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Multiple mesodermal lineage differentiation of Apodemus sylvaticus embryonic stem cells in vitro
Tao Wang, Frank Mao, Wenyu Lai, Weiqiang Li, Weihua Yu, Zifei Wang, Lirong Zhang, Jinli Zhang, Jin Niu, Xiuming Zhang, Bruce T Lahn, Andy Xiang
BMC Cell Biology , 2010, DOI: 10.1186/1471-2121-11-42
Abstract: Herein we report the generation of multiple mesodermal AS-ES1 lineages via embryoid body (EB) formation by the hanging drop method and the addition of particular reagents and factors for induction at the stage of EB attachment. The AS-ES1 cells generated separately in vitro included: adipocytes, osteoblasts, chondrocytes and cardiomyocytes. Histochemical staining, immunofluorescent staining and RT-PCR were carried out to confirm the formation of multiple mesodermal lineage cells.The appropriate reagents and culture milieu used in mesodermal differentiation of mouse ES cells also guide the differentiation of in vitro AS-ES1 cells into distinct mesoderm-derived cells. This study provides a better understanding of the characteristics of AS-ES1 cells, a new species ES cell line and promotes the use of Apodemus ES cells as a complement to mouse ES cells in future studies.Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocyst-stage embryos [1]. The abilities of ES cells to undergo indefinite self-renewal in vitro and to produce derivative lineages of all three embryonic germ layers in vitro and in vivo make them highly prized in both clinical and research settings [2]. ES, or ES-like, cells have thus far been derived from a number of mammalian species, including the mouse [3], rat [4], bovine [5], sheep [6], pig [7], rhesus macaque [8], crab-eating macaque [9], marmoset [10] and human [11].Apodemus sylvaticus is a common rodent species found throughout Europe. A. sylvaticus has a gross appearance similar to that of the laboratory mouse. The rearing conditions are also quite similar to those of the mouse. However, the superficial resemblance between A. sylvaticus and the laboratory mouse belies the rather deep evolutionary divide separating these two species. The combination of these properties--that is, the similar rearing conditions and large evolutionary divergence--makes A. sylvaticus highly attractive as a potential model organi
Efficient Differentiation of Embryonic Stem Cells into Mesodermal Precursors by BMP, Retinoic Acid and Notch Signalling  [PDF]
Josema Torres, Javier Prieto, Fabrice C. Durupt, Simon Broad, Fiona M. Watt
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0036405
Abstract: The ability to direct differentiation of mouse embryonic stem (ES) cells into specific lineages not only provides new insights into the pathways that regulate lineage selection but also has translational applications, for example in drug discovery. We set out to develop a method of differentiating ES cells into mesodermal cells at high efficiency without first having to induce embryoid body formation. ES cells were plated on a feeder layer of PA6 cells, which have membrane-associated stromal-derived inducing activity (SDIA), the molecular basis of which is currently unknown. Stimulation of ES/PA6 co-cultures with Bone Morphogenetic Protein 4 (BMP4) both favoured self-renewal of ES cells and induced differentiation into a Desmin and Nestin double positive cell population. Combined stimulation with BMP4 and all-trans Retinoic Acid (RA) inhibited self-renewal and resulted in 90% of cells expressing Desmin and Nestin. Quantitative reverse transcription-polymerase chain reaction (qPCR) analysis confirmed that the cells were of mesodermal origin and expressed markers of mesenchymal and smooth muscle cells. BMP4 activation of a MAD-homolog (Smad)-dependent reporter in undifferentiated ES cells was attenuated by co-stimulation with RA and co-culture with PA6 cells. The Notch ligand Jag1 was expressed in PA6 cells and inhibition of Notch signalling blocked the differentiation inducing activity of PA6 cells. Our data suggest that mesodermal differentiation is regulated by the level of Smad activity as a result of inputs from BMP4, RA and the Notch pathway.
