Limits...
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.

Show MeSH

Related in: MedlinePlus

Expression and Timing Requirement of med14 for Development(A) Worsening of the log mutant phenotype by injection of morpholino (MO) targeting med14 (red arrowhead denotes somite defect).(A′ and A′′) Somite structure of 3.5 dpf rhodamine phalloidin (RhPh) stained WT and MO-injected mutant embryos. Defects in muscle fiber patterns of mutant embryos injected with MO are shown (yellow arrowheads).(B–D) RNA ISH analysis shows that med14 is broadly expressed at the one- and two-cell stage and at 12 hpf (“WT/MUT” denotes unknown genotype). At 24 hpf, broad med14 expression is undetectable in zygotic med14 mutants.(E–K) Temporal rescue of the log mutant phenotype using Tg(hsp70:med14, α-crystallin:EGFP), with initial heat shock performed at the specified time, and then every 12 hr following until 5 dpf. Scale bars, 0.5 mm.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4400641&req=5

fig3: Expression and Timing Requirement of med14 for Development(A) Worsening of the log mutant phenotype by injection of morpholino (MO) targeting med14 (red arrowhead denotes somite defect).(A′ and A′′) Somite structure of 3.5 dpf rhodamine phalloidin (RhPh) stained WT and MO-injected mutant embryos. Defects in muscle fiber patterns of mutant embryos injected with MO are shown (yellow arrowheads).(B–D) RNA ISH analysis shows that med14 is broadly expressed at the one- and two-cell stage and at 12 hpf (“WT/MUT” denotes unknown genotype). At 24 hpf, broad med14 expression is undetectable in zygotic med14 mutants.(E–K) Temporal rescue of the log mutant phenotype using Tg(hsp70:med14, α-crystallin:EGFP), with initial heat shock performed at the specified time, and then every 12 hr following until 5 dpf. Scale bars, 0.5 mm.

Mentions: As we expected that loss of Med14 would have global effects on transcription, we next examined whether med14 had a maternal function. Injection of med14 morpholino (which would affect maternal med14 transcript, but not protein) worsened the log mutant phenotype (Figure 3A), notably inducing defects in skeletal muscle fibers (Figures 3A′ and 3A′′). med14 transcript was maternally deposited (Figure 3B) and expressed broadly later in development (Figure 3C; data not shown). ISH analysis in m628 allele mutants revealed a loss of med14 expression by 24hpf, suggesting nonsense-mediated decay of mutant transcript and degradation of maternally deposited WT transcript by this time point (Figure 3D). Efforts to deplete WT maternal med14 transcript by making maternal zygotic mutants through a germline replacement strategy (Ciruna et al., 2002) were not successful (results not shown). To determine whether the log mutant phenotype could be alleviated by prolonged expression of med14, we generated a transgenic line expressing WT med14 RNA under control of the inducible hsp70 promoter, which had no apparent effects on WT development (Figure 3E). In log mutants, overexpression of med14 every 12 hr beginning at 12 hpf until 120 hpf resulted in maximal rescue (Figure 3F). Further analysis revealed that initiation of med14 overexpression at 24 hpf or later in log mutants resulted in progressively more severe phenotypes, with initiation after 48 hpf resulting in no discernable rescue (Figures 3G–3K). These results suggest that maternal Med14 function alleviates the severity of early phenotypes in log mutants.


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)

Expression and Timing Requirement of med14 for Development(A) Worsening of the log mutant phenotype by injection of morpholino (MO) targeting med14 (red arrowhead denotes somite defect).(A′ and A′′) Somite structure of 3.5 dpf rhodamine phalloidin (RhPh) stained WT and MO-injected mutant embryos. Defects in muscle fiber patterns of mutant embryos injected with MO are shown (yellow arrowheads).(B–D) RNA ISH analysis shows that med14 is broadly expressed at the one- and two-cell stage and at 12 hpf (“WT/MUT” denotes unknown genotype). At 24 hpf, broad med14 expression is undetectable in zygotic med14 mutants.(E–K) Temporal rescue of the log mutant phenotype using Tg(hsp70:med14, α-crystallin:EGFP), with initial heat shock performed at the specified time, and then every 12 hr following until 5 dpf. Scale bars, 0.5 mm.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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

fig3: Expression and Timing Requirement of med14 for Development(A) Worsening of the log mutant phenotype by injection of morpholino (MO) targeting med14 (red arrowhead denotes somite defect).(A′ and A′′) Somite structure of 3.5 dpf rhodamine phalloidin (RhPh) stained WT and MO-injected mutant embryos. Defects in muscle fiber patterns of mutant embryos injected with MO are shown (yellow arrowheads).(B–D) RNA ISH analysis shows that med14 is broadly expressed at the one- and two-cell stage and at 12 hpf (“WT/MUT” denotes unknown genotype). At 24 hpf, broad med14 expression is undetectable in zygotic med14 mutants.(E–K) Temporal rescue of the log mutant phenotype using Tg(hsp70:med14, α-crystallin:EGFP), with initial heat shock performed at the specified time, and then every 12 hr following until 5 dpf. Scale bars, 0.5 mm.
Mentions: As we expected that loss of Med14 would have global effects on transcription, we next examined whether med14 had a maternal function. Injection of med14 morpholino (which would affect maternal med14 transcript, but not protein) worsened the log mutant phenotype (Figure 3A), notably inducing defects in skeletal muscle fibers (Figures 3A′ and 3A′′). med14 transcript was maternally deposited (Figure 3B) and expressed broadly later in development (Figure 3C; data not shown). ISH analysis in m628 allele mutants revealed a loss of med14 expression by 24hpf, suggesting nonsense-mediated decay of mutant transcript and degradation of maternally deposited WT transcript by this time point (Figure 3D). Efforts to deplete WT maternal med14 transcript by making maternal zygotic mutants through a germline replacement strategy (Ciruna et al., 2002) were not successful (results not shown). To determine whether the log mutant phenotype could be alleviated by prolonged expression of med14, we generated a transgenic line expressing WT med14 RNA under control of the inducible hsp70 promoter, which had no apparent effects on WT development (Figure 3E). In log mutants, overexpression of med14 every 12 hr beginning at 12 hpf until 120 hpf resulted in maximal rescue (Figure 3F). Further analysis revealed that initiation of med14 overexpression at 24 hpf or later in log mutants resulted in progressively more severe phenotypes, with initiation after 48 hpf resulting in no discernable rescue (Figures 3G–3K). These results suggest that maternal Med14 function alleviates the severity of early phenotypes in log mutants.

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