tachykinins and intestinal motility in different fish groups

GENERAL AND COMPARATIVE ENDOCRINOLOGY 83, 388-396 (1991)

Tachykinins and Intestinal Motility in Different Fish Groups

JORGEN JENSEN AND SUSANNE HOLMGREN

Comparative Neuroscience Unit, Department of Zoophysiology, University of Giiteborg, Box 250 59, S-400 31 Giiteborg. Sweden

Accepted October 16, 1990

The presence and function of tachykinins were studied in the intestine of hagtish (Myxine glutinosa), lamprey (Lampetra jluviatilis), starry ray (Raja radiata), hmgtish (Lepidosiren paradoxa), bichir (Polypterus senegalensis), and rainbow trout (Oncorhynchus mykiss), which represent different systematic groups of fish. Immunohistochemistry revealed cells containing substance P (SP)-like material in the intestine of lamprey and lungfish, and in the stomach of the ray. The intestinal motility was studied using isolated muscle strip preparations. SP had no effect on hagfish or lamprey intestine. In the other four species SP produced intestinal contractions. In ray, bichir, and lungtish the tachykinins may be released from endocrine cells and act, at least in the bichir and lungtish. directly onto the smooth muscle cells. In the rainbow trout intestine, where SP-like material may be released from both nerve tibres and endocrine cells, it is indicated that the contractile effect is in part direct upon the smooth muscle and in part via stimulation of cholinergic and serotonergic neurons. 0 1991 Academic Press, Inc.

The tachykinins share the C-terminal Phe-X-Gly-Leu-Met-NH,, the biologically active part of the peptide. Several mammalian tachykinins have been isolated and sequenced, including substance P (SP), neurokinin A and neurokinin B (Chang and Leeman, 1971; Kangawa et al., 1983; Kimura et al., 1983), and numerous tachykinins from amphibian skin have been structurally characterized (see Erspamer, 1981). However, the exact identity of the tachykinins present in the gut of nonmammalian vertebrates is, with few exceptions, unknown. Nerve fibers and endocrine cells containing SP-like material occur in the gut wall in representative species of all the ma- jor vertebrate groups (see Jensen, 1989).

SP contracts gastrointestinal smooth muscle in many vertebrates. Early studies on fish demonstrated that crude extracts of a SP-like peptide from fish intestine or SP purified from mammalian intestine had an excitatory effect on intestinal preparations from Pleuronectes platessa, Labrus berg- gylta, and Ruja batis (von Euler and

ijstlund, 1957). More recently, this contractile effect of SP on the fish gut has been confirmed. However, the mechanism un- derlying the excitation seems to vary between species and between different parts of the alimentary canal. Thus, in the gut of the elasmobranch, Squalus acanthias, SP has a direct effect on the smooth muscle cells (Holmgren, 1985), while there are ad- ditional pathways via cholinergic and/or serotonergic neurons in teleosts (Holmgren et al., 1985; Jensen et al., 1987; Kitazawa et al., 1988). In guinea pig and dog ileum the contractile effect of SP is in part produced by a stimulation of cholinergic neurons and in part by a direct action on the smooth muscle cells (Rose11 et al., 1977; Holzer and Lembeck, 1980; Yau, 1978; Daniel et al., 1982).

Previous investigations on the effect of tachykinins on the fish gut have only con- cerned a few species of teleosts and elas- mobranchs, and the function of tachykinins in other types of fishes is not known. The aim of the present study was to examine

388 0016~6480/91 $1.50 Copyright 0 1991 by Academic Press Inc. All rights of reproduction in any form reserved

TACHYKININS IN FISH INTESTINE 389

actions of SP on the intestine of species rep- resenting different classes and orders of fish to elucidate evolutionary trends in the mechanisms of action of SP. In addition to the physiological experiments, immunohistochemical studies were carried out to reveal the location of tachykinins in the intestinal wall.

