1. Developmental Biology
  2. Microbiology and Infectious Disease
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Region-specific regulation of stem cell-driven regeneration in tapeworms

  1. Tania Rozario  Is a corresponding author
  2. Edward B Quinn
  3. Jianbin Wang
  4. Richard E Davis
  5. Phillip A Newmark  Is a corresponding author
  1. Morgridge Institute for Research, United States
  2. University of Colorado School of Medicine, United States
  3. Howard Hughes Medical Institute, United States
  4. University of Wisconsin–Madison, United States
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Cite this article as: eLife 2019;8:e48958 doi: 10.7554/eLife.48958

Abstract

Tapeworms grow at rates rivaling the fastest-growing metazoan tissues. To propagate they shed large parts of their body; to replace these lost tissues they regenerate proglottids (segments) as part of normal homeostasis. Their remarkable growth and regeneration are fueled by adult somatic stem cells that have yet to be characterized molecularly. Using the rat intestinal tapeworm, Hymenolepis diminuta, we find that regenerative potential is regionally limited to the neck, where head-dependent extrinsic signals create a permissive microenvironment for stem cell-driven regeneration. Using transcriptomic analyses and RNA interference, we characterize and functionally validate regulators of tapeworm growth and regeneration. We find no evidence that stem cells are restricted to the regeneration-competent neck. Instead, lethally irradiated tapeworms can be rescued when cells from either regeneration-competent or regeneration-incompetent regions are transplanted into the neck. Together, the head and neck tissues provide extrinsic cues that regulate stem cells, enabling region-specific regeneration in this parasite.

https://doi.org/10.7554/eLife.48958.001

eLife digest

Many worms have remarkable abilities to regrow and repair their bodies. The parasitic tapeworms, for example, can reach lengths of several meters and grow much more quickly than tissues in humans and other complex animals. This growth allows tapeworms to counteract the continual loss of the segments that make up their bodies, known as proglottids – a process that happens throughout their lives.

The capacity to regenerate thousands of lost body segments and maintain an overall body length suggests that tapeworms have groups of stem cells in their body which can grow and divide to produce the new body parts. Yet, regeneration in tapeworms has not been closely studied.

Rozario et al. have now examined Hymenolepsis diminuta, the rat tapeworm, and identified the neck of the tapeworm as crucial for its ability to regrow lost body segments. Further analysis identified two genes, zmym3 and pogzl, that are essential for cell division during tapeworm growth. However, Rozario et al. showed that these genes are active elsewhere in the worm’s body and that it is the conditions found specifically in the tapeworm’s neck that create the right environment for stem cells to enable regeneration of new segments.

Tapeworms provide a valuable example for studying the growth of stem cells and these findings highlight the important role that the cells’ surroundings play in driving stem cell activity. These findings could also lead to new insights into how stem cells behave in other animals and could potentially lead to new approaches to prevent or treat tapeworm infections.

https://doi.org/10.7554/eLife.48958.002

Introduction

Tapeworms are parasitic flatworms that infect humans and livestock, causing lost economic output, disease, and in rare cases, death (Del Brutto, 2013). These parasites are well known for their ability to reach enormous lengths. For example, humans infected with the broad or fish tapeworm, Diphyllobothrium latum, harbor parasites that average 6 m in length (Craig and Ito, 2007). It is less commonly appreciated that tapeworms can regenerate to accommodate their multi-host life cycle. Adult tapeworms in their host intestines develop proglottids (segments) that are gravid with embryos. Tapeworms pinch off the posterior and gravid sections of their body, which exit with the host excrement, to be eaten by a suitable intermediate host that supports larval tapeworm development. Despite losing large body sections, the tapeworm does not progressively shorten; instead, it regenerates proglottids, allowing the worms to maintain an equilibrium length. Despite this remarkable biology, tapeworms are an unexplored animal model in the study of regenerative behaviors.

Up to the 1980s the rat intestinal tapeworm, Hymenolepis diminuta, had been a favorite model organism among parasitologists. H. diminuta grows rapidly–within the first 15 days of infection, it produces up to 2200 proglottids, increases in length by up to 3400 times, and weight by up to 1.8 million times (Roberts, 1980)–and is easily propagated in the laboratory. Foundational work on their biochemistry, ultrastructure, and developmental biology enriched our understanding of these tapeworms (Arai, 1980). However, with the dawn of the molecular age and the rise of genetic model organisms, H. diminuta was essentially left behind. Here, we show that H. diminuta is an excellent, tractable model for the study of stem cells and regeneration, with the power to inform us about parasite physiology.

As an obligate endoparasite, adult H. diminuta will expire once its host rat dies. However, the lifespan of H. diminuta can be greatly increased via regeneration. A single adult tapeworm can be serially amputated and transplanted into a new host intestine, where the fragment can regenerate into a mature tapeworm even after 13 rounds of amputation over 14 years (Read, 1967). These observations have led to speculation that H. diminuta may be inherently immortal. This situation is reminiscent of the free-living cousins of tapeworms: freshwater planarians like Schmidtea mediterranea, which reproduce indefinitely by fission, and can regenerate their whole body from tiny fragments (Newmark and Sánchez Alvarado, 2002).

Planarian immortality and regeneration are enabled by adult somatic stem cells called neoblasts (Newmark and Sánchez Alvarado, 2002; Reddien, 2018; Baguñà, 2012). These stem cells are the only dividing undifferentiated cells within the soma. Like planarians, H. diminuta maintains a population of neoblast-like adult somatic stem cells (Roberts, 1980) that are likely responsible for their growth and regenerative ability. Recently, stem cells of multiple species of parasitic flatworms have been described (Collins et al., 2013; Koziol et al., 2014; Koziol et al., 2015; Wang et al., 2013; Koziol et al., 2010). Stem cells play crucial roles in parasite development, transmission, homeostasis, and even disease. For example, stem cells enable prolific reproduction and longevity (Collins, 2017), mediate host-parasite interactions (Collins et al., 2016), and allow metastatic parasite transmission in host tissues (Brehm and Koziol, 2014). How stem cells may regulate regeneration in parasites such as tapeworms is largely unexplored and the subject of this study.

We use H. diminuta, to investigate the molecular basis of tapeworm regeneration. We have established and refined experimental tools such as transcriptomics, in vitro parasite culture, whole-mount and fluorescent RNA in situ hybridization (WISH and FISH), cycling-cell tracing with thymidine analogs, RNA interference (RNAi), and cell transplantation, all described in this work. We determine that the ability to regenerate is regionally limited to the neck of adult H. diminuta. However, regeneration from the neck is finite without signals from the tapeworm head. Using RNA sequencing (RNA-seq), we identify and characterize various markers of the somatic cycling-cell population, which includes tapeworm stem cells. Using RNAi, we functionally validate molecular regulators of growth and regeneration. However, our analyses failed to uncover a neck-specific stem cell population that explains the regional regenerative ability displayed by H. diminuta. Instead, we show that cells from both regeneration-competent and regeneration-incompetent regions of H. diminuta have stem cell ability and can restore viability to lethally irradiated tapeworms. Our results show that extrinsic signals present in the tapeworm neck, rather than specialized stem cells, confer region-specific regenerative ability in this tapeworm.

Results

The anatomy of adult H. diminuta consists of a head with four suckers, an unsegmented neck, and a body with thousands of proglottids/segments that grow and mature in an anterior-to-posterior direction (Roberts, 1980; Rozario and Newmark, 2015) (Figure 1a). What regions of the tapeworm body are competent to regenerate? In order to test regeneration competency, it is necessary to grow tapeworms in vitro instead of in the intestine, where the suckers are required to maintain parasites in vivo. We established H. diminuta in vitro culture conditions modified from Schiller's method (Schiller, 1965) and tested the regeneration competence of 1 cm amputated fragments (Figure 1b–c). The anterior-most fragments (head+neck+body) were competent to regenerate, confirming in vivo observations using amputation and transplantation (Read, 1967; Goodchild, 1958). Anterior fragments that were first decapitated (neck+body) were also competent to regenerate. In contrast, ‘body only’ fragments failed to regenerate proglottids. All amputated fragments could grow in length (Figure 1d), differentiate mature reproductive structures, and mate. Despite the failure to regenerate, ‘body only’ fragments could grow because each existing proglottid increased in length as it progressively matured (Figure 1—figure supplement 1a–b). However, only fragments that retained the neck were able to regenerate new proglottids over time. The neck of 6-day-old tapeworms used in this study is typically 2–3 mm long when observed after DAPI staining and widefield fluorescent microscopy. By amputating 2 mm ‘neck only’ fragments, we find that the neck is sufficient to regenerate an average of 383 proglottids (SD = 138, N = 4, n = 20) after 12 days in vitro (Figure 1e). In no case did we observe head regeneration. Furthermore, amputated heads alone could not regenerate in vitro (Figure 1—figure supplement 1c) nor in vivo (Read, 1967). Thus, neither the head nor body can regenerate proglottids, but the neck is both necessary and sufficient for proglottid-specific regeneration in H. diminuta.

Figure 1 with 2 supplements see all
Regeneration competence of H. diminuta.

(a) Schematic of H. diminuta adults. (b) DAPI-stained 1 cm fragments grown in vitro. (c–d) Quantification of proglottid number and growth in length from (b). Error bars = SD, N = 2–5, n = 7–21; one-way ANOVA with Dunnett’s multiple comparison test, compared to day 0. (e) Representative DAPI-stained ‘neck only’ fragment regeneration. (f–g) 2 mm anterior fragments, with or without the head, grown in vitro for 12–15 days and then re-amputated serially. Error bars = SD, +head: one-way ANOVA with Tukey’s multiple comparison test, -head: Student’s t-test. (h–i) DAPI-stained 1 mm fragments from the anterior, middle, and posterior of the neck grown in vitro. Error bars = SD, N = 3, n = 22–29, one-way ANOVA with Tukey’s multiple comparison test.

https://doi.org/10.7554/eLife.48958.003

Previous in vivo studies have shown that H. diminuta can regenerate after serial rounds of amputation and transplantation for over a decade (Read, 1967) and perhaps indefinitely. Using in vitro culture, we confirmed that anterior fragments of H. diminuta can regenerate after at least four rounds of serial amputation (Figure 1f–g). Decapitated (-head) fragments regenerated proglottids after the first amputation; however, re-amputation abrogated regeneration (Figure 1f–g). After decapitation, a definitive neck could not be maintained and eventually, the whole tissue was comprised of proglottids (Figure 1—figure supplement 2). Without the head, proglottid regeneration from the neck is finite. Thus, while the neck is necessary and sufficient for proglottid regeneration, the head is required to maintain an unsegmented neck and for persistent regeneration.

If signals from the head regulate regeneration, is regenerative potential asymmetric across the anterior-posterior (A-P) axis of the neck? We subdivided the neck into three 1 mm fragments and found that the most-anterior neck fragments regenerated more proglottids than the middle or posterior neck fragments (Figure 1h–i). Thus, regeneration potential is asymmetric across the neck A-P axis with a strong anterior bias.

