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The role of Importin-?s in the maintenance and lineage commitment of mouse embryonic stem cells.

ABSTRACT: Members of the Importin-? family recognize nuclear localization signals (NLS) and nuclear export signals (NES). These proteins play important roles in various nucleocytoplasmic transport processes in cells. Here, we examined the expression patterns of 21 identified Importin-? genes in mouse embryonic stem cells (mESCs), mouse embryonic fibroblast (MEF) and mESCs differentiated into neural ectoderm (NE) or mesoendoderm (ME). We observed striking differences in the Importin-? mRNA expression levels within these cell types. We also found that knockdown of selected Importin-? genes led to suppression of Nanog, and altered the balance of Oct4/Sox2 expression ratio, which is important for NE/ME lineage choice. Furthermore, we demonstrated that knockdown of XPO4, RanBP17, RanBP16, or IPO7 differentially affected the lineage selection of differentiating mESCs. More specifically, knockdown of XPO4 selectively stimulated the mESC differentiation towards definitive endoderm, while concomitantly inhibiting NE differentiation. RanBP17 knockdown also promoted endodermal differentiation with no effect on NE differentiation. RanBP16 knockdown caused differentiation into ME, while IPO7 knockdown inhibited NE differentiation, without obvious effects on the other lineages. Collectively, our results suggest that Importin-?s play important roles in cell fate determination processes of mESCs, such as in the maintenance of pluripotency or selection of lineage during differentiation.

SUBMITTER: Sangel P

PROVIDER: S-EPMC3907685 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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The role of Importin-βs in the maintenance and lineage commitment of mouse embryonic stem cells.

Sangel Percival P Oka Masahiro M Yoneda Yoshihiro Y

FEBS open bio 20140106

Members of the Importin-β family recognize nuclear localization signals (NLS) and nuclear export signals (NES). These proteins play important roles in various nucleocytoplasmic transport processes in cells. Here, we examined the expression patterns of 21 identified Importin-β genes in mouse embryonic stem cells (mESCs), mouse embryonic fibroblast (MEF) and mESCs differentiated into neural ectoderm (NE) or mesoendoderm (ME). We observed striking differences in the Importin-β mRNA expression level ...[more]

PMID: 24490135

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Project description:Catecholamine release is known to modulate cardiac output by increasing heart rate. Although much is known about catecholamine function and regulation in adults, little is known about the presence and role of catecholamines during heart development. The present study aimed therefore to evaluate the effects of different catecholamines on early heart development in an in vitro setting using embryonic stem (ES) cell-derived cardiomyocytes. Effects of catecholamine depletion induced by reserpine were examined in murine ES cells (line D3, ?PIG44) during differentiation. Cardiac differentiation was assessed by immunocytochemistry, qRT-PCR, quantification of beating clusters, flow cytometry and pharmacological approaches. Proliferation was analyzed by EB cross-section measurements, while functionality of cardiomyocytes was studied by extracellular field potential (FP) measurements using microelectrode arrays (MEAs). To further differentiate between substance-specific effects of reserpine and catecholamine action via ?- and ?-receptors we proved the involvement of adrenergic receptors by application of unspecific ?- and ?-receptor antagonists. Reserpine treatment led to remarkable down-regulation of cardiac-specific genes, proteins and mesodermal marker genes. In more detail, the average ratio of ?40% spontaneously beating control clusters was significantly reduced by 100%, 91.1% and 20.0% on days 10, 12, and 14, respectively. Flow cytometry revealed a significant reduction (by 71.6%, n?=?11) of eGFP positive CMs after reserpine treatment. By contrast, reserpine did not reduce EB growth while number of neuronal cells in reserpine-treated EBs was significantly increased. MEA measurements of reserpine-treated EBs showed lower FP frequencies and weak responsiveness to adrenergic and muscarinic stimulation. Interestingly we found that developmental inhibition after ?- and ?-adrenergic blocker application mimicked developmental changes with reserpine. Using several methodological approaches our data suggest that reserpine inhibits cardiac differentiation. Thus catecholamines play a critical role during development.

Dataset Information

The role of Importin-?s in the maintenance and lineage commitment of mouse embryonic stem cells.

Publications

The role of Importin-βs in the maintenance and lineage commitment of mouse embryonic stem cells.

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