Invertebrate Reproduction and Development, 35~2 (1999) 109-125Balaban, Philadelphia/Rehovot0168-8170/99/$05.00 0 1999 Balaban

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Relationship between musculature and nervous system in theregenerating pharynx in Girardia tigrina (Plathelminthes)

NATALIA D. KRESHCHENKO’, M. REUTER**, I.M. SHEIMAN’, D.W. HALTON3, R.N. JOHNSTON3,C. SHAW3 and M.K.S. GUSTAFSSON*

‘Russian Comparative Neuroendocrinologv Research Group, Institute of Cell Biophysics, Russian Academy of Sciences,Pushchino, Moscow Region 142292, Russia

2Department of Biologv Abe Akademi University, Artillerigatan 6, FIN-20520 Abe, FinlandTel. +358 (02) 215-4258; Fax +358 (02) 215-4748; email: mreuterL&a.abo.$

‘Comparative Neuroendocrinologv Research Group, Schools of Clinical Medicine and Biology h Biochemistry,The Queen ‘s University of Berfast, Berfast BT 7 INN, UK

Received 21 September 1998; Accepted 28 November 1998

Summary

The study shows that the regenerating pharynx of Girardia tigrina forms a simple and valuablemodel system for studies of pattern formation in the nervous system and its relationship to thedeveloping muscles. Immunocytochemistry was used, with antisera raised against 5-HT and thenative planarian neuropeptide GYIRFamide. We studied the sequential development of theinnervation in the regenerating pharynx, and using TRITC-labelled phalloidin we followed thecorresponding differentiation and maturation of pharynx musculature. The peptidergic andaminergic neuronal cell types develop according to different time schedules and differentmodes. Throughout the process, the GYIRFarnide-IR elements are in contact with the old partsof the nervous system, while the apical 5-HT-IR elements develop de novo. In the regeneratingpharynx the GYIRFamide-IR nerves develop in a proximodistal direction. The first 5-HT-IR cellbodies appear in the tip of the pharynx and are symmetrically placed. They have no contact tothe rest of the nervous system. From these cell bodies processes grow disto-proximally andfasciculate with tibres from the proximal part. A striking parallelism between the appearance ofGYIRFamide-IR nerves and muscle fibres stained with TRITC-phalloidin was observed. TheGYIRFamide-IR nerves cling to the muscle fibres. These results suggest that the contactbetween muscle Iibres and GYIRF-IR nerves is essential for the function of pharynx. Thedelayed appearance of 5-HT-IR nervous elements is discussed in terms of the influence of 5-HTon sprouting of nerve fibres and synapse formation. The development of the pharynx in tail partsafter fission is compared with that after pharynx amputation. The faster rate observed forpharynx regenerating after amputation in comparison with that in regenerating tail partsindicates the importance of the remains of the old nervous system in the pharynx structure.

Key wora!s: Girardia tigrina, pharynx, regeneration, neuronal pattern, musculature

*Corresponding author.

110 N.D. Kreshchenko /IRD 35 (1999) 109-125

Introduction

Pattern formation during development is a centralissue in biology and is studied in many model systems.Pattern formation of the nervous system is an area ofintense research. A simple model system for thesephenomena is provided by the planarian Girurdiu(Dugesia) tigrina (Girard, 1850; De Vries and Sluys,1991) (Plathelminthes, Tricladida). G. tigrina has avery high capacity of regeneration. An excised piece ofany region of the planarian body is able to regeneratethe entire animal (see Bronsted, 1969; Baguii?t, 1998).Cell proliferation and differentiation, cell lineage, axialpolarity and patterning of structures take place duringplanarian regeneration. The presence of a population ofundifferentiated, self-renewing cells, called neoblasts,is the prerequisite for planarian regeneration. Theneoblasts give rise to all types of differentiated cells inthe worm. They occur scattered throughout theplanarian body (for review see Bagutia et al., 1990).

