most efficient tetraploid production method for porcine embryos and investigated whether tetraploid blastomere aggregation could enhance the quality of tetraploid embryos. Three methods were investigated to produce tetraploid embryos: First, tetraploid embryos were produced using electro-fusion of two-cell stage parthenogenetic blastomere (FUTP). Second, somatic cell was injected into the mature oocyte and fused to produce tetraploid embryos. Third, oocytes were matured with Cytochalasin B (CB) for the late 22 h of in vitro maturation to inhibit the first polar body (PB1). Following that, non-PB1 oocytes were treated with CB for 4 h after parthenogenetic activation. There was no significant difference in the blastocyst development rate and tetraploid production rate of the embryos produced through the three methods. However, FUTP-derived blastocysts had a significantly lower percentage of apoptotic cells compared to other methods. The developmental competence of embryos, expression of trophectoderm cell marker genes, and distribution of YAP1 protein were investigated in tetraploid embryos produced using the FUTP method. The FUTP method most effectively prevented apoptosis during porcine tetraploid embryo formation. Tetraploid aggregation-derived blastocysts have a high proportion of trophectoderm with increased expression of the CDX2 mRNA and high YAP1 intensity. High-quality blastocysts derived from a tetraploid embryo aggregation can serve as suitable source material for testing the differentiation potential of pluripotent stem cells for blastocyst complementation in pigs.
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Affiliation(s)
- Joohyeong Lee
- Department of Companion Animal Industry, College of Healthcare and Biotechnology, Semyung University, Jecheon, Republic of Korea
| | - Lian Cai
- Laboratory of Veterinary Embryology and Biotechnology (VETEMBIO), Veterinary Medical Center and College of Veterinary Medicine, Chungbuk National University, Cheongju, Republic of Korea
- Institute of Stem Cell and Regenerative Medicine (ISCRM), Chungbuk National University, Cheongju, Republic of Korea
- Graduate School of Veterinary Biosecurity and Protection, Chungbuk National University, Cheongju, Republic of Korea
| | - Mirae Kim
- Laboratory of Veterinary Embryology and Biotechnology (VETEMBIO), Veterinary Medical Center and College of Veterinary Medicine, Chungbuk National University, Cheongju, Republic of Korea
- Institute of Stem Cell and Regenerative Medicine (ISCRM), Chungbuk National University, Cheongju, Republic of Korea
| | - Hyerin Choi
- Laboratory of Veterinary Embryology and Biotechnology (VETEMBIO), Veterinary Medical Center and College of Veterinary Medicine, Chungbuk National University, Cheongju, Republic of Korea
- Institute of Stem Cell and Regenerative Medicine (ISCRM), Chungbuk National University, Cheongju, Republic of Korea
| | - Dongjin Oh
- Laboratory of Veterinary Embryology and Biotechnology (VETEMBIO), Veterinary Medical Center and College of Veterinary Medicine, Chungbuk National University, Cheongju, Republic of Korea
- Institute of Stem Cell and Regenerative Medicine (ISCRM), Chungbuk National University, Cheongju, Republic of Korea
| | - Ali Jawad
- Laboratory of Veterinary Embryology and Biotechnology (VETEMBIO), Veterinary Medical Center and College of Veterinary Medicine, Chungbuk National University, Cheongju, Republic of Korea
- Institute of Stem Cell and Regenerative Medicine (ISCRM), Chungbuk National University, Cheongju, Republic of Korea
| | - Eunsong Lee
- College of Veterinary Medicine, Kangwon National University, Chuncheon, Republic of Korea
| | - Sang-Hwan Hyun
- Laboratory of Veterinary Embryology and Biotechnology (VETEMBIO), Veterinary Medical Center and College of Veterinary Medicine, Chungbuk National University, Cheongju, Republic of Korea
- Institute of Stem Cell and Regenerative Medicine (ISCRM), Chungbuk National University, Cheongju, Republic of Korea
- Graduate School of Veterinary Biosecurity and Protection, Chungbuk National University, Cheongju, Republic of Korea
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3
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Abstract
Polyploids generated by natural whole genome duplication have served as a dynamic force in vertebrate evolution. As evidence for evolution, polyploid organisms exist generally, however there have been no reports of polyploid organisms in mammals. In mice, polyploid embryos under normal culture conditions normally develop to the blastocyst stage. Nevertheless, most tetraploid embryos degenerate after implantation, indicating that whole genome duplication produces harmful effects on normal development in mice. Most previous research on polyploidy has mainly focused on tetraploid embryos. Analysis of various ploidy outcomes is important to comprehend the effects of polyploidization on embryo development. The purpose of this present study was to discover the extent of the polyploidization effect on implantation and development in post-implantation embryos. This paper describes for the first time an octaploid embryo implanted in mice despite hyper-polyploidization, and indicates that these mammalian embryos have the ability to implant, and even develop, despite the harmfulness of extreme whole genome duplication.
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4
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Paim LMG, FitzHarris G. Tetraploidy causes chromosomal instability in acentriolar mouse embryos. Nat Commun 2019; 10:4834. [PMID: 31645568 PMCID: PMC6811537 DOI: 10.1038/s41467-019-12772-8] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2019] [Accepted: 09/20/2019] [Indexed: 11/16/2022] Open
Abstract
Tetraploidisation is considered a common event in the evolution of chromosomal instability (CIN) in cancer cells. The current model for how tetraploidy drives CIN in mammalian cells is that a doubling of the number of centrioles that accompany the genome doubling event leads to multipolar spindle formation and chromosome segregation errors. By exploiting the unusual scenario of mouse blastomeres, which lack centrioles until the ~64-cell stage, we show that tetraploidy can drive CIN by an entirely distinct mechanism. Tetraploid blastomeres assemble bipolar spindles dictated by microtubule organising centres, and multipolar spindles are rare. Rather, kinetochore-microtubule turnover is altered, leading to microtubule attachment defects and anaphase chromosome segregation errors. The resulting blastomeres become chromosomally unstable and exhibit a dramatic increase in whole chromosome aneuploidies. Our results thus reveal an unexpected mechanism by which tetraploidy drives CIN, in which the acquisition of chromosomally-unstable microtubule dynamics contributes to chromosome segregation errors following tetraploidisation.
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Affiliation(s)
- Lia Mara Gomes Paim
- Centre de Recherche du Centre Hospitalier de l'Université de Montréal, H2X 0A9, Montreal, QC, Canada
| | - Greg FitzHarris
- Centre de Recherche du Centre Hospitalier de l'Université de Montréal, H2X 0A9, Montreal, QC, Canada.
- Département d'Obstétrique-Gynécologie, Université de Montréal, H3T 1C5, Montreal, QC, Canada.
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5
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Abstract
Human germ cell tumours (GCTs) are derived from stem cells of the early embryo and the germ line. They occur in the gonads (ovaries and testes) and also in extragonadal sites, where migrating primordial germ cells are located during embryogenesis. This group of heterogeneous neoplasms is unique in that their developmental potential is in effect determined by the latent potency state of their cells of origin, which are reprogrammed to omnipotent, totipotent or pluripotent stem cells. Seven GCT types, defined according to their developmental potential, have been identified, each with distinct epidemiological and (epi)genomic features. Heritable predisposition factors affecting the cells of origin and their niches likely explain bilateral, multiple and familial occurrences of the different types of GCTs. Unlike most other tumour types, GCTs are rarely caused by somatic driver mutations, but arise through failure to control the latent developmental potential of their cells of origin, resulting in their reprogramming. Consistent with their non-mutational origin, even the malignant tumours of the group are characterized by wild-type TP53 and high sensitivity for DNA damage. However, tumour progression and the rare occurrence of treatment resistance are driven by embryonic epigenetic state, specific (sub)chromosomal imbalances and somatic mutations. Thus, recent progress in understanding GCT biology supports a comprehensive developmental pathogenetic model for the origin of all GCTs, and provides new biomarkers, as well as potential targets for treatment of resistant disease.
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Affiliation(s)
- J Wolter Oosterhuis
- Laboratory for Experimental Patho-Oncology, Department of Pathology, Erasmus MC Cancer Institute, Rotterdam, Netherlands.
| | - Leendert H J Looijenga
- Laboratory for Experimental Patho-Oncology, Department of Pathology, Erasmus MC Cancer Institute, Rotterdam, Netherlands
- Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands
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Soares MJ, Varberg KM, Iqbal K. Hemochorial placentation: development, function, and adaptations. Biol Reprod 2019; 99:196-211. [PMID: 29481584 DOI: 10.1093/biolre/ioy049] [Citation(s) in RCA: 115] [Impact Index Per Article: 19.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2018] [Accepted: 02/21/2018] [Indexed: 11/12/2022] Open
Abstract
Placentation is a reproductive adaptation that permits fetal growth and development within the protected confines of the female reproductive tract. Through this important role, the placenta also determines postnatal health and susceptibility to disease. The hemochorial placenta is a prominent feature in primate and rodent development. This manuscript provides an overview of the basics of hemochorial placental development and function, provides perspectives on major discoveries that have shaped placental research, and thoughts on strategies for future investigation.
