REGULAR ARTICLE

Effects of neuropeptide F on regenerationin Girardia tigrina (Platyhelminthes)

Natalia D. Kreshchenko & Zakhar Sedelnikov &

Inna M. Sheiman & Maria Reuter & Aaron G. Maule &

Margaretha K. S. Gustafsson

Received: 3 April 2007 /Accepted: 13 September 2007 / Published online: 20 December 2007# Springer-Verlag 2007

Abstract The effects of neuropeptide F (NPF; fromMoniezia expansa) on the regeneration of Girardia tigrinawere studied. The animals were decapitated and incubatedin water (control) or NPF. The dynamics of the proliferationof the neoblasts in the developing tissue were studiedduring the course of regeneration by monitoring the mitoticindex (MI). The effects of incubation in FMRFamide andGYIRFamide on the MI were also tested. The course ofcephalic regeneration was followed with in vivo computer-assisted morphometry for up to 7 days. The developmentof the regenerating nervous system and the musculaturewas visualised by immunostaining with a primary antise-rum to the C-terminal decapeptide of NPF (YFAIIGRPRFa)and tetramethylrhodamine-isothiocyanate-conjugatedphalloidin, which stains F-actin in muscle filaments. Thestudy showed that NPF had a stimulatory effect on the mito-tic activity of the neoblasts. FMRFamide and GYIRFamidedid not have this effect. NPF also stimulated the growth of

the regenerating head and the growing nervous system andmusculature. NPF is postulated to have a morphogeneticaction in the regenerating animals.

Keywords Regeneration . Neoblasts . Mitotic index .

Neuropeptides .Girardia tigrina (Platyhelminthes)

Introduction

Several types of complex morphogenetic processes, such ascell proliferation, cell differentiation and pattern formation,take place during regeneration. Free-living flatworms,belonging to the phylum Platyhelminthes, taxon Tricladida,are one of the favoured model organisms for the study ofmechanisms of morphological and functional restoration. Amorphogenetic plasticity and high regeneration abilitycharacterise these relatively simple organisms. Regenera-tion in triclads is a global process involving all bodyregions by a combination of morphallactic and epimorphicmechanisms and is based upon a population of totipotentstem cells, the neoblasts (Baguñà 1981). Several publica-tions on regenerating triclads have recently been published(Reddien and Sanchez Alvarado 2004; Bode et al. 2006;Sanchez Alvarado 2006; Saló 2006; Nimeth et al. 2007).

Although intensive studies on regeneration in tricladshave been performed, the mechanisms underlying thephenomenon are not fully understood. Attempts to isolatethe natural endogenous substance(s), which activate regen-eration in triclads, have led to the identification of thepeptidergic nature of the activator and inhibitor of regen-eration (Friedl and Webb 1979). Several neuropeptides andgrowth factors have been detected immunocytochemicallyin free-living and parasitic flatworms (for references, seeGustafsson and Halton 2001; Reuter and Halton 2001;

Cell Tissue Res (2008) 331:739–750DOI 10.1007/s00441-007-0519-y

This work was supported by two grants from the Finnish Academy ofScience (nos. 202685, 2004) and (no. 112090, 2006) to M.G., anRFBR grant (07-04-00452a) to N.K. and a Wellcome Trust grant(069411) to A.G.M. for which we express our gratitude.

N. D. Kreshchenko : Z. Sedelnikov : I. M. SheimanInstitute of Cell Biophysics,Pushchino, Moscow Region 142290, Russia

M. Reuter :M. K. S. Gustafsson (*)Department of Biology, Åbo Akademi University,Artillerigatan 6,FIN-20520 Åbo, Finlande-mail: magustaf@abo.fi

A. G. MauleParasitology Research Group, School of Biological Sciences,Queen’s University Belfast,97 Lisburn Road,Belfast BT9 7BL, UK

Gustafsson et al. 2002; Reuter and Kreshchenko 2004).Some of these neuropeptides and growth factors, such assubstance P (SP) and substance K (SK), Hydra headactivator, epidermal growth factor, fibroblast growth factorand somatostatin, have been shown to modulate the rate ofregeneration and the mitotic activity in regenerating triclads(Baguñà et al. 1989; Sheiman et al. 1989; Tiras et al. 1990).