The regenerating pharynx can be used as a modelsystem for studies of the relationship betweendeveloping nerves and muscles and for patternformation of the nervous system. Since the beginningof this century, studies on regeneration of the pharynxin planaria have been made (see Kreshchenko, 1993;Kreshchenko and Sheiman, 1994; Sheiman andKreshchenko, 1995). Most of these studies concernhistological differentiation, but some also provideinformation on the functional recovery of the pharynx(Kishida and Asai, 1980; Asai, 1990; Kreshchenko,1993). The function of the pharynx and its dependenceon the presence of neuroactive substances, such as 5-HT, has been studied by Kabotyanski et al. (1990).

Planarians form a new pharynx on three occasions:(1) in the tail part after fission, (2) in tail fragments ifthe worm has been cut in two, and (3) after amputationof the pharynx. As multiplication by fission is thenormal way of multiplication, formation of the pharynxafter fission must be considered a normal event. Itoccurs without the contribution of any remains of anold pharynx and cannot be considered as regeneration.However, after pharynx amputation, real regenerationtakes place, starting from remains of the old pharynx.

When studying pattern formation, migrating anddifferentiating cells are followed. Cellular markers aretherefore of utmost importance. A cellular marker, theposition-specific monoclonal antibody TCEN49, wasintroduced by Bueno et al. (1996) and the spatial andtemporal variations during regeneration in G. tigrinawere followed. Similarly Bueno et,al. (1997) followedthe sequence of events in the development of pharynxin tail fragments of G. tigrina. Monoclonal antibodies

for epithelial, secretory and muscle cells were used butthe development of the nervous system was notstudied.

The key role of neuroactive substances in thecontrol of regeneration and fission in planarians hasbeen reviewed by Webb (1988) and Villar andSchaeffer (1993). So far, next to nothing is knownabout the innervation of the regenerating pharynx andthe role of neuronal substances for the process ofregeneration. A convenient tool for following thesequence of events in the development of the nervoussystem during regeneration is the immunocyto-chemical (ICC) method, combined with confocal laserscanning microscopy. The development of themusculature in the pharynx can be followed by stainingwith TRITC-labelled phalloidin. By using doublestaining, the relationship between the developingnerves and muscles can be followed. The rationalebehind the phalloidin staining method is that F-actinfilaments, which occur predominantly in muscle fibres,specifically bind phallotoxins (Wieland, 1977).

A noteworthy difference between the establishmentof the aminergic and peptidergic neuronal cells in theregenerating cerebral ganglion and the growingpharynx has been noted. In the regenerating cerebralganglion, the 5-HT immunoreactive (IR) cells appearfirst and cells IR to the neuropeptide NPF later (Reuteret al., 1995, 1996). In the growing pharynx, however,the order is the opposite, the peptidergic immuno-reactivity develops first and immunoreactivity to 5-HTlater. Different functional roles for the aminergic andpeptidergic neuronal cells in the cerebral ganglion andpharynx were thus suggested.

In order to follow the development of theinnervation in the regenerating pharynx of G. tigrina,an ICC investigation was carried out. We studied indetail the development of the aminergic andpeptidergic innervation of the regenerating pharynxand correlated it to the development of the pharynxmusculature and to the ability of the pharynx tofunction, i.e., to capture food. For comparison, thespontaneous development of pharynx in tail parts wasstudied.

Materials and Methods

Species and culture conditions

Specimens of an asexual strain of G. tigrina Girard,1850 were obtained from a stock culture in the Instituteof Cell Biophysics, Pushchino, Moscow Region,Russia. The worms were kept in tap water at roomtemperature (20&l “C) and fed with chironomid larvae.

N.D. Kreshchenko / IRD 35 (I 999) 109-125 111

Prior to experimentation the worms were starved for7 days.

Experimental design

Regeneration after pharynx amputation.

The planarians were placed on ice covered withfilter paper. The pharynx was amputated by a thinscalpel through a small incision in the dorsal surface.The operated animals were placed into glass jars withtap and distilled water (2:1), kept at 2O*l”C andallowed to regenerate.

Development of pharynx in tail parts

Development of the pharynx in tail parts afterfission was followed at 21*1 “C, using tap and distilledwater (2:l) as above. The worms were observed dailyand samples were fixed every day.

