CAR expression in human embryos and hESC illustrates its role in pluripotency and tight junctions

Reproduction ◽  
2014 ◽  
Vol 148 (5) ◽  
pp. 531-544 ◽  
Author(s):  
M Krivega ◽  
M Geens ◽  
H Van de Velde
Keyword(s):  
Outer Layer ◽  
Transmembrane Domain ◽  
Splice Variants ◽  
Embryonic Stem ◽  
Cell Types ◽  
Preimplantation Development ◽  
Coxsackie Virus ◽  
Preimplantation Embryos ◽  
Human Embryos ◽  
Adenovirus Receptor

Coxsackie virus and adenovirus receptor,CXADR(CAR), is present during embryogenesis and is involved in tissue regeneration, cancer and intercellular adhesion. We investigated the expression of CAR in human preimplantation embryos and embryonic stem cells (hESC) to identify its role in early embryogenesis and differentiation. CAR protein was ubiquitously present during preimplantation development. It was localised in the nucleus of uncommitted cells, from the cleavage stage up to the precursor epiblast, and corresponded with the presence of solubleCXADR3/7splice variant. CAR was displayed on the membrane, involving in the formation of tight junction at compaction and blastocyst stages in both outer and inner cells, and CAR corresponded with the full-length CAR-containing transmembrane domain. In trophectodermal cells of hatched blastocysts, CAR was reduced in the membrane and concentrated in the nucleus, which correlated with the switch in RNA expression to theCXADR4/7andCXADR2/7splice variants. The cells in the outer layer of hESC colonies contained CAR on the membrane and all the cells of the colony had CAR in the nucleus, corresponding with the transmembraneCXADRandCXADR4/7. Upon differentiation of hESC into cells representing the three germ layers and trophoblast lineage, the expression ofCXADRwas downregulated. We concluded thatCXADRis differentially expressed during human preimplantation development. We described various CAR expressions: i) solubleCXADRmarking undifferentiated blastomeres; ii) transmembrane CAR related with epithelial-like cell types, such as the trophectoderm (TE) and the outer layer of hESC colonies; and iii) soluble CAR present in TE nuclei after hatching. The functions of these distinct forms remain to be elucidated.

scholarly journals Spatiotemporal dynamics of OCT4 protein localization during preimplantation development in mice

Reproduction ◽  
2016 ◽  
Vol 152 (5) ◽  
pp. 417-430 ◽  
Author(s):  
Atsushi Fukuda ◽  
Atsushi Mitani ◽  
Toshiyuki Miyashita ◽  
Hisato Kobayashi ◽  
Akihiro Umezawa ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Splice Variants ◽  
Protein Localization ◽  
Preimplantation Development ◽  
Preimplantation Embryos ◽  
Sequencing Analysis ◽  
Dependent Manner ◽  
Cell Stage ◽  
Protein Levels ◽  
Oct4 Protein

Spatiotemporal expression of transcription factors is crucial for genomic reprogramming. Pou5f1 (Oct4) is an essential transcription factor for reprogramming. A recent study reported that OCT4A, which is crucial for establishment and maintenance of pluripotent cells, is expressed in oocytes, but maternal OCT4A is dispensable for totipotency induction. Whereas another study reported that OCT4B, which is not related to pluripotency, is predominantly expressed instead of OCT4A during early preimplantation phases in mice. To determine the expression states of OCT4 in murine preimplantation embryos, we conducted in-depth expression and functional analyses. We found that pluripotency-related OCT4 mainly localizes to the cytoplasm in early preimplantation phases, with no major nuclear localization until the 8–16-cell stage despite high expression in both oocytes and early embryos. RNA-sequencing analysis using oocytes and early preimplantation embryos could not identify the splice variants creating alternative forms of OCT4 protein. Forced expression of OCT4 in zygotes by the injection of polyadenylated mRNA clearly showed nuclear localization of OCT4 protein around 3–5-fold greater than physiological levels and impaired developmental competency in a dose-dependent manner. Embryos with modest overexpression of OCT4 could develop to the 16-cell stage; however, more than 50% of the embryos were arrested at this stage, similar to the results for OCT4 depletion. In contrast, extensive overexpression of OCT4 resulted in complete arrest at the 2-cell stage accompanied by downregulation of zygotically activated genes and repetitive elements related to the totipotent state. These results demonstrated that OCT4 protein localization was spatiotemporally altered during preimplantation development, and strict control of Oct4 protein levels was essential for proper totipotential reprogramming.

scholarly journals Structure and expression of a gene encoding a putative GTP-binding protein identified by provirus integration in a transgenic mouse strain.

