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

Abstract

Planarian adult stem cells (pASCs) or neoblasts represent an ideal system to study the evolution of stem cells and pluripotency as they underpin an unrivaled capacity for regeneration. We wish to understand the control of differentiation and pluripotency in pASCs and to understand how conserved, convergent or divergent these mechanisms are across the Bilateria. 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.

Keywords: Differentiation; Methyl binding domain; Pluripotency; Regeneration.

Fig. 1

mbd2/3 Is expressed in stem cells and the germline. (A) mbd2/3 is expressed cells throughout the parenchyma but absent from the pharynx (black asterisk) and anterior to the eyes (black arrows) and is lost by irradiation similar to the pASC marker gene Smedwi-2. Smed-GluR (glutamate receptor) expression is unaffected by irradiation. Smed-mbd2/3 is expressed in the germline, shown is expression in testes primordia of juvenile animals in paraffin sections (B) and in the testes whole mount in mature sexual animals (red arrows). Scale bars, 50 µm in (B) 500 µm. in (C). (D) Expression of mbd2/3 is absent from the regeneration blastema through 3 days of regeneration, but faint expression can be detected in 5 day old blastema and later the worms restore normal mbd2/3 distribution. Dashed lines show the boundary between old and new tissue. Scale bar, 500 µm.

Fig. 2

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.

Fig. 3

mbd2/3(RNAi) Leads to disruption of tissue homeostasis. Most mbd2/3(RNAi) animals start to regress anterior regions by 2 weeks varying from mild to more extreme phenotypes (A). Head regression correlates with loss of cephalic ganglia (B) and the anterior gut branches (C) and (D). At 3 weeks almost all animals have completely lost anterior regions (red arrow-head), although a few display only mild anterior regression (E). By three weeks posterior gut branches have also started to be lost (F)–(H). While gut branches (blue arrows in (G)) and the pharynx (stars in (H) and (J)) are lost, the VNCs (white arrows in (J)) are maintained (F)–(H) and (J). Animals at 4 weeks have regressed further, but are mostly still alive (I). Scale bars 500 µm, except in C and G 200 µm. Panels in G represent regions in white boxes depicted in panel F.

Fig. 4

mbd2/3(RNAi) Does not affect pASC maintenance or proliferation. (A) The distribution of pASCs assessed by Smedwi-2 expression found to be consistent between mbd2/3(RNAi) animals and their controls during homeostasis even in animals with regressed anterior regions. Smed-H2B expressing cells are also maintained in regenerating and animals undergoing normal homeostasis (B) and (C). Numbers of Smed-H2B expressing cells are not significantly different from controls in these animals (D). Numbers of mitotic cells are not significantly different from controls during regeneration or homeostasis, except at 5 days of regeneration (E) and (F). Scale bars 500 µm, accept in (C) and (D) 100 µm. ^⁎ Indicates p<0.05, determined by Student's t-test. Standard error bars are shown.

Fig. 5

mbd2/3(RNAi) Leads to an accumulation of NB.21.11e⁺ cells and a decline Smed-AGAT-1⁺ during regeneration. Double FISH on middle pieces shows maintenance of Smed-NB.21.11e⁺ cells and loss of Smed-AGAT-1⁺ cells during regeneration in mbd2/3(RNAi) animals (A). Anterior to the left. Scale bar, 200 µm. mbd2/3(RNAi) leads a significant increase in NB.21.11e⁺ cells and a significant decrease in Smed-AGAT-1⁺ cells (B), which is quantification of (A); n=3. Standard error bars are shown. ^⁎ Indicates p<0.05. Accumulation of NB.21.11e⁺ cells and a decline in AGAT-1⁺ cells is apparent throughout regenerating head, middle and tail pieces (C) and (D) Scale bar 500 µm. RT-qPCR analyses of both progeny marker transcripts correlates with the accumulation and loss of early and late stem cell progeny (E). The alternate progeny marker Smed-CYP1A1-1 is also depleted during regeneration by mbd2/3(RNAi) during regeneration (F). Scale bar, 200 µm.

Fig. 6

mbd2/3(RNAi) Leads to an accumulation of NB.21.11e⁺ cells and a decline Smed-AGAT-1⁺ during homeostasis. Smed-NB.21.11e expressing early progeny cells during homeostasis are maintained and they accumulate at sites of anterior regression (black arrow) in mbd2/3(RNAi) animals (A). Reduction in the number of Smed-AGAT-1 expression late pASC progeny cells during tissue homeostasis in mbd2/3(RNAi) animals, with loss in an anterior to posterior direction (B). Scale bars, 500 µm. (C) Double FISH on intact animals during homeostasis to visualize neoblast progeny cell formation. +: eyes. Scale bar, 200 µm. Quantification of (C) with n=3. ^⁎⁎ Indicates p<0.01 as determined by Student's t-test. (D) The alternate early progeny marker Smed-NB.32.1g is also maintained while the alternate late progeny marker Smed-CYP1A1-1 is depleted (E). Scale bars, 500 µm.

Fig. 7

Lack of evidence for a role for DNA methylation in pASC biology Smed-dnmt-2 is expressed in the germ line and germ line stem cells (A) Scale bars, 500 µm. gDNA digestion with the methylation dependent restriction enzyme McrBC does not indicate any DNA cytosine methylation in the Schmidtea mediterranea genome as compared with that of humans. −: no enzyme; +: with enzyme (B). Control chromatogram after HPLC-MS displaying the sensitivity of nucleosides detection, in particular for 5^mC. (C) A representative chromatogram of S. mediterranea nucleoside analysis indicating the lack of 5^mC (D).