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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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Smed-med14 Is Required for Tissue Homeostasis and Regeneration in Planarians(A–F) med14(RNAi) animals show defects in homeostasis, with head regression (yellow arrowheads) and ventral curling apparent in 100% of cases by 3fd10.(G and H) By 3fd12–15, head and tail regression (yellow and red arrowheads) and the beginnings of lysis are apparent in 100% of med14(RNAi) animals, similar to what is observed following irradiation-mediated depletion of the stem cell pool.(I–L) In trunk fragments that are regenerating a head (yellow arrowhead) and a tail (red arrowhead), no regeneration is observed by 7 dpa in med14(RNAi) animals.Scale bars, 100 μm.
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fig5: Smed-med14 Is Required for Tissue Homeostasis and Regeneration in Planarians(A–F) med14(RNAi) animals show defects in homeostasis, with head regression (yellow arrowheads) and ventral curling apparent in 100% of cases by 3fd10.(G and H) By 3fd12–15, head and tail regression (yellow and red arrowheads) and the beginnings of lysis are apparent in 100% of med14(RNAi) animals, similar to what is observed following irradiation-mediated depletion of the stem cell pool.(I–L) In trunk fragments that are regenerating a head (yellow arrowhead) and a tail (red arrowhead), no regeneration is observed by 7 dpa in med14(RNAi) animals.Scale bars, 100 μm.

Mentions: To further decipher Med14 function, we pursued med14 knockdown in the freshwater planarian S. mediterranea. A BLAST search of S. mediterranea genome and transcriptomes (Labbé et al., 2012) with both human and zebrafish Med14 sequences revealed a single planarian ortholog, Smed-med14 (med14 in this manuscript). When intact planarians were subjected to med14 RNAi, 100% of animals displayed a ventral curling phenotype by 10 days after the third feeding (3fd10) (Figures 5A–5D). By 3fd15, head and tail regression phenotypes became pronounced in med14(RNAi) animals (Figures 5E and 5F), with lysis of the epidermis following (Figure 5G), similar to what is observed following irradiation (Figure 5H). As ventral curling and lysis are hallmarks of a stem cell defect (Reddien et al., 2005), we next examined whether med14(RNAi) animals retained regenerative ability, which depends on stem cell function. Following amputation into thirds at 3fd3, regeneration was severely diminished at both 3 and 7 days post-amputation (dpa) in med14(RNAi) animals (Figures 5I–5L). As seen in zebrafish embryos, med14 appeared to be ubiquitously expressed in planarians (Figure 6A). However, dilution of probe and reduction of staining time resulted in a stem cell-like expression pattern (Figure 6A′), in agreement with transcriptome data showing med14 to be 6.7-fold enriched in stem cells over differentiated tissues (Labbé et al., 2012). Confirmation of med14 expression in (but not limited to) stem cells was obtained by confocal imaging of the head and tail regions following med14 fluorescent RNA ISH and PIWI (a marker of stem cells) antibody staining (Figures 6A′′ and 6A′′′).


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)

Smed-med14 Is Required for Tissue Homeostasis and Regeneration in Planarians(A–F) med14(RNAi) animals show defects in homeostasis, with head regression (yellow arrowheads) and ventral curling apparent in 100% of cases by 3fd10.(G and H) By 3fd12–15, head and tail regression (yellow and red arrowheads) and the beginnings of lysis are apparent in 100% of med14(RNAi) animals, similar to what is observed following irradiation-mediated depletion of the stem cell pool.(I–L) In trunk fragments that are regenerating a head (yellow arrowhead) and a tail (red arrowhead), no regeneration is observed by 7 dpa in med14(RNAi) animals.Scale bars, 100 μm.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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

fig5: Smed-med14 Is Required for Tissue Homeostasis and Regeneration in Planarians(A–F) med14(RNAi) animals show defects in homeostasis, with head regression (yellow arrowheads) and ventral curling apparent in 100% of cases by 3fd10.(G and H) By 3fd12–15, head and tail regression (yellow and red arrowheads) and the beginnings of lysis are apparent in 100% of med14(RNAi) animals, similar to what is observed following irradiation-mediated depletion of the stem cell pool.(I–L) In trunk fragments that are regenerating a head (yellow arrowhead) and a tail (red arrowhead), no regeneration is observed by 7 dpa in med14(RNAi) animals.Scale bars, 100 μm.
Mentions: To further decipher Med14 function, we pursued med14 knockdown in the freshwater planarian S. mediterranea. A BLAST search of S. mediterranea genome and transcriptomes (Labbé et al., 2012) with both human and zebrafish Med14 sequences revealed a single planarian ortholog, Smed-med14 (med14 in this manuscript). When intact planarians were subjected to med14 RNAi, 100% of animals displayed a ventral curling phenotype by 10 days after the third feeding (3fd10) (Figures 5A–5D). By 3fd15, head and tail regression phenotypes became pronounced in med14(RNAi) animals (Figures 5E and 5F), with lysis of the epidermis following (Figure 5G), similar to what is observed following irradiation (Figure 5H). As ventral curling and lysis are hallmarks of a stem cell defect (Reddien et al., 2005), we next examined whether med14(RNAi) animals retained regenerative ability, which depends on stem cell function. Following amputation into thirds at 3fd3, regeneration was severely diminished at both 3 and 7 days post-amputation (dpa) in med14(RNAi) animals (Figures 5I–5L). As seen in zebrafish embryos, med14 appeared to be ubiquitously expressed in planarians (Figure 6A). However, dilution of probe and reduction of staining time resulted in a stem cell-like expression pattern (Figure 6A′), in agreement with transcriptome data showing med14 to be 6.7-fold enriched in stem cells over differentiated tissues (Labbé et al., 2012). Confirmation of med14 expression in (but not limited to) stem cells was obtained by confocal imaging of the head and tail regions following med14 fluorescent RNA ISH and PIWI (a marker of stem cells) antibody staining (Figures 6A′′ and 6A′′′).

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