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. 2016 Aug 18;3(3):168-78.
doi: 10.1002/reg2.58. eCollection 2016 Jun.

The NuRD Complex Component p66 Suppresses Photoreceptor Neuron Regeneration in Planarians

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Free PMC article

The NuRD Complex Component p66 Suppresses Photoreceptor Neuron Regeneration in Planarians

Constanza Vásquez-Doorman et al. Regeneration (Oxf). .
Free PMC article

Abstract

Regeneration involves precise control of cell fate to produce an appropriate complement of tissues formed within a blastema. Several chromatin-modifying complexes have been identified as required for regeneration in planarians, but it is unclear whether this class of molecules uniformly promotes the production of differentiated cells. We identify a function for p66, encoding a DNA-binding protein component of the NuRD (nucleosome remodeling and deacetylase) complex, as well as the chromodomain helicase chd4, in suppressing production of photoreceptor neurons (PRNs) in planarians. This suppressive effect appeared restricted to PRNs because p66 inhibition did not influence numbers of eye pigment cup cells (PCCs) and decreased numbers of brain neurons and epidermal progenitors. PRNs from p66(RNAi) animals differentiated with some abnormalities but nonetheless produced arrestin+ projections to the brain. p66 inhibition produced excess ovo+otxA+ PRN progenitors without affecting numbers of ovo+otxA- PCC progenitors, and ovo and otxA were each required for the p66(RNAi) excess PRN phenotype. Together these results suggest that p66 acts through the NuRD complex to suppress PRN production by limiting expression of lineage-specific transcription factors.

Keywords: Differentiation; NuRD complex; eye regeneration; neoblasts; p66; photoreceptor neurons; planarian; stem cells.

Figures

Figure 1
Figure 1
p66 and the NuRD complex suppress formation of photoreceptor neurons (PRNs) in regeneration. (A)−(C) p66 RNAi prevented formation of visible eyes but caused production of excess PRNs without affecting numbers of pigment cup cells (PCCs). (A) Images of live animals treated with p66 RNAi regenerated unpigmented blastemas with apparently no eyes. (B) In situ hybridization to detect PRN marker opsin and PCC marker tyrosinase after the indicated treatments, with quantification of eye cell numbers and PRN/PCC ratio shown to the right. Error bars show standard deviations from at least 10 eyes for each condition and P values were computed from two‐tailed t tests. (C) In situ hybridization to detect PRN progenitor marker otxA and PCC progenitor marker sp6‐9 after the indicated treatments. (D)−(E) Attenuated chd4 RNAi causes production of supernumerary PRNs. chd4, encoding a core component of the NuRD complex, is known to be required for blastema formation and survival due to its necessity in producing progenitor cells of the epidermal cell lineage. To circumvent this requirement and investigate possible functions related to eye formation, animals were fed with a mixture of bacteria expressing chd4 (as indicated) and control dsRNA to produce weakened effects of chd4 inhibition. (D) Images of live animals treated with chd4 RNAi failed to regenerate (6/12 animals) or regenerated very small blastemas (6/12 animals). Attenuated chd4 RNAi dosing formed no eye pigmentation (13/15 50% chd4 animals, 12/16 25% chd4 animals), no blastema pigmentation and failed to regenerate tails, reminiscent of effects of p66 RNAi. (E) Animals treated with 100% chd4 dsRNA failed to form a blastema or eyes (10/16 animals), but those treated with 50% or 25% dsRNA formed 1–2 eyes with excess PRNs (5/11 did not regenerate, 4/11 one enlarged opsin+ cluster, 2/11 two eyes 50% chd4 animals; and 5/8 two enlarged opsin+ clusters and few tyro+ cells, 3/8 two eyes 25% chd4 animals). (A)−(E) Scoring shown in the lower left of the panels indicates the number of animals presenting each phenotype versus total number examined. Cartoons show surgeries (red) and enlarged regions (green). Anterior, top. Bars: 300 μm (A), 25 μm (B, C, E), or 500 μm (D).
Figure 2
Figure 2
p66 inhibition causes incomplete terminal differentiation. (A) Photoreceptors from p66(RNAi) produce processes that project toward the brain (8/12 animals), as determined by immunostaining with an anti‐arrestin antibody (VC‐1). p66(RNAi) animals had projections with normal orientations (8/12 animals), projections disoriented away from the brain (2/12 animals), or no projections (2/12 animals). (B) In situ hybridizations to examine the effect of p66 RNAi on expression of other genes marking differentiated PRNs. p66(RNAi) animals had strongly reduced expression of soxB, eye53‐1, and eye53‐2. White arrowheads indicate PRN expressing otxA and these factors. (C) Transverse views from z‐stack confocal projections showing ventral displacement and separation of PRNs with respect to PCCs in p66(RNAi) animals. (D) opsin+ PRNs from regenerating p66(RNAi) animals do not express neoblast marker smedwi‐1. p66(RNAi) animals showed a delay in PRN (opsin+ cells) regeneration; 4 days after head removal, p66(RNAi) animals presented no PRN (8/9 animals). Cartoons show surgeries (red) and enlarged regions (green). Anterior, top except in (C) (D, dorsal; V, ventral). Bars: 75 μm (B), 50 μm (C), or 25 μm (D).
Figure 3
Figure 3
p66 influences eye differentiation by limiting numbers of ovo+ and otxA+ eye progenitors. (A) In situ hybridization to detect ovo expression during regeneration after p66 or control RNAi, with quantification of cell numbers at days 2 and 3 shown on the right. Error bars are standard deviations and P values are computed from a two‐tailed t test. At least three animals were scored for each condition. Numbers of ovo+ cells are elevated early in regeneration by day 2–3. (B) Double FISH to detect ovo and otxA expression early in regeneration. otxA+ cells appear earlier in regeneration of p66(RNAi) versus control RNAi animals and otxA expression is stronger in p66 RNAi animals. Two days after head removal, control animals presented no or low ovo expression (5/8 animals), or two ovo+ cell clusters (3/8 animals); none of them expressed otxA. In contrast, p66(RNAi) animals presented several (3/8 animals) or many (5/8 animals) ovo+ cells, and some of these animals weakly expressed otxA (4/8 animals). (C) otxA or ovo RNAi suppresses the p66(RNAi) extra PRN phenotype. Animals were injected with pairwise combinations of control, p66, otxA, and ovo dsRNA as indicated for 3 days, then amputated to remove heads or tails, injected with dsRNA again 12 days later, then scored at day 8 for appearance of pigmented eyes, and stained by double FISH for expression of opsin and tyrosinase. Numbers of PRNs and PCCs were counted in at least 10 eyes and displayed in the boxplots. (D) Model of eye differentiation showing p66 and the NuRD complex as acting to suppress numbers of otxA+ ovo+ photoreceptor neuron progenitors and, consequently, numbers of mature PRNs. Cartoon shows surgeries (red) and enlarged regions (green). Anterior, top. Bars: 400 μm (A), 75 μm (B), 500 μm (C, left), or 100 μm (C, right).

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