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Esrrb is a direct Nanog target gene that can substitute for Nanog function in pluripotent cells.

Festuccia N, Osorno R, Halbritter F, Karwacki-Neisius V, Navarro P, Colby D, Wong F, Yates A, Tomlinson SR, Chambers I - Cell Stem Cell (2012)

Bottom Line: Moreover, Esrrb can reprogram Nanog(-/-) EpiSCs and can rescue stalled reprogramming in Nanog(-/-) pre-iPSCs.Finally, Esrrb deletion abolishes the defining ability of Nanog to confer LIF-independent ESC self-renewal.These findings are consistent with the functional placement of Esrrb downstream of Nanog.

View Article: PubMed Central - PubMed

Affiliation: MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, 5 Little France Drive, Edinburgh EH16 4UU, Scotland.

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Esrrb Overexpression Confers LIF and Nanog-Independent Self-Renewal(A) lifr−/−:PyLT+ LRK1 cells were transfected with episomal plasmids encoding the indicated ORF (EV; empty vector) and the number of AP-positive colonies was determined after clonal density plating in the absence of IL-6/sIL6R. Error bars: standard deviation (n = 3).(B) Schematic representation of EfEsrrb ESCs.(C) Colony morphology (top) and AP staining (bottom) of EfEsrrb c1 cultured in the presence of hLIF-05.(D) E14Tg2a, Nanog-, and Esrrb-overexpressing cells before and after Cre reversion were plated at clonal density and cultured in the presence or absence of LIF or hLIF-05 for 7 days, and the number of AP-positive colonies was counted. Error bars: standard deviation (n = 3).(E) Chimeras generated after injection into C57BL/6 blastocysts of EfEsrrb-Cre ESCs passaged twice at clonal density in the presence of hLIF-05 and transfected with a Cre expression vector to excise the Esrrb transgene.(F) Schematic representation of the genetic manipulations used to make ESΔN-iNanog or ESΔN-iEsrrb cells.(G) Colony morphology of ESΔN-iNanog (iN) or ESΔN-iEsrrb (iE) cells plated at clonal density and cultured in the presence of hLIF-05 (+/− doxycycline) for 8 days. Right hand panels: AP staining of colonies formed in the presence of doxycycline.(H) Number of AP-positive colonies formed after clonal density plating of ESΔN-iNanog (iN) or ESΔN-iEsrrb (iE) cells in the presence of LIF or hLIF-05 and cultured (+/− doxycycline) for 8 days. Error bars: standard deviation (n = 3).(I) ESΔN-iNanog (iN) and ESΔN-iEsrrb (iE) cells in a neural differentiation protocol, without (top rows) or with (bottom rows) doxycycline for 9 days. Cells were fixed, stained for βIII-Tubulin (Tuj), and analyzed by fluorescence microscopy.See also Figures S3 and S4 and Table S2.
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fig2: Esrrb Overexpression Confers LIF and Nanog-Independent Self-Renewal(A) lifr−/−:PyLT+ LRK1 cells were transfected with episomal plasmids encoding the indicated ORF (EV; empty vector) and the number of AP-positive colonies was determined after clonal density plating in the absence of IL-6/sIL6R. Error bars: standard deviation (n = 3).(B) Schematic representation of EfEsrrb ESCs.(C) Colony morphology (top) and AP staining (bottom) of EfEsrrb c1 cultured in the presence of hLIF-05.(D) E14Tg2a, Nanog-, and Esrrb-overexpressing cells before and after Cre reversion were plated at clonal density and cultured in the presence or absence of LIF or hLIF-05 for 7 days, and the number of AP-positive colonies was counted. Error bars: standard deviation (n = 3).(E) Chimeras generated after injection into C57BL/6 blastocysts of EfEsrrb-Cre ESCs passaged twice at clonal density in the presence of hLIF-05 and transfected with a Cre expression vector to excise the Esrrb transgene.(F) Schematic representation of the genetic manipulations used to make ESΔN-iNanog or ESΔN-iEsrrb cells.(G) Colony morphology of ESΔN-iNanog (iN) or ESΔN-iEsrrb (iE) cells plated at clonal density and cultured in the presence of hLIF-05 (+/− doxycycline) for 8 days. Right hand panels: AP staining of colonies formed in the presence of doxycycline.(H) Number of AP-positive colonies formed after clonal density plating of ESΔN-iNanog (iN) or ESΔN-iEsrrb (iE) cells in the presence of LIF or hLIF-05 and cultured (+/− doxycycline) for 8 days. Error bars: standard deviation (n = 3).(I) ESΔN-iNanog (iN) and ESΔN-iEsrrb (iE) cells in a neural differentiation protocol, without (top rows) or with (bottom rows) doxycycline for 9 days. Cells were fixed, stained for βIII-Tubulin (Tuj), and analyzed by fluorescence microscopy.See also Figures S3 and S4 and Table S2.