MATERIALS AND METHODS

IO Atlantic hagfish (M.vxine ylutinosa-Agnatha-Cy- clostomata), IO lamprey (Lampefra f/uviatilis- Agnatha-Cyclostomata). IO starry ray (Ruju rudiaru-

Chondrichthyes-Elasmobranchii-Batoidea), 7 South American lungfish (Lepidosiren paradoxa-Oste- ichthyes-Sarcopterygii-Dipnoi), 7 bichir (Polyprerus

sene&ensis-Osteichthyes-Actinopterygii-Chon- drostei), and 20 rainbow trout (Oncorhynchus mykiss-

Osteoichthyes-Actinopterygii-Teleostei) were used in the experiments. The classification is based on Romer (1962).

Rainbow trout were supplied from a local hatchery and lampreys were captured in a river in the north- eastern part of Sweden. Both species were kept in tanks with aerated fresh water at IO”. Hagtish and starry ray were captured off the Swedish west coast and kept in the laboratory in aerated sea water at IO”. Bichir were obtained from a dealer in tropical fish and kept in fresh water at 25”. Lungtish were captured in Brazil and kept in aquaria at approximately 25” at the university of Rio Claro, Brazil, where the physiological experiments were also performed.

Rainbow trout were killed by a sharp blow to the head. while the other species were killed by sectioning of the spine just behind the head, followed by pithing.

The intestine was rapidly dissected out and then pre- pared for motility studies or immunohistochemistry.

Motility Studies

Pieces of approximately 2 X IO mm were cut from the proximal part of the intestine in the longitudinal or circular direction from all species except from the lungfish. where preparations were cut out along the circular axis only.

The preparations were mounted in organ baths containing 10 ml of Ringer’s solution. The Ringer’s solution for rainbow trout and lamprey contained (mM): NaCl 130.6. KCI 4.8, CaClz I .2. MgSO, I .2. NaHCO, 23.8, NaH?PO, 2.8, glucose 5.5, pH 7.4 (Holmgren 1983): the solution for hagtish contained (mM): NaCl 474. KCI 8.0. CaClz 5.0. MgSO, 2.9, MgCI, 9.0, NaHCO, 15.0. NaH,PO, 0.5. glucose 5.5. pH 7.2 (modified from Holmgren and FBnge 1981): the solution for starry ray contained (mM): NaCl 231.0, KCI

4.8, CaClz 4.8, MgSO, 2.4, NaHCO, 1 I .9, NaH2P0, 1.6, glucose 5.5, urea 400, pH 7.5 (Nilsson et al., 1975): and the solution for bichir contained (mm: NaCl 85.6, KC1 3.8, CaCI, 2.2, MgSO, 0.8, NaHCO, 23.8, NaH?PO, 2.8, glucose 5.5, pH 7.4 (modified from Abrahamsson et al., 1979). All the above Ringer’s so- lutions were continuously bubbled with a mixture of OZ (97%) and CO? (3%). The Ringer’s solution used for lung&h contained (mm: NaCl 107.5, KCI 3.8, CaClz 2.2, MgSO, 0.8, NaH?PO, 0.6. glucose 5.5 (modified from Abrahamsson er al., 1979). pH 7.5 and was bubbled with air.

The muscle strip preparations were attached to Grass FTO3 transducers connected to a Grass poly- graph Model 7 for isometric recordings of tension. An initial tension of IO mN was applied. To obtain a stable baseline tonus the preparations were left for at least I hr before any drugs were added.

Immunohistochemistty

Pieces of the gut were dissected out and cut open, pinned flat to pieces of dental wax, and fixed floating in 15% picric acid and 2% formaldehyde in 0.1 M phosphate buffer (pH 7.3) for 18 hr at 4”. The tissues were then repeatedly rinsed in 80% ethanol, dehydrated, xy- lene treated for 30 min. and rehydrated. Pieces (4 x 4 mm) were put into phosphate buffer containing 30% sucrose for 24 hr, frozen in liquid nitrogen, and cut in IO-pm sections, which were collected on chrom-alum coated slides and incubated with antiserum. The re- maining tissues were separated into thin sheets of mucosa and muscle layers by peeling, and 4 x 6 mm pieces were incubated with antiserum (Costa et ul.,

1980). Incubations were made with the primary antiserum

for 16-20 hr in a moist chamber at room temperature. The preparations were rinsed for 3 x IO min in phosphate buffered saline (PBS) containing 0.15 M NaCl for whole mounts and 0.5 M NaCl for sections. The preparations were then incubated with the secondary antiserum (Swine anti rabbit IgG; diluted I: IO, Dako- patts) conjugated with fluorescein-iso-thiocyanate (FITC) for I .5 hr in a moist chamber at room temperature.