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Affiliation(s)
- Michael J Soares
- Institute for Reproduction and Perinatal Research and the Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, Kansas, USA.,Department of Pediatrics, University of Kansas Medical Center, Kansas City, Kansas, USA and the Center for Perinatal Research, Children΄s Research Institute, Children΄s Mercy, Kansas City, Missouri, USA
| | - Kaela M Varberg
- Institute for Reproduction and Perinatal Research and the Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, Kansas, USA
| | - Khursheed Iqbal
- Institute for Reproduction and Perinatal Research and the Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, Kansas, USA
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7
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Imai H, Fujii W, Kusakabe KT, Kiso Y, Kano K. Aggregation recovers developmental plasticity in mouse polyploid embryos. Reprod Fertil Dev 2019; 31:404-411. [DOI: 10.1071/rd18093] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2018] [Accepted: 07/21/2018] [Indexed: 11/23/2022] Open
Abstract
Tetraploid embryos normally develop into blastocysts and embryonic stem cells can be established from tetraploid blastocysts in mice. Thus, polyploidisation does not seem to be so harmful during preimplantation development. However, the mechanisms by which early mammalian development accepts polyploidisation are poorly understood. In this study, we aimed to elucidate the effect of polyploidisation on early mammalian development and to further comprehend its tolerance using hyperpolyploid embryos produced by repetitive whole genome duplication. We successfully established several types of polyploid embryos (tetraploid, octaploid and hexadecaploid) and studied their developmental potential invitro. We demonstrated that all types of these polyploid embryos maintained the ability to develop to the blastocyst stage, which implies that mammalian cells might have basic cellular functions in implanted embryos, despite polyploidisation. However, the inner cell mass was absent in hexadecaploid blastocysts. To complement the total number of cells in blastocysts, a fused hexadecaploid embryo was produced by aggregating several hexadecaploid embryos. The results indicated that the fused hexadecaploid embryo finally recovered pluripotent cells in the blastocyst. Thus, our findings suggest that early mammalian embryos may have the tolerance and higher plasticity to adapt to hyperpolyploidisation for blastocyst formation, despite intense alteration of the genome volume.
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8
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Abstract
The electrofusion of 2-cell embryos proves to be a simple and efficient way of generating mammalian tetraploid embryos. Many factors affect the fusion efficiency, such as fusion medium, electric field intensity, and fusion pulse length. In mice, production of tetraploid embryos by electrofusion has already been investigated; however, the investigation to produce porcine tetraploid embryos is seldom reported. In this chapter, we will describe oocytes in vitro maturation, in vitro fertilization, and the optimum conditions for electrofusion of 2-cell embryos to produce tetraploid embryos in pig.
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9
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Menon T, Nair S. Transient window of resilience during early development minimizes teratogenic effects of heat in zebrafish embryos. Dev Dyn 2018; 247:992-1004. [PMID: 29806169 PMCID: PMC6099245 DOI: 10.1002/dvdy.24640] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2017] [Revised: 03/13/2018] [Accepted: 05/12/2018] [Indexed: 12/13/2022] Open
Abstract
Background: Transient heat shock during early development is an established experimental paradigm for doubling the genome of the zebrafish zygote, which has practical applications in expedited identification of recessive mutations in genetic screens. Despite the simplicity of the strategy and the genetic tractability of zebrafish, heat shock has not been used for genome doubling since the proof‐of‐principle experiments done in the 1980s. This is because of poor survival of embryos that ensue from transient heat shocks and gross developmental abnormalities in the few survivors, which is incompatible with phenotype driven screens. Results: We show that heat shocks during early zebrafish development uncouple the second cycle of DNA and centrosome duplication. Interestingly, the developmental time of the heat shock that triggers the dissociation between DNA and centrosome duplication cycles significantly affect the potential of embryos to survive and attain normal morphology. The potential to develop normally after a heat shock alters in a developmental time span of 2 min in zebrafish embryos, a phenomenon that has not been reported in any species. Conclusions: The existence of heat resilient developmental windows and reduced heat teratogenicity during these windows could be an effective step forward in practical application of transient heat for experimental manipulation of ploidy in zebrafish. More broadly, heat resilience before zygotic genome activation suggests that metazoan embryos may possess innate protective features against heat beyond the canonical heat shock response. Developmental Dynamics 247:992–1004, 2018. © 2018 Wiley Periodicals, Inc.
Zebrafish embryos at the end of pronuclear fusion and before initiation of zygotic mitosis are resistant to teratogenic effects of heat. The teratogenic heat resilient window exists transiently during the maternally controlled phase of development. Heat shock during the teratogenic heat resilient window enables generation of morphologically normal zebrafish tetraploids. Diploidization of haploids by transient heat shocks during the teratogenic heat resilient windows aids in effective generation of gynogenic diploids.
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Affiliation(s)
- Triveni Menon
- Department of Biological Sciences, Tata Institute of Fundamental Research, Colaba, Mumbai, India
| | - Sreelaja Nair
- Department of Biological Sciences, Tata Institute of Fundamental Research, Colaba, Mumbai, India
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10
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Yaguchi K, Yamamoto T, Matsui R, Shimada M, Shibanuma A, Kamimura K, Koda T, Uehara R. Tetraploidy-associated centrosome overduplication in mouse early embryos. Commun Integr Biol 2018; 11:e1526605. [PMID: 30534347 PMCID: PMC6284596 DOI: 10.1080/19420889.2018.1526605] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2018] [Accepted: 09/12/2018] [Indexed: 01/08/2023] Open
Abstract
Recently, we observed that tetraploidization of certain types of human cancer cells resulted in upregulation of centrosome duplication cycles and chronic generation of the extra centrosome. Here, we investigated whether tetraploidy-linked upregulation of centrosome duplication also occurs in non-cancer cells using tetraploidized parthenogenetic mouse embryos. Cytokinesis blockage at early embryonic stage before de novo centriole biogenesis provided the unique opportunity in which tetraploidization can be induced without transient doubling of centrosome number. The extra numbers of the centrioles and the centrosomes were observed more frequently in tetraploidized embryos during the blastocyst stage than in their diploid counterparts, demonstrating the generality of the newly found tetraploidy-driven centrosome overduplication in mammalian non-cancer systems.
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Affiliation(s)
- Kan Yaguchi
- Graduate School of Life Science, Hokkaido University, Sapporo, Japan
| | - Takahiro Yamamoto
- Graduate School of Life Science, Hokkaido University, Sapporo, Japan
| | - Ryo Matsui
- Graduate School of Life Science, Hokkaido University, Sapporo, Japan
| | - Masaya Shimada
- Graduate School of Life Science, Hokkaido University, Sapporo, Japan
| | - Atsuko Shibanuma
- Creative Research Institution, Hokkaido University, Sapporo, Japan
| | - Keiko Kamimura
- Graduate School of Life Science, Hokkaido University, Sapporo, Japan
| | - Toshiaki Koda
- Graduate School of Life Science, Hokkaido University, Sapporo, Japan.,Faculty of Advanced Life Science, Hokkaido University, Sapporo, Japan
| | - Ryota Uehara
- Graduate School of Life Science, Hokkaido University, Sapporo, Japan.,Faculty of Advanced Life Science, Hokkaido University, Sapporo, Japan
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11
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Clarke EK, Rivera Gomez KA, Mustachi Z, Murph MC, Schvarzstein M. Manipulation of Ploidy in Caenorhabditis elegans. J Vis Exp 2018. [PMID: 29608173 PMCID: PMC5931776 DOI: 10.3791/57296] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
Mechanisms that involve whole genome polyploidy play important roles in development and evolution; also, an abnormal generation of tetraploid cells has been associated with both the progression of cancer and the development of drug resistance. Until now, it has not been feasible to easily manipulate the ploidy of a multicellular animal without generating mostly sterile progeny. Presented here is a simple and rapid protocol for generating tetraploid Caenorhabditis elegans animals from any diploid strain. This method allows the user to create a bias in chromosome segregation during meiosis, ultimately increasing ploidy in C. elegans. This strategy relies on the transient reduction of expression of the rec-8 gene to generate diploid gametes. A rec-8 mutant produces diploid gametes that can potentially produce tetraploids upon fertilization. This tractable scheme has been used to generate tetraploid strains carrying mutations and chromosome rearrangements to gain insight into chromosomal dynamics and interactions during pairing and synapsis in meiosis. This method is efficient for generating stable tetraploid strains without genetic markers, can be applied to any diploid strain, and can be used to derive triploid C. elegans. This straightforward method is useful for investigating other fundamental biological questions relevant to genome instability, gene dosage, biological scaling, extracellular signaling, adaptation to stress, development of resistance to drugs, and mechanisms of speciation.
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Affiliation(s)
- Erlyana K Clarke
- Brooklyn College, Biology Department, City University of New York
| | - Katherine A Rivera Gomez
- Brooklyn College, Biology Department, City University of New York; The Graduate Center, Biology Department, City University of New York
| | - Zaki Mustachi
- Brooklyn College, Biology Department, City University of New York
| | - Mikaela C Murph
- Brooklyn College, Biology Department, City University of New York; Advanced Science Research Center, City University of New York
| | - Mara Schvarzstein
- Brooklyn College, Biology Department, City University of New York; The Graduate Center, Biology Department, City University of New York; Advanced Science Research Center, City University of New York;
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12
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Alternative dominance of the parental genomes in hybrid cells generated through the fusion of mouse embryonic stem cells with fibroblasts. Sci Rep 2017; 7:18094. [PMID: 29273752 PMCID: PMC5741742 DOI: 10.1038/s41598-017-18352-4] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2017] [Accepted: 12/11/2017] [Indexed: 01/01/2023] Open
Abstract
For the first time, two types of hybrid cells with embryonic stem (ES) cell-like and fibroblast-like phenotypes were produced through the fusion of mouse ES cells with fibroblasts. Transcriptome analysis of 2,848 genes differentially expressed in the parental cells demonstrated that 34-43% of these genes are expressed in hybrid cells, consistent with their phenotypes; 25-29% of these genes display intermediate levels of expression, and 12-16% of these genes maintained expression at the parental cell level, inconsistent with the phenotype of the hybrid cell. Approximately 20% of the analyzed genes displayed unexpected expression patterns that differ from both parents. An unusual phenomenon was observed, namely, the illegitimate activation of Xist expression and the inactivation of one of two X-chromosomes in the near-tetraploid fibroblast-like hybrid cells, whereas both Xs were active before and after in vitro differentiation of the ES cell-like hybrid cells. These results and previous data obtained on heterokaryons suggest that the appearance of hybrid cells with a fibroblast-like phenotype reflects the reprogramming, rather than the induced differentiation, of the ES cell genome under the influence of a somatic partner.
