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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 Necessary for the Maintenance of Adult Stem Cells(A) ISH for med14 in wild-type intact animals showing ubiquitous staining.(A′) A stem cell like expression pattern is evident with reduced staining.(A′′ and A′′′) Confocal image at 25× magnification of med14 fluorescent RNA ISH (red) and PIWI antibody staining (green) in the planarian head (white dashed box in A′) and tail (black dashed box in A′) respectively. The boxed area in each is enlarged for clarification. med14 is expression in, but not limited to, the stem cell population.(B–C′′′) ISH analysis using a stem cell specific riboprobe (piwi-1) during a time course of med14(RNAi). By 3fd12, the stem cell population is largely absent in med14(RNAi) animals. The remaining piwi-1+ cells at 3fd12 (C′′′) may represent primordial germ cells (red arrowheads in M).(D–E′) Loss of proliferative phosphorylated histone H3 (H3P) +’ve cells in med14(RNAi) animals by 3fd9 (E′).(F–I) Expression of S-phase markers h2b and pcna in WT and med14 RNAi animals at 3fd12.(J–K′) By 3fd3, the progenitor cell population in med14(RNAi) animals (marked by prog-1 expression) is reduced compared with controls and completely absent by 3fd12.(L–M′) Increased cell death by 3fd9 as observed by whole-mount TUNEL analysis in med14(RNAi) animals.(N–Y) Normal expression of markers of differentiated cell types in med14(RNAi) animals as evident for the nervous system (pc2), gut (porcn), muscle (collagen), pharynx (laminin), eyes (ovo), and protonephridia (cavii-1). Head regression is evident in some treated worms (black arrow heads in O and Q). Eye progenitors (red arrow head in V) are not observed in med14(RNAi) animals despite ovo expression in the eye spots (black arrow heads in V and W).Scale bars, 100 μm. See also Figure S2.
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fig6: Smed-med14 Is Necessary for the Maintenance of Adult Stem Cells(A) ISH for med14 in wild-type intact animals showing ubiquitous staining.(A′) A stem cell like expression pattern is evident with reduced staining.(A′′ and A′′′) Confocal image at 25× magnification of med14 fluorescent RNA ISH (red) and PIWI antibody staining (green) in the planarian head (white dashed box in A′) and tail (black dashed box in A′) respectively. The boxed area in each is enlarged for clarification. med14 is expression in, but not limited to, the stem cell population.(B–C′′′) ISH analysis using a stem cell specific riboprobe (piwi-1) during a time course of med14(RNAi). By 3fd12, the stem cell population is largely absent in med14(RNAi) animals. The remaining piwi-1+ cells at 3fd12 (C′′′) may represent primordial germ cells (red arrowheads in M).(D–E′) Loss of proliferative phosphorylated histone H3 (H3P) +’ve cells in med14(RNAi) animals by 3fd9 (E′).(F–I) Expression of S-phase markers h2b and pcna in WT and med14 RNAi animals at 3fd12.(J–K′) By 3fd3, the progenitor cell population in med14(RNAi) animals (marked by prog-1 expression) is reduced compared with controls and completely absent by 3fd12.(L–M′) Increased cell death by 3fd9 as observed by whole-mount TUNEL analysis in med14(RNAi) animals.(N–Y) Normal expression of markers of differentiated cell types in med14(RNAi) animals as evident for the nervous system (pc2), gut (porcn), muscle (collagen), pharynx (laminin), eyes (ovo), and protonephridia (cavii-1). Head regression is evident in some treated worms (black arrow heads in O and Q). Eye progenitors (red arrow head in V) are not observed in med14(RNAi) animals despite ovo expression in the eye spots (black arrow heads in V and W).Scale bars, 100 μm. See also Figure S2.

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 Necessary for the Maintenance of Adult Stem Cells(A) ISH for med14 in wild-type intact animals showing ubiquitous staining.(A′) A stem cell like expression pattern is evident with reduced staining.(A′′ and A′′′) Confocal image at 25× magnification of med14 fluorescent RNA ISH (red) and PIWI antibody staining (green) in the planarian head (white dashed box in A′) and tail (black dashed box in A′) respectively. The boxed area in each is enlarged for clarification. med14 is expression in, but not limited to, the stem cell population.(B–C′′′) ISH analysis using a stem cell specific riboprobe (piwi-1) during a time course of med14(RNAi). By 3fd12, the stem cell population is largely absent in med14(RNAi) animals. The remaining piwi-1+ cells at 3fd12 (C′′′) may represent primordial germ cells (red arrowheads in M).(D–E′) Loss of proliferative phosphorylated histone H3 (H3P) +’ve cells in med14(RNAi) animals by 3fd9 (E′).(F–I) Expression of S-phase markers h2b and pcna in WT and med14 RNAi animals at 3fd12.(J–K′) By 3fd3, the progenitor cell population in med14(RNAi) animals (marked by prog-1 expression) is reduced compared with controls and completely absent by 3fd12.(L–M′) Increased cell death by 3fd9 as observed by whole-mount TUNEL analysis in med14(RNAi) animals.(N–Y) Normal expression of markers of differentiated cell types in med14(RNAi) animals as evident for the nervous system (pc2), gut (porcn), muscle (collagen), pharynx (laminin), eyes (ovo), and protonephridia (cavii-1). Head regression is evident in some treated worms (black arrow heads in O and Q). Eye progenitors (red arrow head in V) are not observed in med14(RNAi) animals despite ovo expression in the eye spots (black arrow heads in V and W).Scale bars, 100 μm. See also Figure S2.
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fig6: Smed-med14 Is Necessary for the Maintenance of Adult Stem Cells(A) ISH for med14 in wild-type intact animals showing ubiquitous staining.(A′) A stem cell like expression pattern is evident with reduced staining.(A′′ and A′′′) Confocal image at 25× magnification of med14 fluorescent RNA ISH (red) and PIWI antibody staining (green) in the planarian head (white dashed box in A′) and tail (black dashed box in A′) respectively. The boxed area in each is enlarged for clarification. med14 is expression in, but not limited to, the stem cell population.(B–C′′′) ISH analysis using a stem cell specific riboprobe (piwi-1) during a time course of med14(RNAi). By 3fd12, the stem cell population is largely absent in med14(RNAi) animals. The remaining piwi-1+ cells at 3fd12 (C′′′) may represent primordial germ cells (red arrowheads in M).(D–E′) Loss of proliferative phosphorylated histone H3 (H3P) +’ve cells in med14(RNAi) animals by 3fd9 (E′).(F–I) Expression of S-phase markers h2b and pcna in WT and med14 RNAi animals at 3fd12.(J–K′) By 3fd3, the progenitor cell population in med14(RNAi) animals (marked by prog-1 expression) is reduced compared with controls and completely absent by 3fd12.(L–M′) Increased cell death by 3fd9 as observed by whole-mount TUNEL analysis in med14(RNAi) animals.(N–Y) Normal expression of markers of differentiated cell types in med14(RNAi) animals as evident for the nervous system (pc2), gut (porcn), muscle (collagen), pharynx (laminin), eyes (ovo), and protonephridia (cavii-1). Head regression is evident in some treated worms (black arrow heads in O and Q). Eye progenitors (red arrow head in V) are not observed in med14(RNAi) animals despite ovo expression in the eye spots (black arrow heads in V and W).Scale bars, 100 μm. See also Figure S2.
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