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