Histological observationsRegenerating planarians were observed on glass

slides each day for 6 days. In order to relax the worms,a drop of warm (36°C) water with 2% HNO, and 0.5%novokain was added, immediately followed by a dropof formalin (37-40 Oh) at 36°C. The specimens werefixed in formalin/ethanol/acetic acid (10:85:5) (Lillie,1965) for 30-40 min and transferred to 70% ethanolfor 30 min. They were dehydrated in a rising series ofpolyethylenglycol (PEG) according to the method ofEfimov (1993). They were then embedded in PEG with10% celloidin for 30 min, mounted on blocks andsectioned at 6-7mm. The sections were mounted onslides and stained with hematoxylin (Erlich) for3-5 min, washed in tap water, brought through a risingseries of ethanol (70%, 96 %, 1 OO%, 100%) and xylol(2x) for 0.5-l min and mounted in Canada balsamunder cover glasses.

Immunocytochemistry

Worms were fixed flat on ice, under a cover glass,in Stefanini’s fixative (2% paraformaldehyde and 15%picric acid in 0.1 M sodium phosphate buffer) at pH 7.6and stored for l-7 days at 4”C, and rinsed for 2448 hin 0.1 M sodium phosphate buffer (pH 7.6) containing10% sucrose. The worms were embedded in TissueTek and sectioned at 10-20pm on a Bright cryostat.Frontal and transverse sections were cut, placed ongelatine-coated glass slides, allowed to dry, and frozenat - 70°C. Prior to staining, the sections were thawedand immersed in 0.01 M phosphate buffer 0.2% Triton

X-100 (PBST). Immunostaining was performedaccording to the indirect immunofluorescence methodof Coons et al. (1955).

Double-staining with two primary antibodies

The concentration of the primary antisera used inall stainings was the 1:500. Simultaneous incubationswere performed with antibodies to the flatwormneuropeptide GYIRFamide (Bdelloura candida) raisedin guinea pigs by Johnston et al. (1995) and rabbit anti-5HT (INCSTAR) for 48 h. Thereafter, the sectionswere rinsed 3 x5 min in PBST and sequentially incu-bated for l-2 h with the secondary antibody, FITC-labelled goat anti-guinea pig (Cappel) and TRITCswine anti-rabbit (DAKO) (dilution 1:30). The sectionswere rinsed 3x5min in PBST, mounted in 50%glycerol in PBS and stored in the dark.

Controls

The controls for specificity included: (1) omittingthe primary antibody, and (2) using non-immuneserum.

Phalloidin staining of musculature

In order to study the development of themusculature in the regenerating pharynx, sections werestained with TRITC-conjugated phalloidin (Sigma)(1:200) for 20 min at 4°C. The phalloidin staining wasperformed on the same sections that had been stainedwith anti-GYIRFamide and anti-SHT, studied in theconfocal microscope and photographed.

Microscopy

The sections were examined in a Leitz Orthoplanmicroscope combined with filter-blocks 12 and N2.Photomicrographs were produced by the Olympusautomatic photomicrographic system, model PMIOADS. Kodak 5052 TMX 100 film was used. Aconfocal scanning laser microscope (LEICA TCS 4D)was used to better visualize the differentiating nervecells, the growing nerve processes and the develop-ment of the musculature.

-‘

Results *

The structure of the intact pharynxFig. 1A and B show frontal sections stained with

heamatoxylin-eosin and phalloidin, respectively. Thepharynx is shaped like a young mushroom with a cap

114 N.D. Kreshchenko /IRD 35 (1999) 109-125

Regeneration of amputated pharynxDAY1

The wound on the dorsal side resulting from theamputation of the pharynx is closed. Inside the worm atthe intestinal level the old pharynx cavity is present. Atthe ventral surface of the worm, the round mouthopening to the pharynx cavity is present. Numerousbasophilic cells with strongly stained nuclei haveaccumulated at the anterior end of this cavity, forminga small flattened bud or blastema, the pharynxprimordium. At the posterior pole ofthe pharynx cavitya bud of varying size occurs. The epithelium coveringthe primordium and the posterior bud is columnar.Phalloidin staining reveals the remains of the capstructure. Strong staining is observed at the borderbetween the flattened blastemal lining and the rest ofthe fold between the former pharynx stalk and the oldpharynx cavity. Anchoring filaments radiate from theremaining fold to the body musculature (Fig. 4A).Actin filaments are also present along the lateral sidesof the pharynx cavity.