1991 ◽  
Vol 11 (2) ◽  
pp. 886-893 ◽  
Author(s):  
K Mooslehner ◽  
U Müller ◽  
U Karls ◽  
L Hamann ◽  
K Harbers
Keyword(s):  
Mouse Strain ◽  
Embryonal Carcinoma ◽  
Germ Line ◽  
Leukemia Virus ◽  
Embryonic Stem ◽  
Mutagenic Effect ◽  
Cell Types ◽  
Transcription Unit ◽  
Preimplantation Embryos ◽  
Gtp Binding

The Mov-10 mouse strain was derived by infection of preimplantation embryos with the Moloney murine leukemia virus and carries one copy of the provirus in its germ line. Here we show that the provirus has integrated into an evolutionarily conserved gene that can code for a protein of 110 kDa containing the three consensus elements characteristic for GTP-binding proteins. The Mov-10 locus was expressed in a variety of cell types, including embryonal carcinoma and embryonic stem cells. Transcription of the gene was down-regulated about 10-fold when F9 embryonal carcinoma cells are differentiated into parietal endodermlike cells and about 2-fold when they are differentiated into visceral endodermlike cells. High levels of Mov-10 transcripts were also found at different stages of embryonal development and in the testes and thymus of adult animals. Expression was cell cycle controlled, with steady-state RNA levels significantly higher in growth-arrested than in growth-stimulated cells. The results suggest that the Mov-10 locus has an important function in development and/or control of cell proliferation. The provirus was shown to have integrated into intron 1 of the gene without disrupting expression, indicating that integration into intronic sequences of a transcription unit does not necessarily affect transcription. This result together with previous results from the Mov-13 mouse strain suggested that proviruses exert their mutagenic effect only by integration in specific sites, such as cis-regulatory DNA elements.

scholarly journals Characterization and target genes of nine human PRD-like homeobox domain genes expressed exclusively in early embryos

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Elo Madissoon ◽  
Eeva-Mari Jouhilahti ◽  
Liselotte Vesterlund ◽  
Virpi Töhönen ◽  
Kaarel Krjutškov ◽  
...  
Keyword(s):  
Stem Cells ◽  
Transcription Factors ◽  
Target Genes ◽  
Genomic Sequence ◽  
Embryonic Stem ◽  
Homeobox Genes ◽  
Preimplantation Embryos ◽  
Human Embryos ◽  
Preimplantation Embryo Development ◽  
Genome Activation

Abstract PAIRED (PRD)-like homeobox genes belong to a class of predicted transcription factor genes. Several of these PRD-like homeobox genes have been predicted in silico from genomic sequence but until recently had no evidence of transcript expression. We found recently that nine PRD-like homeobox genes, ARGFX, CPHX1, CPHX2, DPRX, DUXA, DUXB, NOBOX, TPRX1 and TPRX2, were expressed in human preimplantation embryos. In the current study we characterized these PRD-like homeobox genes in depth and studied their functions as transcription factors. We cloned multiple transcript variants from human embryos and showed that the expression of these genes is specific to embryos and pluripotent stem cells. Overexpression of the genes in human embryonic stem cells confirmed their roles as transcription factors as either activators (CPHX1, CPHX2, ARGFX) or repressors (DPRX, DUXA, TPRX2) with distinct targets that could be explained by the amino acid sequence in homeodomain. Some PRD-like homeodomain transcription factors had high concordance of target genes and showed enrichment for both developmentally important gene sets and a 36 bp DNA recognition motif implicated in Embryo Genome Activation (EGA). Our data implicate a role for these previously uncharacterized PRD-like homeodomain proteins in the regulation of human embryo genome activation and preimplantation embryo development.

scholarly journals Epigenetic dynamics during preimplantation development

Reproduction ◽  
2015 ◽  
Vol 150 (3) ◽  
pp. R109-R120 ◽  
Author(s):  
Chelsea Marcho ◽  
Wei Cui ◽  
Jesse Mager
Keyword(s):  
Genetic Material ◽  
Cell Types ◽  
Dna Demethylation ◽  
Preimplantation Development ◽  
Human Embryos ◽  
Mammalian Development ◽  
Sequencing Technologies ◽  
Active Dna Demethylation ◽  
Generation Sequencing ◽  
Short Days

Successful mammalian development requires descendants of single-cell zygotes to differentiate into diverse cell types even though they contain the same genetic material. Preimplantation dynamics are first driven by the necessity of reprogramming haploid parental epigenomes to reach a totipotent state. This process requires extensive erasure of epigenetic marks shortly after fertilization. During the few short days after formation of the zygote, epigenetic programs are established and are essential for the first lineage decisions and differentiation. Here we review the current understanding of DNA methylation and histone modification dynamics responsible for these early changes during mammalian preimplantation development. In particular, we highlight insights that have been gained through next-generation sequencing technologies comparing human embryos to other models as well as the recent discoveries of active DNA demethylation mechanisms at play during preimplantation.