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13
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Xiang J, Cao S, Zhong L, Wang H, Pei Y, Wei Q, Wen B, Mu H, Zhang S, Yue L, Yue G, Lim B, Han J. Pluripotent stem cells secrete Activin A to improve their epiblast competency after injection into recipient embryos. Protein Cell 2017; 9:717-728. [PMID: 29027123 PMCID: PMC6053354 DOI: 10.1007/s13238-017-0470-y] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2017] [Accepted: 08/22/2017] [Indexed: 11/10/2022] Open
Abstract
It is not fully clear why there is a higher contribution of pluripotent stem cells (PSCs) to the chimera produced by injection of PSCs into 4-cell or 8-cell stage embryos compared with blastocyst injection. Here, we show that not only embryonic stem cells (ESCs) but also induced pluripotent stem cells (iPSCs) can generate F0 nearly 100% donor cell-derived mice by 4-cell stage embryo injection, and the approach has a “dose effect”. Through an analysis of the PSC-secreted proteins, Activin A was found to impede epiblast (EPI) lineage development while promoting trophectoderm (TE) differentiation, resulting in replacement of the EPI lineage of host embryos with PSCs. Interestingly, the injection of ESCs into blastocysts cultured with Activin A (cultured from 4-cell stage to early blastocyst at E3.5) could increase the contribution of ESCs to the chimera. The results indicated that PSCs secrete protein Activin A to improve their EPI competency after injection into recipient embryos through influencing the development of mouse early embryos. This result is useful for optimizing the chimera production system and for a deep understanding of PSCs effects on early embryo development.
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Affiliation(s)
- Jinzhu Xiang
- State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, 100193, China
| | - Suying Cao
- Animal Science and Technology College, Beijing University of Agriculture, Beijing, 102206, China
| | - Liang Zhong
- State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, 100193, China
| | - Hanning Wang
- State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, 100193, China.,Beijing Advanced Innovation Center for Food Nutrition and Human Health, China Agricultural University, Beijing, 100193, China
| | - Yangli Pei
- State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, 100193, China.,State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, 100193, China
| | - Qingqing Wei
- State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, 100193, China
| | - Bingqiang Wen
- State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, 100193, China
| | - Haiyuan Mu
- State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, 100193, China
| | - Shaopeng Zhang
- State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, 100193, China
| | - Liang Yue
- State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, 100193, China
| | - Genhua Yue
- Temasek Life Sciences Laboratory, National University of Singapore, Singapore, 117604, Singapore
| | - Bing Lim
- Stem Cell and Developmental Biology, Genome Institute of Singapore, Singapore, 138672, Singapore
| | - Jianyong Han
- State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, 100193, China.
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14
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CDK inhibitor SU9516 induces tetraploid blastocyst formation from parthenogenetically activated porcine embryos. Biotechnol Lett 2017; 39:951-957. [DOI: 10.1007/s10529-017-2321-6] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2016] [Accepted: 03/09/2017] [Indexed: 11/26/2022]
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15
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Wen B, Li R, Cheng K, Li E, Zhang S, Xiang J, Wang Y, Han J. Tetraploid embryonic stem cells can contribute to the development of chimeric fetuses and chimeric extraembryonic tissues. Sci Rep 2017; 7:3030. [PMID: 28596585 PMCID: PMC5465063 DOI: 10.1038/s41598-017-02783-0] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2016] [Accepted: 04/19/2017] [Indexed: 01/22/2023] Open
Abstract
Our study examined the in vivo chimeric and survival capacities of chimeras created by injecting tetraploid embryonic stem cells (ESCs) expressing green fluorescent protein (GFP) into diploid embryos. At 3.5 days post-coitum (dpc) and 4.5 dpc, the tetraploid ESCs were able to contribute to the inner cell mass (ICM) just as diploid ESCs tagged with GFP. At 6.5 dpc, 8.0 dpc and 10.5 dpc, the tetraploid ESCs manifested in the same location as the diploid ESCs. The GFP cells in the extraembryonic tissues and fetuses of tetraploid ESC chimeras were tetraploid as determined by fluorescence activated cell sorting (FACS). Furthermore, tetraploid ESCs contributed to the development of the placenta, embryolemma and umbilical cord at 13.5 dpc and 16.5 dpc; however, very less GFP cells were found in the fetuses of tetraploid ESC chimeras. We further found that the proliferation of tetraploid ESCs was slower than that of diploid ESCs. In addition, the relative mRNA expression in the three germ layers and the trophoblast was abnormal in the EBs of tetraploid ESCs compared with diploid ESCs. In short, slower proliferation and abnormal differentiation potential of tetraploid ESCs might be two of the reasons for their poor survival and chimeric capacities.
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Affiliation(s)
- Bingqiang Wen
- State Key Laboratory for Agro biotechnology, College of Biological Sciences, China Agricultural University, Beijing, 100193, People's Republic of China
| | - Ruiqi Li
- Reproductive Medicine Centre, Department of Obstetrics and Gynecology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, 510120, People's Republic of China
| | - Keren Cheng
- Department of Biology, The University of Texas at San Antonio, UTSA one Circle, San Antonio, TX 78249, United States
| | - Enhong Li
- State Key Laboratory for Agro biotechnology, College of Biological Sciences, China Agricultural University, Beijing, 100193, People's Republic of China
| | - Shaopeng Zhang
- State Key Laboratory for Agro biotechnology, College of Biological Sciences, China Agricultural University, Beijing, 100193, People's Republic of China
| | - Jinzhu Xiang
- State Key Laboratory for Agro biotechnology, College of Biological Sciences, China Agricultural University, Beijing, 100193, People's Republic of China
| | - Yanliang Wang
- State Key Laboratory for Agro biotechnology, College of Biological Sciences, China Agricultural University, Beijing, 100193, People's Republic of China
| | - Jianyong Han
- State Key Laboratory for Agro biotechnology, College of Biological Sciences, China Agricultural University, Beijing, 100193, People's Republic of China.
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16
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Simmet K, Reichenbach M, Reichenbach HD, Wolf E. Phytohemagglutinin facilitates the aggregation of blastomere pairs from Day 5 donor embryos with Day 4 host embryos for chimeric bovine embryo multiplication. Theriogenology 2015; 84:1603-10. [DOI: 10.1016/j.theriogenology.2015.08.012] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2015] [Revised: 08/03/2015] [Accepted: 08/22/2015] [Indexed: 11/16/2022]
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17
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Tetraploid Embryonic Stem Cells Maintain Pluripotency and Differentiation Potency into Three Germ Layers. PLoS One 2015; 10:e0130585. [PMID: 26091100 PMCID: PMC4474668 DOI: 10.1371/journal.pone.0130585] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2015] [Accepted: 05/21/2015] [Indexed: 01/06/2023] Open
Abstract
Polyploid amphibians and fishes occur naturally in nature, while polyploid mammals do not. For example, tetraploid mouse embryos normally develop into blastocysts, but exhibit abnormalities and die soon after implantation. Thus, polyploidization is thought to be harmful during early mammalian development. However, the mechanisms through which polyploidization disrupts development are still poorly understood. In this study, we aimed to elucidate how genome duplication affects early mammalian development. To this end, we established tetraploid embryonic stem cells (TESCs) produced from the inner cell masses of tetraploid blastocysts using electrofusion of two-cell embryos in mice and studied the developmental potential of TESCs. We demonstrated that TESCs possessed essential pluripotency and differentiation potency to form teratomas, which differentiated into the three germ layers, including diploid embryonic stem cells. TESCs also contributed to the inner cell masses in aggregated chimeric blastocysts, despite the observation that tetraploid embryos fail in normal development soon after implantation in mice. In TESCs, stability after several passages, colony morphology, and alkaline phosphatase activity were similar to those of diploid ESCs. TESCs also exhibited sufficient expression and localization of pluripotent markers and retained the normal epigenetic status of relevant reprogramming factors. TESCs proliferated at a slower rate than ESCs, indicating that the difference in genomic dosage was responsible for the different growth rates. Thus, our findings suggested that mouse ESCs maintained intrinsic pluripotency and differentiation potential despite tetraploidization, providing insights into our understanding of developmental elimination in polyploid mammals.
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18
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Horii T, Yamamoto M, Morita S, Kimura M, Nagao Y, Hatada I. p53 suppresses tetraploid development in mice. Sci Rep 2015; 5:8907. [PMID: 25752699 PMCID: PMC4354145 DOI: 10.1038/srep08907] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2014] [Accepted: 02/09/2015] [Indexed: 11/09/2022] Open
Abstract
Mammalian tetraploid embryos die in early development because of defects in the epiblast. Experiments with diploid/tetraploid chimeric mice, obtained via the aggregation of embryonic stem cells, clarified that while tetraploid cells are excluded from epiblast derivatives, diploid embryos with tetraploid extraembryonic tissues can develop to term. Today, this method, known as tetraploid complementation, is usually used for rescuing extraembryonic defects or for obtaining completely embryonic stem (ES) cell-derived pups. However, it is still unknown why defects occur in the epiblast during mammalian development. Here, we demonstrated that downregulation of p53, a tumour suppressor protein, rescued tetraploid development in the mammalian epiblast. Tetraploidy in differentiating epiblast cells triggered p53-dependent cell-cycle arrest and apoptosis, suggesting the activation of a tetraploidy checkpoint during early development. Finally, we found that p53 downregulation rescued tetraploid embryos later in gestation.
