Cell-Specific Interaction of Retinoic Acid Receptors with Target Genes in Mouse Embryonic Fibroblasts and Embryonic Stem Cells

ABSTRACT All-trans retinoic acid (RA) induces transforming growth factor beta (TGF-β)-dependent autocrine growth of mouse embryonic fibroblasts (MEFs). We have used chromatin immunoprecipitation to map 354 RA receptor (RAR) binding loci in MEFs, most of which were similarly occupied by the RARα and RARγ receptors. Only a subset of the genes associated with these loci are regulated by RA, among which are several critical components of the TGF-β pathway. We also show RAR binding to a novel series of target genes involved in cell cycle regulation, transformation, and metastasis, suggesting new pathways by which RA may regulate proliferation and cancer. Few of the RAR binding loci contained consensus direct-repeat (DR)-type elements. The majority comprised either degenerate DRs or no identifiable DRs but anomalously spaced half sites. Furthermore, we identify 462 RAR target loci in embryonic stem (ES) cells and show that their occupancy is cell type specific. Our results also show that differences in the chromatin landscape regulate the accessibility of a subset of more than 700 identified loci to RARs, thus modulating the repertoire of target genes that can be regulated and the biological effects of RA.

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Differences in Gene Expression between Wild Type and Hoxa1 Knockout Embryonic Stem Cells after Retinoic Acid Treatment or Leukemia Inhibitory Factor (LIF) Removal

Journal of Biological Chemistry ◽

10.1074/jbc.m414397200 ◽

2005 ◽

Vol 280 (16) ◽

pp. 16484-16498 ◽

Cited By ~ 59

Author(s):

Eduardo Martinez-Ceballos ◽

Pierre Chambon ◽

Lorraine J. Gudas

Keyword(s):

Gene Expression ◽

Cell Proliferation ◽

Retinoic Acid ◽

Leukemia Inhibitory Factor ◽

Target Genes ◽

Hox Genes ◽

Es Cells ◽

Embryonic Stem ◽

Inhibitory Factor ◽

Hoxa Cluster

Homeobox (Hox) genes encode a family of transcription factors that regulate embryonic patterning and organogenesis. In embryos, alterations of the normal pattern of Hox gene expression result in homeotic transformations and malformations. Disruption of theHoxa1gene, the most 3′ member of the Hoxa cluster and a retinoic acid (RA) direct target gene, results in abnormal ossification of the skull, hindbrain, and inner ear deficiencies, and neonatal death. We have generated Hoxa1-/-embryonic stem (ES) cells (named Hoxa1-15) from Hoxa1-/-mutant blastocysts to study the Hoxa1 signaling pathway. We have characterized in detail these Hoxa1-/-ES cells by performing microarray analyses, and by this technique we have identified a number of putative Hoxa-1 target genes, including genes involved in bone development (e.g. Col1a1,Postn/Osf2, and the bone sialoprotein gene orBSP), genes that are expressed in the developing brain (e.g. Nnat,Wnt3a,BDNF,RhoB, andGbx2), and genes involved in various cellular processes (e.g. M-RAS,Sox17,Cdkn2b,LamA1,Col4a1,Foxa2,Foxq1,Klf5, andIgf2). Cell proliferation assays and Northern blot analyses of a number of ES cell markers (e.g. Rex1,Oct3/4,Fgf4, andBmp4) suggest that the Hoxa1 protein plays a role in the inhibition of cell proliferation by RA in ES cells. Additionally, Hoxa1-/-ES cells express high levels of various endodermal markers, includingGata4andDab2, and express much lessFgf5after leukemia inhibitory factor (LIF) withdrawal. Finally, we propose a model in which the Hoxa1 protein mediates repression of endodermal differentiation while promoting expression of ectodermal and mesodermal characteristics.

