Adhesive organ regeneration in Macrostomum lignano
© The Author(s). 2016
Received: 4 February 2016
Accepted: 23 May 2016
Published: 2 June 2016
Flatworms possess pluripotent stem cells that can give rise to all cell types, which allows them to restore lost body parts after injury or amputation. This makes flatworms excellent model systems for studying regeneration. In this study, we present the adhesive organs of a marine flatworm as a simple model system for organ regeneration. Macrostomum lignano has approximately 130 adhesive organs at the ventral side of its tail plate. One adhesive organ consists of three interacting cells: one adhesive gland cell, one releasing gland cell, and one modified epidermal cell, called an anchor cell. However, no specific markers for these cell types were available to study the regeneration of adhesive organs.
We tested 15 commercially available lectins for their ability to label adhesive organs and found one lectin (peanut agglutinin) to be specific to adhesive gland cells. We visualized the morphology of regenerating adhesive organs using lectin- and antibody staining as well as transmission electron microscopy. Our findings indicate that the two gland cells differentiate earlier than the connected anchor cells. Using EdU/lectin staining of partially amputated adhesive organs, we showed that their regeneration can proceed in two ways. First, adhesive gland cell bodies are able to survive partial amputation and reconnect with newly formed anchor cells. Second, adhesive gland cell bodies are cleared away, and the entire adhesive organ is build anew.
Our results provide the first insights into adhesive organ regeneration and describe ten new markers for differentiated cells and tissues in M. lignano. The position of adhesive organ cells within the blastema and their chronological differentiation have been shown for the first time. M. lignano can regenerate adhesive organs de novo but also replace individual anchor cells in an injured organ. Our findings contribute to a better understanding of organogenesis in flatworms and enable further molecular investigations of cell-fate decisions during regeneration.
KeywordsLectin Flatworm Duo-gland system Marine adhesion TEM Superresolution microscopy
Flatworms (Platyhelminthes) possess the extraordinary capacity of regeneration. Recently, the molecular foundation of the stem-cell-based regeneration process has been intensely studied in planarians [1–4]. Several studies have addressed regeneration and stem cell regulation for the basal, free-living flatworm Macrostomum lignano [5–8]. M. lignano is able to regenerate its anterior-most region and any tissue posterior to the pharynx [5, 6]. After amputation, regeneration of the tail plate completes within 6 to 10 days . In previous studies, the number of differentiated adhesive organs has been used as a marker for complete tail-plate regeneration [6, 9]. M. lignano is a small marine flatworm that was first described in 2005 . The animal possesses approximately 130 adhesive organs in a half-moon shaped arc at the ventral side of its tail plate [9, 10]. Each organ consists of three cell types : an adhesive gland cell, which secretes the glue to adhere animals to any substrate, and a releasing gland cell, which expels a releasing factor for detachment, both gland cells secreting their vesicles through a modified epidermal cell (the anchor cell). We use the term “adhesive organ” to refer to a cluster of one adhesive gland cell, one releasing gland cell, and one anchor cell, as defined by Tyler . The simplicity of the system—i.e. three interacting cells, a fast regeneration time, and restricted localization in the tail—makes adhesive organs an optimal system for analysing regeneration. After tail-amputation, it is obvious that all adhesive organs have to be completely rebuilt from stem cells. This process requires the coordinated spatial and temporal differentiation of the three cell types. Furthermore, the outgrowing necks of one adhesive gland cell and one releasing gland cell must pair and together penetrate a newly forming anchor cell . This has to occur independently about 130 times. Finally, the anchor cells must position themselves in a horseshoe-shaped arc at the ventral side of the tail plate. Such a developmental mechanism raises the question of whether M. lignano, and perhaps flatworms in general, have a defined developmental program for adhesive organ formation. This hypothesis leads to the conclusion that direct cellular interaction and an encompassing regulatory program are required for the formation of a functional adhesive organ. Alternatively, flatworms may show developmental plasticity with respect to adhesive organ formation. Thereby, flatworms must be able to integrate a newly differentiating stem cell into a partially injured organ. One problem in addressing this question is the absence of cell type-specific markers. Apart from some tissue- and cell type-specific antibodies for M. lignano [7, 13, 14], adhesive cell type-specific labelling is missing. In studies of several invertebrate species, lectins have been used as a marker for specific tissues [15–17]. Lectins are proteins with a high binding specificity to the oligosaccharide moieties found in glycoproteins, and they are widely used in biomedical research . Moreover, lectins were successfully applied in the planarian flatworm Schmidtea mediterranea  and the sea star Asterias rubens  to label secretory adhesive cells. Therefore, we tested commercially available lectins for their ability to label M. lignano secretory cells.