Transcriptomic and phenotypic analysis of murine embryonic stem cell derived BMP2+ lineage cells: an insight into mesodermal patterning
Michael Doss, Shuhua Chen, Johannes Winkler, Rita Hippler-Altenburg, Margareta Odenthal, Claudia Wickenhauser, Sridevi Balaraman, Herbert Schulz, Oliver Hummel, Norbert Hübner, Nandini Ghosh-Choudhury, Isaia Sotiriadou, Jürgen Hescheler, Agapios Sachinidis
Genome Biology , 2007, DOI: 10.1186/gb-2007-8-9-r184
Abstract: We generated a transgenic BMP2 embryonic stem (ES) cell lineage expressing both puromycin acetyltransferase and enhanced green fluorescent protein (EGFP) driven by the BMP2 promoter. Puromycin resistant and EGFP positive BMP2+ cells with a purity of over 93% were isolated. Complete transcriptome analysis of BMP2+ cells in comparison to the undifferentiated ES cells and the control population from seven-day-old embryoid bodies (EBs; intersection of genes differentially expressed between undifferentiated ES cells and BMP2+ EBs as well as differentially expressed between seven-day-old control EBs and BMP2+ EBs by t-test, p < 0.01, fold change >2) by microarray analysis led to identification of 479 specifically upregulated and 193 downregulated transcripts. Transcription factors, apoptosis promoting factors and other signaling molecules involved in early embryonic development are mainly upregulated in BMP2+ cells. Long-term differentiation of the BMP2+ cells resulted in neural crest stem cells (NCSCs), smooth muscle cells, epithelial-like cells, neuronal-like cells, osteoblasts and monocytes. Interestingly, development of cardiomyocytes from the BMP2+ cells requires secondary EB formation.This is the first study to identify the complete transcriptome of BMP2+ cells and cell phenotypes from a mesodermal origin, thus offering an insight into the role of BMP2+ cells during embryonic developmental processes in vivo.Bone morphogenetic protein (BMP)2 is a member of the transforming growth factor (TGF)-β superfamily and plays a crucial role in early embryonic patterning as shown by gene ablation studies [1,2]. It is normally expressed in lateral plate mesoderm and extraembryonic mesoderm [1,3]. BMP2+ mesodermal cells at this stage comprise a subset of mesoderm, the lateral plate cardiogenic mesoderm [4]. BMP2 expression immediately follows the transient expression of T-Brachyury in the nascent mesoderm. Interestingly, administration of soluble BMP2 to chick embryo explant cult
Regulation and Functions of the lms Homeobox Gene during Development of Embryonic Lateral Transverse Muscles and Direct Flight Muscles in Drosophila  [PDF]
Dominik Müller,Teresa Jagla,Ludivine Mihaila Bodart,Nina J?hrling,Hans-Ulrich Dodt,Krzysztof Jagla,Manfred Frasch
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0014323
Abstract: Patterning and differentiation of developing musculatures require elaborate networks of transcriptional regulation. In Drosophila, significant progress has been made into identifying the regulators of muscle development and defining their interactive networks. One major family of transcription factors involved in these processes consists of homeodomain proteins. In flies, several members of this family serve as muscle identity genes to specify the fates of individual muscles, or groups thereof, during embryonic and/or adult muscle development. Herein, we report on the expression and function of a new Drosophila homeobox gene during both embryonic and adult muscle development.
The L-type Ca2+ Channels Blocker Nifedipine Represses Mesodermal Fate Determination in Murine Embryonic Stem Cells  [PDF]
Filomain Nguemo, Bernd K. Fleischmann, Manoj K. Gupta, Tomo ?ari?, Daniela Malan, Huamin Liang, Kurt Pfannkuche, Wilhelm Bloch, Heribert Schunkert, Jürgen Hescheler, Michael Reppel
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0053407
Abstract: Dihydropyridines (DHP), which nifedipine is a member of, preferentially block Ca2+ channels of different cell types. Moreover, influx of Ca2+ through L-type Ca2+ channels (LTCCs) activates Ca2+ signaling pathways, which in turn contribute to numerous cellular processes. Although LTCCs are expressed in undifferentiated cells, very little is known about its contributions to the transcriptional regulation of mesodermal and cardiac genes. This study aimed to examine the contribution of LTCCs and the effect of nifedipine on the commitment of pluripotent stem cells toward the cardiac lineage in vitro. The murine embryonic stem (ES, cell line D3) and induced pluripotent stem (iPS, cell clone 09) cells were differentiated into enhanced green fluorescence protein (EGFP) expressing spontaneously beating cardiomyocytes (CMs). Early treatment of differentiating cells with 10 μM nifedipine led to a significant inhibition of the cardiac mesoderm formation and cardiac lineage commitment as revealed by gene regulation analysis. This was accompanied by the inhibition of spontaneously occurring Ca2+ transient and reduction of LTCCs current density (ICaL) of differentiated CMs. In addition, nifedipine treatment instigated a pronounced delay of the spontaneous beating embryoid body (EB) and led to a poor surface localization of L-type Ca2+ channel α1C (CaV1.2) subunits. Contrary late incubation of pluripotent stem cells with nifedipine was without any impact on the differentiation process and did not affect the derived CMs function. Our data indicate that nifedipine blocks the determined path of pluripotent stem cells to cardiomyogenesis by inhibition of mesodermal commitment at early stages of differentiation, thus the proper upkeep Ca2+ concentration and pathways are essentially required for cardiac gene expression, differentiation and function.