A crescent of GYIRFamide-IR nerve fibres occursbasally (Fig. 4B). These nervous elements represent theremains of the crescent observed close to the cap in theintact animals. This remaining part of the old pharynxwill form the basal part of the new pharynx. The lateralsides of the old pharynx cavity are innervated. In theposterior bud the basal part is innervated by strongGYIRFamide-IR nerve fibres (Fig. 4D). 5-HTimmunoreactivity occurs in the wide crescent of fibresbasally to the flattened thin blastema and in a networkof fibres innervating the lateral sides of the pharynxcavity (Fig. 4C). 5-HT-IR occurs in the basal part ofthe posterior bud (Fig. 4E).

DAY2

The ventral body surface and the mouth have thesame appearance as on day 1 (Fig. 4F). In the pharynxcavity, the anterior bud has grown posteriorly into theold pharynx cavity, forming an elongated outpocketingwith a small centrally located triangular slit in the basalpart. This slit represents the first indication of the newpharynx lumen and is covered with a thick epithelium.The outer epithelium of the pharynx bud hasdifferentiated. The cells in the center of the pharynxare irregularly distributed. The bud in the posterior endof the pharynx cavity is slightly thinner and longer.The filaments radiating from the border between theremaining cap structure and the pharynx cavity arestrongly stained with phalloidin (Fig. 5A).

In the primordium, GYIRFamide-IR fibres extend

from nervous elements in the cap structure between theneoblasts in the blastema to the level of the nascentpharynx lumen (Fig. 5B). Short branches of 5-HT-IRfibres extend from nervous elements in the capstructure into the basal part of primordium (Fig. 5C).

DAY3

The newly formed pharynx continues to grow,tilling the old pharynx cavity. The lumen of the newpharynx is slightly enlarged and in some preparations,its posterior end opens into the pharynx cavity andanteriorly connects to the anterior branch of the gut.The flattened outer epithelium of pharynx is welldeveloped. The cells in the inner epithelium appearmore or less balloon-shaped with wide intercellularspaces. Irregularly arranged longitudinal and circularmuscles occur. The bud in the posterior end of thepharynx cavity is still present. Weak phalloidinstaining was observed in thin, irregularly orientatedfilaments in the regenerating pharynx primordium(Fig. 5D). The filaments originate at the surface of thelumen. Strong staining occurs in filaments of the rest ofthe cap structure.

The intensity ofthe GYIRFamide-immunoreactivityhas increased in fibres running distally from neuronalelements in the cap structure into the center of thepharynx. A few weakly stained fibres extend into theblastema where most fibres were observed in theperipheral parts, thus probably belonging to the outerpharynx nerve plexus (Fig. 5E). The number of 5-HT-IR fibres has increased in the cap structure and theynow extend deeper into the basal part of the pharynx(Fig. 5F). Sometimes a short, weakly stained fibrestump was observed in the most apical part and once avery weakly stained cell body was noted in the apex ofthe pharynx.

DAY4

The length of regenerating pharynx has markedlyincreased. The pharynx lumen is well developed(Fig. 6A). Muscle cells are easily distinguished; theinner columnar epithelium of the pharynx lumen is stillnot fully differentiated; the outer epithelium is flat(Fig. 6B). A small posterior bud showing unchangedimmunoreactivity patterns is still present. Weakphalliodin staining was observed in thiqradiating andslightly thicker longitudinal filaments along thepharynx tube. Some of the radiating fibres are single,some double (Fig. 6C). The radiating fibres insertbetween the balloon-shaped epithelial cells of thelumen. In the basal part strongly stained filaments form

N.D. KreshchenkoiIRD 35 (1999) 109-125 119

the new pharynx. Strongly stained, pear-shaped 5-HT-IR cells were regularly observed near the apex of thepharynx (Fig. 6F). No contact exists between these cellbodies and the 5-HT-IR fibres growing into thepharynx from the basal side. The genesis of 5-HT-IRneurons thus differs from that of GYIRFamide-IRneurons and from the genesis of muscle fibres.

