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Planarian MBD2/3 is required for adult stem cell pluripotency independently of DNA methylation.

Jaber-Hijazi F, Lo PJ, Mihaylova Y, Foster JM, Benner JS, Tejada Romero B, Chen C, Malla S, Solana J, Ruzov A, Aziz Aboobaker A - Dev. Biol. (2013)

Bottom Line: Here we show the planarian methyl-CpG Binding Domain 2/3 (mbd2/3) gene is required for pASC differentiation during regeneration and tissue homeostasis.The genome does not have detectable levels of 5-methylcytosine (5(m)C) and we find no role for a potential DNA methylase.We conclude that MBD proteins may have had an ancient role in broadly controlling animal stem cell pluripotency, but that DNA methylation is not involved in planarian stem cell differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Zoology, Tinbergen Building, South Parks Road, University of Oxford, Oxford OX1 3PS, United Kingdom.

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mbd2/3(RNAi) Leads to the disruption of various differentiated tissues during regeneration. (A) mbd2/3(RNAi) effect at 7 days of regeneration showing defects in pharynx (white asterisk) and eye (white arrows) formation. (B)–(F) mbd2/3(RNAi) affects the formation of the eyes (B), pharynx (C), anterior and posterior gut branches (black arrow heads) (D) and (E), brain ganglia (F) and (G); yellow arrows in (G), cells in and around the CNS (H) and secretory gland cells around the pharynx (I) during regeneration. However, the VNCs (G), blue arrows), cilia and secretory cell types are formed during regeneration. White dashed lines represent the boundary between the old and new tissue. Immunostaining was performed in (B), (E), (G) and (J), in situ hybridisation in (C), (D), (F), (H) and (I). 3C11: anti-SYNORF-1.
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f0010: mbd2/3(RNAi) Leads to the disruption of various differentiated tissues during regeneration. (A) mbd2/3(RNAi) effect at 7 days of regeneration showing defects in pharynx (white asterisk) and eye (white arrows) formation. (B)–(F) mbd2/3(RNAi) affects the formation of the eyes (B), pharynx (C), anterior and posterior gut branches (black arrow heads) (D) and (E), brain ganglia (F) and (G); yellow arrows in (G), cells in and around the CNS (H) and secretory gland cells around the pharynx (I) during regeneration. However, the VNCs (G), blue arrows), cilia and secretory cell types are formed during regeneration. White dashed lines represent the boundary between the old and new tissue. Immunostaining was performed in (B), (E), (G) and (J), in situ hybridisation in (C), (D), (F), (H) and (I). 3C11: anti-SYNORF-1.

Mentions: We performed mbd2/3(RNAi) and checked the efficacy with qRT-PCR (Supplementary Fig. 2A–C). We observed mbd2/3 transcripts in mbd2/3(RNAi) animals levels were reduced to between 2 and 9% of control(RNAi) levels. A summary of phenotypic progression during both regeneration and homeostasis is presented in Supplementary Tables 5 and 6. We found that animals were able to form both anterior and posterior blastemas (Fig. 2A, Supplementary Fig. 2D). However, both trunks (77%) and tail fragments (91%) failed to make any visible eyes after 7 days of regeneration (Fig. 2A, Supplementary Fig. 2D). After 14 days of regeneration some eye pigment cells were observed in 60% of middle pieces and 38% of tail pieces in the still un-pigmented head region, but in all cases these were greatly reduced (when present at all) compared to conrol(RNAi) controls. To confirm the specificity of this phenotype we also performed RNAi with a second region of the gene and observed the same phenotype (Supplementary Fig. 2E). We confirmed our observation of only partial eye regeneration by staining with the VC-1 monoclonal antibody, which labeled just small set of eye specific cells (7/15 regenerating middle pieces, no staining in remainder) in mbd2/3(RNAi) animals but labeled completely regenerated visual neurons in control(RNAi) animals (Fig. 2B).