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Affiliation(s)
- Takuro Horii
- Laboratory of Genome Science, Biosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-machi, Maebashi, Gunma 371-8512, Japan
| | - Masamichi Yamamoto
- Advanced Scientific Research Leaders Development Unit, Gunma University, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511, Japan
| | - Sumiyo Morita
- Laboratory of Genome Science, Biosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-machi, Maebashi, Gunma 371-8512, Japan
| | - Mika Kimura
- Laboratory of Genome Science, Biosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-machi, Maebashi, Gunma 371-8512, Japan
| | - Yasumitsu Nagao
- Medical Research Center, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan
| | - Izuho Hatada
- Laboratory of Genome Science, Biosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-machi, Maebashi, Gunma 371-8512, Japan
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19
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Abstract
Production of the germline-competent chimeras using genetically modified ES cell lines is an essential step in the establishment of novel mouse models. In addition chimeras provide a powerful tool to study the cell lineage and to analyze complex phenotypes of mutant mice. Mouse chimeras with tetraploid embryos are used to rescue extraembryonic defects, to analyze an impact of gene function on specific lineage, to study the interaction between embryonic and extraembryonic tissues, and to produce mutant embryos and mice for the phenotype analysis. Tetraploid embryos are generated by the fusion of two blastomeres of the mouse embryo. The applications of tetraploid complementation assay and the protocol are described below.
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Affiliation(s)
- Marina Gertsenstein
- Toronto Centre for Phenogenomics (TCP), Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 25 Orde, Toronto, ON, Canada, M5T 3H7,
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20
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Matveeva NM, Kizilova EA, Serov OL. Generation of mouse chimeras with high contribution of tetraploid embryonic stem cells and embryonic stem cell-fibroblast hybrid cells. Methods Mol Biol 2015; 1313:61-71. [PMID: 25947656 DOI: 10.1007/978-1-4939-2703-6_4] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/04/2023]
Abstract
The in vitro long-term cultivation of embryonic stem (ES) cells derived from pre-implantation embryos offers the unique possibility of combining ES cells with pre-implantation embryos to generate chimeras, thus facilitating the creation of a bridge between in vitro and in vivo investigations. Genomic manipulation using ES cells and homologous recombination is one of the most outstanding scientific achievements, resulting in the generation of animals with desirable genome modifications. As such, the generation of ES cells with different ploidy via cell fusion also deserves much attention because this approach allows for the production of chimeras that contain somatic cells with various ploidy. Therefore, this is a powerful tool that can be used to study the role of polyploidy in the normal development of mammals.
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Affiliation(s)
- Natalia M Matveeva
- Institute of Cytology and Genetics, Lavrentiev Prospect, 10, Novosibirsk, 630090, Russia
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21
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Pfeiffer MJ, Esteves TC, Balbach ST, Araúzo-Bravo MJ, Stehling M, Jauch A, Houghton FD, Schwarzer C, Boiani M. Reprogramming of two somatic nuclei in the same ooplasm leads to pluripotent embryonic stem cells. Stem Cells 2014; 31:2343-53. [PMID: 23922292 DOI: 10.1002/stem.1497] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2013] [Revised: 06/24/2013] [Accepted: 07/07/2013] [Indexed: 11/10/2022]
Abstract
The conversion of the nuclear program of a somatic cell from a differentiated to an undifferentiated state can be accomplished by transplanting its nucleus to an enucleated oocyte (somatic cell nuclear transfer [SCNT]) in a process termed "reprogramming." This process achieves pluripotency and occasionally also totipotency. Exploiting the obstacle of tetraploidy to full development in mammals, we show that mouse ooplasts transplanted with two somatic nuclei simultaneously (double SCNT) support preimplantation development and derivation of novel tetraploid SCNT embryonic stem cells (tNT-ESCs). Although the double SCNT embryos do not recapitulate the expression pattern of the pluripotency-associated gene Oct4 in fertilized embryos, derivative tNT-ESCs have characteristics of genuine pluripotency: in vitro they differentiate into neurons, cardiomyocytes, and endodermal cells; in vivo, tNT-ESCs form teratomas, albeit at reduced rates compared to diploid counterparts. Global transcriptome analysis revealed only few specific alterations, for example, in the quantitative expression of gastrulation-associated genes. In conclusion, we have shown that the oocyte's reprogramming capacity is in excess of a single nucleus and that double nucleus-transplanted embryos and derivative ESCs are very similar to their diploid counterparts. These results have key implications for reprogramming studies based on pluripotency: while reprogramming in the tetraploid state was known from fusion-mediated reprogramming and from fetal and adult hepatocyte-derived induced pluripotent stem cells, we have now accomplished it with enucleated oocytes.
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22
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Panopoulos A, Pacios-Bras C, Choi J, Yenjerla M, Sussman MA, Fotedar R, Margolis RL. Failure of cell cleavage induces senescence in tetraploid primary cells. Mol Biol Cell 2014; 25:3105-18. [PMID: 25143403 PMCID: PMC4196863 DOI: 10.1091/mbc.e14-03-0844] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Induction of tetraploidy through cleavage failure induces G1 arrest and senescence in primary mammalian cells but not in immortal cells. Induction of senescence occurs without DNA damage, and the capacity to become senescent appears to be a prerequisite of tetraploid arrest. Tetraploidy can arise from various mitotic or cleavage defects in mammalian cells, and inheritance of multiple centrosomes induces aneuploidy when tetraploid cells continue to cycle. Arrest of the tetraploid cell cycle is therefore potentially a critical cellular control. We report here that primary rat embryo fibroblasts (REF52) and human foreskin fibroblasts become senescent in tetraploid G1 after drug- or small interfering RNA (siRNA)-induced failure of cell cleavage. In contrast, T-antigen–transformed REF52 and p53+/+ HCT116 tumor cells rapidly become aneuploid by continuing to cycle after cleavage failure. Tetraploid primary cells quickly become quiescent, as determined by loss of the Ki-67 proliferation marker and of the fluorescent ubiquitination-based cell cycle indicator/late cell cycle marker geminin. Arrest is not due to DNA damage, as the γ-H2AX DNA damage marker remains at control levels after tetraploidy induction. Arrested tetraploid cells finally become senescent, as determined by SA-β-galactosidase activity. Tetraploid arrest is dependent on p16INK4a expression, as siRNA suppression of p16INK4a bypasses tetraploid arrest, permitting primary cells to become aneuploid. We conclude that tetraploid primary cells can become senescent without DNA damage and that induction of senescence is critical to tetraploidy arrest.
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Affiliation(s)
- Andreas Panopoulos
- Tumor Initiation and Maintenance Program, Sanford-Burnham Medical Research Institute, La Jolla, CA 92037
| | - Cristina Pacios-Bras
- Department of Immunology and Oncology, Centro Nacional de Biotecnologia, Consejo Superior de Investigaciones Científicas, E-28049 Madrid, Spain
| | - Justin Choi
- Tumor Initiation and Maintenance Program, Sanford-Burnham Medical Research Institute, La Jolla, CA 92037
| | - Mythili Yenjerla
- Tumor Initiation and Maintenance Program, Sanford-Burnham Medical Research Institute, La Jolla, CA 92037
| | - Mark A Sussman
- San Diego Heart Research Institute and Department of Biology; San Diego State University, San Diego, CA 92182
| | - Rati Fotedar
- Tumor Initiation and Maintenance Program, Sanford-Burnham Medical Research Institute, La Jolla, CA 92037
| | - Robert L Margolis
- Tumor Initiation and Maintenance Program, Sanford-Burnham Medical Research Institute, La Jolla, CA 92037
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23
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Condic ML. Totipotency: what it is and what it is not. Stem Cells Dev 2014; 23:796-812. [PMID: 24368070 PMCID: PMC3991987 DOI: 10.1089/scd.2013.0364] [Citation(s) in RCA: 79] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2013] [Accepted: 12/23/2013] [Indexed: 02/03/2023] Open
Abstract
There is surprising confusion surrounding the concept of biological totipotency, both within the scientific community and in society at large. Increasingly, ethical objections to scientific research have both practical and political implications. Ethical controversy surrounding an area of research can have a chilling effect on investors and industry, which in turn slows the development of novel medical therapies. In this context, clarifying precisely what is meant by "totipotency" and how it is experimentally determined will both avoid unnecessary controversy and potentially reduce inappropriate barriers to research. Here, the concept of totipotency is discussed, and the confusions surrounding this term in the scientific and nonscientific literature are considered. A new term, "plenipotent," is proposed to resolve this confusion. The requirement for specific, oocyte-derived cytoplasm as a component of totipotency is outlined. Finally, the implications of twinning for our understanding of totipotency are discussed.