Recently, special interest has focused on neuropeptidesthat have been isolated from flatworms, such as theFMRFamide (FMRFa)-related peptides (GNFFRFa,GYIRFa, YIRFa, RYIRFa) and the neuropeptide-F-likepeptides (NPFs) (Day and Maule 1999; Halton and Maule2004; McVeigh et al. 2005). NPF was originally purifiedfrom the tapeworm Moniezia expansa by Maule et al.(1991) and the free-living flatworm Artioposthia triangulata(= Arthurdendyus triangulatus) by Curry et al. (1992). Twoother flatworm neuropeptides have been characterised bymeans of molecular methods from the blood flukes,Schistosoma mansoni and S. japonicum. These neuropep-tides differ from each other by only a single amino acid(Humphries et al. 2004). Moreover, an enzyme involved inC-terminal amidation has been characterised from S.mansoni (Mair et al. 2004). The NPF precursor gene (npf)has been identified and characterised from M. expansa (Mairet al. 2000) and A. triangulatus (Dougan et al. 2002). NPFshave been isolated from several insects and molluscs(Rajpara et al. 1992; Brown et al. 1999; de Jong-Brinket al. 2001; Garczynski et al. 2005). These NPFs varybetween 36 and 40 amino acids in length and displaysequence resemblance to the vertebrate neuropeptide Y(NPY) family of peptides (Tatemoto et al. 1982). Inmammals, NPY and the related peptides, peptide YY andPP, comprise the NPY/PP super family and have a pivotalaction in the regulation of food intake, enzyme secretion,motility and other physiological processes. In the centralnervous system (CNS), NPY is an inhibitory neurotrans-mitter and exhibits anxiolytic, anti-stress, anti-depressant,anti-convulsant and anti-nociceptive actions in addition toits hypertensive, potent appetite-stimulating effects andits capacity to shift circadian rhythms (for references, seeBrain and Cox 2006). Data have accumulated to supporta possible mitogenic and growth-promoting function ofNPY-like peptides in vertebrates (Nie and Selbie 1998;Hansel et al. 2001; Pedrazzini 2004). Several reportsindicate a hormonal function of NPFs in invertebrates,especially in association with reproduction and development(Cerstiaens et al. 1999; de Jong-Brink et al. 2001;Huybrechts et al. 2004).

Little is known about the role of NPF-like neuropeptidesin biochemical and physiological processes in flatworms.Specific NPF immunostaining (NPF-IS) has been observedin the CNS and peripheral nervous system of manyflatworms (Day and Maule 1999). NPF-immunoreactive

(NPF-IR) nerves often occur close to the reproductivesystem. Only a few studies describe the function ofNPF in flatworms. Marks et al. (1996) have found thatthe C-fragment of NPF elicits contractile responses inmuscle preparations of the liver fluke, Fasciola hepatica.Hrchková et al. (2004) have shown that NPF stimulates themotility of the larvae of the tapeworm, Mesocestoidesvogae. Kreshchenko et al. (2001) have presented a pre-liminary report on the effects of FMRFa-related peptidesand NPF on regeneration in the fresh-water triclad Girardiatigrina: NPF (10−6 M) stimulates the formation of the headblastema and pharyngeal regeneration in G. tigrina, where-as FMRFa (10−6 M) stimulates pharyngeal regeneration,but GYIRFa (10−6 M) had no significant effect.

The three most important strategies in connection withthe growth and regeneration of an organism are cellproliferation, cell growth and the increase of the extracel-lular matrix (Wolpert et al. 1999). The three strategies mustbe investigated with different methods. In this study, theeffects of NPF on the growing blastema in G. tigrina havebeen investigated by using three approaches. The animalswere first decapitated and incubated with either NPF orwater (control) for up to 7 days. The three approaches were:(1) the dynamics of the proliferation of the neoblasts in thepost-blastema region was studied by following the mitoticindex (MI) after treatment with colchicine and staining witha DNA-binding dye (Hoechst 333 42), together with theeffects of incubation in FMRFa and GYIRFa on the MI;(2) the course of regeneration of the head region was fol-lowed with in vivo computer-assisted morphometry; (3) thedevelopment of the regenerating nervous system (NS) andthe musculature was visualised by immunostaining with aprimary antiserum to the C-terminal decapeptide of NPF(YFAIIGRPRFa) and tetramethylrhodamine isothiocyanate(TRITC)-conjugated phalloidin, which stains F-actin inmuscle filaments. The study showed that NPF had astimulating effect on the mitotic activity of the neoblasts.FMRFa and GYIRFa did not have this effect. NPF alsostimulated the growth of the regenerating head and thegrowing NS and musculature. A morphogenetic action ofNPF is postulated.

Materials and methods

Animals

Specimens of an asexual strain of Girardia tigrina Girard1850 (Platyhelminthes, Tricladida) were obtained from theInstitute of Cell Biophysics, Pushchino, Moscow Region,Russia. The animals were kept in large glass aquaria insemi-darkness in tap water. They were fed with mosquitolarvae (Chironomide) or frozen beef liver once or twice a

740 Cell Tissue Res (2008) 331:739–750

week. The animals were starved for at least 1 week beforethe experiments, which were carried out at 20±1°C.

The rationale behind the order of experiments was thefollowing:

1. In 1999, a stimulating effect NPF at 10−6 M on thegrowth of the NS in regenerating G. tigrina was foundin immunocytochemical studies performed in ourlaboratory in UK.

2. In 2000, a stimulating effect of NPF at 10−6 M on therestoration of the pharynx function during its regener-ation in anterior and tail fragments of G. tigrina wasfound in a study performed by us in Russia. NPF at10−6 M was thus chosen as the starting concentrationfor the following experiments.

3. The experiments were continued by testing the effectsof NPF at 10−6 M, but also lower concentrations. Now,the increase in the MI and the growth of the blastemawas followed by using colchicine treatment in ourlaboratories in UK (2002) and in Finland (2004) and invivo computer-assisted morphometry in our laboratoryin Russia (2006), respectively.

