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An in vivo requirement for the mediator subunit med14 in the maintenance of stem cell populations.

Burrows JT, Pearson BJ, Scott IC - Stem Cell Reports (2015)

Bottom Line: In planarians, RNAi knockdown demonstrated a requirement for med14 and many other Mediator components in adult stem cell maintenance and regeneration.Multiple stem/progenitor cell populations were observed to be reduced or absent in zebrafish med14 mutant embryos.Taken together, our results show a critical, evolutionarily conserved, in vivo function for Med14 (and Mediator) in stem cell maintenance, distinct from a general role in transcription.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada; Program in Developmental and Stem Cell Biology, The Hospital for Sick Children, Toronto, ON M5G 0A4, Canada.

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A Conserved Requirement for med14 in the Maintenance of Stem Cell Populations(A–D) Expression of retinal stem cell marker mz98 (arrowhead) is present, but reduced in log mutants at 2.25 dpf, and absent at 3.25 dpf.(E–H) Expression of the hematopoietic stem cell marker cmyb is initiated in 2.25 dpf log mutant embryos (arrowhead), but largely absent by 3.25 dpf.(I and J) Expression of the putative gut stem cell marker lgr4 is not observed in 3.25 dpf log mutant embryos (arrowhead, note expression in WT).(K and L) Robust tail fin regeneration in WT as compared with log mutant embryos at 4 dpf following amputation (at area of dotted line) at 2 dpf.(M–R) o-Dianisidane staining of red blood cells in trunks of WT and log mutant embryos. Scale bars, 0.5 mm.(S and T) Confocal projections of 2.5 dpf Tg(cmcl2:nlsDsRedExpress) WT and log morphant hearts.(U) Quantification of myocardial cell number in WT and morphant hearts shows a significant decrease in morphants (p = 0.0017, n = 6 for both conditions).(V) Cardiac edema and heart defects remain in 3.5 dpf Tg (cmlc2:med14, α-crystallin:EGFP) log mutant embryos.(W and X) Expression of the second heart field marker ltbp3 in the arterial pole of the heart (arrowhead) is reduced in log mutants at 48 hpf.
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fig7: A Conserved Requirement for med14 in the Maintenance of Stem Cell Populations(A–D) Expression of retinal stem cell marker mz98 (arrowhead) is present, but reduced in log mutants at 2.25 dpf, and absent at 3.25 dpf.(E–H) Expression of the hematopoietic stem cell marker cmyb is initiated in 2.25 dpf log mutant embryos (arrowhead), but largely absent by 3.25 dpf.(I and J) Expression of the putative gut stem cell marker lgr4 is not observed in 3.25 dpf log mutant embryos (arrowhead, note expression in WT).(K and L) Robust tail fin regeneration in WT as compared with log mutant embryos at 4 dpf following amputation (at area of dotted line) at 2 dpf.(M–R) o-Dianisidane staining of red blood cells in trunks of WT and log mutant embryos. Scale bars, 0.5 mm.(S and T) Confocal projections of 2.5 dpf Tg(cmcl2:nlsDsRedExpress) WT and log morphant hearts.(U) Quantification of myocardial cell number in WT and morphant hearts shows a significant decrease in morphants (p = 0.0017, n = 6 for both conditions).(V) Cardiac edema and heart defects remain in 3.5 dpf Tg (cmlc2:med14, α-crystallin:EGFP) log mutant embryos.(W and X) Expression of the second heart field marker ltbp3 in the arterial pole of the heart (arrowhead) is reduced in log mutants at 48 hpf.

Mentions: We next re-examined the zebrafish log mutant phenotype, with a focus on stem/progenitor cells and regeneration. Using RNA ISH, we first assayed expression of the retinal stem cell marker mz98 (Cerveny et al., 2010) and found that while present at 2.25 dpf, expression was lost by 3.25 dpf in log mutants (Figures 7A–7D). Analysis of the hematopoietic stem cell marker cmyb (Bertrand et al., 2008) in the ventral trunk/tail region of the embryo revealed a loss of expression in log mutants by 3.25 dpf (Figures 7E–7H). Similarly, the putative gut stem cell marker lgr4 (Hirose et al., 2011) showed a severe reduction in expression in log mutants at 3.25 dpf (Figures 7I and 7J). To assess a possible role for Med14 in vertebrate regeneration, we employed amputation of the zebrafish embryonic tailfin (Kawakami et al., 2004). Following resection of the tailfin at 2 dpf, we observed no appreciable regrowth of log mutant fins by 4 dpf (Figures 7K and 7L).