DAYS

At this stage, some of the experimental animals hadrecovered their ability to protrude their pharynx. Ingeneral the structure of the new pharynx is very similarto that of the intact one. The epithelium, musculatureand parenchyma have differentiated. The newly formedpharynx has the same proportions as the intact pharynxbut is generally smaller. Phalloidin staining wasobserved in radial and longitudinal muscle fibres. Thenumber of radial filaments has increased (Fig. 7A).Single radial filaments are seen between doublefilaments. The double filaments insert on the luminalside with fork-like branches (Fig. 7B). The intensityand thickness of the stained filaments are still not thesame as in the body filaments. Thick filaments connectthe mushroom-shaped pharynx to the bodymusculature.

GYIRFamide-IR fibres and cells occur in all partsof pharynx (Fig. 7C). The number of fibres is greaterand the immunoreaction stronger in the basal parts.The number of small bi- or multipolar cells along thepharynx has increased. Cells were observed with bothlongitudinal and radial nerve libres. Double stainingshows that GYIRFamide-IR fibres run very close to thephalloidin stained filaments. Several uni- or bipolar5-HT-IR cells were observed in the region of the newpharynx. At the base an increase in the number offtbres was also noted. Radial tibres connect the outerand inner cylindrical nerve plexuses. Thus, the contactbetween the outer and inner nerve cylinder isestablished (Fig. 7D).

DAY 6-9

During these days the animals are able to feed. Thepharynx is, however, still smaller than the matureorgan. The pattern of actin filaments corresponds tothat of the fully developed pharynx. The inner andouter nerve plexuses have increased in thickness, butare still not fully developed.

On day 7 the GYIRFamide-IR fibres do not reachthe epithelium of the pharynx lumen, but on day 9 theydo so. The pattern of S-HT-IR differs in intensity incomparison to that of fully developed pharynx. On day

7 the 5-HT-IR fibres still do not reach the epitheliallining of the lumen, whereas in the intact pharynx theydo.

Development of pharynx in tail parts

DAY1

Phalloidin staining shows that the pharynx cavityhas developed and the pharynx primordium protrudesinto the cavity. GYIRFamide- and 5-HT-IR fibresoccur at the margins of the pharynx cavity. TheseIibres are in contact with the MCs and with the surfacenerve net. Nerve fibres from\ the margins sendprocesses into the primordium forming an archsurrounding elements intruding from the looseintestinal tissue (Fig. SA,B). The GYIRFamide-IRfibres are more numerous and more intensely stainedthan the 5-HT-IR fibres. Outside these nervouselements a tissue of more compact columnar cells wasobserved.

DAY2

Phalloidin staining shows that the primordium hasgrown. No slit indicating development of the pharynxlumen is present. In the basal part of the pharynxprimordium both GYIRFamide- and 5-HT-IR fibresform a tangled masse (Fig. 8C,D).

DAY3

The pharynx primordium has increased in lengthand a pharynx lumen has appeared. Weak phalloidinstaining occurs in radial filaments along the pharynxtube; longitudinal filaments are also observed.However, neither filaments forming a cap structure norfilaments attaching the new pharynx to the bodymusculature have developed.

Several bipolar GYIRFamide-IR cells occur at thebase and in the middle part of the pharynx primordium(Fig. 8E). Short 5-HT-IR fibre stumps occur in thebasal part of the pharynx primordium and in its apicalend (Fig. 8F). So far no contact between these two partof 5-HT-IR elements has been established.

DAY45 -

The pharynx primordium has increased in lengthand a lumen has opened into the pharynx cavity.Phalloidin stained filaments occur in the pharynx tube,but still no cap structure or radiating libres connectingpharynx to the body muscles can be detected.