Here, we present ten new markers for differentiated M. lignano cell types and tissues, nine lectins, and one cell-type specific antibody. We describe the morphology of regenerating adhesive organs using two of these markers (one lectin and the antibody), as well as with EdU staining and transmission electron microscopy. We show that adhesive gland and releasing gland cells differentiate earlier than their connected anchor cell. Before the anchor cell migrates to the epidermal surface and forms microvilli, it surrounds the necks of two fully differentiated gland cells. Partial amputation of anchor cells revealed that some adhesive gland cell bodies survive this injury and reconnect with a newly formed anchor cell. Our findings pave the way for further molecular analyses of cell-fate decisions during adhesive organ regeneration.
Lectins as markers for differentiated cells and tissues of Macrostomum lignano
Lectin labelling of different cells and tissues in Macrostomum lignano
Frontal glands 1, 2, 4
Frontal glands 3
Lens culinaris agglutinin
n = 25
Phaseolus vulgaris erythro agglutinin
n = 28
Phaseolus vulgaris leuco agglutinin
n = 27
Succinylated wheat germ agglutinin
n = 17
Griffonia (Bandeiraea) simplicifolia lectin I
n = 23
n = 45
n = 48
Ricinus communis agglutinin
n = 24
n = 32
Wheat germ agglutinin
n = 23
Sambucus nigra agglutinin
n = 16
Maackia amurensis lectin II
n = 18
Dolichos bilforus agglutinin
n = 21
Sophora Japonica agglutinin
n = 20
Ulex europaeus agglutinin 1
n = 12
neg. control (without lectin)
n = 36
Soybean agglutinin (SBA) labelled frontal glands 1, 2, and 4, as well as the pharyngeal glands, the female antrum, the cement glands, and developing eggs in the antrum (Fig. 1b and Additional file 3: Figure S2). In most of the specimen (37 out of 45), SBA additionally labelled the prostate glands (Additional file 3: Figure S2). Griffonia simplicifolia lectin I (GSL I) led to a staining in the pharyngeal glands, the female antrum, and frontal glands 1, 2, and 4 (Fig. 1c and Additional file 4: Figure S3). In contrast to SBA, no staining was observed in the cement glands or prostate glands. Succinylated wheat germ agglutinin (sWGA) labelled the pharyngeal glands and frontal glands type 3 (Fig. 1d and Additional file 5: Figure S4). The necks of frontal gland type 3 proceeded in parallel with rhammites through the neuropil and the rostrum (Additional file 5: Figure S4B-C). Ricinus communis agglutinin (RCA) resulted in a ubiquitous staining of the whole animal, excluding the epidermis (Fig. 1e and Additional file 6: Figure S5). Some tissues, such as the female antrum, developing eggs, the prostate glands, and the secretory gland cells of the adhesive organs, appeared to be more strongly labelled than the rest of the animal (Fig. 1e and Additional file 6: Figure S5). Out of 15 lectins, four labelled the epidermal layer of the animals. Lens culinaris agglutinin (LCA) stained the outline of the epidermal cells, probably representing the epidermal cell junctions (Fig. 1f and Additional file 7: Figure S6A1-B3). Concanavalin A (Con A) led to an overall staining of the epidermal layer (Fig. 1g and Additional file 7: Figure S6C1-D3). Phaseolus vulgaris erythro (PHA-E) and leuco (PHA-L) agglutinins led to the same speckled staining of the epidermis (Fig. 1h-i). At higher magnification, hair-like structures on the epidermal surface became obvious, most likely representing the glycocalyx of epidermal microvilli (Additional file 8: Figure S7A-D). The rhabdite gland openings, which penetrate the epidermis, remained unstained (Additional file 8: Figure S7B). At the ventral side of the tail plate, a clear staining of the specialized microvilli of the adhesive organs was visible (Fig. 1i inset and Additional file 8: Figure S7D), representing their glycocalyx. The glycocalyx covers the epidermal surface, including microvilli and adhesive organs (Additional file 8: Figure S7C). Out of the tested lectins, the Arachis hypogaea peanut agglutinin (PNA) resulted in specific labelling of the adhesive gland cells in the tail plate, along with other tissues (described in the next section).