An in vivo requirement for the mediator subunit med14 in the maintenance of stem cell populations.

Burrows JT, Pearson BJ, Scott IC - Stem Cell Reports (2015)

A Conserved Requirement for med14 in the Maintenance of Stem Cell Populations(A–D) Expression of retinal stem cell marker mz98 (arrowhead) is present, but reduced in log mutants at 2.25 dpf, and absent at 3.25 dpf.(E–H) Expression of the hematopoietic stem cell marker cmyb is initiated in 2.25 dpf log mutant embryos (arrowhead), but largely absent by 3.25 dpf.(I and J) Expression of the putative gut stem cell marker lgr4 is not observed in 3.25 dpf log mutant embryos (arrowhead, note expression in WT).(K and L) Robust tail fin regeneration in WT as compared with log mutant embryos at 4 dpf following amputation (at area of dotted line) at 2 dpf.(M–R) o-Dianisidane staining of red blood cells in trunks of WT and log mutant embryos. Scale bars, 0.5 mm.(S and T) Confocal projections of 2.5 dpf Tg(cmcl2:nlsDsRedExpress) WT and log morphant hearts.(U) Quantification of myocardial cell number in WT and morphant hearts shows a significant decrease in morphants (p = 0.0017, n = 6 for both conditions).(V) Cardiac edema and heart defects remain in 3.5 dpf Tg (cmlc2:med14, α-crystallin:EGFP) log mutant embryos.(W and X) Expression of the second heart field marker ltbp3 in the arterial pole of the heart (arrowhead) is reduced in log mutants at 48 hpf.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4400641&req=5

fig7: A Conserved Requirement for med14 in the Maintenance of Stem Cell Populations(A–D) Expression of retinal stem cell marker mz98 (arrowhead) is present, but reduced in log mutants at 2.25 dpf, and absent at 3.25 dpf.(E–H) Expression of the hematopoietic stem cell marker cmyb is initiated in 2.25 dpf log mutant embryos (arrowhead), but largely absent by 3.25 dpf.(I and J) Expression of the putative gut stem cell marker lgr4 is not observed in 3.25 dpf log mutant embryos (arrowhead, note expression in WT).(K and L) Robust tail fin regeneration in WT as compared with log mutant embryos at 4 dpf following amputation (at area of dotted line) at 2 dpf.(M–R) o-Dianisidane staining of red blood cells in trunks of WT and log mutant embryos. Scale bars, 0.5 mm.(S and T) Confocal projections of 2.5 dpf Tg(cmcl2:nlsDsRedExpress) WT and log morphant hearts.(U) Quantification of myocardial cell number in WT and morphant hearts shows a significant decrease in morphants (p = 0.0017, n = 6 for both conditions).(V) Cardiac edema and heart defects remain in 3.5 dpf Tg (cmlc2:med14, α-crystallin:EGFP) log mutant embryos.(W and X) Expression of the second heart field marker ltbp3 in the arterial pole of the heart (arrowhead) is reduced in log mutants at 48 hpf.
Mentions: We next re-examined the zebrafish log mutant phenotype, with a focus on stem/progenitor cells and regeneration. Using RNA ISH, we first assayed expression of the retinal stem cell marker mz98 (Cerveny et al., 2010) and found that while present at 2.25 dpf, expression was lost by 3.25 dpf in log mutants (Figures 7A–7D). Analysis of the hematopoietic stem cell marker cmyb (Bertrand et al., 2008) in the ventral trunk/tail region of the embryo revealed a loss of expression in log mutants by 3.25 dpf (Figures 7E–7H). Similarly, the putative gut stem cell marker lgr4 (Hirose et al., 2011) showed a severe reduction in expression in log mutants at 3.25 dpf (Figures 7I and 7J). To assess a possible role for Med14 in vertebrate regeneration, we employed amputation of the zebrafish embryonic tailfin (Kawakami et al., 2004). Following resection of the tailfin at 2 dpf, we observed no appreciable regrowth of log mutant fins by 4 dpf (Figures 7K and 7L).

Bottom Line: In planarians, RNAi knockdown demonstrated a requirement for med14 and many other Mediator components in adult stem cell maintenance and regeneration.Multiple stem/progenitor cell populations were observed to be reduced or absent in zebrafish med14 mutant embryos.Taken together, our results show a critical, evolutionarily conserved, in vivo function for Med14 (and Mediator) in stem cell maintenance, distinct from a general role in transcription.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada; Program in Developmental and Stem Cell Biology, The Hospital for Sick Children, Toronto, ON M5G 0A4, Canada.

Show MeSH
Related in: MedlinePlus