Since the neck is the only region competent to regenerate, are stem cells preferentially confined to the neck? In lieu of specific molecular markers for stem cells, we examined the distribution of all cycling cells in adult tapeworms. In flatworms, it has been repeatedly shown that the only proliferative somatic cells are undifferentiated cells with stem cell morphology and/or function; these cells have been termed neoblasts, adult somatic stem cells, or germinative cells, depending on the organism (Collins et al., 2013; Koziol et al., 2014; Baguñà et al., 1989; Newmark and Sánchez Alvarado, 2000; Ladurner et al., 2000). In H. diminuta, proliferation does not occur in regions comprised solely of differentiated cells (muscle and tegument/parasite skin at the animal edge) (Bolla and Roberts, 1971). Instead, proliferation is only detected in regions where undifferentiated cells with the typical morphology of stem cells can be distinguished (Bolla and Roberts, 1971; Sulgostowska, 1972). Thus, cycling somatic cells in H. diminuta would not include differentiated cells, but would include stem cells and any dividing progeny. To label cycling cells, we used two methods: (i) uptake of the thymidine analog F-ara-EdU (Neef and Luedtke, 2011) to mark cells in S-phase and (ii) FISH to detect cell cycle-regulated transcripts, such as the replication licensing factor minichromosome maintenance complex component 2 (mcm2) and histone h2b (h2b), which are conserved cycling-cell markers in free-living and parasitic flatworms (Collins et al., 2013; Solana et al., 2012). We detected cycling somatic cells throughout the tapeworm body (Figure 2a–b). Contrary to previous results (Bolla and Roberts, 1971), we also detected cycling cells in the head, though in small numbers (Figure 2a). The scarcity of these cells may be the reason they were originally missed. Taken together, cycling cells are present in all regions, regardless of regeneration competence.

Figure 2 with 2 supplements see all
Cycling somatic cells are distributed throughout the tapeworm body and are irradiation sensitive.

(a-b) Maximum-intensity projections of confocal sections showing distribution of cycling cells by 2 hr uptake of F-ara-EdU (a) or FISH for mcm2 (b). Fewer cycling cells were found in the head (box), while abundant cycling cells were observed in both somatic and gonadal tissues throughout the body. t = testis, o = ovary. (c) Maximum-intensity projections of tile-stitched confocal sections after 1 hr uptake of F-ara-EdU (green) 3 days post-irradiation. (d) Quantification of F-ara-EdU+ cell inhibition from (c). Error bars = SD, N = 2, n = 11 and 9, Student’s t-test. (e) RNA-seq strategy to identify genes expressed in cycling cells. (Nuclei are counterstained with DAPI (gray) in this and all subsequent figures.).

https://doi.org/10.7554/eLife.48958.006

To further our understanding of how tapeworm stem cells are distributed and regulated, we sought to identify stem cell markers. Stem cell genes have been discovered previously in flatworms by identifying transcripts downregulated after exposure to irradiation, which depletes cycling cells (Collins et al., 2013; Solana et al., 2012; Eisenhoffer et al., 2008). Exposing H. diminuta to 200 Gy x-irradiation reduced the number of cycling cells by 91 ± 6% after 3 days (Figure 2c–d) and abrogated both growth and regeneration (Figure 2—figure supplement 1a–b). This dosage is lethal; all fragments from worms exposed to 200 Gy x-irradiation degenerated after 1 month (Figure 2—figure supplement 1c–d). We leveraged the sensitivity of H. diminuta to lethal irradiation in order to identify new molecular markers of cycling somatic cells by RNA-seq (Figure 2e). A de novo transcriptome of 14,346 transcripts was assembled (see Materials and methods) to which sequencing reads were mapped. We identified 683 transcripts that were irradiation sensitive (downregulated; FDR ≤ 0.05) (Supplementary file 1a). Expression of irradiation-sensitive transcripts by WISH was indeed reduced after exposure to irradiation, validating our RNA-seq approach (Figure 2—figure supplement 2).

Two rounds of expression screening were then applied to hone in on cycling-cell transcripts from our irradiation-sensitive dataset (Figure 2e). The position of cycling cells in the neck is spatially restricted in a conserved pattern (Koziol and Castillo, 2011) (Figure 3a): cycling cells reside in the neck parenchyma bounded by the nerve cords and are absent from the animal edge where muscle and tegument are located (Bolla and Roberts, 1971). Among 194 irradiation-sensitive transcripts that displayed clear WISH patterns, 63% were expressed in the neck parenchyma, though in a variety of patterns (Figure 3—figure supplement 1). 13% showed similar patterns to h2b and mcm2 (Figure 3b–c, Figure 3—figure supplement 1b). These include the predicted nucleic acid binding factors Zn finger MYM type 3 (zmym3) and pogo transposable element with ZN finger domain-like (pogzl), as well as NAB co-repressor domain two superfamily member (nab2) and nuclear lamina component laminB receptor (lbr). 25% of irradiation-sensitive transcripts, were expressed in a minority of cells in the neck parenchyma (Figure 3—figure supplement 1c). 24% were expressed within the parenchyma and more broadly toward the animal edge (Figure 3—figure supplement 1d). The remainder represented transcripts expressed at segment boundaries or in differentiated tissues (Figure 3—figure supplement 1e–f). All transcripts that were expressed in the neck parenchyma were also found throughout the worm body, even in the most posterior proglottids (Figure 3—figure supplement 1b–c). In conclusion, irradiation-sensitive transcripts identified by RNA-seq likely represent markers for stem cells, progenitors, and even differentiated cells that were lost or compromised following irradiation.

Figure 3 with 3 supplements see all
Expression screening for cycling cell markers.

(a) Confocal section of a tapeworm anterior. Cycling cells (mcm2: magenta) in the neck parenchyma are between the nerve cords (cadherin: green). s: sucker, nc: nerve cord, oc: osmoregulatory canal, t: tegument, m: muscle, and p: parenchyma. (b) WISH of known cycling-cell markers h2b and mcm2. sc: scolex (head) and n: neck. (c) WISH for irradiation-sensitive transcripts expressed in the neck parenchyma. (d) Confocal sections of dFISH for irradiation-sensitive transcripts (green) with h2b or mcm2 (magenta) from neck parenchyma. Cyan arrowheads indicate cells magnified at the far right.

https://doi.org/10.7554/eLife.48958.009

To focus on transcripts with enriched expression in cycling cells, we performed double FISH (dFISH) with irradiation-sensitive candidates and either h2b or mcm2, which we used interchangeably as they are co-expressed in the neck parenchyma (Figure 3—figure supplement 2). After dFISH for 53 candidates, 72% of transcripts tested were co-expressed in cycling cells (Figure 3—figure supplement 3a, Supplementary file 1b). The irradiation-sensitive transcripts from Figure 3c were indeed colocalized in cycling somatic cells (Figure 3d). One transcript, the homeobox factor prospero (prox1), was expressed exclusively in a subset of cycling cells (Figure 3—figure supplement 3b). We confirmed that genes with expression that only partially overlapped in the neck parenchyma, such as the Zn finger-containing gene HDt_10981 and palmitoyl-protein thioesterase 1 (ppt1), were expressed in both cycling cells and non-cycling cells (Figure 3—figure supplement 3c). We propose that these genes likely represent lineage-committed stem cells or progenitors for tissues such as muscle, neurons, tegument, or protonephridia. 28% of irradiation-sensitive transcripts were predominantly expressed in non-cycling cells that were juxtaposed to cycling cells (Figure 3—figure supplement 3d). The transcriptional heterogeneity detected in the cycling-cell compartment is reminiscent of similar observations made in the regenerative planarian S. mediterranea (Reddien, 2018). A comparative analysis between verified tapeworm cycling-cell transcripts and their putative planarian homologs revealed a number of transcripts with conserved expression in cycling-cell populations from these distantly related flatworms (Supplementary file 1c) (see Discussion). In summary, our analysis revealed a heterogeneous and complex mixture of cell types or states in the neck parenchyma as well as within the cycling-cell population.

What role(s) do the newly identified cycling-cell genes play during regeneration? We performed RNAi of target genes, confirmed knockdown by quantitative PCR (Figure 4—figure supplement 1), and assayed for defects in growth and regeneration (Figure 4a). As a proof of principle, we knocked down h2b, which should compromise growth due to cycling cell loss, as observed in other flatworms (Collins et al., 2016; Solana et al., 2012). Knockdown of h2b, zmym3, and pogzl each resulted in diminished growth and regeneration (Figure 4b–c). The number of proglottids regenerated was also reduced, but could not be quantified as many RNAi worms were so thin and frail (Figure 4b) that proglottid definition was lost.

Figure 4 with 2 supplements see all
RNAi to identify genes required for growth and regeneration in H. diminuta.

(a) Schematic of RNAi paradigm. (b) DAPI-stained worms after RNAi knockdown of h2b, zmym3, and pogzl. (c) Quantification of worm lengths after RNAi. Error bars = SD, N = 3–4, n = 26–37, one-way ANOVA with Dunnett’s multiple comparison test compared to control. (d-e) Maximum-intensity projections (d) and quantification (e) of cycling-cell inhibition after 1 hr F-ara-EdU uptake following RNAi. Worms with degenerated necks were excluded from analysis. Error bars = SD, N = 3, n = 11–14, one-way ANOVA with Dunnett’s multiple comparison test compared to control. (f) mcm2 WISH on worm anteriors after RNAi. (g) WISH of zmym3 and pogzl sampled from anterior to posterior of adult 6-day-old worms. ga: genital anlagen; t: testis; o: ovary.

https://doi.org/10.7554/eLife.48958.013

Are these RNAi-induced failures in growth and regeneration due to defects in the cycling-cell population? RNAi knockdown of h2b, zmym3, and pogzl severely reduced the number of proliferative cells in the neck that could incorporate F-ara-EdU (Figure 4d–e). We also observed fewer mcm2+ cells after RNAi (Figure 4f). Taken together, these results indicate that the cycling-cell population is either lost or dysregulated. Therefore, h2b, zmym3, and pogzl are necessary for the maintenance and/or proper function of cycling cells, likely including stem cells, in H. diminuta.

Although we have identified heterogeneity within the cycling-cell population of the neck parenchyma and uncovered genes that are required for growth and regeneration, it remains unclear why regeneration competence is restricted to the neck. By WISH and FISH, all cycling-cell transcripts including zmym3 and pogzl were detected throughout the whole tapeworm body (Figure 4g, Figure 3—figure supplement 1b–c). In the tapeworm posterior, zmym3 and pogzl were expressed in gonadal tissues (which contain mitotic germ cells) but also in somatic cells within the parenchyma (Figure 4—figure supplement 2). If zmym3 and pogzl mark stem cells, this suggests that stem cells reside even in posterior tissues that are not competent to regenerate. Since zmym3 and pogzl label all cycling cells, it is possible that stem cells of limited potential exist in the posterior, but an elusive subpopulation of pluripotent stem cells is confined to the neck.

Since we observed an anterior bias in regenerative ability (Figure 1h–i), we hypothesized that RNA-seq may reveal an anteriorly biased stem cell distribution that may point us to a pluripotent stem cell subpopulation. Thus, we performed RNA-seq of 1 mm anterior, middle, and posterior neck fragments (Figure 1h), and identified 461 anterior-enriched and 241 anterior-depleted transcripts (Supplementary file 1d). By WISH, anterior-enriched and anterior-depleted transcripts were often detected in corresponding gradients (Figure 5a), but in patterns that were excluded from the neck parenchyma. When we overlaid the anterior-enriched and -depleted datasets with our irradiation-sensitive dataset, the majority of anterior-enriched transcripts (88%) were not irradiation sensitive (Figure 5b). Our results suggest that the A-P polarized signals across the neck region arise predominantly within the non-cycling compartments.

RNA-seq identifies anterior-enriched transcripts that are expressed predominantly in non-cycling cells.