Peanut agglutinin as an adhesive gland cell marker
An intermediate filament-specific antibody as an anchor cell marker
Adhesive organ regeneration after tail plate amputation
Can adhesive gland cell necks regenerate and penetrate a novel anchor cell?
To clarify whether the regrowth of other gland necks is a common ability in M. lignano, we amputated animals anteriorly to the brain and visualized the regeneration of the gland cell necks using the lectin sWGA (Additional file 11: Figure S10). After amputation, the gland cell bodies remained, and their necks elongated with the regenerating rostrum (Additional file 11: Figure S10). Therefore, we conclude that outgrowth of gland cell necks can be a general feature of the regenerating tissues of M. lignano.
Lectins as new markers in Macrostomum lignano
Macrostomum lignano is an emerging model system for studies of stem cell dynamics and regeneration [5, 7–9, 20–28]. Recently, the genome and transcriptomes of M. lignano were published, facilitating molecular and genetic studies [29, 30]. However, the visualization of cell types is limited to in situ hybridization  and a few specific antibodies [7, 13, 14]. With this study, we add commercially available lectins as simple-to-use and inexpensive labelling reagents to the methodical toolbox of M. lignano. Five lectins label various types of secretory gland cells throughout the animal. Additionally, lectin labelling revealed four new types of frontal glands in the anterior part of the animal. The function of these gland cells and the composition of their secretions is currently unknown. In Schmidtea mediterranea, the proteinaceous component of secreted mucus was identified , and the involvement of the secretions in locomotion, innate immunity, adhesion, and protection against environmental reactive oxygen species was predicted. Secreted proteins showed a high similarity between parasitic- and free living flatworms  and may also be conserved in M. lignano. Lectins used in combination with in situ hybridization could help to map the expression of secreted proteins to a specific gland cell type and thereby reveal their function. Additionally, we showed that lectins can be combined with EdU labelling and therefore enable the visualization of cell turn-over and renewal. These approaches will further enable the investigation of gene function during differentiation and regeneration processes.
PNA labelling of adhesive gland cells
Many marine organisms rely on adhesive secretions to attach themselves temporary or permanently to the substrate . Most of these adhesives consist of various proteins, either alone or in combination with other components. Glycoproteins have been identified in the adhesives of several marine organisms, such as barnacles [33, 34], the sea star Asterias rubens [19, 35], the green mussel Perna viridis [36, 37], and the green alga Ulva [38, 39]. In the sea star A. rubens, 11 lectins labelled the disc epidermis at the level of adhesive cells, and four (DBA, WGA, RCA, and Con A) additionally labelled the secreted adhesive material . In M. lignano, PNA and RCA labelled the adhesive gland cells, along with other structures. The labelling of the adhesive gland cells with PNA was restricted to their secretory vesicles, suggesting that the PNA labelled glycoconjugate is secreted and part of the adhesive. The subcellular localization of the RCA labelling could not be visualized. Both PNA and RCA also label the subepidermal marginal adhesive glands in the planarian S. mediterranea . Whether the corresponding glycoconjugates are conserved and involved in the adhesive glue is currently unknown. There are eight additional lectins leading to a staining in the adhesive glands of S. mediterranea. Four of these (DBA, WGA, sWGA, and SBA) were also tested in M. lignano and led to a different staining result.