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Gene Targeting of Cdc42 and Cdc42GAP Affirms the Critical Involvement of Cdc42 in Filopodia Induction, Directed Migration, and Proliferation in Primary Mouse Embryonic Fibroblasts

Molecular Biology of the Cell ◽

10.1091/mbc.e06-05-0466 ◽

2006 ◽

Vol 17 (11) ◽

pp. 4675-4685 ◽

Cited By ~ 111

Author(s):

Linda Yang ◽

Lei Wang ◽

Yi Zheng

Keyword(s):

Gene Targeting ◽

Cell Lines ◽

Cell Cycle Progression ◽

Cell Model ◽

Es Cells ◽

Embryonic Stem ◽

Mouse Embryonic Fibroblasts ◽

Embryonic Fibroblasts ◽

Membrane Ruffling ◽

Primary Mouse

Recent studies in Cdc42 knockout mouse embryonic stem (ES) cells and ES-derived fibroblastoid cell lines raise concern on a body of literature derived by dominant mutant expression approach in a variety of cell lines implicating mammalian Cdc42 as a key regulator of filopodia induction, directional migration and cell cycle progression. To resolve the physiological function of mammalian Cdc42, we have characterized the Cdc42−/− and Cdc42GAP−/− primary mouse embryonic fibroblasts (MEFs) produced by gene targeting as the Cdc42 loss- or gain-of-activity cell model. The Cdc42−/− cells were defective in filopodia formation stimulated by bradykinin and in dorsal membrane ruffling stimulated by PDGF, whereas the Cdc42GAP−/− cells displayed spontaneous filopodia. The Cdc42 loss- or gain-of-activity cells were defective in adhesion to fibronectin, wound-healing, polarity establishment, and migration toward a serum gradient. These defects were associated with deficiencies of PAK1, GSK3β, myosin light chain, and FAK phosphorylation. Furthermore, Cdc42−/− cells were defective in G1/S-phase transition and survival, correlating with deficient NF-κB transcription and defective JNK, p70 S6K, and ERK1/2 activation. These results demonstrate a different requirement of Cdc42 activity in primary MEFs from ES or ES-derived clonal fibroblastoid cells and suggest that Cdc42 plays cell-type–specific signaling roles.

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Embryonic Fibroblasts from Mice Lacking Tgif Were Defective in Cell Cycling

Molecular and Cellular Biology ◽

10.1128/mcb.02156-05 ◽

2006 ◽

Vol 26 (11) ◽

pp. 4302-4310 ◽

Cited By ~ 32

Author(s):

Lynn Mar ◽

Pamela A. Hoodless

Keyword(s):

Retinoic Acid ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Cycle Phase ◽

Embryonic Fibroblasts ◽

Inhibition Of Proliferation ◽

Cellular Analysis ◽

Tale Homeodomain ◽

Acid Pathway ◽

First Time

ABSTRACT Holoprosencephaly (HPE) is the most common structural anomaly of the human brain, resulting from incomplete cleavage of the developing forebrain during embryogenesis. Haploinsufficient mutations in the TG-interacting factor (TGIF) gene were previously identified in a subset of HPE families and sporadic patients, and this gene is located within a region of chromosome 18 that is associated with nonrandom chromosomal aberrations in HPE patients. TGIF is a three-amino-acid loop extension (TALE) homeodomain-containing transcription factor that functions both as a corepressor of the transforming growth factor beta (TGF-β) pathway and as a competitor of the retinoic acid pathway. Here we describe mice deficient in Tgif that exhibited laterality defects and growth retardation and developed kinked tails. Cellular analysis of mutant mouse embryonic fibroblasts (MEFs) demonstrated for the first time that Tgif regulates proliferation and progression through the G1 cell cycle phase. Additionally, wild-type human TGIF was able to rescue this proliferative defect in MEFs. In contrast, a subset of human Tgif mutations detected in HPE patients was unable to rescue the proliferative defect. However, an absence of Tgif did not alter the normal inhibition of proliferation caused by treatment with TGF-β or retinoic acid. Developmental control of proliferation by Tgif may play a role in the pathogenesis of HPE.

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Activin/Nodal signalling before implantation: setting the stage for embryo patterning

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2013.0539 ◽

2014 ◽

Vol 369 (1657) ◽

pp. 20130539 ◽

Cited By ~ 8

Author(s):

Costis Papanayotou ◽

Jérôme Collignon

Keyword(s):

Transforming Growth Factor Beta ◽

Target Genes ◽

Transforming Growth Factor ◽

Embryonic Stem ◽

Signalling Pathway ◽

Loss Of Function ◽

Embryo Patterning ◽

Developmental Progression ◽

Growth Factor Beta

Activins and Nodal are members of the transforming growth factor beta (TGF-β) family of growth factors. Their Smad2/3-dependent signalling pathway is well known for its implication in the patterning of the embryo after implantation. Although this pathway is active early on at preimplantation stages, embryonic phenotypes for loss-of-function mutations of prominent components of the pathway are not detected before implantation. It is only fairly recently that an understanding of the role of the Activin/Nodal signalling pathway at these stages has started to emerge, notably from studies detailing how it controls the expression of target genes in embryonic stem cells. We review here what is currently known of the TGF-β-related ligands that determine the activity of Activin/Nodal signalling at preimplantation stages, and recent advances in the elucidation of the Smad2/3-dependent mechanisms underlying developmental progression.