(a) WISH of tapeworm anteriors for transcripts that were anterior-enriched (FC ≥1.5, FDR ≤ 0.05) or -depleted (FC ≤−1.5, FDR ≤ 0.05) by RNA-seq. Panels oriented anterior facing left. (b) Differential gene expression analyses of 1 mm anterior, middle, and posterior neck fragments overlaid with irradiation-sensitive transcripts. (c) WISH of transcripts that were anterior-enriched and irradiation-sensitive by RNA-seq that showed expression in a subset of cells in the neck parenchyma. (d) Confocal sections from dFISH of anterior-enriched transcripts (green) and mcm2 (magenta). Cyan arrowheads indicate cells that are magnified at the far right.

https://doi.org/10.7554/eLife.48958.016

Since our RNA-seq analysis identified 57 transcripts that were anterior enriched and irradiation sensitive, we examined expression patterns within this category. We found 15 transcripts expressed in a subset of cells within the neck parenchyma (Figure 5c) and initially hypothesized that these transcripts may represent subsets of stem cells. We were able to successfully test eight candidates by dFISH with cycling-cell markers and found that the majority (7/8) were not expressed in cycling cells (Figure 5d, Supplementary file 1b). Only prox1 was co-expressed in cycling cells (Figure 3—figure supplement 3b). At present, the identity and function of prox1+ cells is unknown. Furthermore, prox1 is expressed throughout the tapeworm body (Figure 3—figure supplement 1). Thus, our analyses have not revealed an anteriorly biased subpopulation of stem cells that confer regenerative ability.

With no evidence for a unique neck-specific subpopulation of stem cells, we hypothesized that stem cells may be distributed throughout the tapeworm, but that extrinsic signals functioning in the neck are necessary to instruct stem cell behavior and/or proglottid regeneration. We designed a functional assay to test populations of cells for the ability to rescue regeneration, modelled after similar experiments performed on planarians (Baguñà, 2012). We exposed tapeworms to a lethal dose of x-irradiation (200 Gy), injected cells from wild-type donors into the neck region, amputated 5 mm anterior fragments, and assayed rescue of lethality and regeneration after 30 days in vitro (Figure 6a). Remarkably, bulk-cell transplants were able to either partially or fully rescue irradiated worms that were destined to die (Figure 6a,c). ‘Full’ rescue was ascribed to worms with normal adult appearance whereas ‘partial’ rescue was assigned to cases in which proglottids were regenerated but the worms displayed abnormalities, like contracted necks (Figure 6—figure supplement 1a). We did not observe any proglottid regeneration in irradiated worms with or without buffer injection (Figure 6a,c).

Figure 6 with 2 supplements see all
Stem cell activity depends on cycling cells but is not confined to cells from the neck.

(a-b) DAPI-stained worms after rescue with cell transplantations from whole-worm donors (a) or sourced from depicted donors (b). (c) Quantification of rescue phenotypes from pooled experiments. Number of animals listed above bars. (d) Model for head-dependent neck maintenance and proglottid formation. (e) Models of head-dependent or -independent stem cell niches.

https://doi.org/10.7554/eLife.48958.017

Is the rescue ability described above dependent on tapeworm cycling cells? We exposed donors to F-ara-EdU for 1 hr, to label cycling cells prior to transplantation into irradiated hosts (Figure 6—figure supplement 1b). Though bulk-cell transplants were performed, injection sites contained 0, 1, or small groups of F-ara-EdU+ cells immediately after transplantations (Figure 6—figure supplement 1c), likely due to technical challenges. Despite this issue, we were able to detect large colonies of F-ara-EdU+ cells 3 days post-transplantation (Figure 6—figure supplement 1d). We also observed that some labeled cells were incorporated into terminally differentiated tissues at the animal edge (Figure 6—figure supplement 1d: inset). Thus, cycling cells from donors are able to become established and differentiate inside the irradiated host.

To test if the cycling-cell population is necessary to rescue lethally irradiated tapeworms, we depleted cycling cells from donor worms using 50 mM hydroxyurea (HU), which resulted in 96 ± 3% loss of cycling cells after 6 days (Figure 6—figure supplement 1e–f). Cycling cells are essential for rescue of regeneration as injected cells from HU-treated donors rescued only 1% of the time, compared to 26% rescue using cells sourced from sister donors that did not receive the drug (Figure 6b–c). HU was used to deplete cycling cells instead of irradiation in order to avoid inducing DNA damage in the transplanted cells. Cells transplanted from HU-treated donors had otherwise comparable morphology to untreated cells (Figure 6—figure supplement 1g). Our results suggest that tapeworm cycling cells contain bona fide stem cell activity.

With this functional assay in hand, we examined the rescue ability of cells from anterior donor tissues (including the regeneration-competent neck) compared to donor tissues from the most posterior termini of 6-day-old tapeworms (which are regeneration incompetent and exclusively comprised of proglottids). Cells from either region were able to rescue regeneration in lethally irradiated tapeworms (Figure 6b–c). Thus, cells from posterior proglottids were competent to receive signals from the head and neck region that regulate regenerative ability. Interestingly, using pulse-chase experiments with F-ara-EdU, we find that the cycling cells of posterior proglottids can give rise to multiple differentiated cell types like muscle/tegument at the animal edge as well as flame cells of the protonephridial system marked by anti-acetylated α-tubulin antibodies (Rozario and Newmark, 2015) (Figure 6—figure supplement 2). Thus, the cycling cells from tapeworm posteriors show hallmarks of stem cell activity, despite the fact that this tissue is not competent to regenerate.

Taken together, the results of our study support the idea that the regeneration competence of the neck is due to extrinsic signals that regulate regeneration, rather than intrinsic properties of stem cells in the neck region (see Discussion). It appears that in tapeworms, location matters enormously: the head and neck environment provide cues that regulate the ability of stem cells to regenerate proglottids, even though cycling cells (and likely stem cells), are not anatomically confined.

Discussion

Across the flatworm phylum, both free-living and parasitic worms maintain stem cells throughout adulthood but display a range of regenerative abilities. The freshwater planarian S. mediterranea can regenerate its whole body from tiny amputated fragments (Newmark and Sánchez Alvarado, 2002). The blood fluke Schistosoma mansoni cannot regenerate after amputation, though it does employ adult somatic stem cells in other ways, such as to repair injury (Collins and Collins, 2016) and maintain its tegument (Collins et al., 2016; Wendt et al., 2018). Prior to this study, the regenerative ability of tapeworms had never been tested comprehensively. Although it was known that anterior fragments containing the head, neck, and immature proglottids could regenerate into fully mature tapeworms once transplanted into a rat intestine (Read, 1967; Goodchild, 1958), fragments lacking heads could not be tested for regenerative ability using transplantation. Attempts were made to suture H. diminuta fragments with mutilated or removed heads into a rat intestine but these fragments were invariably flushed out (Goodchild, 1958). Here we employ a robust in vitro culture system that allowed us to test regeneration of any amputated H. diminuta fragment for the first time. We show that the neck is both necessary and sufficient for proglottid regeneration, though this regenerative ability is ultimately finite without regulatory signals that depend on the presence of the head. H. diminuta is an intriguing model to discover signals that both drive and limit regenerative ability.

During homeostasis, the neck of H. diminuta serves as a ‘growth zone’ from which proglottids are thought to bud one at a time (Lumsden and Specian, 1980), thus, it makes intuitive sense that this tissue would retain the ability to regenerate proglottids post-amputation. Furthermore, cells with the typical morphology of stem cells reside in the neck (Bolla and Roberts, 1971). However, we find that cycling cells are present in all regions regardless of regenerative competence. Thus, it was necessary to embark on a more thorough characterization of tapeworm cycling cells to understand how H. diminuta may regulate stem cells and enable proglottid regeneration.

We depleted cycling cells in H. diminuta using irradiation and employed RNA-seq to uncover potential stem cell regulators in an unbiased fashion. Though irradiation may have secondary effects beyond stem cell depletion (Solana et al., 2012), this approach allowed us to generate an initial list of candidate tapeworm stem cell genes. Using dFISH, we were able to verify 38 transcripts that were expressed at least partially in cycling cells, providing the first molecular characterization of this population in H. diminuta.

Adult somatic stem cells in free-living flatworms have already been well described molecularly, and share many conserved regulators. However, parasitic flatworms have lost some stem cell genes (e.g. piwi, vasa, and tudor) (Tsai et al., 2013) but retained others (e.g. argonaute, fgfr) (Collins et al., 2013; Koziol et al., 2014). How do the cycling-cell transcripts we identified in H. diminuta compare to stem cells in free-living planarians? (Fincher et al., 2018; Plass et al., 2018; Labbé et al., 2012; Rozanski et al., 2019) (Supplementary file 1c). Of 38 verified tapeworm cycling-cell transcripts, 28 had putative planarian homologs (tblasx E-value <10−10) though not all were reciprocal blast hits. 16 of these planarian transcripts were designated as cluster-defining genes in the Fincher et al. single-cell sequencing dataset and 6/16 are neoblast cluster-defining genes. Plass et al. also performed single-cell sequencing of planarians but most of the putative planarian homologs of tapeworm cycling-cell transcripts that we identified were not found in their dataset. However, 8/28 transcripts showed enriched expression in neoblast clusters. We also compared the expression of these planarian transcripts in RNA-seq of three cell populations sorted by fluorescence-activated cell sorting (FACS): 1) X1 (neoblasts in G2/M), 2) X2 (G1 neoblasts and progenitors), and 3) Xins (differentiated cells) (Labbé et al., 2012; Rozanski et al., 2019). We find that 22/28 putative planarian homologs of tapeworm cycling-cell transcripts show enriched expression in either the X1 or X2 populations, which contain neoblasts. Thus, despite >500 million years of separation between free-living and parasitic flatworm evolution (Laumer et al., 2015), tapeworm cycling-cell transcripts have conserved signatures with planarian neoblasts.

In multiple species of flatworms, stem cells have been shown to be transcriptionally heterogenous (Solana et al., 2012; Fincher et al., 2018; van Wolfswinkel et al., 2014; Zeng et al., 2018). For example, in larvae of the tapeworm Echinococcus multilocularis, many putative stem cell markers show limited overlapping gene expression patterns (Koziol et al., 2014). In keeping with these findings, we observe transcriptional heterogeneity within the cycling-cell population of H. diminuta. Additionally, we identified 23 transcripts, including zmym3 and pogzl, that label all cycling cells. Importantly, we were able to use RNAi to functionally verify that cycling-cell genes like zmym3 and pogzl are critical for stem cell maintenance and that inhibition of these genes leads to impaired growth and regeneration. Both zmym3 and pogzl are neoblast cluster-defining genes in planarians (Supplementary file 1c) suggesting that their functions in stem cell regulation may be conserved across the two species. In fact, the planarian homolog of tapeworm pogzl, factor initiating regeneration 1 (fir1), was recently shown to be expressed in planarian neoblasts (Han, 2018). RNAi of fir1 resulted in decreased cell division after amputation and failure to regenerate blastemas (Han, 2018). On the other hand, the function of zmym3 in regeneration is not known, but in other systems, zmym3 regulates cell cycle progression (Hu et al., 2017) and DNA repair (Leung et al., 2017), two essential functions for stem cells. Coincidentally, both zmym3 and pogzl are Zn finger proteins with predicted DNA-binding activity and could function as transcriptional regulators of stem cells. Thus, it would be interesting to further understand the mechanism of action of zmym3 and pogzl in stem cells of parasitic and free-living flatworms.

In this study, we showed the first use of RNAi in H. diminuta. RNAi has been demonstrated previously in other tapeworm species (Pierson et al., 2010; Mizukami et al., 2010; Spiliotis et al., 2010), though it has not been widely adopted for studying tapeworm biology due to technical challenges like poor knockdown efficacy, inefficient penetrance, and the difficulty of in vitro culture. Taking advantage of the robust in vitro culture of H. diminuta, our RNAi scheme can be expanded to ascertain functions for many parasitic flatworm genes that thus far have been refractory to functional analyses.