Adhesive organs as model for organogenesis
Flatworms are well known for their capacity to regenerate large body parts. The potential to investigate naturally occurring organ regeneration in an in vivo system led to several regeneration studies within the last few years (reviewed in ). In the past years, the central nervous system was the favourite model system for whole organ regeneration (reviewed in ). Yet the broad range of cell types and differently expressed genes within this system made analyses challenging . Recently, there has been a trend in flatworm regeneration research to investigate simpler organ systems, such as the excretory system [42, 43], the optic cups [44–47], the pharynx , and the intestine [49, 50]. Most regeneration studies were focused on free-living triclads such as S. mediterranea and Dugesia japonica, whereas the less derived Macrostomorpha [51–53] gained less attention. One great advantage of studying the regeneration of adhesive organs in a species of Macrostomum is its simple structure of only three interacting cells . Few systems are equally simple and accessible but still with the complexity of multi-cellular organs. Even in closely related families of the Macrostomida, the adhesive organs exhibit a higher complexity. For example, in the family of Microstomum, the releasing glands form branches; in Bradynectes, the microvilli number and arrangement vary highly between single organs; and in Myozona, two adhesive glands are associated with one releasing gland and one anchor cell [12, 54, 55]. In higher orders of Platyhelminthes, including Proseriata, Tricladida, and Rhabdocoela, the adhesive organs form a relatively large adhesive field consisting of numerous anchor cells and gland neck openings. The gland necks are highly branched and penetrate several anchor cells. In contrast to M. lignano, adhesive papillae are also present on the lateral sides of the body or even encircle it [12, 55].
Regeneration of adhesive organs
In earlier studies of M. lignano, the number of adhesive papillae was used as an indication for complete tail plate regeneration [6, 9, 27]. However, as no suitable markers were available, no further investigations on adhesive organ regeneration or differentiation were performed. With the lectin PNA and the Macif1 antibody, we visualized the location of differentiated adhesive gland cells and anchor cells during regeneration. Thus far, no marker for releasing gland cells could be identified. To remove the complete tail plate, we used the same cutting level as has been used in previous studies [9, 27]. The regeneration of adhesive organs requires the differentiation of stem cells towards adhesive- and releasing gland cells and anchor cells. First, one adhesive- and one releasing gland cell differentiate in a temporally and spatially coordinated manner to form the gland cell pair of one adhesive organ. Second, the outgrowing gland cell necks of the adhesive and the releasing glands, which are in direct contact with each other, penetrate an undifferentiated anchor cell. At this point, the future anchor cell is still located within the regeneration blastema and has not reached the epidermal surface and no intermediate filaments are expressed. Finally, the anchor cell integrates into the epidermis and forms junctional complexes with the neighbouring epidermal cells . Then they establish the intermediate filament network in their cytoplasm, with connection to the tail plate ECM occurring via hemidesmosomes . After 10 days of regeneration, all adhesive organs are formed and conjointly give rise to the horse-shoe shaped adhesive system of the tail plate .
After amputation of the anchor cells on one side of the tail plate, the majority (68.2 %) of the adhesive gland cells were EdU negative at the amputated site. In contrast, 96 h after amputation of the whole tail plate, all PNA labelled adhesive gland cells had an EdU-positive nucleus. This indicates that some adhesive gland cell bodies were able to survive the partial amputation of their necks and reconnected with a newly formed anchor cell. In M. lignano, a pool of neoblasts is present that does not undergo cell division but is ready to differentiate into the required cell types [25, 26]. In addition, neoblasts arrested in G2-phase were predicted [25, 26]. It is possible that some of the EdU-negative adhesive gland cells differentiated from these neoblasts, which did not undergo a round of cell division and are EdU negative. However, it is unlikely that all EdU-negative gland cells originate from this pool of stem cells since their overall number would be too low. The amputation of the anchor cells led to a decrease in PNA labelling after 24 and 48 h at the area of the cutting. As the PNA labelling was restricted to the adhesive gland vesicles, we speculate that after the amputation of their necks, the adhesive gland cells stopped the production of vesicles until their reconnection to a new anchor cell.