After rinsing for 3 x 10 min in PBS (0.15 M or 0.5 M NaCI), the preparations were mounted in carbonate- buffered glycerol (I: I, pH 8.4) and viewed in a Leitz Dialux fluorescence microscope. Photography was performed with a Leitz orthomat camera on Kodak Tmax film. Specificity of positive reactions was tested by incubation with antisera preincubated with syn- thetic substance P l-l I (IO nmoliml).

The following primary antisera were used: GIO (J6n- sson) raised against substance P l-11, diluted I:400 (Jensen et ~1.. 1987). RMSPI (Murphy), RMSP4 (Mur- phy). and CRB-SP (Cambridge Research Biochemicals Ltd., Cambridge, England). all diluted I: 100.

390 JENSEN AND HOLMGREN

Drugs

The following drugs were used: acetylcholine chlo- ride (Sigma Chemical Co., St. Louis, MO), atropine sulphate (Sigma), carbachol (Sigma), eledoisin (Penin- sula Laboratories Europe, Ltd., Merseyside, En- gland). 5hydroxytryptamine (Sigma), kassinin (Bachem Feinkemikalien AG. Bubendorf, Switzer- land). methysergide (a gift from Sandoz AG), neurokinin A (Bachem), neurokinin B (Bachem), physalaemin (Peninsula), substance P (CRB), and tetrodotoxin (Sigma).

Calculations

Concentration-response curves were obtained from the muscle strip preparations with respect to SP. acetylcholine, or .5-hydroxytryptamine, and pD,-values ( -log EC,,; van Rossum 1963) were calculated. When SP was active, consecutive curves were made before and during treatment with atropine, methysergide, or tetrodotoxin. Wilcoxon matched-pairs, signed-ranks test was used for statistical evaluation of the results. Differences where P < 0.05 were regarded as statisti- cally significant.

Values are presented as means 2 S.E.M.

RESULTS

Ha&h, Myxine glutinosa

Immunohistochemistry with the antisera

employed did not reveal immunoreactivity in either nerves or endocrine cells. SP (lop9 - 3x lo-’ M) had no effect on the tension of longitudinal or circular muscle strip preparations from the intestine of the hagfish (n = 5), nor did the muscle strips respond to the other tachykinins tested, eledoisin (n = 3), kassinin (n = 3), and physalaemin (n = 4) in the concentration range lop9 - 3 x lo-’ M. To verify viability of the preparations, the cholinergic agonist carbachol was added to the organ baths. Carbachol (10-6-10~5 A4) produced an increase in tonus in all preparations tested (n = 4).

Lamprey, Lampetra jluviatilis

The antisera RMSPl and RMSP4 gave a moderate density of immunoreactive endocrine cells in the intestinal mucosa (Fig. la). The immunoreaction was attenuated by preincubation of the antisera with substance P.

Intestinal preparations from the lamprey were in some cases spontaneously active. SP (lo-i0 - 3x10-’ M) produced no

FIG. 1. SP-like immunoreactivity in endocrine cells of the gut mucosa. (a) Lampetra .fhvia~i~is. intestine; (b) Ruju rudiaru, pyloric stomach: (c) Lepidosiren purudoxu, intestine. Calibration bar in all figures = 25 urn.


change in tonus or in frequency or amplitude of the rhythmic contractions when added to the circular or longitudinal muscle preparations (n = 5). The preparations were unaffected by neurokinin A (n = 4), eledoisin (n = 3), or physalaemin (n = 4) in concentrations of 10e7 or 3 X lo-’ M. Car- bachol, which was found to be excitatory on intestinal preparations from several of the other species used in this study, did not stimulate the lamprey intestine. In contrast, an inhibition of the muscular activity was occasionally observed when carbachol was added to spontaneously active circular preparations. To test the ability of the preparations to show an excitatory response, the potassium concentration in the Ringer solution was raised, which in all preparations caused an increase in tonus and/or an increase in frequency and amplitude of contraction (n = 5).