Our screening strategy allowed us to verify genes with enriched expression in some or all cycling cells; however, none of these genes were expressed exclusively in the neck. Since we had observed that regenerative ability was anteriorly biased across the neck, we attempted to leverage this observation and query whether a subpopulation of pluripotent cycling cells may be asymmetrically distributed across the neck and would be identifiable by RNA-seq. Through A-P transcriptional profiling of the neck, we identified 461 anterior-enriched transcripts, but the vast majority of them were neither irradiation-sensitive nor detected in cycling cells by dFISH. Thus, a subpopulation of neck-resident pluripotent stem cells, seems unlikely to explain the region-specific regenerative ability of tapeworms. Nonetheless, our study does not exclude the existence of a subpopulation of pluripotent stem cells that may be stably maintained in the adult. Future studies using single-cell RNA sequencing are likely to provide a thorough characterization of adult somatic stem cells in H. diminuta, as has been the case for planarians (Fincher et al., 2018; Plass et al., 2018; Zeng et al., 2018).

Is the neck competent to regenerate because of a unique stem cell population that has yet to be identified, or because of signals extrinsic to stem cells that make the neck permissive for regeneration? Tapeworms exposed to a lethal dose of irradiation prior to amputation are not competent to regenerate and will eventually degenerate and die. However, transplantation of cells from wild-type donors into the necks of irradiated tapeworms rescued lethality and regeneration. This rescue ability is severely compromised if donor worms are first depleted of cycling cells using drug treatment with HU, suggesting that some or all cycling cells have stem cell ability. Interestingly, stem cell ability is not restricted to cells from regeneration-competent regions: cells from posterior tissues that do not regenerate proglottids are still able to rescue regeneration when transplanted into the neck. These data strongly suggest that the microenvironment within the neck confers regenerative ability to this region.

The interplay between intrinsic and extrinsic stem cell regulatory signals has been shown to play important roles in regeneration. Head regeneration was induced in three naturally regeneration-deficient planarian species by manipulating the gradient of Wnt signaling by RNAi (Sikes and Newmark, 2013; Liu et al., 2013; Umesono et al., 2013). These planarians maintain pluripotent stem cells but do not normally regenerate heads from posterior tissues due to inappropriately high levels of Wnt signaling, which inhibit anterior regeneration. As in planarians, gradients of Wnt signaling delineate A-P polarity in tapeworms (Koziol et al., 2016). Our transcriptional profiling of the neck A-P axis has already revealed hundreds of candidate genes with polarized expression profiles. Future experiments will help clarify how Wnt signaling and other A-P axis regulation in the neck impacts tapeworm regeneration.

Several plausible models can explain region-specific regeneration in H. diminuta. Head-dependent signals may create gradients across the neck that inhibit proglottidization and are necessary to maintain the neck as an unsegmented tissue. Proglottids can only form once the inhibitory signals are sufficiently diminished (Figure 6d). In this model, the neck is competent to regenerate because of its juxtaposition to the head. After decapitation, the head-dependent signals eventually dissipate and segmentation signals dominate at the expense of the neck. The cellular source of the head-dependent signals and their molecular identity will be exciting avenues for future research.

In addition to its function in maintaining the neck, the head may also play a role in stem cell regulation (Figure 6e). The head may regulate a niche (directly or indirectly) that is necessary for the maintenance of pluripotency in the neck. In this model, stem cells are collectively pluripotent only when they receive head-dependent niche signals, thus limiting regenerative potential to the neck. Alternatively, stem cells may depend on a local niche that is independent of the head. In this model, stem cells have the capacity to form all cell lineages from any amputated fragment; however, the extrinsic signals that activate proglottid formation are restricted to the posterior neck region. Identifying the stem cell niche and its relationship to the head and neck microenvironment will provide crucial insights into our understanding of tapeworm regeneration.

Conclusion

Our study shows that H. diminuta is a powerful developmental model for understanding intrinsic and extrinsic regulation of stem cells and regeneration. The regionally limited regenerative biology of H. diminuta and the technical advances put forth in this work show that we can exploit this tapeworm to understand the complexities of stem cell regulation in parasites. We defined heterogenous stem cells that are collectively pluripotent but that require extrinsic head-dependent signals to enable persistent proglottid regeneration. Understanding how the stem cell niche we describe is regulated may have broad implications for elucidating stem cell biology in parasitic flatworms, as well as other regenerative animals.

Materials and methods

Key resources table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional
information
Strain, (Hymenolepis diminuta)BioSample accession SAMN11958994Carolina BiologicalsCat# 132232
Antibodyanti-Oregon Green 488-HRP antibody (rabbit polyclonal)InvitrogenA21253IF(1:1000)
Antibodyanti-DIG-AP (sheep polyclonal)Sigma AldrichCat# 11093274910IF(1:2000)
Antibodyanti-DIG-POD (sheep polyclonal)Sigma AldrichCat#: 11207733910IF(1:2000)
Antibodyanti-DNP-HRP (rabbit polyclonal)Vector LaboratoriesCat#: MB-0603IF(1:2000)
Antibodyanti-acetylated α-tubulin (mouse monoclonal)Santa CruzCat#: sc-23950IF(1:500)
Sequence-based reagentPCR primersThis paperSupplementary file 1E
Sequence-based reagentTranscriptome Shotgun Assembly (Hymenolepis diminuta)DDB/ENA/GenbankGHNR01000000
Sequence-based reagentSequence Read Archives for transcriptome assemblyDDB/ENA/GenbankPRJNA546290SRX6045715- SRX6045719
Sequence-based reagentSequence Read Archives for differential gene expressionDDB/ENA/GenbankPRJNA546293SRX6064929- SRX6064933
Recombinant DNA reagentPlasmid- pJC53.2Addgene26536
Chemical compound, drugF-ara-EdUSigma AldrichT5112930.1 μM (in 1% final DMSO concentration)
Chemical compound, drugOregon green 488-azideInvitrogenO10180100 μM
Chemical compound, drugHydroxyureaSigma AldrichCat#: H862750 mM

Animal care and use

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Infective H. diminuta cysts were obtained from Carolina Biological (132232). To obtain adult tapeworms, 100–400 cysts were fed to Sprague-Dawley rats by oral gavage in ~0.5 mL of 0.85% NaCl. Rats were euthanized in a CO2 chamber 6 days post-gavage, tapeworms were flushed out of the small intestine, and washed in 1X Hanks Balanced Salt Solution (HBSS; Corning) (140 mg/L CaCl2, 100 mg/L MgCl2.6H2O, 100 mg/L MgSO4.7H2O, 400 mg/L KCl, 60 mg/L KH2PO4, 350 mg/L NaHCO3, 8 g/L NaCl, 48 mg/L Na2HPO4, 1 g/L D-glucose, no phenol red). Rodent care was in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Wisconsin-Madison (M005573).

In vitro parasite culture

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Biphasic parasite cultures were prepared based on the Schiller method (Schiller, 1965). Briefly, the solid phase was made in 50 mL Erlenmeyer flasks by mixing 30% heat-inactivated defibrinated sheep blood (Hemostat) with 70% agar base for 10 mL blood-agar mixture per flask. Fresh blood was heat-inactivated at 56°C for 30 min then kept at 4°C and used repeatedly for one week by first warming the blood to 37°C. The agar base was prepared from 8 g Difco nutrient agar and 1.75 g NaCl in 350 mL water, autoclaved, and stored at 4°C. Before use, the agar base was microwaved to liquify, and cooled to below 56°C before mixing with warmed blood. After the blood-agar mixture solidified, 10 mL of Working Hanks 4 (WH4; 1X HBSS/4 g/L total glucose/1X antibiotic-antimycotic (Sigma)) was added. Each flask was topped with a gas-permeable stopper (Jaece Identi-plug) and pre-incubated at 37°C in hypoxia (3% CO2/5% O2/92% N2) overnight before use. Before tapeworms were transferred into the flasks, the liquid phase was adjusted to pH7.5 with 200 μL 7.5% NaHCO3 (Corning). Tapeworms were first washed in WH4 for 10 mins at 37°C in petri dishes pre-coated with 0.5% BSA to inhibit sticking. Transfers to pre-cultured flasks were performed by gently lifting the worms with a stainless-steel hook (Moody Tools) and immersing them in the liquid phase. Tapeworms were grown in hypoxia and transferred to fresh cultures every 3–4 days.

Fixation and DAPI staining

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Tapeworms were heat-killed by swirling in 75°C water for a few seconds until the worms relaxed and elongated, then fixative (4% formaldehyde in Phosphate Buffered Saline with 0.3% TritonX-100 (PBSTx)) was added immediately for 30 min-2hr at room temperature or overnight at 4°C. For DAPI staining, samples were incubated in 1 μg/mL DAPI (Sigma) in PBSTx overnight at 4°C and cleared in 80% glycerol/10 mM Tris pH7.5/1 mM EDTA overnight at room temperature before mounting.

F-ara-EdU uptake and staining

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For F-ara-EdU pulse, tapeworms were incubated in 0.1 μM F-ara-EdU (Sigma) in 1% DMSO at 37°C in WH4. Tapeworms were heat-killed (above) and fixed in 4% formaldehyde/10% DMSO/1% NP40/PBSTx. Large tissues/worms were permeabilized by incubating in PBSTx at room temp for several days. Additional permeabilization was achieved by treatment with 10 μg/mL Proteinase-K/0.1% SDS/PBSTx for 10–30 min at room temperature, fixed in 4% formaldehyde/PBSTx for 10 min before samples were cut into small pieces or retained whole in PBSTx. Samples were further permeabilized in PBSTx/10% DMSO/1% NP40 for 20 min-1 hr (depending on size) before performing the click-it reaction (Salic and Mitchison, 2008) with Oregon Green 488 azide (Invitrogen). Signal was detected using anti-Oregon Green 488-HRP antibody (1:1000; Invitrogen) in K-block (5% Horse serum/0.45% fish gelatin/0.3% Triton-X/0.05% Tween-20/PBS) (Collins et al., 2011) followed by 10–20 min Tyramide Signal Amplification (TSA) reaction (King and Newmark, 2013). Tiled confocal z-stacks through the anterior of the worms were taken and cell numbers were counted using background subtraction on Imaris software. F-ara-EdU+ cells were normalized to worm area from maximum projections of the DAPI stain. Flame cells were stained using an anti-acetylated α-tubulin mouse antibody at 1:500 (sc-23950, Santa Cruz) as described previously (Rozario and Newmark, 2015).

Irradiation

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Most irradiation was performed using a CellRad irradiator (Faxitron Bioptics) at 200 Gy (150 kV, 5 mA) with two exceptions. Due to instrument failure, a cesium irradiator was used for one rescue experiment with donors + /- HU (Figure 6b) at 400 Gy (92 ± 5% cycling cell loss 3 days post-irradiation). The rescue experiment with + /- HU donors was performed a third time once we gained access to an x-irradiator (Xstrahl RS225 Cell Irradiator), where the lethal dose was 200 Gy (63 ± 10% cycling cell loss 3 days post-irradiation). All three experiments gave similar results despite the use of different irradiators. In all cases, lethal irradiation was determined as the dosage at which tapeworms degenerated, had 0 proglottids, and were inviable after 30 days in culture. Irradiation was performed in WH4 in BSA-coated petri dishes.