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Genes involved in cell adhesion and signaling: a new repertoire of retinoic acid receptor target genes in mouse embryonic fibroblasts

Journal of Cell Science ◽

10.1242/jcs.131946 ◽

2013 ◽

Vol 127 (3) ◽

pp. 521-533 ◽

Cited By ~ 22

Author(s):

Z. Al Tanoury ◽

A. Piskunov ◽

D. Andriamoratsiresy ◽

S. Gaouar ◽

R. Lutzing ◽

...

Keyword(s):

Cell Adhesion ◽

Retinoic Acid ◽

Target Genes ◽

Retinoic Acid Receptor ◽

Mouse Embryonic Fibroblasts ◽

Embryonic Fibroblasts ◽

Acid Receptor

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The TGFβ Family in Human Placental Development at the Fetal-Maternal Interface

Biomolecules ◽

10.3390/biom10030453 ◽

2020 ◽

Vol 10 (3) ◽

pp. 453

Author(s):

Susana M. Chuva de Sousa Lopes ◽

Marta S. Alexdottir ◽

Gudrun Valdimarsdottir

Keyword(s):

Stem Cell ◽

Transforming Growth Factor Beta ◽

Molecular Mechanisms ◽

Transforming Growth Factor ◽

Cell Model ◽

Embryonic Stem ◽

Model Systems ◽

Special Focus ◽

Placental Development

Emerging data suggest that a trophoblast stem cell (TSC) population exists in the early human placenta. However, in vitro stem cell culture models are still in development and it remains under debate how well they reflect primary trophoblast (TB) cells. The absence of robust protocols to generate TSCs from humans has resulted in limited knowledge of the molecular mechanisms that regulate human placental development and TB lineage specification when compared to other human embryonic stem cells (hESCs). As placentation in mouse and human differ considerably, it is only with the development of human-based disease models using TSCs that we will be able to understand the various diseases caused by abnormal placentation in humans, such as preeclampsia. In this review, we summarize the knowledge on normal human placental development, the placental disease preeclampsia, and current stem cell model systems used to mimic TB differentiation. A special focus is given to the transforming growth factor-beta (TGFβ) family as it has been shown that the TGFβ family has an important role in human placental development and disease.

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Retinoic acid signaling is critical during the totipotency window in early mammalian development

Nature Structural & Molecular Biology ◽

10.1038/s41594-021-00590-w ◽

2021 ◽

Author(s):

Ane Iturbide ◽

Mayra L. Ruiz Tejeda Segura ◽

Camille Noll ◽

Kenji Schorpp ◽

Ina Rothenaigner ◽

...

Keyword(s):

Retinoic Acid ◽

Regulatory Networks ◽

Es Cells ◽

Embryonic Stem ◽

Mammalian Development ◽

Cell Stage ◽

Cell Potential ◽

Cellular Models ◽

Early Embryos ◽

Genome Activation

AbstractTotipotent cells hold enormous potential for regenerative medicine. Thus, the development of cellular models recapitulating totipotent-like features is of paramount importance. Cells resembling the totipotent cells of early embryos arise spontaneously in mouse embryonic stem (ES) cell cultures. Such ‘2-cell-like-cells’ (2CLCs) recapitulate 2-cell-stage features and display expanded cell potential. Here, we used 2CLCs to perform a small-molecule screen to identify new pathways regulating the 2-cell-stage program. We identified retinoids as robust inducers of 2CLCs and the retinoic acid (RA)-signaling pathway as a key component of the regulatory circuitry of totipotent cells in embryos. Using single-cell RNA-seq, we reveal the transcriptional dynamics of 2CLC reprogramming and show that ES cells undergo distinct cellular trajectories in response to RA. Importantly, endogenous RA activity in early embryos is essential for zygotic genome activation and developmental progression. Overall, our data shed light on the gene regulatory networks controlling cellular plasticity and the totipotency program.