Mentions: Different ESC lines in a Nanog mutant series (Chambers et al., 2003, 2007) showed a correlation between Nanog expression and levels of Esrrb mRNA (Figure 1B) and protein (Figure 1C). These variations in Esrrb mRNA levels reflect transcriptional control of Esrrb by Nanog rather than RNA stabilization, since differences in mRNA level (Figure S1C) were also seen for the pre-mRNA (Figure S1D). Furthermore, tamoxifen-induced elimination of Nanog from ESCs (Chambers et al., 2007) results in decreased Esrrb mRNA expression, an effect not attributable to differentiation as shown by stable Oct4 levels (Figure S1E). To investigate the dynamics of Nanog control of Esrrb transcription, we measured Esrrb mRNA levels in TβC44Cre6 Nanog−/− ESCs expressing a tamoxifen-regulatable Nanog-ERT2 fusion protein (ESΔN-NERT, Figure S2A). In these cells Nanog nuclear relocalization is induced within 15 min of tamoxifen addition (Figure 1D). Three independent ESΔN-NERT lines induced Esrrb mRNA and protein at levels that correlated to the level of Nanog-ERT2 mRNA expression (Figures S1F and S1G). Tamoxifen treatment of ESΔN-NERT cells resulted in self-renewal in the absence of LIF to an extent comparable to that induced by wild-type Nanog expression (Figure S1I) in an identical Nanog−/− background (Figures 2F and S2A), indicating that Nanog-ERT2 is fully functional.


Esrrb is a direct Nanog target gene that can substitute for Nanog function in pluripotent cells.

Festuccia N, Osorno R, Halbritter F, Karwacki-Neisius V, Navarro P, Colby D, Wong F, Yates A, Tomlinson SR, Chambers I - Cell Stem Cell (2012)