Transcriptome assembly

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RNA was collected from five regions: 1) head and neck, 2) immature proglottids, 3) mature reproductive proglottids, 4) gravid proglottids, and 5) mixed larval stages isolated from beetles. The first three regions covered the entirety of 3.5-week-old adult tapeworms. Gravid proglottids were taken from posteriors of 10-week-old tapeworms. Paired-end libraries were constructed with 2 × 150 bp reads from a HiSeq2500 chip. 2 x ~ 30 million reads were obtained for each sample. The transcriptome was assembled from three components: 1) map-based assembly, 2) de novo assembly, and 3) Maker predictions from Wormbase Parasite. The map-based assembly was performed using TopHat2 with the 2014 H. diminuta draft genome courtesy of Matt Berriman (Wellcome Sanger Institute, UK). 15,859 transcripts were assembled using TopHat. De novo assembly was performed using Velvet/Oases and resulted in 144,682 transcripts. There were 11,275 predicted Maker transcripts and 73.2% matched (>95% along the length) to the TopHat transcripts. The remaining predicted transcripts that were not represented in the TopHat dataset were added for a combined TopHat/predicted set of 17,651 transcripts. Most of the Oases transcripts matched to the TopHat/predicted set but 35,300 or 24.4% of the Oases transcripts did not (>75% match cut-off). These transcripts could be transcripts missed in the genome, transcription noise, non-coding transcripts, or contamination. We found significant contamination from beetle tissue in the larval tapeworm sample (more below). Initial filtering for contamination excluded 1388 transcripts (from beetle, rat, bacterial, and viral sources). At this point 51,563 transcripts were retained from the three methodologies described above and were processed for further filtering.

There was significant contamination from beetle tissues that had adhered to the tapeworm larvae, which produced transcripts with best hits to beetle proteins (Ixodes scapularis, Harpegnathos saltator, Monodelphis domestica, Nasonia vitripennis, Pediculus humanus corporis, Solenopsis invicta, Tenebrio molitor, or Tribolium castaneum). Most of the transcripts were from the Oases de novo assembly and did not match the H. diminuta genome. Furthermore, they were strongly over-represented in the larval sample only. To filter out beetle contamination, we removed 11,918 transcripts from the Oases assembly without matches to the H. diminuta genome that showed >90% expression (by RPKM) in the larval sample.

To the remaining 39,645 transcripts, we applied additional filters: 1) Remove transcripts if average RPKM <1 unless the transcript is long (>1000 bp), has a long ORF (>500 bp) or is annotated. 11,615 transcripts were removed as they met none of these criteria. 2) A stringent expression cut-off was applied to the remaining Oases transcripts; transcripts were discarded if average RPKM <5 and maximum RPKM <10 unless the transcripts were long (>1000 bp), had long ORFs (>500 bp) or were annotated. 8027 transcripts were removed. 3) 51 transcripts were removed because they are mitochondrial or rRNAs. 4) An ORF size filter was applied to remove all transcripts with ORF <300 bp unless they are annotated. 5331 transcripts were removed. 5) For the Maker predicted transcripts, expression and size filters were applied to remove transcripts with expression <1 RPKM and size <500 bp. 275 transcripts were removed.

Our final transcriptome is comprised of 14,346 transcripts (84.9% TopHat, 8.4% Maker predictions, 6.1% Oases with match to genome, and 0.6% Oases without match to genome). The total transcriptome size is 34 Mb with average transcript length of 2,354 bp. This Transcriptome Shotgun Assembly project has been deposited at DDB/ENA/Genbank under the accession GHNR00000000. The version described in this paper is the first version, GHNR01000000. All sequence reads are available at GenBank Bioproject PRJNA546290.

RNA-seq for differential gene expression analyses

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Tissue was collected and immediately frozen on dry ice in 100 μL Trizol (Life Technologies) before RNA extraction. Tissue homogenization was performed as the mixture was in a semi-frozen state using RNase-free pestles and a pestle motor. RNA was purified using the Direct-zol RNA MiniPrep kit (Zymo). RNA quality was assessed using Bioanalyzer, libraries were prepared with TruSeq Stranded mRNAseq Sample Prep kit (Illumina), and sequenced on two lanes on a HiSeq2500 chip. We performed paired-end sequencing and obtained ~20 million reads per sample. Samples were obtained in triplicate. To identify irradiation-sensitive transcripts, 2 mm anterior tapeworm fragments were cut from 10 worms after 3 days in vitro. To identify differentially expressed transcripts across the neck A-P axis, 1 mm fragments were cut from 20 freshly obtained 6-day-old tapeworms. Paired-end reads were mapped to the transcriptome (above) using default settings on CLC Genomics Workbench 6 (Qiagen) except that read alignments were done with a relaxed length fraction of 0.5. Differential gene expression analysis was done with the same software using estimate tagwise dispersions on total read counts and a total count filter cut-off of 5 reads. All sequence reads used for differential gene expression analyses are available at GenBank Bioproject PRJNA546293.

Cloning

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Target genes were amplified using PCR with Platinum Taq (Life Technologies) from cDNA generated from RNAs extracted from tapeworm anteriors to enrich for neck transcripts. PCR products were inserted via TA-mediated cloning into the previously described vector pJC53.2 (Collins et al., 2010) pre-digested with Eam11051. Anti-sense riboprobes could be generated by in vitro transcription with SP6 or T3 RNA polymerases. For RNAi, dsRNA was generated using T7 RNA polymerase. For sequences and primers, refer to Supplementary file 1e.

In situ hybridization

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WISH and FISH protocols were modified from previously published methods for planarians (King and Newmark, 2013) and the mouse bile-duct tapeworm Hymenolepis microstoma (Olson et al., 2018). Tapeworms were heat killed and fixed in 4% formaldehyde/10% DMSO/1% NP40/PBSTx for 30 min at room temperature before washing and dehydration into methanol. Dehydrated samples were frozen at −30°C for at least 2 days. After rehydration, samples were permeabilized in 10 μg/mL Proteinase-K/0.1% SDS/PBSTx for 30 min, washed into 0.1 M Triethanolamine pH7-8 (TEA), 2.5 μL/mL acetic anhydride was added for 5 min with vigorous swirling, acetic anhydride step was repeated, washed in PBSTx, and post-fixed in 4% formaldehyde/PBSTx for 10 min. Probe synthesis, hybridization, and staining were performed as previously described (King and Newmark, 2013) using probe concentrations at ~50 ng/mL for 16–48 hr at 56°C. All probes were synthesized with either DIG or DNP haptens and detected using the following antibodies, all at 1:2000: anti-DIG-AP (Sigma), anti-DIG-POD (Sigma), anti-DNP-HRP (Vector Labs). Colorimetric development was done using NBT (Roche)/BCIP (Sigma) or with Fast-Blue (Sigma) (Currie et al., 2016). Fluorescent signal was visualized after 10–20 min TSA reaction (King and Newmark, 2013). DAPI staining and mounting were performed as described above.

Imaging

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Confocal imaging was performed on a Zeiss LSM 880 with the following objectives: 20X/0.8 NA Plan-APOCHROMAT, 40X/1.3 NA Plan-APOCHROMAT, and 63X/1.4 NA Plan-APOCHROMAT. WISH samples and whole-mount DAPI-stained worms were imaged using Zeiss AxioZoom V16 macroscope. Image processing was performed using ImageJ for general brightness/contrast adjustments, maximum-intensity projections, and tile stitching (Preibisch et al., 2009).

RNAi

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dsRNA was synthesized as previously described (Rouhana et al., 2013) and resuspended at concentrations ~ 1.5–2 μg/μL. For control injections, 1.5 kb dsRNA derived from ccdB and camR-containing insert of the pJC53.2 vector was used (Collins et al., 2010). 6-day-old tapeworms were obtained and microinjected with dsRNA using femtotips II via the Femtojet injection system (Eppendorf) to obtain spreading across the first ~3–4 mm anterior of the tapeworm. The spread of injected fluids could be detected by a temporary increase in opacity. 500 hPa injection pressure for 0.3–1 s was used per injection site. Whole tapeworms were cultured in vitro for 3 days, 2 mm anterior fragments were amputated, worms were re-injected with dsRNA on day 6, and cultured in vitro for an additional 9 days before termination.

qPCR for target gene knockdown efficacy

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Whole worms (6 days old) were injected with dsRNA throughout and frozen in Trizol on dry ice after 6 days in vitro for RNA extraction according to manufacturer’s protocol and DNAse (Promega) treatment for 30 min at 37°C. cDNA synthesis was performed using SuperScriptIII First-Strand Synthesis System (Invitrogen) with Oligo(dT)20 primers followed by iScript cDNA Synthesis Kit (Bio-Rad). qPCR was performed using GoTaq Mastermix (Promega) on a StepOnePlus real-time PCR machine (Applied Biosystems). 60S ribosomal protein L13 (60Srpl13) was used as an internal normalization control. For primers refer to Supplementary file 1e.

Hydroxyurea (HU) treatment

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Tapeworms were treated with HU (Sigma) or HBSS (for controls) every day for a total of 6 days. HU stock solution was made fresh every day at 2 M in HBSS. 250 μL was added to each flask of tapeworms for final concentration of 50 mM. HU is unstable at 37°C so worms were transferred into fresh HU-containing media every two days, and fresh HU was added every other day.

Cell transplantations

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For dissociated cell preparations, tapeworms were placed in a drop of calcium-magnesium free HBSS (CMF HBSS, Gibco), minced into small pieces with a tungsten needle, incubated in 3X Trypsin-EDTA (Sigma) in CMF HBSS for 30 min at 37°C and dissociated using a dounce homogenizer (Kontes). Cells were pelleted by centrifugation at 250 g for 5 min. The cell pellet was washed in CMF HBSS and passed through cell strainers at 100 μm, 40 μm, 20 μm, and 10 μm (Falcon and Sysmex) with one spin and wash in between. Cells were pelleted and resuspended in 200–400 μL WH4 with 0.05% BSA. Cell injections were performed using the Cell Tram Oil four injection system (Eppendorf) into the necks of irradiated worms. For + /- HU donors, cell concentrations were measured using a hemocytometer and normalized (to ~108 cells/mL) to ensure equal numbers of cells were injected. For all rescue experiments, cells were injected into irradiated hosts on the same day that the hosts were irradiated. After 3 days in vitro, 5 mm anterior fragments were amputated and grown for an additional 27 days.

Statistical analysis

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Statistical analyses were performed using Prism7 software (GraphPad Prism). All experiments were repeated at least twice. All measurements were taken from distinct samples. Error bars, statistical tests, number of replicates (N) and sample sizes (n) are indicated in corresponding figure legends. Either Dunnett’s or Tukey’s multiple comparison tests were used for one-way ANOVAs. SD = standard deviation. P-values: ns = not significant, *=p ≤ 0.5, ****=p ≤ 0.0001.

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Decision letter

  1. Yukiko M Yamashita
    Reviewing Editor; University of Michigan, United States
  2. Marianne E Bronner
    Senior Editor; California Institute of Technology, United States
  3. Yukiko M Yamashita
    Reviewer; University of Michigan, United States
  4. Peter W Reddien
    Reviewer; Whitehead Institute for Biomedical Research, United States

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your article "Region-specific regulation of stem cell-driven regeneration in tapeworms" for consideration by eLife. Your article has been reviewed by three peer reviewers, including Yukiko M Yamashita as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Marianne Bronner as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Peter W Reddien (Reviewer #3).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

This work is the first comprehensive study to analyze regeneration of tapeworm H. diminuta at a molecular level and establishes it as a model system to study striking regeneration capacity. Accordingly, all reviewers agreed that this study represents an important advancement in the field of regenerative biology. Essentially all comments raised by reviewers are technical ones and are straightforward to address.

Therefore, we would like to invite you to submit a revised version that addresses the reviewers' comments below. We look forward to receiving the revised manuscript.

Reviewer #1:

This work establishes tapeworm (H. diminuta) as a model system for molecular studies on stem cell-based tissue homeostasis. Given their capacity to live for extremely long (if not infinite), understanding the underlying mechanisms of their regeneration will be of significant interest and importance. The most important conclusion from this paper is that regenerative capacity is confined within neck, but it requires head (likely as the 'niche') to maintain long-term regeneration. Head is only the niche, but does not have stem cells on its own. And this niche appears to play a dominant role as all regions (including body) appear to contain 'proliferative' cells.

