Long-range neural and gap junction protein-mediated cues control polarity during planarian regeneration

Abstract

Having the ability to coordinate the behavior of stem cells to induce regeneration of specific large-scale structures would have far-reaching consequences in the treatment of degenerative diseases, acute injury, and aging. Thus, identifying and learning to manipulate the sequential steps that determine the fate of new tissue within the overall morphogenetic program of the organism is fundamental. We identified novel early signals, mediated by the central nervous system and 3 innexin proteins, which determine the fate and axial polarity of regenerated tissue in planarians. Modulation of gap junction-dependent and neural signals specifically induces ectopic anterior regeneration blastemas in posterior and lateral wounds. These ectopic anterior blastemas differentiate new brains that establish permanent primary axes re-established during subsequent rounds of unperturbed regeneration. These data reveal powerful novel controls of pattern formation and suggest a constructive model linking nervous inputs and polarity determination in early stages of regeneration.

FIGURE 1. Neural cues mediate polarity during regeneration

(A) Schematic of transverse amputations in the presence of octanol. The incidence of bipolar head phenotype gradually increases toward posterior areas. Inset is representative of bipolar-head regenerate (white arrows indicate heads). Red dotted lines indicate level of amputation. Data are presented as mean +/− confidence intervals (95%). (B) Schematic representation of transverse amputations along the A/P axis to obtain fragments with a pre-existing head (left). Fragments generated after amputations exhibited very few bipolar-head regenerate. (C) Planarians were dissected in different ways (illustrative representation is shown for each case within columns) and the regeneration pattern was recorded after 2 weeks of regeneration. Animals reestablishing the original pattern were considered normal, while worms developing abnormalities were classified into seven categories by macroscopic observations, A/P polarity, and CNS morphological abnormalities. Seven abnormalities were: middle head; side head; bipolar + middle head; side protrusion; bipolar head and double side head (color-coded at the top of the graph). Percentage of animals regenerating each pattern is displayed along with the number of animals (within parentheses) assayed in each condition (Roman numbers). Untreated animals always regenerated normally (not shown for simplicity). Most cases (3/4) where dissections did not disrupt VNC integrity at pre-pharyngeal level regenerated 100% normal animals (conditions I, II, VII and X). Conversely, almost all cases (7/8) where VNC integrity was disrupted in pre or post-pharyngeal area led to regeneration of abnormalities characterized by alterations in CNS patterning and polarity (conditions III-IX). These results suggest that VNC integrity and GJC play important role in regenerative patterning and polarity. Since all animals regenerated tissues and developed blastemas, treatment with GJ inhibitor does not alter normal response to wound damage but rather instructively influences the identity of the newly formed tissue. Importantly only animals with disrupted VNC integrity and octanol treated led to novel phenotypes (D) characterized by multiple heads (white arrows) and pharynxes (gray arrows). Representative images of immunostaining with anti-synapsin antibody (green signal) over a pseudocolored red background are shown. In all cases original anterior end is to the top. Bars represent 200μm.

FIGURE 2. CNS-mediated inputs and GJC are required early during phases of regeneration

(A) The percentage of bipolar-head regeneration was recorded from post-pharyngeal fragments in which octanol treatment began at different time points post-amputation. The incidence of bipolar-head phenotype was sharply reduced when GJ blocker (octanol) treatment began a few hours post-amputation, suggesting that critical decisions to identify missing parts and set A/P polarity take place early (within first 3-6 hours) during regeneration. (B) Planarians were amputated at post-pharyngeal level, treated with octanol, and subjected to disruption of VNC at anterior areas (red dotted line in pre-pharyngeal area) at different time points after initial post-pharyngeal cut (time 0). Significant A/P duplications were observed when VNC contiguity was disrupted prior to 12 hours after amputation. Numbers in parenthesis indicate the sequence of amputations, being first post-pharyngeal and then VNC disruption at pre-pharyngeal level; all animals were treated with octanol. For each time point n≥ 10 animals. In all cases data are represented as mean +/− confidence intervals (95%) and curve fitting is shown with dotted line.

FIGURE 3. Loss-of-function by RNAi of three innexin genes led to A/P polarity phenotype during homeostasis and regeneration