4. In addition, the effects of FMRFa at 10−6 M andGYIRFa at 10−6 M, but also at lower concentrations, onthe MI were tested by us in UK and Finland.

Decapitation

G. tigrina of 9–10 mm in length were chosen fordecapitation. For anaesthesia, the animals were placed onice covered with filter paper and decapitated behind theauricles with a thin eye scalpel under a stereomicroscope(Fig. 1a). Because the regeneration process is dependent onthe temperature, the changing seasons and other unaccount-able conditions, each set of experiments had an individualcontrol group. The decapitated animals were transferred tobeakers with water (control group = C-group) or solutionsof peptides (experimental group = E-group).

Neuropeptides

Synthetic NPF was obtained from Immunogenetics (USA),GYIRFa from Genosys Biotechnology (UK) and FMRFafromSigma (USA). The amino acid sequence of the 39-residueNPF peptide from M. expansa is PDKDFIVNPSDLVLDNKAALRDYLRQINEYFAIIGRPRF-NH2. The peptides weredissolved in double-distilled water and the stock solution(10−4 M) was kept frozen in small aliquots.

MI in intact and regenerating G. tigrina

To obtain a base line for mitotic activity, the MI in intactanimals was counted in tissue strips from three body

regions (“neck”, pre-pharyngeal and tail; Fig. 1a). In orderto observe the accumulation of mitosis and the possibletoxic effects of 0.02% colchicine, the dynamics of the MIwas followed in tissue strips from the “neck”, pre-pharyngeal and tail regions of intact animals after incuba-tion for 1, 2, 3, 8, 12, 24 and 48 h. For the proliferationstudy, the decapitated animals were kept in small groups(10–20) in tap water (C-group) or in solutions of thevarious neuropeptides (E-group). The following peptideswere used: NPF, FMRFa and GYIRFa (10−6–10−8 M). Asan active control, the effects of a mixture of amino acids at10−5–10−6 M, comprising the neuropeptides studied (NPF,FMRFa and GYIRFa), were examined. In all experiments,colchicine was added to the incubation medium (finalconcentration 0.02%) to arrest cells in metaphase. Themitotic activity was followed in 7–10 animals of the C-group and the E-group at regular time points (2, 4, 6 or 8,12, 24 and 48 h of regeneration). Fragments (1–1.5 mm inlength) containing the post-blastema tissue were dissectedfrom the cut end with a small eye scalpel (Fig. 1b).

Cell dissociation was performed according to thefollowing protocol. (1)The samples were placed in smallEppendorf tubes together with a solution (100 μl) contain-ing methanol, glacial acetic acid, glycerol, and distilledwater (2:1:1:12) for 30 min at room temperature. (2) After30 min, the tubes with the samples were gently shaken.(3) To assess the number of mitotic figures, 10 μl 0.1 mMHoechst 333 42 fluorescent dye for chromatin was added.(4) A few minutes later, the cell suspension was fixedwith formaldehyde (4% final concentration). (5) The cellsuspension was placed on slides, air-dried at room tem-

Fig. 1 Representations of Girardia tigrina (shaded regions position atwhich mitoses were counted). a Intact G. tigrina showing the level ofdecapitation (cut). The “neck” region (neck) in the intact animalscorresponds to the post-blastema region (post-bl) in the regeneratinganimals. Counts were made in the prepharyngeal region (pre-ph),pharynx (ph), tail. b Regenerating G. tigrina (bl blastema, post-blpost-blastema region)

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perature and covered by 20% glycerol in phosphate-buffered saline (PBS, pH 7.3) and a cover glass.

Microscopy

The slides were examined with a Fluoval (Qarl Zeiss, Jena)or Leica DM RXA fluorescent microscope. A charge-coupled device camera (Leica DC 300F) was used formicrophotography. The number of mitotic nuclei in allstages of the mitotic cycle (from early prophase totelophase) per 1,000 nuclei was scored on 8–10 fields ofview on each slide. At least three to four repetitions pertime point were performed on separate slides. This gave3,000–4,000 cells per time point. When needed, repeatedcounting of the same slides was performed on separate daysand the data were accumulated and averaged. The MI(number of mitosis per 100 cells) was calculated.

Statistical analysis

MIs were calculated with mean and standard errors andthe MIs of control and experimental groups were com-pared. The results were statistically analysed by using theGraphPad Prizm statistical program with an unpaired two-tailed Student’s t-test. Most experiments were repeatedseveral times.