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Molecular cloning of gene sequences regulated during squamous differentiation of tracheal epithelial cells and controlled by retinoic acid.

Molecular and Cellular Biology ◽

10.1128/mcb.7.11.4017 ◽

1987 ◽

Vol 7 (11) ◽

pp. 4017-4023 ◽

Cited By ~ 44

Author(s):

H L Smits ◽

E E Floyd ◽

A M Jetten

Keyword(s):

Retinoic Acid ◽

Epithelial Cells ◽

Cdna Library ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Squamous Differentiation ◽

Squamous Cells ◽

Tracheal Epithelial Cells ◽

Molecular Events ◽

Tracheal Epithelial

A cDNA library was constructed from polyadenylated RNA present in squamous differentiated rabbit tracheal epithelial cells. Screening of the cDNA library was aimed at identifying RNAs that were abundant in squamous cells and expressed at low levels in undifferentiated cells. Two different recombinants were obtained containing inserts, 0.86 and 0.77 kilobases (kb) in size, that hybridized to mRNAs 1.0 and 1.25 kb in length. These RNAs were present at approximately 50-fold higher levels in squamous cells than in proliferative or confluent retinoic acid-treated cells. The increase in the levels of the 1.0- and 1.25-kb RNAs correlated closely with the onset of squamous differentiation and was not related to induction of terminal cell division. Treatment of rabbit tracheal epithelial cells with transforming growth factor beta, which induces squamous differentiation in these cells, also resulted in elevated levels of the 1.0- and 1.25-kb RNAs. The increased levels of these RNAs in squamous cells appeared to a large extent to be regulated at a posttranscriptional level. Retinoic acid not only inhibited the increase in the levels of the 1.0- and 1.25-kb RNAs but also reversed the expression of these RNAs in squamous cells. These results suggest that retinoic acid affects, directly or indirectly, molecular events that induce alterations in the posttranscriptional processing of the transcripts corresponding to the 1.0- and 1.25-kb RNAs.

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Steroidogenic Factor-1 (SF-1)-Driven Differentiation of Murine Embryonic Stem (ES) Cells into a Gonadal Lineage

Endocrinology ◽

10.1210/en.2011-0219 ◽

2011 ◽

Vol 152 (7) ◽

pp. 2870-2882 ◽

Cited By ~ 24

Author(s):

Unmesh Jadhav ◽

J. Larry Jameson

Keyword(s):

Target Genes ◽

De Novo ◽

Embryoid Body ◽

Es Cells ◽

Embryonic Stem ◽

Cell Lineage ◽

Culture Conditions ◽

Steroidogenic Factor 1 ◽

Lineage Differentiation ◽

And Function

Steroidogenic factor 1 (SF-1) is essential for the development and function of steroidogenic tissues. Stable incorporation of SF-1 into embryonic stem cells (SF-1-ES cells) has been shown to prime the cells for steroidogenesis. When provided with exogenous cholesterol substrate, and after treatment with retinoic acid and cAMP, SF-1-ES cells produce progesterone but do not produce other steroids such as cortisol, estradiol, or testosterone. In this study, we explored culture conditions that optimize SF-1-mediated differentiation of ES cells into defined steroidogenic lineages. When embryoid body formation was used to facilitate cell lineage differentiation, SF-1-ES cells were found to be restricted in their differentiation, with fewer cells entering neuronal pathways and a larger fraction entering the steroidogenic lineage. Among the differentiation protocols tested, leukemia inhibitory factor (LIF) removal, followed by prolonged cAMP treatment was most efficacious for inducing steroidogenesis in SF-1-ES cells. In this protocol, a subset of SF-1-ES cells survives after LIF withdrawal, undergoes morphologic differentiation, and recovers proliferative capacity. These cells are characterized by induction of steroidogenic enzyme genes, use of de novo cholesterol, and production of multiple steroids including estradiol and testosterone. Microarray studies identified additional pathways associated with SF-1 mediated differentiation. Using biotinylated SF-1 in chromatin immunoprecipitation assays, SF-1 was shown to bind directly to multiple target genes, with induction of binding to some targets after steroidogenic treatment. These studies indicate that SF-1 expression, followed by LIF removal and treatment with cAMP drives ES cells into a steroidogenic pathway characteristic of gonadal steroid-producing cells.

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