Starry Ray, Raja radiata

All antisera used revealed SP-immunoreactivity in gastric nerve fibres and endocrine cells (Fig. lb), while the intestine ap- peared devoid of such features. The endocrine cells were especially frequent in the mucosa of the lower (pyloric) part of the stomach.

SP (lo- lo - 3~ 10e7 M; n = 6) induced a contractile response in a few of the circular muscle strip preparations from the intestine of the starry ray. An excitatory effect was more consistent when SP was added to longitudinal muscle preparations (Fig. 2; n = 6). The response was, however, seldom concentration-dependent. Occasional excitatory effects were also obtained with eledoisin (n = 6), neurokinin A (n = 3), neurokinin B (n = 6), and physalaemin (n = 6) in a concentration of lop7 or 3 x 1O-7 M.

Carbachol (IO-’ M) increased the tonus and often also initiated rhythmic contractile activity in the muscle preparations (n = 6).

Lun&ll, Lepidmiren paradoxa

Only a few, very weakly stained immu-

STARRY RAY

“1

LUNGFISH 4

mN

n 1

-10 -9 -8 -7 -65

BICHIR

:1.... OJ

-10 -9 -8 -7 -65 -6

RAINBOW TROUT IO

mN

I OJ

-10 -9 -8 -7 -6 5 LOG MOLAR CONC SUBSTANCE P - 5 min

FIG. 2. Recordings showing the effect of SP on longitudinal intestinal muscle strip preparations from the starry ray, the bichir, and the rainbow trout, and on a circular intestinal preparation from the lungfish.

noreactive fibres were observed in the intestinal valves, while a moderately high number of SP-immunoreactive endocrine cells were present in the mucosa of the intestine and rectum (Fig. Ic).

Most circular muscle strip preparations of the lungfish intestine showed spontane- ous rhythmic contractions at the start of the experiments. SP (10m9 - 3X lo-’ M) produced a dose-dependent increase in frequency and/or amplitude of the rhythmic contractions and often also increased the tonus (Fig. 2). The pD,-value was 8.0 ? 0.2 (n = 5).

To examine the possible involvement of enteric neurons in the mediation of the response to SP, a comparison was made between the concentration-response curves produced by SP before and during treatment with antagonists. No significant


changes in pD,-value or maximal response to SP could be demonstrated after treatment with the cholinergic antagonist atropine (lop6 M; 12 = 5), the serotonergic antagonist methysergide (10e6 M; n = 5), or the sodium channel blocker tetrodotoxin (lo-6 M; II = 5).

Bichir, Polypterus senegalensis

Longitudinal muscle preparations of the bichir intestine were spontaneously active with a high frequency of contractions. Ex- posure to SP (10-l’ - 10e6 M) mostly induced a dose-dependent increase in tonus and sometimes also an increase in frequency and amplitude of contractions (Fig. 2; pD, = 7.7 + 0.3; n = 7). The response was not reduced after treatment with tetrodotoxin (lop6 M; n = 5).

Circular preparations were only occasionally (3 out of 7) stimulated by SP (lo-lo - 1o-6 M).

Rainbow Trout, Oncorhynchus mykiss

SP only occasionally contracted circular muscle strip preparations from the rainbow trout intestine and then only in high concentrations (IO-’ - 3~ lo-’ M; n = 6). Longitudinal muscle strips were, on the other hand, contracted by SP (lo-” - 3~ lo-’ M) in a dose dependent manner (Fig. 2; pD, = 8.0 + 0.1; n = 10).

When two consecutive concentration- response curves for SP on the same preparation were compared no significant difference could be found in pD,-values or maximal response (Fig. 3). Treatment of the preparations with tetrodotoxin ( lop6 M) significantly reduced the maximal response to SP by 41.8 ? 8.8% (n = 10; Fig. 3). Similarly, both methysergide (lo-” M) and atropine ( 1O-6 M) significantly reduced the maximal response to SP by 35.3 + I1 .O% (n = 10; Fig. 3) and 32.7 ? 6.2% (n = 9; Fig. 3), respectively.