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Affiliation(s)
- Maureen L Condic
- Department of Neurobiology, School of Medicine, University of Utah , Salt Lake City, Utah
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24
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Wen D, Saiz N, Rosenwaks Z, Hadjantonakis AK, Rafii S. Completely ES cell-derived mice produced by tetraploid complementation using inner cell mass (ICM) deficient blastocysts. PLoS One 2014; 9:e94730. [PMID: 24733255 PMCID: PMC3986396 DOI: 10.1371/journal.pone.0094730] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2014] [Accepted: 03/18/2014] [Indexed: 01/15/2023] Open
Abstract
Tetraploid complementation is often used to produce mice from embryonic stem cells (ESCs) by injection of diploid (2n) ESCs into tetraploid (4n) blastocysts (ESC-derived mice). This method has also been adapted to mouse cloning and the derivation of mice from induced pluripotent stem (iPS) cells. However, the underlying mechanism(s) of the tetraploid complementation remains largely unclear. Whether this approach can give rise to completely ES cell-derived mice is an open question, and has not yet been unambiguously proven. Here, we show that mouse tetraploid blastocysts can be classified into two groups, according to the presence or absence of an inner cell mass (ICM). We designate these as type a (presence of ICM at blastocyst stage) or type b (absence of ICM). ESC lines were readily derived from type a blastocysts, suggesting that these embryos retain a pluripotent epiblast compartment; whereas the type b blastocysts possessed very low potential to give rise to ESC lines, suggesting that they had lost the pluripotent epiblast. When the type a blastocysts were used for tetraploid complementation, some of the resulting mice were found to be 2n/4n chimeric; whereas when type b blastocysts were used as hosts, the resulting mice are all completely ES cell-derived, with the newborn pups displaying a high frequency of abdominal hernias. Our results demonstrate that completely ES cell-derived mice can be produced using ICM-deficient 4n blastocysts, and provide evidence that the exclusion of tetraploid cells from the fetus in 2n/4n chimeras can largely be attributed to the formation of ICM-deficient blastocysts.
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Affiliation(s)
- Duancheng Wen
- Ansary Stem Cell Institute and Department of Genetic Medicine, Weill Cornell Medical College, New York, New York, United States of America
- Ronald O. Perelman and Claudia Cohen Center for Reproductive Medicine, Weill Cornell Medical College, New York, New York, United States of America
- * E-mail: (DW); (SR)
| | - Nestor Saiz
- Developmental Biology Program, Sloan Kettering Institute, New York, New York, United States of America
| | - Zev Rosenwaks
- Ronald O. Perelman and Claudia Cohen Center for Reproductive Medicine, Weill Cornell Medical College, New York, New York, United States of America
| | | | - Shahin Rafii
- Ansary Stem Cell Institute and Department of Genetic Medicine, Weill Cornell Medical College, New York, New York, United States of America
- * E-mail: (DW); (SR)
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25
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He W, Kong Q, Shi Y, Xie B, Jiao M, Huang T, Guo S, Hu K, Liu Z. Generation and developmental characteristics of porcine tetraploid embryos and tetraploid/diploid chimeric embryos. GENOMICS PROTEOMICS & BIOINFORMATICS 2013; 11:327-33. [PMID: 24120753 PMCID: PMC4357820 DOI: 10.1016/j.gpb.2013.09.007] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 08/03/2013] [Revised: 09/05/2013] [Accepted: 09/16/2013] [Indexed: 01/15/2023]
Abstract
The aim of this study was to optimize electrofusion conditions for generating porcine tetraploid (4n) embryos and produce tetraploid/diploid (4n/2n) chimeric embryos. Different electric field intensities were tested and 2 direct current (DC) pulses of 0.9 kV/cm for 30 μs was selected as the optimum condition for electrofusion of 2-cell embryos to produce 4n embryos. The fusion rate of 2-cell embryos and the development rate to blastocyst of presumably 4n embryos, reached 85.4% and 28.5%, respectively. 68.18% of the fused embryos were found to be 4n as demonstrated by fluorescent in situ hybridization (FISH). Although the number of blastomeres in 4n blastocysts was significantly lower than in 2n blastocysts (P<0.05), there was no significant difference in developmental rates of blastocysts between 2n and 4n embryos (P>0.05), suggesting that the blastocyst forming capacity in 4n embryos is similar to those in 2n embryos. Moreover, 4n/2n chimeric embryos were obtained by aggregation of 4n and 2n embryos. We found that the developmental rate and cell number of blastocysts of 4-cell (4n)/4-cell (2n) chimeric embryos were significantly higher than those of 2-cell (4n)/4-cell (2n), 4-cell (4n)/8-cell (2n), 4-cell (4n)/2-cell (2n) chimeric embryos (P<0.05). Consistent with mouse chimeras, the majority of 4n cells contribute to the trophectoderm (TE), while the 2n cells are mainly present in the inner cell mass (ICM) of porcine 4n/2n chimeric embryos. Our study established a feasible and efficient approach to produce porcine 4n embryos and 4n/2n chimeric embryos.
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Affiliation(s)
- Wenteng He
- College of Life Science, Northeast Agricultural University of China, Harbin 150030, China
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26
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Battulin NR, Khabarova AA, Boyarskikh UA, Menzorov AG, Filipenko ML, Serov OL. Reprogramming somatic cells by fusion with embryonic stem cells does not cause silencing of the Dlk1-Dio3 region in mice. World J Stem Cells 2012; 4. [PMID: 23189213 PMCID: PMC3506971 DOI: 10.4252/wjsc.v4.i8.87] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM To examine the imprinted Dlk1-Dio3 locus in pluripotent embryonic stem (ES) cell/fibroblast hybrid cells. METHODS Gtl2, Rian, and Mirg mRNA expression in mouse pluripotent ES cell/fibroblast hybrid cells was examined by real-time reverse transcription-polymerase chain reaction. Pyrosequencing and bisulfate sequencing were used to determine the DNA methylation level of the Dlk1-Dio3 locus imprinting control region. RESULTS The selected hybrid clones had a near-tetraploid karyotype and were highly pluripotent judging from their capacity to generate chimeric embryos and adult chimeras. Our data clearly demonstrate that Gtl2, Rian, and Mirg, which are imprinted genes within the Dlk1-Dio3 locus, are active in all examined ES cell/fibroblast hybrid clones. In spite of interclonal variability, the expression of the imprinted genes is comparable to that of ES cells and fibroblasts. Quantitative analysis of the DNA methylation status of the intergenic differentially methylated region (IG DMR) within the Dlk1-Dio3 locus by pyrosequencing and bisulfite sequencing clearly showed that the DNA methylation status of the imprinted region in the tested hybrid clones was comparable to that of both ES cells and fibroblasts. CONCLUSION Reprogramming process in a hybrid cell system is achieved without marked alteration of the imprinted Dlk1-Dio3 locus.
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Affiliation(s)
- Nariman R Battulin
- Nariman R Battulin, Anna A Khabarova, Aleksey G Menzorov, Oleg L Serov, Institute of Cytology and Genetics SD RAS, Lavrentyeva 10, Novosibirsk 630090, Russian
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27
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Serov OL, Matveeva NM, Khabarova AA. Reprogramming mediated by cell fusion technology. INTERNATIONAL REVIEW OF CELL AND MOLECULAR BIOLOGY 2012; 291:155-90. [PMID: 22017976 DOI: 10.1016/b978-0-12-386035-4.00005-7] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/20/2023]
Abstract
This review is focused on recent advances in fusion-based reprogramming of cells of different pluripotent statuses or lineage origins. Recent findings are discussed from standpoints of both the developmental potency of hybrid cells generated by fusion of pluripotent embryonic stem (ES) cells, embryonal carcinoma (EC) cells, and somatic cells and epigenetic mechanisms and other aspects involved in the reprogramming process. Complete reprogramming occurs at least 5-7 days after fusion and includes at least two steps. (i) initiation at the heterokaryon stage and choice of the direction of reprogramming using an "all-or-none principle" to establish the dominance of one parental genome and (ii) "fixation" of the newly acquired expression profile by epigenetic mechanisms. The first step is realized without cell division, whereas the second requires cell proliferation. Reprogramming in hybrid cells is rapid and complete. Thus, cell fusion is a powerful tool for reprogramming.
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Affiliation(s)
- Oleg L Serov
- Institute of Cytology and Genetics, Academy of Sciences of Russia, Siberian Branch, Novosibirsk, Russia
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28
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Park MR, Hwang KC, Bui HT, Cho SG, Park C, Song H, Oh JW, Kim JH. Altered gene expression profiles in mouse tetraploid blastocysts. J Reprod Dev 2012; 58:344-52. [PMID: 22362217 DOI: 10.1262/jrd.11-110m] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
In this study, it was demonstrated that tetraploid-derived blastocyst embryos had very few Oct4-positive cells at the mid-blastocyst stage and that the inner cell mass at biomarkers Oct4, Sox2 and Klf4 was expressed at less than 10% of the level observed in diploid blastocysts. In contrast, trophectoderm-related gene transcripts showed an approximately 10 to 40% increase. Of 32,996 individual mouse genes evaluated by microarray, 50 genes were differentially expressed between tetraploid or diploid and parthenote embryos at the blastocyst stage (P<0.05). Of these 50 genes, 28 were more highly expressed in tetraploid-derived blastocysts, whereas 22 were more highly downregulated. However, some genes involved in receptor activity, cell adhesion molecule, calcium ion binding, protein biosynthesis, redox processes, transport, and transcription showed a significant decrease or increase in gene expression in the tetraploid-derived blastocyst embryos. Thus, microarray analysis can be used as a tool to screen for underlying defects responsible for the development of tetraploid-derived embryos.