Lectins have been shown to be highly useful tools as markers for tissues and organs in diverse organisms. We now show that a collection of nine lectins can be used to stain specific cell types in Macrostomum lignano. We used PNA-based labelling of adhesive gland cells to study the regeneration of adhesive organs in this species. In combined staining with an anchor cell specific antibody, we explored the spatial and temporal formation of the adhesive system upon amputation. Furthermore, we examined the ability of gland cells to regrow amputated gland cell necks. With respect to the adhesive organs, this required the integration of a newly differentiated anchor cell into the rebuilding adhesive organ, as compared to the complete de novo formation of adhesive organs after whole tail amputation. Overall, our data can provide a foundation for understanding cell differentiation, cellular interactions, and organ formation.
Macrostomum lignano  cultures of the inbred line DV1  were kept in petri dishes with nutrient enriched artificial seawater (Guillard’s f/2 medium)  and were fed ad libitum with the diatom Nitzschia curvilineata. Animals were maintained in a climate chamber with 20 °C, 60 % humidity and a 14:10 day-night cycle.
Animals were relaxed with 7.14 % MgCl2 hexahydrate and then fixed in 4 % formaldehyde (made from paraformaldehyde) in PBS (PFA) for 1 h. Afterwards the specimen were washed six times 10 min in Tris-buffered saline (pH 8.0) supplemented with 5 mM CaCl2 and 0.1 % Triton (TBS-T). Buffers were additionally supplemented with 5 mM MnCl2 for Con A, LCA, PSA, and PHA-L, and 5 mM MgCl2 for GSL I. Unspecific background staining was blocked by pre-incubation in TBS-T containing 3 % (w/v) bovine serum albumin (BSA-T) overnight at 4 °C. Biotinylated lectins were diluted in BSA-T to a final concentration of 25 μg/ml and applied to the specimen for 2 h at room temperature. After six washes of 10 min each in TBS-T, the specimen were either incubated for 2 h in Texas-Red-conjugated streptavidin (Vector Laboratories) diluted 1:100 in BSA-T or for 1 h in Dylight488-conjugated-streptavidin (Vector Laboratories) diluted 1:300 in BSA-T at room temperature. After several washing steps in TBS-T, the specimen were mounted in Vectashield and analysed using a Zeiss Axioscope A1 microscope or a Leica SP5 II confocal scanning microscope. Control reactions for PNA labelling were performed by pre-incubating the lectin with its inhibitory monosaccharide D-galactose (0.2 M) for 2 h at 4 °C. For super resolution microscopy, the labelled specimen were mounted in Mowiol and examined with a Leica SP8 gSTED microscope system.
Double labelling of adhesive gland cells and anchor cells
Anti-Macif1 antibodies were raised in rabbits against the peptide CERSRDQKEIKRLRDE (aa 212 - 226) by Eurogentec. For regeneration experiments, the animals were relaxed with 7.14 % MgCl2 hexahydrate, cut at the desired level using a razor blade and immediately transferred to f/2-medium. After different times of regeneration, animals were relaxed with 7.14 % MgCl2 hexahydrate and then fixed in 4 % PFA for 1 h. After six washing steps of 10 min each with TBS-T, unspecific background staining was blocked by pre-incubation in 3 % BSA-T for 30 min at room temperature. The specimen were incubated with 1:300 diluted biotinylated lectin PNA (Vector Laboratories) in 3 % BSA-T for 2 h at room temperature. After three 10 min washes in TBS-T, the specimen were incubated for 1 h in Dylight488-conjugated-streptavidin (Vector Laboratories) diluted 1:300 in BSA-T at room temperature. The specimen were washed several times with TBS-T and re-fixed with 4 % PFA for 20 min at room temperature. After several washes with TBS-T, specimen were heated overnight in a 1:10 diluted epitope retrieval solution (DakoCytomation K5336) at 80 °C. After several washing steps with TBS-T, the specimen were blocked in 3 % BSA-T for 4 h at 4 °C. The specimen were then incubated with 1:1000 diluted polyclonal Rabbit-α-macif1 antibody in 3 % BSA-T overnight at 4 °C. After six washes of 10 min each with TBS-T, the specimen were incubated for 1 h in a goat-α-rabbit-TRITC antibody diluted 1:600 in BSA-T at room temperature. After several washing steps with TBS-T, the specimen were mounted in Vectashield and analysed using a Leica SP5 II confocal scanning microscope. Stacks were acquired sequentially and z-projected.