The effects of 5-hydroxytryptamine (5- HT) and acetylcholine on the rainbow trout

intestine were also studied. 5-HT (IO-* - 3 x lop4 M) contracted the longitudinal muscle strip preparations (pD, = 5.7 ? 0.1; n = 5). The effect was not significantly al- tered by tetrodotoxin (n = 5) or atropine (n = 5). However, methysergide (1O-6 M) al- most completely blocked the response to 5-HT (n = 5).

Acetylcholine (lo-’ - 3 x 1O-3 h4) also caused contractions of the rainbow trout intestine (pD, = 5.2 2 0.2; n = 5). The excitation was not affected by tetrodotoxin (n = 5) or methysergide (n = 5), while atropine (10e6 M) inhibited the response to acetylcholine (n = 5).

DISCUSSION

The presence of SP-like material has been demonstrated in the alimentary tract of several teleost and elasmobranch fishes. Immunoreactive nerve fibres are found in the myenteric plexus, in the submucosa, and in the muscle layers of the stomach and intestine, the distribution varying between species. Endocrine cells are often present in the gut mucosa (see Bjenning and Holm- gren, 1988; Jensen, 1989).

The elasmobranch tachykinins scyliorhinin I and scyliorhinin II show sequence variations compared to mammalian tachykinins (Conlon et al., 1986; Conlon and Thim, 1988), and it is likely that the tachykinins in the gut of other fish species are different from the mammalian tachykinins to some degree. In all immunohistochemical studies so far performed in fish, antisera raised against the mammalian SP have been used; discrepant results may reflect different abilities of the antisera to interact with the fish tachykinins. This possibility must always be taken into account when using antisera raised against mammalian peptides in nonmammalian species.

The lack of SP-like material in the hagfish intestine agrees with a previous immunohistochemical study, in which no nerves containing a tachykinin could be detected


% RESPONSE

100

50

0 -10 -9 -a -7 -6

% RESPONSE

9: lO(

5(

C

6RE :SPONSE % RESPONSE

LOG MOLAR CONC SUBSTANCE P

-9 -a -7 -6


-10 -9 -a -7 -6

LOG MOLAR CCNC SUBSTANCE P

MET

-10 -9 -a -7 -6


FIG. 3. Concentration-response curves for SP on longitudinal muscle strip preparations from the intestine of the rainbow trout. The upper left panel shows the curve for SP (SPl) compared to a second consecutive curve for SP (SP2). In the other panels curves are shown for SP before (-O-) and after treatment (-O-) with tetrodotoxin (TTX), methysergide (MET), or atropine (ATR). Solid lines show the mean individual sensitivity in the range 1040% of the maximal response (At&s and Simonis, 1961). The dotted curves (....A....) show the calculated difference between the curves before and after treatment with antagonists. Horizontal and vertical bars indicate SEM.

in the hagfish alimentary canal (Bjenning testine by radioimmunoassay (Lembeck et and Holmgren, 1988). However, Reinecke al., 1985; Conlon and Falkmer. 1989). Us- (1987) found SP-like immunoreactivity in ing C-terminal SP and neurokinin A anti- endocrine cells of the intestinal surface ep- sera on intestinal extract purified on ithelium, being most frequent in the caudal HPLC, Conlon and Falkmer (1989) found part of the intestine. SP-like material was multiple forms of tachykinins in the hagfish demonstrated in extracts of the hagtish in- intestine. The hagfish tachykinins are, how-


ever, probably not identical to SP, since no IR was detected with an antiserum directed towards the N-terminal of SP.

The absence or low number of nerve fibres in the intestine of lungfishes and lampreys agrees with the situation in other “ancient” species such as Myxine and Polypterus (P. Burkhardt-Holm and S. Holmgren, unpublished observations). However, the presence of immunoreactive endocrine cells in the gut mucosa of both lungfish and lamprey is similar to most other fish species, and may represent a more “primitive” state.