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Affiliation(s)
- Mi-Ryung Park
- Department of Animal Biotechnology, College of Animal Bioscience and Technology, Konkuk University, Seoul 143-701, Republic of Korea
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29
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Suárez-Villota EY, Vargas RA, Marchant CL, Torres JE, Köhler N, Núñez JJ, de la Fuente R, Page J, Gallardo MH. Distribution of repetitive DNAs and the hybrid origin of the red vizcacha rat (Octodontidae). Genome 2012; 55:105-17. [PMID: 22272977 DOI: 10.1139/g11-084] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Great genome size (GS) variations described in desert-specialist octodontid rodents include diploid species ( Octomys mimax and Octodontomys gliroides ) and putative tetraploid species ( Tympanoctomys barrerae and Pipanacoctomys aureus ). Because of its high DNA content, elevated chromosome number, and gigas effect, the genome of T. barrerae is claimed to have resulted from tetraploidy. Alternatively, the origin of its GS has been attributed to the accumulation of repetitive sequences. To better characterize the extent and origin of these repetitive DNA, self-genomic in situ hybridization (self-GISH), whole-comparative genomic hybridization (W-CGH), and conventional GISH were conducted in mitotic and meiotic chromosomes. Self-GISH on T. barrerae mitotic plates together with comparative self-GISH (using its closest relatives) discriminate a pericentromeric and a telomeric DNA fraction. As most of the repetitive sequences are pericentromeric, it seems that the large GS of T. barrerae is not due to highly repeated sequences accumulated along chromosomes arms. W-CGH using red-labeled P. aureus DNA and green-labeled O. mimax DNA simultaneously on chromosomes of T. barrerae revealed a yellow-orange fluorescence over a repetitive fraction of the karyotype. However, distinctive red-only fluorescent signals were also detected at some centromeres and telomeres, indicating closer homology with the DNA sequences of P. aureus. Conventional GISH using an excess of blocking DNA from either P. aureus or O. mimax labeled only a fraction of the T. barrerae genome, indicating its double genome composition. These data point to a hybrid nature of the T. barrerae karyotype, suggesting a hybridization event in the origin of this species.
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Affiliation(s)
- E Y Suárez-Villota
- Institute of Ecology and Evolution, Universidad Austral de Chile, Valdivia, Chile
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30
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Lin J, Shi L, Zhang M, Yang H, Qin Y, Zhang J, Gong D, Zhang X, Li D, Li J. Defects in trophoblast cell lineage account for the impaired in vivo development of cloned embryos generated by somatic nuclear transfer. Cell Stem Cell 2011; 8:371-5. [PMID: 21474101 DOI: 10.1016/j.stem.2011.02.007] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2010] [Revised: 01/09/2011] [Accepted: 02/10/2011] [Indexed: 10/18/2022]
Abstract
The low success rate of somatic nuclear transfer (NT) is hypothesized to be mainly due to functional defects in the trophoblast cell lineage rather than the inner cell mass (ICM); this hypothesis, however, remains to be tested directly. Here we separated the ICMs from cloned blastocysts and aggregated the cloned ICM with two fertilization-derived (FD) tetraploid (4N) embryos. We found that the full-term development of cloned ICMs was dramatically improved after the trophoblast cells in the cloned blastocysts were replaced by cells from tetraploid embryos, thus providing direct evidence that defects in trophoblast cell lineage underlie the low success rate of somatic NT.
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Affiliation(s)
- Jiangwei Lin
- Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yueyang Road, Shanghai, China
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31
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Park MR, Lee AR, Bui HT, Park C, Park KK, Cho SG, Song H, Kim JH, Nguyen VT, Kim JH. Chromosome remodeling and differentiation of tetraploid embryos during preimplantation development. Dev Dyn 2011; 240:1660-9. [PMID: 21547981 DOI: 10.1002/dvdy.22653] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 04/07/2011] [Indexed: 11/07/2022] Open
Abstract
Although it is known that the tetraploid embryo contributes only to the placenta, the question of why tetraploid embryos differentiate into placenta remains unclear. To study the effect of electrofusion on the development of mouse tetraploid oocytes, mouse two-cell embryos were fused and cultured in vitro in Chatot-Ziomek-Bavister medium. After electrofusion, two chromosome sets from the tetraploid blastomere were individually duplicated before nuclear fusion. At 8-10 hr after electrofusion, each chromosome set was condensing and the nuclear membrane was breaking down. Around 12-14 hr after electrofusion, the two chromosome sets had combined together and had reached the second mitotic metaphase, at this point with 8n sets of chromosomes. Interestingly, we discovered that expression of OCT4, an inner cell mass cells biomarker, is lost by the tetraploid expanded blastocysts, but that CDX2, a trophectoderm cells biomarker, is strongly expressed at this stage. This observation provides evidence clarifying why tetraploid embryos contribute only to trophectoderm.
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Affiliation(s)
- Mi-Ryung Park
- Department of Animal Biotechnology, KonKuk University, Seoul, Korea
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Simerly C, McFarland D, Castro C, Lin CC, Redinger C, Jacoby E, Mich-Basso J, Orwig K, Mills P, Ahrens E, Navara C, Schatten G. Interspecies chimera between primate embryonic stem cells and mouse embryos: monkey ESCs engraft into mouse embryos, but not post-implantation fetuses. Stem Cell Res 2011; 7:28-40. [PMID: 21543277 DOI: 10.1016/j.scr.2011.03.002] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/11/2010] [Revised: 03/04/2011] [Accepted: 03/10/2011] [Indexed: 10/18/2022] Open
Abstract
Unequivocal evidence for pluripotency in which embryonic stem cells contribute to chimeric offspring has yet to be demonstrated in human or nonhuman primates (NHPs). Here, rhesus and baboons ESCs were investigated in interspecific mouse chimera generated by aggregation or blastocyst injection. Aggregation chimera produced mouse blastocysts with GFP-nhpESCs at the inner cell mass (ICM), and embryo transfers (ETs) generated dimly-fluorescencing abnormal fetuses. Direct injection of GFP-nhpESCs into blastocysts produced normal non-GFP-fluorescencing fetuses. Injected chimera showed >70% loss of GFP-nhpESCs after 21 h culture. Outgrowths of all chimeric blastocysts established distinct but separate mouse- and NHP-ESC colonies. Extensive endogenous autofluorescence compromised anti-GFP detection and PCR analysis did not detect nhpESCs in fetuses. NhpESCs localize to the ICM in chimera and generate pregnancies. Because primate ESCs do not engraft post-implantation, and also because endogenous autofluorescence results in misleading positive signals, interspecific chimera assays for pluripotency with primate stem cells is unreliable with the currently available ESCs. Testing primate ESCs reprogrammed into even more naïve states in these inter-specific chimera assays will be an important future endeavor.
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Affiliation(s)
- Calvin Simerly
- Division of Developmental and Regenerative Medicine, Department of Obstetrics, Gynecology, and Reproductive Sciences, University of Pittsburgh School of Medicine, Pittsburgh Development Center, Magee-Womens Research Institute and Foundation, 204 Craft Avenue, Pittsburgh, PA, USA
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Artus J, Hadjantonakis AK. Generation of chimeras by aggregation of embryonic stem cells with diploid or tetraploid mouse embryos. Methods Mol Biol 2011; 693:37-56. [PMID: 21080273 DOI: 10.1007/978-1-60761-974-1_3] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/30/2023]
Abstract
From the hybrid creatures of the Greek and Egyptian mythologies, the concept of the chimera has evolved and, in modern day biology, refers to an organism comprises of at least two populations of genetically distinct cells. Mouse chimeras have proven an invaluable tool for the generation of genetically modified strains. In addition, chimeras have been extensively used in developmental biology as a powerful tool to analyze the phenotype of specific mutations, to attribute function to gene products and to address the question of cell autonomy versus noncell autonomy of gene function. This chapter describes a simple and economical technique used to generate mouse chimeras by embryo aggregation. Multiple aggregation combinations are described each of which can be tailored to answer particular biological questions.
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Affiliation(s)
- Jérôme Artus
- Developmental Biology Program, Sloan-Kettering Institute, New York, NY, USA
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Cell Fusion and Tissue Regeneration. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2011; 713:161-75. [DOI: 10.1007/978-94-007-0763-4_10] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
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Wei Y, Zhu J, Huan Y, Liu Z, Yang C, Zhang X, Mu Y, Xia P, Liu Z. Aberrant expression and methylation status of putatively imprinted genes in placenta of cloned piglets. Cell Reprogram 2010; 12:213-22. [PMID: 20677935 DOI: 10.1089/cell.2009.0090] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
Unlike embryos derived from fertilization, most cloned embryos die during postimplantation development, and those that survive to term are frequently defective. Many of the observed defects involve placenta. Abnormal placentation has been described in several cloned species. Imprinted genes are important regulators of placenta growth, and may be subjected to faulty reprogramming during somatic cell nuclear transfer. We aimed to determine the expression levels and methylation patterns of imprinted genes in placentas of live cloned piglets and dead ones. Quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed that the expression of all four imprinted genes (IGF2, H19, PEG3, and GRB10) was significantly reduced in placentas of dead clones compared with placentas of live cloned piglets and controls (p < 0.05). In contrast, both live and dead cloned piglets exhibited steady-state mRNA levels for these genes within the control range (p > 0.05). Transcript levels for these genes in live clones rarely differed from those of controls in both piglets and placentas. Examination of the methylation status of DMR2 of IGF2 and CTCF3 of H19 genes revealed that both genes exhibited significant high methylation levels in placentas of dead clones compared with placentas of live clones and controls. In contrast, both genes showed a normal differential methylation pattern in live cloned piglets and their placentas compared with controls. Importantly, dead cloned piglets also showed a normal pattern. Our results suggest that abnormal expression of imprinted genes in placenta may contribute to the development failure in pig somatic cell nuclear transfer (SCNT), which may be caused by abnormal methylation patterns in differentially methylated regions (DMRs) of imprinted genes as a result of incomplete reprogramming during SCNT.
