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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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Transcription Is Not Largely Affected in log (med14) Mutants(A–D) Expression of opn1sw1, initiated by 2.5 dpf in WT embryos, reaches WT levels in log mutant embryos by 4.0 dpf.(E) Tg(hsp70:EGFP) log mutant embryos heat shocked at 3.5 dpf show robust GFP signal.(F–H′) log mutant cells transplanted into WT hosts (traced using a β-actin:EGFP transgene) are evident in 15 dpf hosts (including in the semicircular canals, yellow arrowhead in F) and survive until 2 ypf (years post-fertilization, yellow arrowheads).(I) Summary of results of microarray analysis on cDNA from 2.25 dpf WT versus log mutant embryos.(J) Quantification of mRNA per 0.1mg total RNA at 3.25 dpf from WT and log mutant embryos.(K) Normalized qPCR values for med14 expression in mutant relative to WT control embryos (∗p < 0.05 using the one-tailed unpaired Student’s t test).(L) Mean normalized expression (MNE) of β-actin in WT and MUT embryos at 2.25 dpf calculated using a universal reference approach. No significant difference (p = 0.63) was observed between WT (0.107 ± 0.00380) and log MUT (0.123 ± 0.0297) samples (n = 3) by one-tailed unpaired Student’s t test. For (J)–(L), three biological replicates of 450 (J) or 10 (K and L) embryos were used.
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fig4: Transcription Is Not Largely Affected in log (med14) Mutants(A–D) Expression of opn1sw1, initiated by 2.5 dpf in WT embryos, reaches WT levels in log mutant embryos by 4.0 dpf.(E) Tg(hsp70:EGFP) log mutant embryos heat shocked at 3.5 dpf show robust GFP signal.(F–H′) log mutant cells transplanted into WT hosts (traced using a β-actin:EGFP transgene) are evident in 15 dpf hosts (including in the semicircular canals, yellow arrowhead in F) and survive until 2 ypf (years post-fertilization, yellow arrowheads).(I) Summary of results of microarray analysis on cDNA from 2.25 dpf WT versus log mutant embryos.(J) Quantification of mRNA per 0.1mg total RNA at 3.25 dpf from WT and log mutant embryos.(K) Normalized qPCR values for med14 expression in mutant relative to WT control embryos (∗p < 0.05 using the one-tailed unpaired Student’s t test).(L) Mean normalized expression (MNE) of β-actin in WT and MUT embryos at 2.25 dpf calculated using a universal reference approach. No significant difference (p = 0.63) was observed between WT (0.107 ± 0.00380) and log MUT (0.123 ± 0.0297) samples (n = 3) by one-tailed unpaired Student’s t test. For (J)–(L), three biological replicates of 450 (J) or 10 (K and L) embryos were used.

Mentions: To examine effects on Pol II transcription in log mutants, we first assayed expression of opsin1sw1, which is initiated in the eye at 2.5 dpf, after developmental defects are readily apparent (Figures 4A and 4B). Although delayed in log mutants (data not shown), opsin1sw1 expression reached a level comparable to WT by 4.0 dpf (Figures 4C and 4D). To test whether transcription could be induced in log mutants, we heat shocked log mutant hsp70:EGFP embryos at 3.5 dpf, after which a robust EGFP fluorescence was observable at 4.0 dpf (Figure 4E). To examine whether cellular transcription and function could continue in the prolonged absence of Med14 function, we carried out transplantation experiments to place transgenic (constitutive β-actin:EGFP) log mutant cells in WT host embryos, thus circumventing the issue of embryonic lethality. β-actin:EGFP expression in log mutant cells was evident at 15 dpf (Figure 4F, mutant cells seen in 100% of 50 transplants). Interestingly, cells were found to contribute to structures not present in mutant embryos, such as the semicircular canals. In fish aged up to 2 years, EGFP-+’ve log mutant cells persisted, as was evident in many nonpigmented tissues (Figures 4G–4H′). This clearly demonstrated that Med14 was not (at least cell autonomously) required for constitutive transcription or cell survival.