Expression of dopamine and adrenergic receptors in mouse embryonic stem cells and preimplantation embryos

Biologia ◽  
2015 ◽  
Vol 70 (9) ◽  
pp. 1263-1271 ◽  
Author(s):  
Štefan Čikoš ◽  
Dušan Fabian ◽  
Ján Burkuš ◽  
Žofia Janštová ◽  
Juraj Koppel
Keyword(s):  
Dopamine Receptor ◽  
Adrenergic Receptors ◽  
Developmental Stages ◽  
Expression Profiles ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Preimplantation Embryos ◽  
Receptor Subtypes ◽  
Mouse Es Cells

Abstract Using RT-PCR we examined expression of dopamine and adrenergic receptors in undifferentiated and spontaneously differentiating mouse embryonic stem (ES) cells. We also examined expression of dopamine receptor subtypes in mouse ovulated oocytes and preimplantation embryos. Comparing the expression of catecholamine receptors in undifferentiated mouse ES cells and in blastocysts (from which ES cells are derived), we found that transcripts of all five dopamine receptors were expressed in both cell types. In contrast, we detected eight adrenergic receptor subtypes in undifferentiated mouse ES cells, but only three subtypes were found in mouse blastocysts. In three adrenergic receptors (α1D, α2B, β1), we found higher expression in the spontaneously differentiating ES cells than in undifferentiated ES cells, and the α1B adrenoceptor was not even detectable in the undifferentiated cells. These results indicate that genes encoding all types of catecholamine receptors are transcribed in mouse ES cells, and some of them are differentially expressed during ES cell differentiation. We found several profiles of dopamine receptor mRNA expression during the preimplantation period. The DR3 transcript was present in all examined stages (oocytes, 4-cell embryos, 8- to 16-cell embryos, blastocysts). DR1 and DR4 transcripts were not found in oocytes, but we detected them in preimplantation embryos. The DR2 receptor transcript was found in all examined stages except for the 4-cell embryos, and the DR5 receptor transcript was found in all examined stages except for the 8- to 16-cell embryos. The expression profiles of dopamine receptor transcripts suggest different roles of some receptor subtypes in particular preimplantation developmental stages.

scholarly journals Endogenous human stem cell-associated retroviruses

2015 ◽  
Author(s):  
Gennadi Glinsky
Keyword(s):  
Stem Cell ◽  
Malignant Tumors ◽  
Early Stage ◽  
Embryonic Stem ◽  
Preimplantation Embryos ◽  
Human Embryos ◽  
Regulatory Sequences ◽  
Human Stem Cell ◽  
Cancer Phenotypes ◽  
Genome Activation

Recent discoveries of endogenous human stem cell-associated retroviruses (SCARs) revealed consistent activation of specific endogenous retroviral elements in human preimplantation embryos and documented the essential role of the sustained retroviral activities in the maintenance of pluripotency, functional identity and integrity of naïve-state embryonic stem cells, and anti-viral resistance of the early-stage human embryos. SCARs activity have been implicated in seeding thousands’ human-specific regulatory sequences in the hESC genome. Activation of specific SCARs, namely LTR7/HERVH and LTR5_Hs/HERVK, has been demonstrated in patients diagnosed with multiple types of cancer, autoimmune diseases, neurodegenerative disorders and it is likely associated with the emergence of clinically lethal therapy resistant death-from-cancer phenotypes in a sub-set of cancer patients diagnosed with different types of malignant tumors.

scholarly journals BMP-treated human embryonic stem cells transcriptionally resemble amnion cells in the monkey embryo

Biology Open ◽  
2021 ◽  
Author(s):  
Sapna Chhabra ◽  
Aryeh Warmflash
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Single Cell ◽  
Human Embryonic Stem Cells ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Cell Types ◽  
Human Embryos ◽  
Early Human