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Affiliation(s)
- Yanchang Wei
- College of Life Science, Northeast Agricultural University , Harbin, People's Republic of China
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36
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Shapiro JA. Mobile DNA and evolution in the 21st century. Mob DNA 2010; 1:4. [PMID: 20226073 PMCID: PMC2836002 DOI: 10.1186/1759-8753-1-4] [Citation(s) in RCA: 56] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2009] [Accepted: 01/25/2010] [Indexed: 01/05/2023] Open
Abstract
Scientific history has had a profound effect on the theories of evolution. At the beginning of the 21st century, molecular cell biology has revealed a dense structure of information-processing networks that use the genome as an interactive read-write (RW) memory system rather than an organism blueprint. Genome sequencing has documented the importance of mobile DNA activities and major genome restructuring events at key junctures in evolution: exon shuffling, changes in cis-regulatory sites, horizontal transfer, cell fusions and whole genome doublings (WGDs). The natural genetic engineering functions that mediate genome restructuring are activated by multiple stimuli, in particular by events similar to those found in the DNA record: microbial infection and interspecific hybridization leading to the formation of allotetraploids. These molecular genetic discoveries, plus a consideration of how mobile DNA rearrangements increase the efficiency of generating functional genomic novelties, make it possible to formulate a 21st century view of interactive evolutionary processes. This view integrates contemporary knowledge of the molecular basis of genetic change, major genome events in evolution, and stimuli that activate DNA restructuring with classical cytogenetic understanding about the role of hybridization in species diversification.
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Affiliation(s)
- James A Shapiro
- Department of Biochemistry and Molecular Biology, University of Chicago, Gordon Center for Integrative Science W123B, 929 E 57th Street, Chicago, IL 60637, USA.
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Carstea AC, Pirity MK, Dinnyes A. Germline competence of mouse ES and iPS cell lines: Chimera technologies and genetic background. World J Stem Cells 2009; 1:22-9. [PMID: 21607104 PMCID: PMC3097913 DOI: 10.4252/wjsc.v1.i1.22] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/01/2009] [Revised: 12/14/2009] [Accepted: 12/21/2009] [Indexed: 02/06/2023] Open
Abstract
In mice, gene targeting by homologous recombination continues to play an essential role in the understanding of functional genomics. This strategy allows precise location of the site of transgene integration and is most commonly used to ablate gene expression ("knock-out"), or to introduce mutant or modified alleles at the locus of interest ("knock-in"). The efficacy of producing live, transgenic mice challenges our understanding of this complex process, and of the factors which influence germline competence of embryonic stem cell lines. Increasingly, evidence indicates that culture conditions and in vitro manipulation can affect the germline-competence of Embryonic Stem cell (ES cell) lines by accumulation of chromosome abnormalities and/or epigenetic alterations of the ES cell genome. The effectiveness of ES cell derivation is greatly strain-dependent and it may also influence the germline transmission capability. Recent technical improvements in the production of germline chimeras have been focused on means of generating ES cells lines with a higher germline potential. There are a number of options for generating chimeras from ES cells (ES chimera mice); however, each method has its advantages and disadvantages. Recent developments in induced pluripotent stem (iPS) cell technology have opened new avenues for generation of animals from genetically modified somatic cells by means of chimera technologies. The aim of this review is to give a brief account of how the factors mentioned above are influencing the germline transmission capacity and the developmental potential of mouse pluripotent stem cell lines. The most recent methods for generating specifically ES and iPS chimera mice, including the advantages and disadvantages of each method are also discussed.
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Affiliation(s)
- Ana Claudia Carstea
- Ana Claudia Carstea, Genetic Reprogramming Group, Agricultural Biotechnology Center, Szent Györgyi A u. 4, H-2100 Gödöllö, Hungary
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Early alteration of the self-renewal/differentiation threshold in trophoblast stem cells derived from mouse embryos after nuclear transfer. Dev Biol 2009; 334:325-34. [DOI: 10.1016/j.ydbio.2009.07.022] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2009] [Revised: 06/12/2009] [Accepted: 07/09/2009] [Indexed: 02/04/2023]
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KAWAGUCHI J, KANO K, NAITO K. Expression Profiling of Tetraploid Mouse Embryos in the Developmental Stages Using a cDNA Microarray Analysis. J Reprod Dev 2009; 55:670-5. [DOI: 10.1262/jrd.09-127a] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Affiliation(s)
- Jutaro KAWAGUCHI
- Laboratory of Applied Genetics, Graduate School of Agricultural and Life Science, University of Tokyo
| | - Kiyoshi KANO
- Laboratory of Applied Genetics, Graduate School of Agricultural and Life Science, University of Tokyo
| | - Kunihiko NAITO
- Laboratory of Applied Genetics, Graduate School of Agricultural and Life Science, University of Tokyo
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40
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Embryonic stem cell/fibroblast hybrid cells with near-tetraploid karyotype provide high yield of chimeras. Cell Tissue Res 2008; 334:371-80. [PMID: 18941781 DOI: 10.1007/s00441-008-0702-9] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2008] [Accepted: 09/16/2008] [Indexed: 12/18/2022]
Abstract
Ten primary clones of hybrid cells were produced by the fusion of diploid embryonic stem (ES) cells, viz., line E14Tg2aSc4TP6.3 marked by green fluorescent protein (GFP), with diploid embryonic or adult fibroblasts derived from DD/c mice. All the hybrid clones had many characteristics similar to those of ES cells and were positive for GFP. Five hybrid clones having ploidy close to tetraploidy (over 80% of cells had 76-80 chromosomes) were chosen for the generation of chimeras via injection into C57BL blastocysts. These hybrid clones also contained microsatellites marking all ES cell and fibroblast chromosomes judging from microsatellite analysis. Twenty chimeric embryos at 11-13 days post-conception were obtained after injection of hybrid cells derived from two of three clones. Many embryos showed a high content of GFP-positive descendents of the tested hybrid cells. Twenty one adult chimeras were generated by the injection of hybrid cells derived from three clones. The contribution of GFP-labeled hybrid cells was significant and comparable with that of diploid E14Tg2aSc4TP6.3 cells. Cytogenetic and microsatellite analyses of cell cultures derived from chimeric embryos or adults indicated that the initial karyotype of the tested hybrid cells remained stable during the development of the chimeras, i.e., the hybrid cells were mainly responsible for the generation of the chimeras. Thus, ES cell/fibroblast hybrid cells with near-tetraploid karyotype are able to generate chimeras at a high rate, and many adult chimeras contain a high percentage of descendants of the hybrid cells.
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41
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Genome duplication and the origin of the vertebrate skeleton. Curr Opin Genet Dev 2008; 18:387-93. [PMID: 18721879 DOI: 10.1016/j.gde.2008.07.009] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/14/2008] [Revised: 06/26/2008] [Accepted: 07/21/2008] [Indexed: 11/22/2022]
Abstract
During vertebrate embryonic development, tissue patterning and differentiation are regulated by members of multigene families. Evolutionary expansion of these families is thought to have played a role in the evolution of anatomical complexity, including the origins of new cell and tissue types. A defining feature of vertebrates is an endoskeleton, the primary components of which are cartilage and bone. The molecular control of skeletal patterning has been the subject of intensive investigation for over two decades. More recently, comparative studies of organisms at key phylogenetic positions have highlighted the importance of gene duplication in the evolutionary diversification of connective tissues. Understanding the natural histories of gene families involved in skeletogenesis is therefore central to the issue of vertebrate skeletal evolution.
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42
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Epigenetic processes in a tetraploid mammal. Mamm Genome 2008; 19:439-47. [PMID: 18758856 DOI: 10.1007/s00335-008-9131-z] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2008] [Accepted: 07/01/2008] [Indexed: 10/21/2022]
Abstract
Polyploidy has played a most important role in speciation and evolution of plants and animals. It is thought that low frequency of polyploidy in mammals is due to a dosage imbalance that would interfere with proper development in mammalian polyploids. The first tetraploid mammal, Tympanoctomys barrerae (Octodontidae), appears to be an exception to this rule. In this study we investigated X chromosome inactivation (XCI) and genomic imprinting in T. barrerae, two epigenetic processes usually involved in dosage control in mammalian genomes. The imprinting status of the Peg1 gene was determined by Peg1 allelic expression studies. The inactive X chromosome was identified on interphase nuclei by immunofluorescence using specific antisera raised against Met3H3K27 and macroH2A1. Quantitative PCR was used to compare the Peg1/Dmd ratio in T. barrerae and in its most closely related diploid species, Octomys mimax. Our data demonstrate that parental-specific silencing of at least one gene and normal X chromosomal dosage mechanism are conserved in the tetraploid genome. We hypothesize a concerted action of genetic and epigenetic mechanisms during the process of functional diploidization of this tetraploid genome.
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43
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Ohta H, Sakaide Y, Wakayama T. Generation of mice derived from embryonic stem cells using blastocysts of different developmental ages. Reproduction 2008; 136:581-7. [PMID: 18757504 DOI: 10.1530/rep-08-0184] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
We previously showed that increasing the cell number of host tetraploid (4n) embryos by aggregating multiple 4n embryos at two to four-cell stages can improve the birthrate of mice from embryonic stem cells (ES mice). In the present study, we assessed whether in vitro aged blastocysts (e.g., E4.5 or E5.5), where their cell number also increased with development, can be used as hosts for generating ES mice. As expected, the cell number of in vitro aged 4n blastocysts increased with development, i.e., 26.5+/-2.4, 49.6+/-8.4, and 84.9+/-20.9 cells for E3.5, E4.5, and E5.5 respectively. Three independent ES cell lines were injected into 4n aged blastocysts, and their developmental ability was compared with that of E3.5 4n blastocysts commonly used for this procedure. We found that the birthrate of ES mice derived from E4.5 blastocysts were comparable with those of mice generated from E3.5 blastocysts. On the other hand, the birthrates decreased when E5.5 blastocysts were used. These results suggest that not only the cell number but also developmental age is important for producing ES mice. We also discuss a comparison of the present findings with those of our previous study, where ES mice were generated using an aggregation method employing the same ES cell lines.