Morphometric measurements

Before decapitation, the whole body areas of each group ofG. tigrina were measured and compared and, if consideredequal, the groups were chosen for the experiment. For themorphometric studies, the decapitated animals were trans-ferred in groups of 25–30 animals to beakers filled with80 ml water (C-group) or solutions of fresh NPF at10−6–10−9 M (E-group). The morphometric measurementswere carried out by means of a computer-imaging systemwith a WAT-502 camera (Japan) mounted into an ocularpiece of a binocular microscope and coupled to an AverMedia TV Capture 98 with VCR hardware and the pos-sibility of stop-frame. The authors have long experienceand a long lasting tradition of morphometric measurements(Sheiman et al. 1989; Tiras et al. 1990). In order to preventsystematic errors related to elongation and contraction ofthe living animals, the following precautions were taken:(1) single animals were transferred to Petri dishes (12–15 cmin diameter), containing a small amount of water or NPF(10−6–10−9 M); (2) the animals were allowed to relax andswim around on the bottom of the dish; (3) the animalswere observed on a computer screen and photographedwhen they were not stretching or elongating; (4) thisprocedure was repeated until the whole group wasmeasured; (5) after the measurements, the animals were

placed back into their original beakers with water or NPF(10−6–10−9 M); (6) on the next day, the measurements wererepeated; (7) blind experiments were performed, i.e. thebeakers were coded so that only the project leader, and notthe person doing the actual measurements, knew which wasC-group or E-group; (8) all measurements were performedon the same living animals starting from Day 1. Thegrowing blastema areas were measured in the C-group andthe E-group after 1, 1.5, 2, 2.5, 3, 5 and 7 days ofregeneration and are expressed in arbitrary units (a.u.).

The method was tested before the experiment by takingmany photos of the same animal and processing themstatistically. In general, the decapitated and regeneratinganimals moved much more slowly than did the intactanimals, especially during the first 3-4 days of regeneration.

NIH (National Institute of Health, USA) image softwarewas used for the measurements. The results were statisti-cally processed by using Student’s t-test.

Immunocytochemistry

For the immunocytochemistry (ICC) studies, the decapi-tated G. tigrina were kept in groups of 40–50 animals inwater (C-group) or NPF (10−6 M) solutions (E-group). Theregenerating animals were followed for 7 days. Theimmunocytochemical staining of the regenerating animalswas performed on whole-mount preparations by using theindirect immunofluorescence method (Coons et al. 1955).Five regenerating animals from both groups were flat-fixedon Days 1, 1.5, 2, 2.5, 3, 4, 5 and 7 after decapitation.Fixation took place on ice between a glass microscope slideand a cover glass for 2 h in 4% (w/v) paraformaldehyde in0.1 M PBS (pH 7.4). The animals were then transferred to asolution of fresh fixative for 2 h (at room temperature).Following an overnight wash at 4°C in PBS containing0.3% (v/v) Triton X-100 (Sigma), 0.1% sodium azide(NaN3) and 0.1% (w/v) bovine serum albumin (Sigma),viz. antibody diluent, NPF-IS was achieved by using aprimary antiserum against the C-terminal decapeptide ofNPF, YFAIIGRPRFa (code 792.3), raised in rabbit. Theanimals were incubated in the primary antiserum (dilution1:1,000) for 48 h at 4°C, washed for 24 h in antibodydiluent at 4°C, immersed in fluorescein-isothiocyanate-labelled swine anti-rabbit IgG (DAKO; dilution 1:30) for24 h at 4°C, washed for 24 h in antibody diluent at4°C, stained with TRITC-conjugated phalloidin for F-actin (Sigma; dilution 1:100) for 12 h at 4°C, washedin PBS and finally mounted in PBS/glycerol (1:9, v/v).Controls included (1) omission of the primary antise-rum, (2) substitution of non-immune rabbit serum forthe primary antiserum and (3) liquid-phase pre-adsorptionof the primary antiserum with the appropriate antigen(200–1000 ng/ml diluted antiserum).

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Whole-mount preparations were examined with a LeicaTCS NT confocal scanning laser microscope. Some speci-mens were viewed from the dorsal side and some from theventral side. Four to 16 optical sections (1.6–22.9 μm apart)were collected from the specimens. Most of the photos arereconstructions (i.e. max projection) formed by adding fourto eight consecutive optical sections at maximal intensity offluorescence taken through 1.9–9.7 μm. The images werestored as bitmap files.

Results

General

During regeneration after decapitation, the following phaseswere discerned in G. tigrina. (1)During the first few hoursof regeneration, a thin layer of epithelium covered thewound. (2) A regenerating blastema began to form by themigration of neoblasts from the underlying tissue, i.e.the post-blastema region in which all mitotic divisions tookplace. In the blastema itself, no mitoses were observed. (3)Active cell differentiation took place in the regeneratingblastema. Nerve and muscle cells began to develop.

Mitotic cells

The nuclei of all cells were well preserved and easy to dis-tinguish. Cells in all stages of mitosis (prophase, meta-phase, anaphase, telophase) were scored (Fig. 2). Thenuclei at the prophase and metaphase stages were large andround (14–15 μm in diameter) or oval in shape (13 μm×16 μm). The interphase nuclei were much smaller(average size: 7–8 μm in diameter; occasionally up to 10μm).

MI in intact G. tigrina

The base line for the mitotic activity in intact G. tigrina wasdetermined in three body regions (Fig. 1a). No colchicinewas used. No significant differences between the MIs in thethree different body regions were observed (MI in “neck”region=0.684±0.037, n=10; MI in pre-pharyngeal region=0.611±0.06, n=11; MI in tail region=0.699±0.046, n=9).