Experiments are conducted to a highest standard, and this study represents a major advance in the field by establishing tapeworm as a stem cell model system. My comments are mostly about writing: I felt that the writing/data presentation can be improved such that general readers (who are interested in stem cells but are not familiar with tapeworm anatomy – which are the majority of readers).

Specific comments:

- The fourth paragraph of the Results states that the only proliferative somatic cells are undifferentiated in tapeworm, citing Bolla and Roberts, 1971 and Sulgostowska, 1972. But these references are from the 70s, and I am not sure (or more in general, readers will wonder) what kind of methods in the 70s could provide such a conclusion at the accuracy of today's standard. Also, if old studies are that conclusive, this study would sound like not providing much of new insights. Maybe a bit more of elaboration (what was done before, and how it compares to the present study, in providing more accurate information) would be helpful.

- Figure 1B: the lack of regeneration with 'body only' is not obvious due to different scales used between day 0 vs. day 9.

- Figure 2 used irradiation to deplete proliferating cells, which the authors assumed to eliminate stem cells. Whereas I agree that this method has been successfully used to identify neoblast-specific genes in planaria, the formal possibility remains that irradiation induce transient quiescence of stem cells. If so, this experiment will be only identifying 'proliferation-associated' genes, instead of 'stem cell specific genes'. In case of planaria, the use of 'lethal dose' (i.e. no stem cells are left indeed) excluded such a possibility. I am guessing 200 Gy for tapeworm is the same treatment, but it is not explained in the paragraph where this experiment is explained (Results, fifth paragraph). Clarification might help here.

- Figure 3 explains proliferating cells are in neck parenchyma. Abstract/Introduction primed me/readers to think that stem cells are not limited to neck region and are present everywhere. However, in this figure, the authors' description focuses on neck area (starting in the sixth paragraph of the Results, they provide detailed location of cycling cells within the neck region, without mentioning the other area of the worm-like body), which inevitably made me wonder whether other regions also exhibit similar expression patterns or not, and I was quite confused going through the explanation around here. I wonder these questions might not occur to authors, because the images may be self-explanatory to them. But those who are not familiar to tapeworm anatomy cannot quite tell where the neck is/where the body starts in the presented images (labelling those regions in the panels will greatly help).

- Results, ninth paragraph: Reduction in mcm2 is used to conclude that cell populations are gone after RNAi of h2b etc. However, as mcm2 itself is a cell proliferation gene, it is still possible that the cell population still exists while reducing the expression of mcm2. Here I am not asking to do additional experiments to distinguish these possibilities (cell population is gone vs. proliferation is diminished). I am simply pointing out a flaw in interpretation so that they can adjust their statement.

- Figure 5-6 conclude that stem cells are not limited to the neck region. This is based on the lack of any transcripts that are differentially expressed between posterior vs. anterior regions, and the fact that cells from any regions can rescue lethally irradiated animals. Based on these data, the authors propose that head/neck serves to provide extrinsic signals to maintain stem cells, yet there are no intrinsic differences among stem cells. They also nicely show that cycling cells contain the stem cell population (by HU-induced depletion of cycling cells). Whereas the data are striking and clear, the explanation seems to be somewhat confusing (or indicating something is missing). ---upon reading the Discussion, I see that most of the issues (below) are indeed discussed well, but as I read through the result section, the description went on without addressing some major question. It might be helpful to slip in a few sentences also in Result section to prepare readers (instead of making them hang up with their questions). One major issue was: how signal from the head regulates the stem cells, which seems to be everywhere in the body, yet no differential transcripts were found (again, the discussion in the Discussion section was excellent, but none of which were primed in the Results section, so I had to keep reading suspended. Just 'see Discussion' might greatly help the reading).

Reviewer #2:

This paper presents the most comprehensive study of cestode regeneration to date and includes a description of a robust in vitro culture for Hymenolepis diminuta that facilitates the use of growth/anatomical bioassays, and powerful techniques like irradiation, cell transplantation and RNAi. Using this in combination with RNAseq, the authors present a fascinating picture of the regenerative capacity of H. diminuta, showing that cycling cells from multiple regions of the worm can rescue regeneration in irradiated animals. The RNAseq data also adds valuable resources to the broader flatworm stem cell research community.

This review raises only minor points for clarification and suggests some experimental questions that may warrant consideration/discussion.

Specific comments:

Results, first paragraph: The author should clarify their definition of 'regeneration', especially in the context of planarian 'regeneration'. For example, a head neck and body segment would still constitute a worm with fewer proglottids – so would 'regeneration' in the normal definition be the right word here?

Results, first paragraph: Clarification on the difference between growth and regeneration, and what is actually happening to cause the increase in length, if not regeneration.

Results, first paragraph: Could authors clarify what region of the neck these '2mm "neck only" fragments' came from?

Would it be more correct to refer to mcm2 and h2b as 'proliferative cell markers', rather than 'stem cell markers'?

Results, fourth paragraph: EdU labelling would be visible when positive, even if only a few cells were labelled – could the authors propose alternative hypotheses for new observation of presence of cycling cells in head?

Results, eighth paragraph: Could authors refer to Figure 4B when highlighting the thin and frail worms resulting from the RNAi experiments.

Results, ninth paragraph: Loss of mcm2 transcript might mean that there are no cycling cells present, but is it possible that the stem cells are still there in a quiescent state?

Results, eleventh paragraph: Should 'gene' be replaced by 'transcript' when discussing RNAseq and ISH?

Clarification of what "subset of cells within neck parenchyma" means. Were the other transcripts not found in the neck or did these 15 genes just show restricted expression in the neck?

Could authors clarify what "but 7/8 genes tested" means?

Results, eleventh paragraph: Does prox1 not warrant further investigation, or at least discussion?

Results, twelfth paragraph: Although present in the Materials and methods, it would be helpful to reader if the lethal dose was stated here.

Results, twelfth paragraph: Any rationale for 5 mm fragments in this instance considering 2 mm fragments were capable of "regeneration"?

Results, twelfth paragraph: What was the time period between irradiation and injection of cells?

Results, fourteenth paragraph: Although HU concentration is provided in the Materials and methods, again it would be helpful for the reader to state this here.

Clarification of 'posterior donor tissue' – does this means that donor tissues were proglottids?

Discussion, first paragraph: Reference for planarian regeneration?

Subsection “F-ara-EdU31 388 uptake and staining”: For how long was tyramide signal amplification performed? Any difference from planarians?

Subsection “Transcriptome assembly”, third paragraph: RPKM units standardise for length of transcript, so filtering length of transcripts should be unnecessary?

Subsection “RNA-seq for differential gene expression analyses”: Some more detail on exactly how DE analysis was performed would be helpful for reader. Authors refer to expression using RPKM units, although it is common for paired end sequencing data to be referred to using FPKM units.

Other comments:

Did the authors consider the irradiation rescue experiment in decapitated worms?

Did the authors try the irradiation rescue experiment using donor worms having undergone RNAi for one of the cell cycle transcripts (e.g. h2b)?

What happens if irradiated worms have cells transplanted into the head or the proglottids, rather than the neck?

Reviewer #3:

The capacity for immense growth and regeneration is a fundamental problem of parasitology. The authors developed the parasitic tapeworm H. diminuta as a modern molecular genetic system in an impressive technical advance promoted by an in vitro culture system. The authors found that the neck region of the tapeworm was necessary and sufficient for regeneration of proglottids, but that a head could not be regenerated and was necessary for serial rounds of regeneration. Dividing cells, however, were present in all regions of the body. The authors developed a transplantation procedure that showed cycling cells from multiple regions of the body were capable of rescuing lethally irradiated hosts when transplanted into the neck, indicating that the neck harbors a permissive environment for stem cell proliferation. The novelty in the work involves the comprehensive testing of the regenerative ability of tapeworms, the molecular description of the H. diminuta cycling cell population, and in the discovery of the existence of essential proliferating cell-extrinsic anterior cues required for stem cell-driven regeneration.

Results, first paragraph: Please clarify the description of growth without proglottid formation. Show data on "differentiate mature reproductive structures"; there is also a "data not shown" statement about head regeneration which would be better to show.

Some genes were irradiation sensitive and near but not co-expressed with proliferation markers (Figure 3—figure supplement 3D). EdU pulse followed by fixation at different timepoints could support their hypothesis for case study genes that they are expressed in early progeny of cycling cells.

The prominence of signal from gonads makes visualization of proliferating mesenchymal cells difficult in data presented from the posterior. Higher magnification FISH of data such as in Figure 4G or Figure 3A would be helpful.

How far posterior could cells be isolated and still be transplanted and result in successful rescue? The explicit details of the region donor posterior cells came from could be better described, or even further posterior regions could be used in transplants. (i.e., did the cells have to come from near the neck, or is it clear that cells distal to the neck can engraft and support proliferation)?

The authors could more explicitly compare the data obtained about the genes expressed in the cycling cell population of H. diminuta to data from neoblasts in planarians (such as zmym3 and su(Hw) – but ideally systematically with all validated cycling cell markers). A fuller discussion comparing the molecular biology of these cells could add additional depth to the work.

EdU experiments with amputated body fragments could show if posterior cycling cells are capable of producing multiple differentiated cells (with marker double-labeling) in tissue maintenance/growth. This could help in address comments on pluripotency/regeneration models in the Discussion.

https://doi.org/10.7554/eLife.48958.030

Author response

Reviewer #1:

[…] Specific comments:

- The fourth paragraph of the Results states that the only proliferative somatic cells are undifferentiated in tapeworm, citing Bolla and Roberts, 1971 and Sulgostowska, 1972. But these references are from the 70s, and I am not sure (or more in general, readers will wonder) what kind of methods in the 70s could provide such a conclusion at the accuracy of today's standard. Also, if old studies are that conclusive, this study would sound like not providing much of new insights. Maybe a bit more of elaboration (what was done before, and how it compares to the present study, in providing more accurate information) would be helpful.

We have added a more thorough description as suggested.

“In flatworms, it has been repeatedly shown that the only proliferative cells are undifferentiated cells with stem cell morphology and/or function; these cells have been termed neoblasts, adult somatic stem cells, or germinative cells, depending on the organism (Collins et al., 2013; Koziol et al., 2014; Baguñà, Salo and Auladell, 1989; Ladurner, Rieger and Baguñà, 2000). […] Thus, cycling somatic cells in H. diminuta would not include differentiated cells, but would include stem cells and any dividing progeny.”

- Figure 1B: the lack of regeneration with 'body only' is not obvious due to different scales used between day 0 vs. day 9.

We have added Figure 1—figure supplement 1 and text (see below) to describe how the body only fragment increases in length without adding new proglottids. At day 0, the proglottids in the amputated “body only” fragments are small and immature but with time, they grow in size and become reproductively mature. Additionally, since there is no regeneration, they do not add new (and small) proglottids. We show that the mean proglottid length is significantly increased in the “body only” fragments compared to the regeneration-competent fragments. We also show higher magnification images of the most mature proglottids that are observed in the “body only” fragments.

“Despite the failure to regenerate, “body only” fragments could grow because each existing proglottid increased in length as it progressively matured (Figure 1—figure supplement 1A-B).”

- Figure 2 used irradiation to deplete proliferating cells, which the authors assumed to eliminate stem cells. Whereas I agree that this method has been successfully used to identify neoblast-specific genes in planaria, the formal possibility remains that irradiation induce transient quiescence of stem cells. If so, this experiment will be only identifying 'proliferation-associated' genes, instead of 'stem cell specific genes'. In case of planaria, the use of 'lethal dose' (i.e. no stem cells are left indeed) excluded such a possibility. I am guessing 200 Gy for tapeworm is the same treatment, but it is not explained in the paragraph where this experiment is explained (Results, fifth paragraph). Clarification might help here.