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Affiliation(s)
- Hiroshi Ohta
- Laboratory for Genomic Reprogramming, Center for Developmental Biology, RIKEN, Chuo-ku, Kobe 650-0047, Japan.
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44
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Ohta H, Sakaide Y, Yamagata K, Wakayama T. Increasing the cell number of host tetraploid embryos can improve the production of mice derived from embryonic stem cells. Biol Reprod 2008; 79:486-92. [PMID: 18463358 DOI: 10.1095/biolreprod.107.067116] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/01/2022] Open
Abstract
Tetraploid (4n) embryo complementation assay has shown that embryonic stem (ES) cells alone are capable of supporting embryonic development (ES mouse), allowing the recovery of mouse lines directly from cultured ES cell lines. Although the advantages of this technique are well recognized, it remains inefficient for generating ES mice. In the present study, we investigated the effects of cell number of host 4n embryos on the production of ES mice. Four independent ES cell lines (two general ES cell lines and two nuclear transfer-derived ES cell lines) were used, and each cell line was aggregated with single (1X) to triple (3X) host 4n embryos. We found that birth rate of ES mice using 1X 4n embryos was quite low (0-2%) regardless of cell line, whereas except for one cell line, approximately 6-14% of embryos developed to full term in the case of 3X 4n embryos. Contamination of host 4n cells in ES mice was quite rare, being comparable to that generated using general methods even if they were delivered from 3X 4n host embryos. These results demonstrate that the use of 3X 4n embryos is effective for generating ES mice. Our technique described here will be applicable to any ES cell line, including general ES cell lines used for gene targeting.
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Affiliation(s)
- Hiroshi Ohta
- Laboratory for Genomic Reprogramming, Center for Developmental Biology, RIKEN, Kobe, Japan.
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45
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Liu L, Aoki VW, Carrell DT. Evaluation of the developmental competence and chromosomal compliment of mouse oocytes derived from in-vitro growth and maturation of preantral follicles. J Assist Reprod Genet 2008; 25:107-13. [PMID: 18253824 DOI: 10.1007/s10815-008-9201-x] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2007] [Accepted: 01/11/2008] [Indexed: 11/29/2022] Open
Abstract
PURPOSE To evaluate the developmental potential and aneuploidy rates of in-vitro versus in-vivo grown and matured mouse oocytes. METHODS Mice were superovulated to obtain in-vivo matured oocytes. Mouse preantral follicles were also mechanically isolated and cultured in-vitro. In-vitro fertilization (IVF) was performed and fertilization, cleavage, and morula/blastocyst formation rates were compared between groups. Cytogenetic analysis was used to compare oocyte aneuploidy rates and aneuploidy characteristics in the developing embryos. RESULTS In-vivo oocyte maturation resulted in higher IVF fertilization, cleavage, and morula/blastocyst formation rates versus in-vitro follicle culture (96.4% versus 78.5%, p<0.001; 95.3% versus 77.4%, p<0.001; 94.1% versus 76.9%, p<0.001). Total aneuploidy rates were higher in embryos derived from in-vitro matured oocytes versus those grown in-vivo (4.0% versus 1.3%, p<0.05). CONCLUSIONS Results indicate a reduced developmental competency of in-vitro matured oocytes. The data also highlight an increased susceptibility to meiotic errors in early stage follicles undergoing in vitro culture.
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Affiliation(s)
- Lihua Liu
- Andrology and IVF Laboratories, University of Utah School of Medicine, 675 Arapeen Dr. Ste 205, Salt Lake City, UT 84117, USA
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46
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Yang X, Smith SL, Tian XC, Lewin HA, Renard JP, Wakayama T. Nuclear reprogramming of cloned embryos and its implications for therapeutic cloning. Nat Genet 2007; 39:295-302. [PMID: 17325680 DOI: 10.1038/ng1973] [Citation(s) in RCA: 426] [Impact Index Per Article: 23.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023]
Abstract
Therapeutic cloning, whereby somatic cell nuclear transfer (SCNT) is used to generate patient-specific embryonic stem cells (ESCs) from blastocysts cloned by nuclear transfer (ntESCs), holds great promise for the treatment of many human diseases. ntESCs have been derived in mice and cattle, but thus far there are no credible reports of human ntESCs. Here we review the recent literature on nuclear reprogramming by SCNT, including studies of gene expression, DNA methylation, chromatin remodeling, genomic imprinting and X chromosome inactivation. Reprogramming of genes expressed in the inner cell mass, from which ntESCs are derived, seems to be highly efficient. Defects in the extraembryonic lineage are probably the major cause of the low success rate of reproductive cloning but are not expected to affect the derivation of ntESCs. We remain optimistic that human therapeutic cloning is achievable and that the derivation of patient-specific ntESC lines will have great potential for regenerative medicine.
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Affiliation(s)
- Xiangzhong Yang
- Center for Regenerative Biology and Department of Animal Science, University of Connecticut, Storrs, Connecticut 06269, USA.
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47
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Abstract
Murine embryonic stem (ES) cells have become an indispensable tool for investigating genetic function both in vitro and, importantly, in vivo. Recent advances, including tetraploid aggregation, new site-specific recombinases and RNAi, have enabled more sophisticated manipulation of the ES cell genome. For instance, it is now possible to control gene expression in both a temporally and spatially restricted manner. Such new technologies are answering complex questions surrounding the function and interaction of an increasing number of genes. This chapter will review both the history and recent technological progress that has been made in mouse ES cell derivation, genetic manipulation and the generation of ES cell-derived chimaeric animals.
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Affiliation(s)
- J S Draper
- Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada.
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48
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Li XY, Jia Q, Di KQ, Gao SM, Wen XH, Zhou RY, Wei W, Wang LZ. Passage number affects the pluripotency of mouse embryonic stem cells as judged by tetraploid embryo aggregation. Cell Tissue Res 2007; 327:607-14. [PMID: 17216193 DOI: 10.1007/s00441-006-0354-6] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2006] [Accepted: 10/13/2006] [Indexed: 11/28/2022]
Abstract
The aim of this study was to determine whether the number of passages affected the developmental pluripotency of embryonic stem (ES) cells as measured by the attainment of adult fertile mice derived from embryonic stem (ES) cell/tetraploid embryo complementation. Thirty-six newborns were produced by the aggregation of tetraploid embryos and hybrid ES cells after various numbers of passages. These newborns were entirely derived from ES cells as judged by microsatellite DNA, coat-color phenotype, and germline transmission. Although 15 survived to adulthood, 17 died of respiratory failure, and four were eaten by their foster mother. From the 15 mice that reached adulthood and that could reproduce, none arose from ES cells at passage level 15 or more. All 15 arose from cells at passages 3-11. Our results demonstrate that the number of passages affects the developmental pluripotency of ES cells.
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Affiliation(s)
- Xiang-Yun Li
- College of Animal Science and Technology, Agricultural University of Hebei, Baoding, Hebei 071001, People's Republic of China.
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49
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Khare A, Shaulsky G. First among equals: competition between genetically identical cells. Nat Rev Genet 2006; 7:577-83. [PMID: 16702983 DOI: 10.1038/nrg1875] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023]
Abstract
Competition between genetically identical organisms is considered insignificant in evolutionary theory because it is presumed to have little selective consequence. We argue that competition between genetically identical cells could improve the fitness of a multicellular organism by directing fitter cells to the germ line or by eliminating unfit cells, and that cell-competition mechanisms have been conserved in multicellular organisms. We propose that competition between genetically identical or highly similar units could have similar selective advantages at higher organizational levels, such as societies.
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Affiliation(s)
- Anupama Khare
- Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030, USA
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50
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Jouneau A, Zhou Q, Camus A, Brochard V, Maulny L, Collignon J, Renard JP. Developmental abnormalities of NT mouse embryos appear early after implantation. Development 2006; 133:1597-607. [PMID: 16556918 DOI: 10.1242/dev.02317] [Citation(s) in RCA: 68] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
In mammals, cloning by nuclear transfer (NT) into an enucleated oocyte is a very inefficient process, even if it can generate healthy adults. We show that blastocysts derived from embryonic stem (ES) donor cells develop at a high rate, correctly express the pluripotential marker gene Oct4 in ICM cells and display normal growth in vitro. Moreover, the majority of them implant in the uterus of recipient females. We combine embryological studies, gene expression analysis during gastrulation and generation of chimaeric embryos to identify the developmental origin (stage and tissue affected) of NT embryo mortality. The majority died before mid-gestation from defects arising early, either at peri-implantation stages or during the gastrulation period. The first type of defect is a non-cell autonomous defect of the epiblast cells and is rescued by complementation of NT blastocysts with normal ES or ICM cells. The second type of defect affects growth regulation and the shape of the embryo but does not directly impair the initial establishment of the patterning of the embryo. Only chimaeras formed by the aggregation of NT and tetraploid embryos reveal no growth abnormalities at gastrulation. These studies indicate that the trophoblast cell lineage is the primary source of these defects. These embryological studies provide a solid basis for understanding reprogramming errors in NT embryos. In addition, they unveil new aspects of growth regulation while increasing our knowledge on the role of crosstalk between the extra-embryonic and the embryonic regions of the conceptus in the control of growth and morphogenesis.
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Affiliation(s)
- Alice Jouneau
- Unité de Biologie du Développement et de la Reproduction, UMR INRA-ENVA, Institut National de la Recherche Agronomique (INRA 78352, France
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