The effect of 0.02% colchicine in the "neck", pre-pharyngeal and tail regions of intact G. tigrina wasdetermined by following the accumulation of mitoticfigures for 48 h (Table 1). During the first 12 h, theincrease in the MI was modest. However, at 24 h, a clearincrease in the MI was noted in the three regions. In the

Fig. 2 Mitotic figures from G. tigrina stained with Hoechst 333 42.a Prophase in an intact G. tigrina. b Metaphase at 24 h of regenerationand treatment with colchicine. The chromosomes are clearly visible.

c Anaphase at 4 h of regeneration. Interphase nuclei (i). d Telophaseat 4 h of regeneration

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“neck” region, the difference between MI at 12 h (0.657±0.057, n=7) and at 24 h (1.806±0.146, n=8) wasstatistically significant (P<0.0001). After 48 h of incuba-tion in colchicine, a small but non-significant decrease inthe number of mitosis was found in the “neck” and in thetail regions. Occasionally, a few anaphases and telophaseswere observed after 24 h of cultivation.

Effect of NPF on MI in regenerating G. tigrina

The MIs in the post-blastema regions of G. tigrina were ex-amined at 2, 4, 6, 12, 24 and 48 h of regeneration. Figure 3shows a comparison between the MI in the post-blastemaregion in the C-group and two E-groups.

In the C-group, no increase in the MI was observedduring the first 12 h of regeneration after decapitation.However, at 24 h, a clear increase in the MI was noted. Thedifferences between MI at 12 h (0.702±0.103, n=7) andat 24 h (1.362±0.066, n=13), on the one hand, and at24 h (1.362±0.066, n=13) and at 48 h (2.098±0.102, n=4)on the other, were statistically significant (P<0.0001).

In the E-groups, a general stimulating effect of NPF at10−6 M and 10−7 M was observed throughout the wholeseries of cultivation, except at time point 6 h. At time points4, 12 and 24 h, the MIs in the E-groups weresignificantly higher than in the C-group (for 4 h,P=0.045; for 12 h, P=0.0345; for 24 h, P=0.0343).

The effect of NPF at 10−8 M was tested only at 24 h ofregeneration and no significant increase in MI wasobserved. The effect of NPF at 10−9 M was not tested.

Effects of FMRFa, GYIRFa and amino acids on MIin regenerating G. tigrina

In the C-group, a slow increase in the MI was observedduring the first 12 h of regeneration after decapitation.Subsequently, the MI increased clearly (Fig. 4). In the E-groups, no stimulating effects of FMRFa (10−6–10−7 M)were observed (Fig. 4). The effects of GYIRFa (10−6–10−8 M)were determined only at 24 h and 48 h. No stimulatingeffects of GYIRFa were observed (Table 2). The same heldtrue for the mixtures of amino acids corresponding to thepeptides FMRFa (10−6 M) and GYIRFa (10−5 M) and NPF(10−6 M; data not shown).

Morphometric results

Morphometric measurements of the growing head blastemain G. tigrina regenerating in water or in various concen-trations of NPF (10−6–10−9 M) were performed during 7consecutive days after decapitation. NPF had a clearstimulatory effect on the growing blastema. The effectwas greatest after incubation in NPF at 10−9 M. Here, thedifferences between the blastema size in the C-group and

Fig. 3 Development of MI in the post-blastema region of regenerat-ing Girardia tigrina. Control animals compared with animalsincubated with NPF at 10−6 M or 10−7 M. *P<0.05, Student’s t-test.np not performed

Fig. 4 Development of MI in the post-blastema region of regenerat-ing G. tigrina. Control animals compared with animals incubated withFMRFa (10−6 M) or (10−7M). No significantly different results wereobtained within time points, Student’s t-test. np not performed

Table 1 Development of MI(mean±SE) in intact Girardiatigrina treated with 0.02%colchicine

Time of treatment (h) “Neck” region n Pre-pharyngeal region n Tail region n

1 0.760±0.050 5 0.667±0.033 3 0.800±0.141 42 0.800±0.108 4 0.700±0.041 4 0.775±0.025 43 0.680±0.114 5 0.580±0.049 5 0.825±0.085 48 0.950±0.132 4 0.800±0.091 4 0.700±0.058 312 0.657±0.057 7 0.857±0.194 4 0.775±0.144 424 1.806±0.146 8 1.487±0.118 5 1.525±0.134 448 1.600±0.153 3 1.833±0.145 3 1.267±0.233 3

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the E-group were significant at most of the time points ofregeneration (Table 3). At Days 2 and 3, the growth rate ofthe blastema increased. This was approximately the timepoint at which nerve and muscle cells began to differentiate(see immunocytochemical results).

The effects of the stronger concentrations of NPF weretested in separate experiments. Table 4 shows the effects ofNPF at 10−7 M and 10−6 M. In general, the effects weresmaller, with significant differences only at some, usuallylate, time points. When testing NPF at 10−7 M, a smallermagnification was used, as the size of the blastema wassmaller. Individual control groups were measured inseparate sets of experiments.