Clusters of Temporal Discordances Reveal Distinct Embryonic Patterning Mechanisms in Drosophila and Anopheles  [PDF]
Dmitri Papatsenko,Michael Levine,Yury Goltsev
PLOS Biology , 2012, DOI: 10.1371/journal.pbio.1000584
Abstract: Evolutionary innovations can be driven by spatial and temporal changes in gene expression. Several such differences have been documented in the embryos of lower and higher Diptera. One example is the reduction of the ancient extraembryonic envelope composed of amnion and serosa as seen in mosquitoes to the single amnioserosa of fruit flies. We used transcriptional datasets collected during the embryonic development of the fruit fly, Drosophila melanogaster, and the malaria mosquito, Anopheles gambiae, to search for whole-genome changes in gene expression underlying differences in their respective embryonic morphologies. We found that many orthologous gene pairs could be clustered based on the presence of coincident discordances in their temporal expression profiles. One such cluster contained genes expressed specifically in the mosquito serosa. As shown previously, this cluster is redeployed later in development at the time of cuticle synthesis. In addition, there is a striking difference in the temporal expression of a subset of maternal genes. Specifically, maternal transcripts that exhibit a sharp reduction at the time of the maternal-zygotic transition in Drosophila display sustained expression in the Anopheles embryo. We propose that gene clustering by local temporal discordance can be used for the de novo identification of the gene batteries underlying morphological diversity.
Clusters of Temporal Discordances Reveal Distinct Embryonic Patterning Mechanisms in Drosophila and Anopheles  [PDF]
Dmitri Papatsenko,Michael Levine,Yury Goltsev
PLOS Biology , 2011, DOI: 10.1371/journal.pbio.1000584
Abstract: Evolutionary innovations can be driven by spatial and temporal changes in gene expression. Several such differences have been documented in the embryos of lower and higher Diptera. One example is the reduction of the ancient extraembryonic envelope composed of amnion and serosa as seen in mosquitoes to the single amnioserosa of fruit flies. We used transcriptional datasets collected during the embryonic development of the fruit fly, Drosophila melanogaster, and the malaria mosquito, Anopheles gambiae, to search for whole-genome changes in gene expression underlying differences in their respective embryonic morphologies. We found that many orthologous gene pairs could be clustered based on the presence of coincident discordances in their temporal expression profiles. One such cluster contained genes expressed specifically in the mosquito serosa. As shown previously, this cluster is redeployed later in development at the time of cuticle synthesis. In addition, there is a striking difference in the temporal expression of a subset of maternal genes. Specifically, maternal transcripts that exhibit a sharp reduction at the time of the maternal-zygotic transition in Drosophila display sustained expression in the Anopheles embryo. We propose that gene clustering by local temporal discordance can be used for the de novo identification of the gene batteries underlying morphological diversity.