Esrrb Overexpression Confers LIF and Nanog-Independent Self-Renewal(A) lifr−/−:PyLT+ LRK1 cells were transfected with episomal plasmids encoding the indicated ORF (EV; empty vector) and the number of AP-positive colonies was determined after clonal density plating in the absence of IL-6/sIL6R. Error bars: standard deviation (n = 3).(B) Schematic representation of EfEsrrb ESCs.(C) Colony morphology (top) and AP staining (bottom) of EfEsrrb c1 cultured in the presence of hLIF-05.(D) E14Tg2a, Nanog-, and Esrrb-overexpressing cells before and after Cre reversion were plated at clonal density and cultured in the presence or absence of LIF or hLIF-05 for 7 days, and the number of AP-positive colonies was counted. Error bars: standard deviation (n = 3).(E) Chimeras generated after injection into C57BL/6 blastocysts of EfEsrrb-Cre ESCs passaged twice at clonal density in the presence of hLIF-05 and transfected with a Cre expression vector to excise the Esrrb transgene.(F) Schematic representation of the genetic manipulations used to make ESΔN-iNanog or ESΔN-iEsrrb cells.(G) Colony morphology of ESΔN-iNanog (iN) or ESΔN-iEsrrb (iE) cells plated at clonal density and cultured in the presence of hLIF-05 (+/− doxycycline) for 8 days. Right hand panels: AP staining of colonies formed in the presence of doxycycline.(H) Number of AP-positive colonies formed after clonal density plating of ESΔN-iNanog (iN) or ESΔN-iEsrrb (iE) cells in the presence of LIF or hLIF-05 and cultured (+/− doxycycline) for 8 days. Error bars: standard deviation (n = 3).(I) ESΔN-iNanog (iN) and ESΔN-iEsrrb (iE) cells in a neural differentiation protocol, without (top rows) or with (bottom rows) doxycycline for 9 days. Cells were fixed, stained for βIII-Tubulin (Tuj), and analyzed by fluorescence microscopy.See also Figures S3 and S4 and Table S2.
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fig2: Esrrb Overexpression Confers LIF and Nanog-Independent Self-Renewal(A) lifr−/−:PyLT+ LRK1 cells were transfected with episomal plasmids encoding the indicated ORF (EV; empty vector) and the number of AP-positive colonies was determined after clonal density plating in the absence of IL-6/sIL6R. Error bars: standard deviation (n = 3).(B) Schematic representation of EfEsrrb ESCs.(C) Colony morphology (top) and AP staining (bottom) of EfEsrrb c1 cultured in the presence of hLIF-05.(D) E14Tg2a, Nanog-, and Esrrb-overexpressing cells before and after Cre reversion were plated at clonal density and cultured in the presence or absence of LIF or hLIF-05 for 7 days, and the number of AP-positive colonies was counted. Error bars: standard deviation (n = 3).(E) Chimeras generated after injection into C57BL/6 blastocysts of EfEsrrb-Cre ESCs passaged twice at clonal density in the presence of hLIF-05 and transfected with a Cre expression vector to excise the Esrrb transgene.(F) Schematic representation of the genetic manipulations used to make ESΔN-iNanog or ESΔN-iEsrrb cells.(G) Colony morphology of ESΔN-iNanog (iN) or ESΔN-iEsrrb (iE) cells plated at clonal density and cultured in the presence of hLIF-05 (+/− doxycycline) for 8 days. Right hand panels: AP staining of colonies formed in the presence of doxycycline.(H) Number of AP-positive colonies formed after clonal density plating of ESΔN-iNanog (iN) or ESΔN-iEsrrb (iE) cells in the presence of LIF or hLIF-05 and cultured (+/− doxycycline) for 8 days. Error bars: standard deviation (n = 3).(I) ESΔN-iNanog (iN) and ESΔN-iEsrrb (iE) cells in a neural differentiation protocol, without (top rows) or with (bottom rows) doxycycline for 9 days. Cells were fixed, stained for βIII-Tubulin (Tuj), and analyzed by fluorescence microscopy.See also Figures S3 and S4 and Table S2.
Mentions: Different ESC lines in a Nanog mutant series (Chambers et al., 2003, 2007) showed a correlation between Nanog expression and levels of Esrrb mRNA (Figure 1B) and protein (Figure 1C). These variations in Esrrb mRNA levels reflect transcriptional control of Esrrb by Nanog rather than RNA stabilization, since differences in mRNA level (Figure S1C) were also seen for the pre-mRNA (Figure S1D). Furthermore, tamoxifen-induced elimination of Nanog from ESCs (Chambers et al., 2007) results in decreased Esrrb mRNA expression, an effect not attributable to differentiation as shown by stable Oct4 levels (Figure S1E). To investigate the dynamics of Nanog control of Esrrb transcription, we measured Esrrb mRNA levels in TβC44Cre6 Nanog−/− ESCs expressing a tamoxifen-regulatable Nanog-ERT2 fusion protein (ESΔN-NERT, Figure S2A). In these cells Nanog nuclear relocalization is induced within 15 min of tamoxifen addition (Figure 1D). Three independent ESΔN-NERT lines induced Esrrb mRNA and protein at levels that correlated to the level of Nanog-ERT2 mRNA expression (Figures S1F and S1G). Tamoxifen treatment of ESΔN-NERT cells resulted in self-renewal in the absence of LIF to an extent comparable to that induced by wild-type Nanog expression (Figure S1I) in an identical Nanog−/− background (Figures 2F and S2A), indicating that Nanog-ERT2 is fully functional.

Bottom Line: Moreover, Esrrb can reprogram Nanog(-/-) EpiSCs and can rescue stalled reprogramming in Nanog(-/-) pre-iPSCs.Finally, Esrrb deletion abolishes the defining ability of Nanog to confer LIF-independent ESC self-renewal.These findings are consistent with the functional placement of Esrrb downstream of Nanog.

View Article: PubMed Central - PubMed

Affiliation: MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, 5 Little France Drive, Edinburgh EH16 4UU, Scotland.

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Related in: MedlinePlus