Pattern of NPF-IS in intact and regenerating G. tigrina

The brain and the main nerve cords in intact G. tigrinastained strongly with anti-NPF (Fig. 5a). The NPF-IRsubepithelial and submuscular NS formed a dense network(Fig. 5b). Multipolar NPF-IR nerve cell bodies (size: 7×15–20 μm) were observed (Fig. 5c). The NPF-IR nerve fibresran close to the muscle fibres. Bipolar NPF-IR nerve cellbodies were also present.

The patterns of NPF-IS and staining with TRITC-conjugated phalloidin in G. tigrina incubated in water (C-group) or in NPF at 10−6 M (E-group) were comparedduring 7 days of regeneration.

Day 1

In both groups, the wound was covered by a thinepithelium and the regenerating blastema formed a thinlayer of cells, showing no staining for NPF (nerves) or for

phalloidin (muscles). In both groups, NPF-IR nerve fibresfrom the subepithelial nerve net accumulated close to thesurface of the wound. In the E-group, the NPF-IR nervefibres extended towards the anterior end of the body.

Day 1,5

In the C-group, the layer of blastema was thin. In the E-group, the blastema had developed well and formed anelongated bud-like structure. In both groups, numerous thinNPF-IR nerve fibres from the subepithelial nerve netextended into the blastema. The nerve fibres underlyingthe new epithelium had grown as far as the anterior end ofthe blastema. In both groups, the border between theblastema and the old stump of the body was easilydistinguished because of the presence of phalloidin-stainedmuscles in the body. No staining for phalloidin wasobserved in the blastema (Fig. 5d,e).

Day 2

In the C-group, the size of the blastema was smaller thanthat in the E-group. In both groups, strong NPF-IS wasobserved in the cut ends of the “old” nerve cords. Thesubepithelial nerve net formed a “ring-like” structure on theborder between the growing blastema and the old tissue.

Day 2,5

In the C-group, the size of the blastema was still clearlysmaller than that in the E-group (Fig. 5f,g). In both groups,thin NPF-IR nerve fibres from the two cut ends of the “old”main nerve cords had grown towards each other, thus

Table 2 Development of MI (mean±SE) in the post-blastema region of regenerating G. tigrina. Control worms compared with worms incubatedwith GYIRFa (10−6M), (10−7M) or (10−8M)

Period of regeneration (h) Control n GYIRFa (10−6 M) n GYIRFa (10−7 M) n Control n GYIRFa (10−8 M) n

24 1.563±0.132 8 1.228±0.215 5 1.645±0.128 11 1.895±0.061 6 1.848±0.340 448 2.900±0.100 3 2.650±0.210 4 3.033±0.088 3

Table 3 Effects of the NPF at10−8 and 10−9M on growth ofblastema in G. tigrina(area of blastema measuredin arbitrary units ±SE)

**P<0.01, *P<0.05

Period of regeneration(days)

Control n NPF (10−8M) n NPF (10−9M) n

1 1,995±110 29 2,365±140* 29 2,673±188** 271.5 3,426±207 29 3,515±212 28 3,622±196 252 4,714±217 26 5,610±208** 26 5,784±224** 242.5 6,922±279 25 7,453±365 26 8,612±425** 273 11,034±508 25 11,679±547 25 12,364±444 285 23,088±838 27 24,136±933 24 26,096±792** 277 30,267±875 25 33,540±1229* 23 35,275±1056** 28

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forming the first indication of the new brain (Fig. 5f). Inboth groups, new thin muscle fibres had appeared in thegrowing blastema. Some of these delicate muscle fibresfollowed the NPF-IR nerve fibres in the subepithelial layer,thus forming the shape of the head blastema (Fig. 5g).

Day 3

From Day 3 onwards, no differences in the shape of thedeveloping head could be observed between the C-groupand the E-group. In both groups, NPF-IR nerve fibres hadgrown from the cut ends of the old nerve cords and formedtwo delicate arches, indicating the developing brain. Musclefibres were now visible in the centre of the regeneratingbud and beneath the developing brain. In the E-group, thepattern of NPF-IS in the forming brain was morepronounced (Fig. 5h,i).

Day 4

In both groups, the shape of the growing head had nowchanged from round to triangular and become moreelongated. The auricles had formed. The new brain hadbecome thicker. The phalloidin staining of the muscle fibreswas stronger, indicating thicker muscle fibres (Fig. 5j).

Day 5

In both groups, longitudinal, diagonal and circular musclefibres surrounded the new brain. The eyes had formedmedially to the new brain. In the new brain, more nervefibres had developed and the two arches of the NPF-IRnerves were now visible (Fig. 5k).

Day 7

In both groups, the shape of the new head and the newbrain had fully developed (Fig. 5l). However, the newganglion was still smaller than that in the intact planarian(Fig. 5a).

Discussion

The results of the three different methods used in this studycomplement and confirm each other. The study has shownthat different concentrations of NPF have a stimulatoryeffect on different phases of regeneration in G. tigrina. NPFat 10−6 M and 10−7 M stimulates the MI, whereas NPF at10−9 M stimulates the actual growth of the blastema.Generally, the mitotic activity, the growth rate of theblastema and the rate of development of the NS andmusculature are higher in NPF-treated G. tigrina.