Alternative Splicing of NURF301 Generates Distinct NURF Chromatin Remodeling Complexes with Altered Modified Histone Binding Specificities  [PDF]
So Yeon Kwon,Hua Xiao,Carl Wu,Paul Badenhorst
PLOS Genetics , 2009, DOI: 10.1371/journal.pgen.1000574
Abstract: Drosophila NURF is an ISWI–containing chromatin remodeling complex that catalyzes ATP–dependent nucleosome sliding. By sliding nucleosomes, NURF can alter chromatin structure and regulate transcription. NURF301/BPTF is the only NURF–specific subunit of NURF and is instrumental in recruiting the complex to target genes. Here we demonstrate that three NURF301 isoforms are expressed and that these encode functionally distinct NURF chromatin remodeling complexes. Full-length NURF301 contains a C-terminal bromodomain and juxtaposed PHD finger that bind histone H3 trimethylated at Lys4 (H3K4me3) and histone H4 acetylated at Lys16 (H4K16Ac) respectively. However, a NURF301 isoform that lacks these C-terminal domains is also detected. This truncated NURF301 isoform assembles a complex containing ISWI, NURF55, and NURF38, indicating that a second class of NURF remodeling complex, deficient in H3K4me3 and H4K16Ac recognition, exists. By comparing microarray expression profiles and phenotypes of null Nurf301 mutants with mutants that remove the C-terminal PHD fingers and bromodomain, we show that full-length NURF301 is not essential for correct expression of the majority of NURF gene targets in larvae. However, full-length NURF301 is required for spermatogenesis. Mutants that lack full-length NURF exhibit a spermatocyte arrest phenotype and fail to express a subset of spermatid differentiation genes. Our data reveal that variants of the NURF ATP–dependent chromatin remodeling complex that recognize post-translational histone modifications are important regulators of primary spermatocyte differentiation in Drosophila.
Structural Requirements for PACSIN/Syndapin Operation during Zebrafish Embryonic Notochord Development  [PDF]
Melissa A. Edeling,Subramaniam Sanker,Takaki Shima,P. K. Umasankar,Stefan H?ning,Hye Y. Kim,Lance A. Davidson,Simon C. Watkins,Michael Tsang,David J. Owen,Linton M. Traub
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0008150
Abstract: PACSIN/Syndapin proteins are membrane-active scaffolds that participate in endocytosis. The structure of the Drosophila Syndapin N-terminal EFC domain reveals a crescent shaped antiparallel dimer with a high affinity for phosphoinositides and a unique membrane-inserting prong upon the concave surface. Combined structural, biochemical and reverse genetic approaches in zebrafish define an important role for Syndapin orthologue, Pacsin3, in the early formation of the notochord during embryonic development. In pacsin3-morphant embryos, midline convergence of notochord precursors is defective as axial mesodermal cells fail to polarize, migrate and differentiate properly. The pacsin3 morphant phenotype of a stunted body axis and contorted trunk is rescued by ectopic expression of Drosophila Syndapin, and depends critically on both the prong that protrudes from the surface of the bowed Syndapin EFC domain and the ability of the antiparallel dimer to bind tightly to phosphoinositides. Our data confirm linkage between directional migration, endocytosis and cell specification during embryonic morphogenesis and highlight a key role for Pacsin3 in this coupling in the notochord.
REST Regulates Distinct Transcriptional Networks in Embryonic and Neural Stem Cells  [PDF]
Rory Johnson,Christina Hui-leng Teh,Galih Kunarso,Kee Yew Wong,Gopalan Srinivasan,Megan L. Cooper,Manuela Volta,Sarah Su-ling Chan,Leonard Lipovich,Steven M. Pollard,R. Krishna Murthy Karuturi,Chia-lin Wei,Noel J. Buckley,Lawrence W. Stanton
PLOS Biology , 2012, DOI: 10.1371/journal.pbio.0060256
Abstract: The maintenance of pluripotency and specification of cellular lineages during embryonic development are controlled by transcriptional regulatory networks, which coordinate specific sets of genes through both activation and repression. The transcriptional repressor RE1-silencing transcription factor (REST) plays important but distinct regulatory roles in embryonic (ESC) and neural (NSC) stem cells. We investigated how these distinct biological roles are effected at a genomic level. We present integrated, comparative genome- and transcriptome-wide analyses of transcriptional networks governed by REST in mouse ESC and NSC. The REST recruitment profile has dual components: a developmentally independent core that is common to ESC, NSC, and differentiated cells; and a large, ESC-specific set of target genes. In ESC, the REST regulatory network is highly integrated into that of pluripotency factors Oct4-Sox2-Nanog. We propose that an extensive, pluripotency-specific recruitment profile lends REST a key role in the maintenance of the ESC phenotype.
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