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)

Transcription Is Not Largely Affected in log (med14) Mutants(A–D) Expression of opn1sw1, initiated by 2.5 dpf in WT embryos, reaches WT levels in log mutant embryos by 4.0 dpf.(E) Tg(hsp70:EGFP) log mutant embryos heat shocked at 3.5 dpf show robust GFP signal.(F–H′) log mutant cells transplanted into WT hosts (traced using a β-actin:EGFP transgene) are evident in 15 dpf hosts (including in the semicircular canals, yellow arrowhead in F) and survive until 2 ypf (years post-fertilization, yellow arrowheads).(I) Summary of results of microarray analysis on cDNA from 2.25 dpf WT versus log mutant embryos.(J) Quantification of mRNA per 0.1mg total RNA at 3.25 dpf from WT and log mutant embryos.(K) Normalized qPCR values for med14 expression in mutant relative to WT control embryos (∗p < 0.05 using the one-tailed unpaired Student’s t test).(L) Mean normalized expression (MNE) of β-actin in WT and MUT embryos at 2.25 dpf calculated using a universal reference approach. No significant difference (p = 0.63) was observed between WT (0.107 ± 0.00380) and log MUT (0.123 ± 0.0297) samples (n = 3) by one-tailed unpaired Student’s t test. For (J)–(L), three biological replicates of 450 (J) or 10 (K and L) embryos were used.
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Related In: Results  -  Collection

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fig4: Transcription Is Not Largely Affected in log (med14) Mutants(A–D) Expression of opn1sw1, initiated by 2.5 dpf in WT embryos, reaches WT levels in log mutant embryos by 4.0 dpf.(E) Tg(hsp70:EGFP) log mutant embryos heat shocked at 3.5 dpf show robust GFP signal.(F–H′) log mutant cells transplanted into WT hosts (traced using a β-actin:EGFP transgene) are evident in 15 dpf hosts (including in the semicircular canals, yellow arrowhead in F) and survive until 2 ypf (years post-fertilization, yellow arrowheads).(I) Summary of results of microarray analysis on cDNA from 2.25 dpf WT versus log mutant embryos.(J) Quantification of mRNA per 0.1mg total RNA at 3.25 dpf from WT and log mutant embryos.(K) Normalized qPCR values for med14 expression in mutant relative to WT control embryos (∗p < 0.05 using the one-tailed unpaired Student’s t test).(L) Mean normalized expression (MNE) of β-actin in WT and MUT embryos at 2.25 dpf calculated using a universal reference approach. No significant difference (p = 0.63) was observed between WT (0.107 ± 0.00380) and log MUT (0.123 ± 0.0297) samples (n = 3) by one-tailed unpaired Student’s t test. For (J)–(L), three biological replicates of 450 (J) or 10 (K and L) embryos were used.
Mentions: To examine effects on Pol II transcription in log mutants, we first assayed expression of opsin1sw1, which is initiated in the eye at 2.5 dpf, after developmental defects are readily apparent (Figures 4A and 4B). Although delayed in log mutants (data not shown), opsin1sw1 expression reached a level comparable to WT by 4.0 dpf (Figures 4C and 4D). To test whether transcription could be induced in log mutants, we heat shocked log mutant hsp70:EGFP embryos at 3.5 dpf, after which a robust EGFP fluorescence was observable at 4.0 dpf (Figure 4E). To examine whether cellular transcription and function could continue in the prolonged absence of Med14 function, we carried out transplantation experiments to place transgenic (constitutive β-actin:EGFP) log mutant cells in WT host embryos, thus circumventing the issue of embryonic lethality. β-actin:EGFP expression in log mutant cells was evident at 15 dpf (Figure 4F, mutant cells seen in 100% of 50 transplants). Interestingly, cells were found to contribute to structures not present in mutant embryos, such as the semicircular canals. In fish aged up to 2 years, EGFP-+’ve log mutant cells persisted, as was evident in many nonpigmented tissues (Figures 4G–4H′). This clearly demonstrated that Med14 was not (at least cell autonomously) required for constitutive transcription or cell survival.

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