MI in intact G. tigrina

Various aspects of the proliferating stem cells, the neo-blasts, in diverse species of triclads have been studied sincethe early 1970s (Baguñà 1974, 1976a, 1976b, 1981;Baguñà and Romero 1981; Morita and Best 1984; Saló2006; Saló and Baguñà 1984, 1985, 1989, 2002). Accord-ing to Baguñà and Romero (1981), the neoblasts constitute20%–30% of the total cell population in intact G. tigrinaand Dugesia mediterranea. In D. japonica, the cor-responding value has been estimated to be 10%–30% (Oriiet al. 2005). The present study has shown that mitoticdivisions take place in the whole body of the intact G.tigrina. However, the rate of cell divisions is low (about0.6%). This result represents the normal renewal of thetissue and serves as a base line for the study of regenera-tion. According to Ladurner et al. (2000), neoblasts con-stitute about 20% of the cell population in Macrostomumsp., 10% of them being in S-phase and 0.5% in mitosis.Bode et al. (2006) have investigated immunogold-labelledS-phase neoblasts, total neoblasts number, their distributionand evidence for arrested neoblasts in Macrostomumlignano. Three populations of proliferating cells have beenidentified: (1) somatic neoblasts located between theepidermis and gastrodermis, (2) neoblasts located withinthe gastrodermis, and (3) gonadal S-phase neoblasts. Thesomatic neoblasts represent 6.5% of the total number ofcells (Bode et al. 2006).

Table 4 Effects of NPF at 10−7 and 10−6 M on growth of blastema in G. tigrina (area of blastema measured in arbitrary units ±SE)

Period of regeneration (days) Control n NPF (10−7 M) n Control n NPF (10−6 M) n

1 844±58.6 24 785±74.6 25 2,901±169.2 24 3,356±184.8 231.5 1,097±78.7 23 1,107±87.4 24 3,047±198.3 23 3,866±199.8** 242 1,436±81.2 24 1,758±138.9* 24 5,720±295.3 24 5,773±338.3 242.5 1,638±69.9 24 1,881±88.7 22 8,183±320.1 21 8,847±455.7 223 2,117±92.8 24 2,147±71.6 22 12,323±638.2 22 12,521±435.4 225 4,430±150.9 24 4,839±160.0* 23 17,592±488.5 23 19,651±577.9** 227 6,105±163.0 21 6,663±223.7* 23 31,828±659.0 21 36,624±1235* 22

**P<0.01, *P<0.05

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Fig. 5 Pattern of NPF-IR nerves (green) and muscles stained withTRITC-conjugated phalloidin (red) in whole-mount preparations ofintact (a-c) and regenerating (d-l) G. tigrina. C-animal is a controlanimal, whereas E-animal is an animal incubated with NPF at 10−6 M.The border between the regenerating blastema (bl) and the “old” tissueis indicated with white lines on the left and right sides of theregenerating head (a auricles, b brain, c commissures, d day, e eye, mcmain nerve cord, n neuron, nn nerve net, r “ring”-like structure of thenervous plexus, se subepithelial nerve plexus). a Intact G. tigrina withbrain, main nerve cords, eyes and auricle. b Intact G. tigrina withsubepithelial and submuscular nerve net. c Intact G. tigrina with amultipolar neuron having a long fibre running between muscle fibres.

d Day 1.5, C-animal with thin blastema. Main nerve cord. e Day 1.5,E-animal with much larger blastema. Nerve net in blastema. f Day 2.5,C-animal with small blastema. Main nerve cord. g Day 2.5, E-animalwith much larger blastema. Nerve net in blastema. h Day 3, C-animalwith blastema. Muscle fibres begin to form in the blastema. A ring-like structure is formed by the nervous plexus on the border betweenthe “old” tissue and the blastema. Brain. i Day 3, E-animal with blas-tema. Brain and main nerve cords. Muscle fibres begin to form in theblastema. j Day 4, head of a C-animal with submuscular nerve net andauricles. k Day 5, head of a C-animal with brain and main nerve cords.l Day 7, head of a fully developed C-animal with brain, main nervecords, two brain commissures, subepithelial nerve plexus and eyes

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We have tested the effect of 0.02% colchicine in intactG. tigrina. No toxicity has been observed. However, as afew anaphases and telophases are observed after 24 h ofcultivation, some metaphases might proceed throughmitosis. Nimeth et al. (2004) have applied 0.005%colchicine to Macrostomum sp. and observed no dramatictoxic effects up to 24 h. Nevertheless, longer treatment withcolchicine leads to a clear decrease in the number of cellsarrested in metaphase. In our studies, MI continues toincrease during 48 h of regeneration, indicating no dramatictoxic effects.

MI in regenerating G. tigrina

In regenerating G. tigrina, incubation in NPF at 10−6 M and10−7 M clearly stimulates the rate of cell division in thepost-blastema region. NPF at 10−8 M has no stimulatingeffect on mitotic divisions at 24 h. The length of the cellcycle of the neoblasts in G. tigrina is not known. The cellcycle of the neoblasts in Schmidtea mediterranea has beenstudied by labelling with 5′-bromo-2′-deoxyuridine (BrdU)by Newmark and Sanchez Alvarado (2000) who estimatethe G2 phase to last 6 h.