Planarian MBD2/3 is required for adult stem cell pluripotency independently of DNA methylation.

Jaber-Hijazi F, Lo PJ, Mihaylova Y, Foster JM, Benner JS, Tejada Romero B, Chen C, Malla S, Solana J, Ruzov A, Aziz Aboobaker A - Dev. Biol. (2013)

mbd2/3(RNAi) Leads to the disruption of various differentiated tissues during regeneration. (A) mbd2/3(RNAi) effect at 7 days of regeneration showing defects in pharynx (white asterisk) and eye (white arrows) formation. (B)–(F) mbd2/3(RNAi) affects the formation of the eyes (B), pharynx (C), anterior and posterior gut branches (black arrow heads) (D) and (E), brain ganglia (F) and (G); yellow arrows in (G), cells in and around the CNS (H) and secretory gland cells around the pharynx (I) during regeneration. However, the VNCs (G), blue arrows), cilia and secretory cell types are formed during regeneration. White dashed lines represent the boundary between the old and new tissue. Immunostaining was performed in (B), (E), (G) and (J), in situ hybridisation in (C), (D), (F), (H) and (I). 3C11: anti-SYNORF-1.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3824064&req=5

f0010: mbd2/3(RNAi) Leads to the disruption of various differentiated tissues during regeneration. (A) mbd2/3(RNAi) effect at 7 days of regeneration showing defects in pharynx (white asterisk) and eye (white arrows) formation. (B)–(F) mbd2/3(RNAi) affects the formation of the eyes (B), pharynx (C), anterior and posterior gut branches (black arrow heads) (D) and (E), brain ganglia (F) and (G); yellow arrows in (G), cells in and around the CNS (H) and secretory gland cells around the pharynx (I) during regeneration. However, the VNCs (G), blue arrows), cilia and secretory cell types are formed during regeneration. White dashed lines represent the boundary between the old and new tissue. Immunostaining was performed in (B), (E), (G) and (J), in situ hybridisation in (C), (D), (F), (H) and (I). 3C11: anti-SYNORF-1.
Mentions: We performed mbd2/3(RNAi) and checked the efficacy with qRT-PCR (Supplementary Fig. 2A–C). We observed mbd2/3 transcripts in mbd2/3(RNAi) animals levels were reduced to between 2 and 9% of control(RNAi) levels. A summary of phenotypic progression during both regeneration and homeostasis is presented in Supplementary Tables 5 and 6. We found that animals were able to form both anterior and posterior blastemas (Fig. 2A, Supplementary Fig. 2D). However, both trunks (77%) and tail fragments (91%) failed to make any visible eyes after 7 days of regeneration (Fig. 2A, Supplementary Fig. 2D). After 14 days of regeneration some eye pigment cells were observed in 60% of middle pieces and 38% of tail pieces in the still un-pigmented head region, but in all cases these were greatly reduced (when present at all) compared to conrol(RNAi) controls. To confirm the specificity of this phenotype we also performed RNAi with a second region of the gene and observed the same phenotype (Supplementary Fig. 2E). We confirmed our observation of only partial eye regeneration by staining with the VC-1 monoclonal antibody, which labeled just small set of eye specific cells (7/15 regenerating middle pieces, no staining in remainder) in mbd2/3(RNAi) animals but labeled completely regenerated visual neurons in control(RNAi) animals (Fig. 2B).

Bottom Line: Here we show the planarian methyl-CpG Binding Domain 2/3 (mbd2/3) gene is required for pASC differentiation during regeneration and tissue homeostasis.The genome does not have detectable levels of 5-methylcytosine (5(m)C) and we find no role for a potential DNA methylase.We conclude that MBD proteins may have had an ancient role in broadly controlling animal stem cell pluripotency, but that DNA methylation is not involved in planarian stem cell differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Zoology, Tinbergen Building, South Parks Road, University of Oxford, Oxford OX1 3PS, United Kingdom.

Show MeSH
Related in: MedlinePlus