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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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Logelei Results from a Mutation in med14(A) Recombination frequency mapping results. Screening of 1,800 map-cross mutant embryos with markers Z45039 and Z9112 revealed 16 and 39 flanking recombinant embryos, respectively. The zero-recombinant region (light green, contained in two bacterial artificial chromosomes [BACs]) was refined with additional markers. Sequencing of med14 revealed a base pair substitution (arrow) leading to a premature stop codon (∗) in each of the three log alleles.(B–E′′′) Lateral and dorsal views of the otic vesicle and pectoral fin of 3.5 dpf WT (B–B′′′), log mutant (C–C′′′), med14 morpholino-injected (D–D′′′), and log mutant injected with med14 RNA (E–E′′′) embryos.
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fig2: Logelei Results from a Mutation in med14(A) Recombination frequency mapping results. Screening of 1,800 map-cross mutant embryos with markers Z45039 and Z9112 revealed 16 and 39 flanking recombinant embryos, respectively. The zero-recombinant region (light green, contained in two bacterial artificial chromosomes [BACs]) was refined with additional markers. Sequencing of med14 revealed a base pair substitution (arrow) leading to a premature stop codon (∗) in each of the three log alleles.(B–E′′′) Lateral and dorsal views of the otic vesicle and pectoral fin of 3.5 dpf WT (B–B′′′), log mutant (C–C′′′), med14 morpholino-injected (D–D′′′), and log mutant injected with med14 RNA (E–E′′′) embryos.

Mentions: We next sought to determine the causal log mutation, which we had previously localized to linkage group 9 (Jin et al., 2007). Further mapping defined a zero recombination region of approximately 100 kb containing four genes. RT-PCR and sequencing from mutant (s231 and the previously isolated m628 and m673 alleles) and wild-type (WT) cDNA pools identified distinct premature stop codons in med14 in all three alleles (Figure 2A). Injection of med14 morpholino recapitulated otic vesicle, pectoral fin and cardiac phenotypes, while injection of 300 pg of RNA encoding a WT form of Med14 partially rescued these defects in log mutants (Figures 2B–2E′′′). Further, injection of RNA encoding the s231, m628, and m673 forms of med14 failed to affect appreciable rescue of log mutants (results not shown). Taken together, this established that the log phenotype is due to mutation of med14.


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)

Logelei Results from a Mutation in med14(A) Recombination frequency mapping results. Screening of 1,800 map-cross mutant embryos with markers Z45039 and Z9112 revealed 16 and 39 flanking recombinant embryos, respectively. The zero-recombinant region (light green, contained in two bacterial artificial chromosomes [BACs]) was refined with additional markers. Sequencing of med14 revealed a base pair substitution (arrow) leading to a premature stop codon (∗) in each of the three log alleles.(B–E′′′) Lateral and dorsal views of the otic vesicle and pectoral fin of 3.5 dpf WT (B–B′′′), log mutant (C–C′′′), med14 morpholino-injected (D–D′′′), and log mutant injected with med14 RNA (E–E′′′) embryos.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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

fig2: Logelei Results from a Mutation in med14(A) Recombination frequency mapping results. Screening of 1,800 map-cross mutant embryos with markers Z45039 and Z9112 revealed 16 and 39 flanking recombinant embryos, respectively. The zero-recombinant region (light green, contained in two bacterial artificial chromosomes [BACs]) was refined with additional markers. Sequencing of med14 revealed a base pair substitution (arrow) leading to a premature stop codon (∗) in each of the three log alleles.(B–E′′′) Lateral and dorsal views of the otic vesicle and pectoral fin of 3.5 dpf WT (B–B′′′), log mutant (C–C′′′), med14 morpholino-injected (D–D′′′), and log mutant injected with med14 RNA (E–E′′′) embryos.
Mentions: We next sought to determine the causal log mutation, which we had previously localized to linkage group 9 (Jin et al., 2007). Further mapping defined a zero recombination region of approximately 100 kb containing four genes. RT-PCR and sequencing from mutant (s231 and the previously isolated m628 and m673 alleles) and wild-type (WT) cDNA pools identified distinct premature stop codons in med14 in all three alleles (Figure 2A). Injection of med14 morpholino recapitulated otic vesicle, pectoral fin and cardiac phenotypes, while injection of 300 pg of RNA encoding a WT form of Med14 partially rescued these defects in log mutants (Figures 2B–2E′′′). Further, injection of RNA encoding the s231, m628, and m673 forms of med14 failed to affect appreciable rescue of log mutants (results not shown). Taken together, this established that the log phenotype is due to mutation of med14.

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