In our study, the MI in the C-group remains at the samelevel for 12 h. Indeed, the MI is at the same level as in theintact G. tigrina. However, a strong increase in the MI hasbeen observed at 24 h and 48 h after decapitation. This is inagreement with the recent results of Nimeth et al. (2007)who have studied regeneration in Macrostomum lignano.During the first 8 h of regeneration, the mitotic activity inM. lignano is actually reduced. However, at 48 h afteramputation, mitotic activity reaches almost twice the valueof the control animals. According to Baguñà (1976a) andSaló and Baguñà (1984), there is an early mitotic peakduring the initial 4–8 h of regeneration and a secondmaximum at 2–3 days after amputation. The early mitoticpeak has been interpreted as reflecting the existence of alarge population of neoblasts in the G2 phase of the cellcycle (G2-neoblasts), awaiting the appropriate signal toundergo division.

In our study, no early mitotic peak has been detected inthe C-group. However, in the E-group, a clear peak in MI isobserved at 4 h of regeneration, indicating that NPFstimulates G2-neoblasts to undergo mitotic division. At6 h, the MI is equal in both groups, indicating that thepool of G2-neoblasts in the E-group has been emptied. Asecond wave of mitoses follows between 12–48 h. Again,the number of mitoses is higher in the E-group than theC-group. In our study, the stimulating effect of NPF at10−6 M varies between 120%–160% of the control values.According to Baguñà et al. (1989), SP (10−7–10−8 M)stimulates the mitotic activity in intact D. tigrina and D.mediterranea by 300%–400% and, in the post-blastema

regions of regenerating worms, by 150%–200% during24 h and 48 h. SP has a direct effect on the G2-neoblastsas an increase in mitoses is observed as early as 1–2 h ofregeneration. The same authors point out that the increasein the number of mitoses induced by SP is caused by anincreased recruitment of cells for division, and not by ashortening of the cell cycle time.

Growth of blastema

The results of the morphometric measurements of thegrowing blastema have shown that NPF has a clear positiveeffect. Interestingly, the positive effect is greatest with thelowest concentration of NPF, viz. 10−9 M. The morpho-metric results thus seem to contradict the MI results.However, the growth of the blastema depends on at leastfour factors:(1) the rate of mitoses, (2) the enlargement ofthe actual cells, (3) the rate of cell differentiation and (4) theincrease in the extracellular matrix.

From Day 2 onwards, a pronounced increase in thegrowth rate of the blastema has been observed in the E-group. At the same time, the immunocytochemical studyhas shown that new nerve and muscle cells begin to appearin the blastema of the E-group. These observations are inconformity with the hypothesis that not only the mitoticactivity, but also other growth strategies are involved in theregeneration process. Further investigations are needed inorder to clarify which one of the four factors mentionedabove is affected by NPF at 10−9 M. According to Hori(1997) and Hori and Kishida (2003), treatment with SP andSK leads to an acceleration of cell proliferation in the earlystage and to a facilitation of cell differentiation in the latestage of regeneration in D. japonica.

In general, the increase in the number of mitoses in theE-groups varies between 120% and 160% and the increasein the size of the blastema varies between 115% and 130%compared with the C-group.

Influence of NPF on developing nervous and musclesystems

Reuter et al. (1995, 1996) have described the neuroanatomyin D. tigrina and the development of the peptidergic andserotoninergic NS during regeneration after fission anddecapitation. With regard to the C-group, the results of ourstudy are in agreement with those of Reuter et al. (1996).However, incubation in NPF clearly stimulates the growthof the blastema and the development of the NS andmusculature. During the whole experimental series, NPF-IS is stronger in the E-group, indicating the presence ofmore NPF-IR fibres and cells. These results are inagreement with those of Kreshchenko et al. (2001).According to Reuter et al. (1996), new nerve cells

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differentiate from the blastemal neoblasts. Serotonin-IRnerve cells are observed as early as days 3–4, whereas NPF-IR nerve cells appear during days 4–6. A similar processhas been seen in the regenerating pharynx by Kreshchenkoet al. (1999). However, in the pharynx, both GYIRF-IRand serotonin-IR nerve cells are present, even at day 3.Sauzin-Monnot (1975) have described the process ofdifferentiation of nerve cells from blastemal neoblasts inD. gonocephala at the ultrastructural level, reporting that, atday 4, nerve cells are fully developed. By using [3H]-thymidine autoradiography, Gustafsson (1976) has beenable to trace the differentiating nerve cells in the tapewormDiphyllobothrium dendriticum. In this constantly growingworm, the rate of renewal of nerve cells is 16% per day. Inorder to trace the fate of the differentiating neoblasts in G.tigrina, we plan to apply the BrdU-labelling technique(Gschwentner et al. 2001).

Acknowledgements We are grateful for the constructive commentsof the referees.

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