(A) Microinjection schedule of double-stranded RNA (dsRNA) in intact planarians. A total of five injections (arrows between green squares) were performed over 19 days. Injecting a mix (1:1) of Dj-Inx-5 + 13 dsRNA for 3 consecutive days and 2 injections of Dj-Inx-12 ds-RNA 2 weeks after first injections led to strong and reproducible phenotypes ~14 days after last injection. Control animals were always run in parallel and injected with water under same schedule. (B) Representative phenotypes following 30-35 days since first injection. Conditions: control (water injected) and Dj-Inx-5 + 13, −12(RNAi). Control animals (left column) did not display any behavioral or anatomical abnormalities as assayed by markers of CNS (anti-synapsin antibody), mitotic cells (anti-H3P antibody), proliferative neoblasts (Dj-Inx-11 antisense probe), and digestive system (Dj-Inx-7 antisense probe). In contrast, animals subjected to Dj-Inx-5 + 13, −12(RNAi) displayed obvious behavioral and morphological changes (right column). Random contractions, ectopic photoreceptor pigmentation (yellow arrow), and extra pharynxes (blue arrows) were evident, especially in post-pharyngeal areas. Ectopic pharynxes, brain tissue (white arrows) and striking alterations in the distribution of neoblasts and the digestive system were observed. Remarkably, the orientation of ectopic brain and pharynx were sometimes reversed with respect to the original A/P axis, and the distribution of neoblasts and digestive system adopted anatomical changes consistent with the new structures developed (e.g.: no mitotic activity surrounding brain and pharynx and formation of primary intestinal branch in post pharyngeal area followed by posterior bifurcation around ectopic pharynx). Bar is 500 μm. (C) Microinjection dsRNA schedule for worms amputated (red arrow) after ~25-28 days of first injection. (D) To analyze the roles of Dj-Inx-5 + 13, −12(RNAi) during regeneration, injected animals were amputated at different levels along the A/P axis producing 5 fragments. Pharyngeal and post-pharyngeal fragments regenerated animals with polarity problems (inset representative picture). Data are represented as mean +/− confidence intervals (95%) and red dotted line indicates plane of amputation. (E) Summary illustration of phenotypes obtained in intact and regenerating of Dj-Inx-5 + 13, −12(RNAi) animals. In both cases, polarity problems and ectopic organs appeared (as after pharmacological inhibition of GJs). The original anterior ends are shown to the left.

FIGURE 4. GJ-mediated and neural information prevents ectopic and permanent axes formation in lateral and posterior-facing wounds

(A) Post-pharyngeal fragments were cut laterally (once or twice simultaneously) in the presence of octanol or not (control, untreated). In all cases (n>90) treated animals developed A/P polarity problems; when both lateral sides were simultaneously cut (schematics and synaptotagmin immunostaining are shown under graph), the fragment produced bipolar or triple heads. Additionally, lateral amputations disrupting the VNC led some animals (~20%, n=38) to produce anterior structures in all 4 blastemas. Note that untreated (control) animals mostly regenerated single headed worms; in some cases where head duplication was observed, this never affected the polarity of the animal. (B) Representative images of animals with single, bipolar, triple, and quadruple heads (white arrows). Triple immunostaining (CNS, mitotic cells, and visual neurons) in quadruple-headed planarians revealed that additional brains were linked by common VNCs (n>10 each). Further, immunostaining in with anti-H3P antibody (recognizes mitotic cells) indicated that mitotic cells were restricted to the new pre-pharyngeal area of each new axis. Visual neurons connecting photoreceptors with brain (assayed by anti-VC-1 antibody) also differentiated within each new brain. Bars are 500 μm.

FIGURE 5. Morphogenetic changes persist for several rounds of regeneration in absence of GJ inhibitor

Schematic illustrating that short treatment with octanol (3 days, blue area) is able to induce morphogenetic changes that persist for weeks, and across multiple rounds of regeneration. Multi-headed worms were obtained from post-pharyngeal regenerating fragments amputated in presence of octanol. The period of time in which animals were in fresh water is depicted with gray background. About one month after first amputation, multiheaded worms were amputated in different planes (transversally or longitudinally, dotted lines) along the A/P axis in plain water (in absence of octanol). Transverse amputations produced single or multiple simultaneous decapitations and longitudinal cuts divided bipolar worms in two mirror images fragments. In all cases, the ectopic heads were regenerated reconstituting multiple headed worms; from longitudinal cuts, two double-headed worms were formed. Total number of decapitations are represented within parentheses. This procedure was repeated two more times and morphogenetic changes persisted (not shown).

FIGURE 6. Schematic model of GJ-mediated and neural signals during regeneration

(A) Post-pharyngeal amputation generates a regenerating worm with pre-existing head and a posterior wound. Our data suggest that long-range signals –informing about the presence of a head within the fragment-travel from anterior (brain in yellow) to the posterior wound (white arrowheads) through two pathways: ventral nerve (VNC, red line) and GJ-mediated signals associated with VNC (blue line) and parenchymatic cells (light blue background). In all, untreated animals or animals exposed to GJ blocker (octanol) and animals with VNC disruption regeneration of the missing posterior area is recreated without polarity problems. However, in animals where both octanol treatment and VNC disruption take place, signals coming from anterior areas are disrupted, leading to abnormalities in polarity (bipolar animals). (B) Blastema fate determination in regenerating post-pharyngeal fragments with anterior and posterior-facing wounds. Instructive signals from anterior wound travel to posterior wound through the same pathways as in “A” but in this case the information carried is to inform about the anterior blastema formation that instruct neoblasts to form a posterior blastema. In animals exposed to octanol this information is altered and animals regenerate bipolar heads. In the case of VNC disruption alone most animals regenerate without problems. If both VNC are disrupted and GJ are inhibited, animals regenerate four heads. Specific cuts for VNC disruption are represented for each case.