Double labelling of EdU and adhesive gland cells
Amputated and uncut animals were soaked in the thymidine analogue 5-ethynyl-2′-deoxyuridine (EdU; Invitrogen) at a concentration of 100 μM in f/2 medium for 4 days continuously. Afterwards, the animals were washed several times with f/2 medium, relaxed with 7.14 % MgCl2 hexahydrate and fixed in 4 % PFA for 30 min. Lectin labelling was performed as described in the section lectin histochemistry, using Texas-Red-conjugated streptavidin. After lectin labelling, the specimen were washed several times with PBS-T and blocked with Blocking reagent solution I (Applichem) overnight at 4 °C. After several washes in PBS-T, the specimen were incubated in Click-iT® EdU reaction cocktail (concentrations according to manufacturer’s instructions – Invitrogen). DNA was visualized with an addition of DAPI (1 μg/ml in PBS-T) for 30 min at room temperature. After several washes with PBS-T, the specimen were mounted in Vectashield and analysed using a Leica SP5 II confocal scanning microscope. Stacks were acquired sequentially and adhesive gland cells with an EdU-positive and -negative nucleus were counted using ImageJ software.
Chemical fixation of M. lignano for transmission electron microscopy was performed as described in previous studies . Animals were relaxed with 7.14 % MgCl2 hexahydrate and fixed according to . Specimen were dehydrated in an acetone series, embedded in Polybed 812, cut and double stained with uranyl acetate and lead citrate, and examined with a Zeiss Libra 120 TEM (Zeiss, Germany). For preservation of the glycocalyx (Additional file 8: Figure S7) specimen were fixed with 2.5 % glutaraldehyde in 0.1 M cacodylate buffer for 1 h. After washing with cacodylate buffer, specimen were post-fixed with reduced osmium tetroxide (2 % osmium tetroxide + 3 % potassium ferrocyanide in 0.1 M cacodylate buffer). After washing, specimen were treated with 1 % thiocarbohydrazide at 60 °C. After washing with distilled water, specimen were fixed with 2 % osmium tetroxide. After washing, specimen were en-block stained with 1 % uranylacetate overnight and incubated in lead aspartate for 30 min at 60 °C. After dehydration, specimen were embedded in durcupan epoxy resin. Images were made using the Olympus SiS iTEM 5.0 software and a TRS 2048 high speed camera.
EdU, 5-ethynyl-2′-deoxyuridine; gSTED, gated stimulated emission depletion; PNA, Arachis hypogaea peanut agglutinin; TEM, Transmission Electron Microscopy.
The authors are most grateful to Martin Offterdinger and Adi Sandbichler for excellent introduction and help in working on the SP8 gSTED system and to Florian Steiner for helpful comments on the manuscript.
The project is supported by Austrian Science Fund (FWF): [P 25404-B25] and COST Action TD0906. BL is a recipient of a DOC Fellowship of the Austrian Academy of Sciences at the Institute of Zoology, University of Innsbruck. P.F. is Research Director of the Fund for Scientific Research of Belgium (F.R.S.-FNRS).
Availability of data and material
The data supporting the results of this manuscript are included in the body of the manuscript and as supplemental data.
BL and PL conceived and designed the study, performed experiments, and wrote the paper. EH and PF designed lectin labelling experiments and helped interpreting the lectin staining results. WS performed electron microscopy experiments. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
The use of Macrostomum lignano for research purposes does not require ethical approval.
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