Human embryonic stem cells (hESCs) possess an immense potential to generate clinically relevant cell types and unveil mechanisms underlying early human development. However, using hESCs for discovery or translation requires accurately identifying differentiated cell types through comparison with their in vivo counterparts. Here, we set out to determine the identity of much debated BMP-treated hESCs by comparing their transcriptome to recently published single cell transcriptomic data from early human embryos (Xiang et al., 2019). Our analyses reveal several discrepancies in the published human embryo dataset, including misclassification of putative amnion, intermediate and inner cell mass cells. These misclassifications primarily resulted from similarities in pseudogene expression, highlighting the need to carefully consider gene lists when making comparisons between cell types. In the absence of a relevant human dataset, we utilized the recently published single cell transcriptome of the early post implantation monkey embryo to discern the identity of BMP-treated hESCs. Our results suggest that BMP-treated hESCs are transcriptionally more similar to amnion cells than trophectoderm cells in the monkey embryo. Together with prior studies, this result indicates that hESCs possess a unique ability to form mature trophectoderm subtypes via an amnion-like transcriptional state.

scholarly journals Ethics in stem cell research

2012 ◽  
Vol 3 (1) ◽  
pp. 13-18
Author(s):  
Md Fakruddin
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Stem Cell Research ◽  
Human Embryonic Stem Cell ◽  
Ethical Issues ◽  
Embryonic Stem ◽  
Cell Types ◽  
Embryonic Stem Cell Research ◽  
Human Embryos

Stem cells have constituted a revolution in regenerative medicine and cancer therapies by providing the possibility of generating multiple therapeutically useful cell types that could be used for treating some of genetic and degenerative disorders. However, human embryonic stem cell research raises few ethical and political controversies because of its involvement in destruction of human embryos. The ethical issues in human embryonic stem cell research encompasses not only with question of the ethics of destroying human embryos, but also with questions about complicity of researchers in destruction of embryos, moral distinction between creating embryos for research purposes and creating them for reproductive ends and the permissibility of cloning human embryos to harvest stem cells. Bangladesh should formulate its own regulations justifying its stand regarding this matter. DOI: http://dx.doi.org/10.3329/bioethics.v3i1.10867 Bangladesh Journal of Bioethics 2012; 3(1):13-18

scholarly journals Stem cell research: immortality or a healthy old age?

2004 ◽  
pp. U7-12 ◽  
Author(s):  
C Mummery
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Embryonic Stem Cells ◽  
Stem Cell Research ◽  
Adult Stem Cells ◽  
Embryonic Stem ◽  
Basic Research ◽  
Cell Types ◽  
Human Embryos ◽  
The Media

Stem cell research holds the promise of treatments for many disorders resulting from disease or trauma where one or at most a few cell types have been lost or do not function. In combination with tissue engineering, stem cells may represent the greatest contribution to contemporary medicine of the present century. Progress is however being hampered by the debate on the origin of stem cells, which can be derived from human embryos and some adult tissues. Politics, religious beliefs and the media have determined society's current perception of their relative value while the ethical antipathy towards embryonic stem cells, which require destruction of a human embryo for their derivation, has in many countries biased research towards adult stem cells. Many scientists believe this bias may be premature and basic research on both cell types is still required. The media has created confusion about the purpose of stem cell research: treating chronic ailments or striving for immortality. Here, the scientific state of the art on adult and embryonic stem cells is reviewed as a basis for a debate on whether research on embryonic stem cells is ethically acceptable.

scholarly journals High-resolution transcriptional and morphogenetic profiling of cells from micropatterned human ESC gastruloid cultures

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Kyaw Thu Minn ◽  
Yuheng C Fu ◽  
Shenghua He ◽  
Sabine Dietmann ◽  
Steven C George ◽  
...  
Keyword(s):  
Single Cells ◽  
Primordial Germ Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
The Body ◽  
Human Embryos ◽  
Germ Layers ◽  
Distinct Cell ◽  
Evolutionarily Conserved ◽  
Species Comparisons

During mammalian gastrulation, germ layers arise and are shaped into the body plan while extraembryonic layers sustain the embryo. Human embryonic stem cells, cultured with BMP4 on extracellular matrix micro-discs, reproducibly differentiate into gastruloids, expressing markers of germ layers and extraembryonic cells in radial arrangement. Using single-cell RNA sequencing and cross-species comparisons with mouse, cynomolgus monkey gastrulae, and post-implantation human embryos, we reveal that gastruloids contain cells transcriptionally similar to epiblast, ectoderm, mesoderm, endoderm, primordial germ cells, trophectoderm, and amnion. Upon gastruloid dissociation, single cells reseeded onto micro-discs were motile and aggregated with the same but segregated from distinct cell types. Ectodermal cells segregated from endodermal and extraembryonic but mixed with mesodermal cells. Our work demonstrates that the gastruloid system models primate-specific features of embryogenesis, and that gastruloid cells exhibit evolutionarily conserved sorting behaviors. This work generates a resource for transcriptomes of human extraembryonic and embryonic germ layers differentiated in a stereotyped arrangement.

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