200 Gy is a lethal dose of irradiation. 100% of 5 mm anterior fragments amputated from worms exposed to 200 Gy x-irradiation will degenerate after 1 month with no detectable proglottids. We have added a more thorough description to the text and have added supporting data to Figure 2—figure supplement 1.

“Exposing H. diminuta to 200 Gy x-irradiation reduced the number of cycling cells by 91 ± 6% after 3 days (Figure 2C-D) and abrogated both growth and regeneration (Figure 2—figure supplement 1A-B). This dosage is lethal; all fragments from worms exposed to 200 Gy x-irradiation degenerated after 1 month (Figure 2—figure supplement 1C-D).”

- Figure 3 explains proliferating cells are in neck parenchyma. Abstract/Introduction primed me/readers to think that stem cells are not limited to neck region and are present everywhere. However, in this figure, the authors' description focuses on neck area (starting in the sixth paragraph of the Results, they provide detailed location of cycling cells within the neck region, without mentioning the other area of the worm-like body), which inevitably made me wonder whether other regions also exhibit similar expression patterns or not, and I was quite confused going through the explanation around here. I wonder these questions might not occur to authors, because the images may be self-explanatory to them. But those who are not familiar to tapeworm anatomy cannot quite tell where the neck is/where the body starts in the presented images (labelling those regions in the panels will greatly help).

We have added annotations to Figure 3B to help highlight that only a portion of the neck is shown. All genes that were expressed in the neck parenchyma were expressed throughout the whole body. We have added images of in situs at the posterior termini in Figure 3—figure supplement 1.

“All transcripts that were expressed in the neck parenchyma were also found throughout the worm body, even in the most posterior proglottids (Figure 3—figure supplement 1B-C).”

- Results, ninth paragraph: Reduction in mcm2 is used to conclude that cell populations are gone after RNAi of h2b etc. However, as mcm2 itself is a cell proliferation gene, it is still possible that the cell population still exists while reducing the expression of mcm2. Here I am not asking to do additional experiments to distinguish these possibilities (cell population is gone vs. proliferation is diminished). I am simply pointing out a flaw in interpretation so that they can adjust their statement.

We have adjusted the description to more accurately describe our observations.

“Are these RNAi-induced failures in growth and regeneration due to defects in the cycling-cell population? RNAi knockdown of h2b, zmym3, and pogzl severely reduced the number of proliferative cells in the neck that could incorporate F-ara-EdU(Figure 4D-E). […] Therefore, h2b, zmym3, and pogzl are necessary for the maintenance and/or proper function of cycling cells, likely including stem cells, in H. diminuta.”

- Figure 5-6 conclude that stem cells are not limited to the neck region. This is based on the lack of any transcripts that are differentially expressed between posterior vs. anterior regions, and the fact that cells from any regions can rescue lethally irradiated animals. Based on these data, the authors propose that head/neck serves to provide extrinsic signals to maintain stem cells, yet there are no intrinsic differences among stem cells. They also nicely show that cycling cells contain the stem cell population (by HU-induced depletion of cycling cells). Whereas the data are striking and clear, the explanation seems to be somewhat confusing (or indicating something is missing). ---upon reading the Discussion, I see that most of the issues (below) are indeed discussed well, but as I read through the Results section, the description went on without addressing some major question. It might be helpful to slip in a few sentences also in Results section to prepare readers (instead of making them hang up with their questions). One major issue was: how signal from the head regulates the stem cells, which seems to be everywhere in the body, yet no differential transcripts were found (again, the discussion in the Discussion section was excellent, but none of which were primed in the Results section, so I had to keep reading suspended. Just 'see Discussion' might greatly help the reading).

We have added a more thorough description in the Results section:

“With this functional assay in hand, we examined the rescue ability of cells from anterior donor tissues (including the regeneration-competent neck) compared to donor tissues from the most posterior termini of 6 day-old tapeworms (which are regeneration incompetent and exclusively comprised of proglottids). […] It appears that in tapeworms, location matters enormously: the head and neck environment provide cues that regulate the ability of stem cells to regenerate proglottids, even though cycling cells (and likely stem cells), are not anatomically confined.”

Reviewer #2:

[…] Specific comments:

Results, first paragraph: The author should clarify their definition of 'regeneration', especially in the context of planarian 'regeneration'. For example, a head neck and body segment would still constitute a worm with fewer proglottids – so would 'regeneration' in the normal definition be the right word here?

Regeneration is broadly defined as “the replacement of a body part lost through traumatic injury (either amputation or autotomy)” (Bely and Nyberg, 2010). This applies to a broad range of biological levels including regeneration of the whole body, individual anatomical structures, internal organs, tissues and cells. In the first paragraph of the Results section, we describe that we have observed only proglottid regeneration in H. diminuta so that the specific regenerative ability demonstrated by these worms is clear.

We have never come across a definition of regeneration that refers to the animal size. If regenerated tapeworms are cultured for long enough they will achieve the same size and number of proglottids as unamputated worms in vitro. Even in the extreme case of whole-body regeneration exhibited by planarians, the regenerated worm will start off smaller than the original worm. Thus, having fewer proglottids than the original tapeworm at any given time after amputation does not negate that the tapeworm can regenerate proglottids following amputation.

Reference:

Bely, A. E., and Nyberg, K. G. (2010). Evolution of animal regeneration: re-emergence of a field. Trends in Ecology and Evolution, 25(3), 161–170.

Results, first paragraph: Clarification on the difference between growth and regeneration, and what is actually happening to cause the increase in length, if not regeneration.

We have added Figure 1—figure supplement 1 and text (see below) to describe how the body only fragment increases in length without adding new proglottids. At day 0, the proglottids in the amputated “body only” fragments are small and immature but with time, they grow in size and become reproductively mature. Additionally, since there is no regeneration, they do not add new (and small) proglottids. We show that the mean proglottid length is significantly increased in the “body only” fragments compared to the regeneration-competent fragments. We also show higher magnification images of the most mature proglottids that are observed in the “body only” fragments.

“Despite the failure to regenerate, “body only” fragments could grow because each existing proglottid increased in length as it progressively matured (Figure 1—figure supplement 1A-B).”

Results, first paragraph: Could authors clarify what region of the neck these '2mm "neck only" fragments' came from?

The neck of 6 day-old tapeworms used in this study is ~2-3 mm in length. We define the neck as the region between the base of the scolex and the first recognizable proglottid, using DAPI staining and fluorescent microscopy. We amputate the head, then cut the following 2 mm neck tissue under a stereomicroscrope. Thus, the “neck only” tissue comprises all/nearly all of the neck tissue. We’ve added text to clarify this.

“The neck of 6 day-old tapeworms used in this study is typically 2-3 mm long when observed after DAPI staining and widefield fluorescent microscopy. By amputating 2 mm “neck only” fragments, we find that the neck is sufficient to regenerate an average of 383 proglottids (SD=138, N=4, n=20) after 12 days in vitro(Figure 1E).”

Would it be more correct to refer to mcm2 and h2b as 'proliferative cell markers', rather than 'stem cell markers'?

In the references cited they are one and the same. But we have no objection to referring to mcm2 and h2b as cycling cells markers and have made the change (Results, fourth paragraph).

Results, fourth paragraph: EdU labelling would be visible when positive, even if only a few cells were labelled – could the authors propose alternative hypotheses for new observation of presence of cycling cells in head?

We believe that Reviewer #2 is asking why previous studies failed to see cycling cells in the head. In 1972, Bolla and Roberts exposed H. diminuta to tritiated thymidine for 10 min, stained sections, and exposed the autoradiographs for 2 weeks. The sensitivity of this method allowed them to see the abundant cycling cells in the neck but the scarce cycling cells in the head were probably lost to background signal or missed during sectioning. The negative data is not shown in their paper. It seems likely that due to the technical limitations of these early detection methods, they concluded that there are no cycling cells in the head.

Results, eighth paragraph: Could authors refer to Figure 4B when highlighting the thin and frail worms resulting from the RNAi experiments.

Added (Results, eighth paragraph).

Results, ninth paragraph: Loss of mcm2 transcript might mean that there are no cycling cells present, but is it possible that the stem cells are still there in a quiescent state?

We have adjusted the description to more accurately describe our observations.

“Are these RNAi-induced failures in growth and regeneration due to defects in the cycling-cell population? RNAi knockdown of h2b, zmym3, and pogzl severely reduced the number of proliferative cells in the neck that could incorporate F-ara-EdU(Figure 4D-E). […] Therefore, h2b, zmym3, and pogzl are necessary for the maintenance and/or proper function of cycling cells, likely including stem cells, in H. diminuta.”

Results, eleventh paragraph: Should 'gene' be replaced by 'transcript' when discussing RNAseq and ISH?

We have made these substitutions throughout the manuscript.

Clarification of what "subset of cells within neck parenchyma" means. Were the other transcripts not found in the neck or did these 15 genes just show restricted expression in the neck?

Could authors clarify what "but 7/8 genes tested" means?

These 15 transcripts showed expression within the neck parenchyma but only in a subset of cells (Figure 5C). Other anterior-enriched transcripts were broadly expressed in the parenchyma/weak/not predominantly in the neck parenchyma. Of the 15 transcripts, we obtained unambiguous and clear results for 8 dFISH experiments. We have clarified our description in the main text:

“We found 15 transcripts expressed in a subset of cells within the neck parenchyma (Figure 5C) and initially hypothesized that these transcripts may represent subsets of stem cells. We were able to successfully test 8 candidates by dFISH with cycling-cell markers and found that the majority (7/8) were not expressed in cycling cells (Figure 5D, Supplementary file 1B).”

Results, eleventh paragraph: Does prox1 not warrant further investigation, or at least discussion?

We do not have enough data to conclude or speculate about the prox1+ cells though we hope that future experiments will help elucidate their identity and function. RNAi of prox1 did not reveal strong gross morphological defects. If the prox1+ cells are lineage committed stem cells/progenitors, then it is likely no gross defects occur but that loss of specific lineages could be determined if we knew more about the identity of these cells. Furthermore, we are reluctant to interpret this negative result especially since RNAi will only reduce and not eliminate prox1 expression.

We added text to acknowledge that the identity and function of these cells is currently unknown, as well as to emphasize that even prox1 is not expressed in a neck-restricted fashion.

“At present, the identity and function of prox1+ cells is unknown. Furthermore, prox1 is expressed throughout the tapeworm body (Figure 3—figure supplement 1).”

Results, twelfth paragraph: Although present in the Materials and methods, it would be helpful to reader if the lethal dose was stated here.

We have added the dosage (Results, thirteenth paragraph).

Results, twelfth paragraph: Any rationale for 5 mm fragments in this instance considering 2 mm fragments were capable of "regeneration"?

We chose to amputate larger fragments to allow for rescue to occur before too much tissue degeneration happened. Having said that, we did not explicitly test if the rescue would be successful in fragments smaller than 5 mm. Since all 5 mm fragments that were irradiated with a lethal dose degenerated and had no proglottids after 30 days, we deemed this protocol suitable.

Results, twelfth paragraph: What was the time period between irradiation and injection of cells?

Worms were irradiated and then injected with cells on the same day, as soon as the dissociated cell preps were ready. We have added this clarification to the Materials and methods section:

“For all rescue experiments, cells were injected into irradiated hosts on the same day that the hosts were irradiated.”

Results, fourteenth paragraph: Although HU concentration is provided in the Materials and methods, again it would be helpful for the reader to state this here.

Added (Results, fifteenth paragraph).

Clarification of 'posterior donor tissue' – does this means that donor tissues were proglottids?

Yes. We took 5 mm of the most posterior termini of 6 day-old tapeworms, which are exclusively comprised of proglottids. We have added this description in the text.