GYIRFamide-IR cells and fibres occur in both theinner and the outer pharynx nerve plexus (Fig. 9A).

122 ND. Kreshchenko / IRD 35 (1999) 109-125

functioning pharynx is formed within 6-9 days. In tailparts the pharynx stalk forms first and the cap structureand anchoring musculature develop much later.

An uncertainty prevails as to the origin of cellsforming the pharynx. According to Kishida and Asai(1980), in the amputated pharynx a blastema is formedby rearrangement of cells, i.e., by a morphallacticprocess. In contrast, in tail parts a pharynx primordiumis formed by accumulation of undifferentiated cells,i.e., an epimorphic process (Bueno et al., 1997). Ourinvestigation shows that the 5-HT-IR nerve cells andthe phalliodin-stained muscle cells arise de nova, i.e.,through an epimorphic process. To restore properproportions morphallaxis takes place as suggested byBueno et al. (1997).

GYIRFamide-IR cells

Surgical removal of the pharynx results in a lesion.Close to the lesion point very active sprouting ofGYIRFamide-IR nerves was observed on day 2. It is awell documented fact that lesion and especially theabsence of 5-HT stimulates sprouting of nerve fibresboth in invertebrates and vertebrates (Abrous et al.,1993; Zhang et al., 1993; Croll and Baker, 1994;Alonso et al., 1995; Diefenbach et al., 1995; Baker andCroll, 1996). Basally located GYIRFamide-IR cellswere first observed on day 3. Palmberg (1990)followed the differentiation and maturation of nervecells in Microstomum lineare with the aid of[3H]thymidine labelling and ICC staining and reportedthat neoblasts differentiated into peptidergic nervecells.

In G. tigrina no regular pattern of GYIRFamide-IRnerves was observed in the first three days. However,at the same time as the actin filaments start to form aregular pattern with longitudinal and radial musclefibres, GYIRFamide-IR nerve tibres arrange them-selves in an outer and inner pharyngeal nerve plexus.The GYIRFamide-IR nerves cling closely to themuscle fibres. From day 4 onwards the worms wereable to protrude their pharynx. This suggests that thecontact between the muscle fibres and theGYIRFamide-IR nerves is essential for the function ofthe pharynx. Johnston et al. (1996) have demonstratedthat synthetic GYIRFamide causes dose-dependentcontractions of muscle fibres isolated from BdeIIuracandida. Day et al. (1994) have shown that flatwormneuropeptides closely related to GYIRFamide causeconcentration-dependent contractions of muscle fibresin the human blood fluke, Schistosoma mansoni.

5-HT-IR celIs

The appearance de novo of single 5-HT-IR cells atthe apical end of the regenerating and developingpharynx is noteworthy; such an origin was observed byReuter et al. (1996) in the regenerating brain of G.tigrina. 5-HT-IR cells differentiate from neoblasts infront of the commissure and thereafter fasciculate withfibres in the commissure. Reuter et al. (1996) made thefirst observation of symmetrically placed 5-HT-IR cellsin pharynx of fissioned worms on day 4. In the presentstudy, 5-HT-IR cell bodies were observed on day 4both in the regenerating and in the developing pharynx.As these cells lack connections to 5-HT-IR neuronalelements, a de novo differentiation from undiffer-entiated neoblasts must have taken place. Sauzin-Monnot (1975) described the process of differentiationof nerve cells from blastemal neoblasts in D.gonocephalu at the ultrastructural level, reporting thatat 96 h the nerve cells are fully developed. In thepresent study the first 5-HT-IR cells appear on day 4,i.e., after 96 h. The process of neuronal cell differen-tiation was studied in another flatworm, the tapeworm,Diphyllobothrium dendriticum, by Gustafsson (1976a)using autoradiography. The tapeworm strobila growscontinuously, as do its main nerve cords. New nervecells are recruited from the pool of undifferentiatedcells, the germinative cells, in the parenchyma. Theymigrate into the main nerve cords at a rate of 16% perday and differentiate. As nerve cells in the main nervecords do not multiply by mitosis, this is the only way toincrease the population of nerve cells. The rate ofdifferentiation is higher in the tapeworm than in theplanaria due to the fact that tapeworms live inside ahomeothermic host at 38°C.