“With this functional assay in hand, we examined the rescue ability of cells from anterior donor tissues (including the regeneration-competent neck) compared to donor tissues from the most posterior termini of 6 day-old tapeworms (which are regeneration incompetent and exclusively comprised of proglottids).”

Discussion, first paragraph: Reference for planarian regeneration?

Added (Discussion, first paragraph).

Subsection “F-ara-EdU31 388 uptake and staining”: For how long was tyramide signal amplification performed? Any difference from planarians?

10-20 min depending on the size of the tissue. This is similar to planarians. We have added the development time to the Materials and methods section.

Subsection “Transcriptome assembly”, third paragraph: RPKM units standardise for length of transcript, so filtering length of transcripts should be unnecessary?

This was not part of the differential gene expression analysis. This is part of the transcriptome assembly. We used a length filter to reduce potential spuriously assembled contigs if they did not also meet the criteria described in the Materials and methods.

Subsection “RNA-seq for differential gene expression analyses”: Some more detail on exactly how DE analysis was performed would be helpful for reader. Authors refer to expression using RPKM units, although it is common for paired end sequencing data to be referred to using FPKM units.

We have added more description of the DGE analysis, which we performed using the recommended standards in CLC Genomics Workbench 6. Estimated tagwise dispersions were calculated using total read counts after mapping to the transcriptome.

“Paired-end reads were mapped to the transcriptome (above) using default settings on CLC Genomics Workbench 6 (Qiagen) except that read alignments were done with a relaxed length fraction of 0.5. Differential gene expression analysis was done with the same software using estimate tagwise dispersions on total read counts and a total count filter cut-off of 5 reads. All sequence reads used for differential genes expression analyses are available at GenBank Bioproject PRJNA546293.”

Other comments:

Did the authors consider the irradiation rescue experiment in decapitated worms?

After decapitation, the neck is not maintained and the entire tissue becomes proglottidized. Since donor cells from proglottids alone could already rescue lethally irradiated worms, we did not pursue this specific experiment.

Did the authors try the irradiation rescue experiment using donor worms having undergone RNAi for one of the cell cycle transcripts (e.g. h2b)?

The cell dissociation protocol we used in this study was very harsh in order to overcome the integrity of the tegument. As a consequence, very few cycling cells were incorporated despite performing bulk cell transplants (Figure 6—figure supplement 1). Since RNAi of cycling cell transcripts like h2b results in extremely small worms, it would take an enormous number of RNAi worms to perform a rescue experiment. We hope to overcome these technical hurdles with future optimization, but currently, this experiment is too technically challenging.

What happens if irradiated worms have cells transplanted into the head or the proglottids, rather than the neck?

We are currently pursuing these kinds of experiments but they are beyond the scope of this paper.

Reviewer #3:

[…] Results, first paragraph: Please clarify the description of growth without proglottid formation. Show data on "differentiate mature reproductive structures"; there is also a "data not shown" statement about head regeneration which would be better to show.

We have added Figure 1—figure supplement 1 and text (see below) to describe how the body only fragment increases in length without adding new proglottids. At day 0, the proglottids in the amputated “body only” fragments are small and immature but with time, they grow in size and become reproductively mature. Additionally, since there is no regeneration, they do not add new (and small) proglottids. We show that the mean proglottid length is significantly increased in the “body only” fragments compared to the regeneration-competent fragments. We also show higher magnification images of the most mature proglottids that are observed in the “body only” fragments.

“Despite the failure to regenerate, “body only” fragments could grow because each existing proglottid increased in length as it progressively matured (Figure 1—figure supplement 1A-B).”

We have also added data of head fragment regeneration failure (Figure 1—figure supplement 1).

“Furthermore, amputated heads alone could not regenerate in vitro (Figure 1—figure supplement 1C) nor in vivo (Read, 1967).”

Some genes were irradiation sensitive and near but not co-expressed with proliferation markers (Figure 3—figure supplement 3D). EdU pulse followed by fixation at different timepoints could support their hypothesis for case study genes that they are expressed in early progeny of cycling cells.

Unfortunately, we are currently unable to perform in situs in conjunction with F-ara-EdU staining. F-ara-EdU is toxic to tapeworms at concentrations above 1 μM (we use 0.1 μM). In order to detect F-ara-EdU, we need extensive tissue permeabilization (several days in PBSTx at room temperature, proteinase K digestion, and DMSO+detergents) before the click-it reaction. Even then, the signal is only clearly visible after antibody amplification and TSA reaction. Performing the F-ara-EdU staining protocol after our in situ protocol compromises both the in situ and the F-ara-EdU signals. At this stage, this is a technical limitation we have not overcome.

The prominence of signal from gonads makes visualization of proliferating mesenchymal cells difficult in data presented from the posterior. Higher magnification FISH of data such as in Figure 4G or Figure 3A would be helpful.

We have added higher magnification confocal sections of in situs from the animal posterior in Figure 4—figure supplement 2. We have circled the gonads so that the mesenchymal expression of these genes is more obvious.

“By WISH and FISH, all cycling-cell transcripts including zmym3 and pogzl were detected throughout the whole tapeworm body(Figure 4G, Figure 3—figure supplement 1B-C). […] Since zmym3 and pogzl label all cycling cells, it is possible that stem cells of limited potential exist in the posterior, but an elusive subpopulation of pluripotent stem cells is confined to the neck.”

How far posterior could cells be isolated and still be transplanted and result in successful rescue? The explicit details of the region donor posterior cells came from could be better described, or even further posterior regions could be used in transplants. (i.e., did the cells have to come from near the neck, or is it clear that cells distal to the neck can engraft and support proliferation)?

We have added more description of the posterior donor tissue. The tissue used was the most posterior termini comprised exclusively of proglottids. Thus, the cells used were the most distal from the neck at the time (6 day-old worms).

“With this functional assay in hand, we examined the rescue ability of cells from anterior donor tissues (including the regeneration-competent neck) compared to donor tissues from the most posterior termini of 6 day-old tapeworms (which are regeneration incompetent and exclusively comprised of proglottids).”

The authors could more explicitly compare the data obtained about the genes expressed in the cycling cell population of H. diminuta to data from neoblasts in planarians (such as zmym3 and su(Hw) – but ideally systematically with all validated cycling cell markers). A fuller discussion comparing the molecular biology of these cells could add additional depth to the work.

As per the reviewer’s suggestion, we have added a new Supplementary file 1C in which we analyze the verified tapeworm cycling cell markers against planarian neoblast genes described in three studies (Fincher et al., 2018; Plass et al., 2018; Labbe et al., 2012). This analysis was also facilitated by the planarian resource Planmine. We have added text to the Results and Discussion.

Results:

“The transcriptional heterogeneity detected in the cycling-cell compartment is reminiscent of similar observations made in the regenerative planarian S. mediterranea. A comparative analysis between verified tapeworm cycling-cell transcripts and their putative planarian homologs revealed a number of transcripts with conserved expression in cycling-cell populations from these distantly related flatworms (Supplementary file 1C) (see Discussion).”

Discussion:

“How do the cycling-cell transcripts we identified in H. diminuta compare to stem cells in free-living planarians? (Fincher et al., 2018; Plass et al., 2018; Labbé et al., 2012; Rozanski et al., 2019) (Supplementary file 1C). […] Thus, despite >500 million years of separation between free-living and parasitic flatworm evolution (Laumer, Hejnol and Giribet, 2015), tapeworm cycling-cell transcripts have conserved signatures with planarian neoblasts.”

In refence to the potential molecular functions of zmym3 and pogzl, we have added text to the Discussion.

“Both zmym3 and pogzl are neoblast cluster-defining genes in planarians (Supplementary file 1C) suggesting that their functions in stem cell regulation may be conserved across the two species. […] Thus, it would be interesting to further understand the mechanism of action of zmym3 and pogzl in stem cells of parasitic and free-living flatworms.”

EdU experiments with amputated body fragments could show if posterior cycling cells are capable of producing multiple differentiated cells (with marker double-labeling) in tissue maintenance/growth. This could help in address comments on pluripotency/regeneration models in the Discussion.

As described above, F-ara-EdU in combination with in situ hybridizations is currently not feasible. However, we were able to do pulse-chase experiments and use antibody staining as well as anatomical location to identify differentiated cells: anti-acetylated α- tubulin antibodies label flame cells of the protonephridial system and the edge-most nuclei are differentiated muscle and tegument. We were able to pulse amputated posterior fragments of proglottids alone (2mm) and perform 1 hr F-ara-EdU pulse followed by a 3 days chase and found that posterior cycling cells could chase into muscle/tegument and flame cells. The data are presented in Figure 6—figure supplement 2 and in the text.

“Interestingly, using pulse-chase experiments with F-ara-EdU, we find that the cycling cells of posterior proglottids can give rise to multiple differentiated cell types like muscle/tegument at the animal edge as well as flame cells of the protonephridial system marked by anti-acetylated α-tubulin antibodies (Rozario and Newmark, 2015) (Figure 6—figure supplement 2). Thus, the cycling cells from tapeworm posteriors show hallmarks of stem cell activity, despite the fact that this tissue is not competent to regenerate.”

https://doi.org/10.7554/eLife.48958.031

Article and author information

Author details

  1. Tania Rozario

    Morgridge Institute for Research, Madison, United States
    Contribution
    Conceptualization, Supervision, Investigation, Methodology, Writing—original draft, Project administration, Writing—review and editing
    For correspondence
    trozario@morgridge.org
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9971-5211
  2. Edward B Quinn

    Morgridge Institute for Research, Madison, United States
    Contribution
    Investigation, Writing—review and editing
    Competing interests
    No competing interests declared
  3. Jianbin Wang

    RNA Bioscience Initiative, Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, United States
    Contribution
    Investigation, Writing—review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3155-894X
  4. Richard E Davis

    RNA Bioscience Initiative, Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, United States
    Contribution
    Supervision, Methodology, Writing—review and editing
    Competing interests
    No competing interests declared
  5. Phillip A Newmark

    1. Morgridge Institute for Research, Madison, United States
    2. Howard Hughes Medical Institute, Chevy Chase, United States
    3. Department of Integrative Biology, University of Wisconsin–Madison, Madison, United States
    Contribution
    Conceptualization, Supervision, Funding acquisition, Writing—original draft, Project administration, Writing—review and editing
    For correspondence
    pnewmark@morgridge.org
    Competing interests
    Reviewing editor, eLife
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0793-022X

Funding

National Institute of Allergy and Infectious Diseases (R21AI119960)

  • Phillip A Newmark

Howard Hughes Medical Institute

  • Phillip A Newmark

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank: members of the Newmark lab, especially Melanie Issigonis and Umair Khan, for discussions and comments on the manuscript; Alvaro Hernandez (Roy J Carver Biotechnology Center, University of Illinois at Urbana-Champaign) for RNA-seq; and Bret Duffin (Morgridge Institute for Research) for invaluable assistance with irradiation. This work was supported by NIH R21AI119960. PAN is an investigator of the Howard Hughes Medical Institute.

Ethics

Animal experimentation: Rodent care was in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Wisconsin-Madison (M005573).

Senior Editor

  1. Marianne E Bronner, California Institute of Technology, United States

Reviewing Editor

  1. Yukiko M Yamashita, University of Michigan, United States

Reviewers

  1. Yukiko M Yamashita, University of Michigan, United States
  2. Peter W Reddien, Whitehead Institute for Biomedical Research, United States

Publication history

  1. Received: June 1, 2019
  2. Accepted: September 10, 2019
  3. Accepted Manuscript published: September 24, 2019 (version 1)
  4. Version of Record published: October 30, 2019 (version 2)

Copyright

© 2019, Rozario et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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