Reuter and Palmberg (1989) suggest that 5-HTinfluences the start of fission and organ differentiationin asexually reproducing A4. lineare, and Ladumer etal. (1997) reached the same conclusion in relation toother Macrostomida. Saitoh et al. (1997) describe a G-protein-coupled receptor family similar to the 5-HTdrolserotonin receptor of Drosophila. The expression ofthis receptor mRNA increases during planarianregeneration, suggesting that it might be involved inthe regeneration mechanism.

It is known that 5-HT suppresses the sproutingresponse of nerve libres (Croll and Baker, 1994;Diefenbach et al., 1995; Baker and Qoll, 1996).According to Zhu et al. (1995), 5-HT transiently down-regulates synthesis of apCAM in Aplysia. Thisselective decrease in apCAM expression anddistribution on the surface of sensory neurons

N.D. Kreshchenko / IRD 35 (1999) 109-125 123

facilitates interaction between neurites leading toformation of new synaptic connections with the motortarget. The late appearance of the 5-HT-IR cell bodiesin the pharynx of G. tigrina may shape a milieu that isfavorable for sprouting and synapse formation.

5-HT is very important for the function of thepharynx (Kabotyansky et al., 1990), but the maturationof the S-HT-IR nervous system is slow. Our resultsshow that the pharynx can function long before thecomplete maturation of the 5-HT-IR elements. In anintact pharynx the 5-HT-IR nervous system is welldeveloped with many cell bodies and numerous fibresinnervating the inner ciliated epithelial layer. Theinnervation of the epithelial layers develops much laterthan that of the inner and outer pharynx plexa. At28 days the size and proportions of the pharynx arerestored, and the innervation, including the innervationof the epithelial layers, now corresponds to that of theintact pharynx (Kreshchenko, 1993).

Muscle cells

By means of TRITC-labelled phalloidin thestructure and the development of the musculature in thegrowing pharynx has been followed in detail for thefirst time. The structure resembles a young mushroomhanging into the pharynx cavity with radiating musclefilaments attaching it to body muscles (Fig. 1). Riegeret al. (1994) described the pharyngeal musculature inMacrostomum hystricium marinum and observed atransverse fan of short radial fibres at the transitionbetween pharynx and gut, and termed the “pharynx-holding apparatus”. In general our results are inagreement with those of Rieger et al. (1994). Thesphincter muscle between the gut and the pharynx hasnot been described previously.

The general pattern of muscle development is thesame in the regenerating and developing pharynx.Myofibrils appear in a proximo-distal order. The mostdistal part of the growing pharynx is for a long timedevoid of muscle fibres. The pharynx continues togrow for 3 weeks, during which time the muscle fibresincrease in number, with new thin fibres arisingbetween already existing thicker fibres in a zigzagpattern. Sauzin-Monnot (1975) has followed thedifferentiation of muscle cells in regenerating D.gonocephala at the ultrastructural level. Myofibrilsappear gradually in activated neoblasts, and musclecells were observed as early as 72 h, i.e., at the sametime as in our study. De nova myogenesis was alsoobserved in G. tigrina by Pedersen (1972) and in D.

dendriticum by Gustafsson (1967b). Bueno et al.(1997) have followed the formation of muscle fibres inthe pharynx of regenerating tail fragments of G.tigrina. The first muscle cells were observed at thesame time as the pharynx lumen formed. As a possiblemechanism, the authors point out that muscle cellsarise de novo from undifferentiated cells anddifferentiate and mature in a proximo-distal directionby intercalation of new tibres between older ones. Thesame pattern was observed in the body wall (Cebria etal., 1997).

Acknowledgements

This investigation was supported by INTAS 96-1365 and by Academy of Finland. We thank Dr. DianaToivola, Mr. Thomas Bymark, Mr. Johan Hindstromand Mr. Esa Nummelin for technical assistance.

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