SP-immunoreactive nerves and endocrine cells in the stomach but not in the intestine of Raja agrees well with the distribution in Pofypterus (P. Burkhardt-Holm and S. Holmgren, unpublished observations), including a notably high density of endocrine SP-immunoreactive cells in the pyloric region. This is, however, probably not a general feature in elasmobranch fishes; in the spiny dog&h (S. acanthias), numerous endocrine cells and nerve fibers containing SP-like material have been demonstrated in the intestine (Holmgren, 1985). In the rainbow trout, nerve fibers containing a SP-like peptide have been observed in the myenteric plexus and to some extent also in the smooth muscle layers of the intestine. A sparse distribution of immunoreactive endocrine cells in the mucosa of the proximal intestine is also reported (Holm- gren et al., 1982).

The inability of SP to produce an effect on hagfish and lamprey preparations indi- cates that the SP-like peptide present in the endocrine cells (Reinecke, 1987; present study) is involved in the control of func- tions such as intestinal secretion and/or va- soregulation, rather than in the regulation of intestinal motility. A vasoregulatory role of a SP-like peptide has indeed indicated in the hagfish; in vitro preparations of the ven- tral aorta are contracted by low concentrations of SP (Reinecke, 1987).

The occasionai contractile effects of SP

on the ray intestine agrees with previous studies of three other species of Raju (An- drews and Young, 1988). The irregular re- sponses did not allow further mechanism studies in the ray, but in the lung&h circular muscle and the bichir longitudinal muscle the lack of effect of tetrodotoxin sug- gests a direct effect of SP on the intestinal muscle. In neither of these species have immunoreactive nerve libers been found in the intestinal wall (P. Burkhardt-Holm and S. Holmgren, unpublished observations; present study), and the endogenous source of tachykinins acting on the intestine of the lunglish may be the endocrine cells present in the intestinal mucosa, while the source in the ray and the bichir may be the endocrine cells in the pyloric part of the stomach.

A direct effect only of SP on receptors on the smooth muscle has earlier been demonstrated in strip preparations from the spiny dogfish, S. acanthias, which like the lungfish, ray, and bichir has a spiral intestine. However, in the spiny dogfish the effect may be mediated by enteric nerves containing tachykinins (Holmgren, 1985).

The situation seems to be more complex in the teleost gut. In the intestinal bulb of the carp, Cyprinus carpio, it has been suggested that the excitatory effect of SP is, as in mammals, both direct on the smooth muscle and indirect via the stimulation of cholinergic neurons (Kitazawa et al., 1988). The contractions produced by SP on the vascularly perfused intestine of the cod, Gadus morhua, also involve direct and indirect effects. The excitation is inhibited not only by tetrodotoxin and atropine but also by the serotonergic antagonist methysergide, indicating that the indirect effect is mediated by both cholinergic and serotonergic neurons (Jensen et al., 1987). In the stomach of the rainbow trout, SP stimulates the motility by a release of 5-HT and by a direct action on the stomach smooth muscle (Holmgren et al., 1985).

The recorded excitatory effect of SP on longitudinal intestinal muscle of the rain-


bow trout was probably both a direct effect on the smooth muscle and an indirect effect via enteric neurons in the same dose inter- val, which was indicated when the difference between the concentration-response curves before and after tetrodotoxin treatment were plotted (Fig. 3). The effects of atropine and methysergide suggest that both cholinergic and serotonergic neurons are involved in the indirect effect, as in the intestine of the cod. The actions via cholinergic and serotonergic nerves probably represent separate pathways for SP, since contractions induced by acetylcholine were unaffected by methysergide and excitations produced by 5-HT remained unchanged after treatment of the muscle preparations with atropine.

In conclusion, SP-like peptides are present in intestinal endocrine cells of the lamprey and hagtish, which represent two widely separate extant groups of the cyclo- stomes, the most primitive contemporary form of fish. The endogenous tachykinins are, however, probably not involved in the control of intestinal motility in these species. In lungfish, bichir, and ray, SP is excitatory, although only occasionally in the ray. In these species tachykinins may be released from endocrine cells present in the gut mucosa, and have a hormonal influence on the motility. At least in the lungfish and bichir, the effect may be directly on the smooth muscle, as in the elasmobranch Squalus. Teleosts show the most varied mechanisms for tachykinin control of the gut motility. In teleosts, both nerves and endocrine cells may be the source of the tachykinins involved in the control of gut motility. Both direct effects on the muscle cells and indirect effects via enteric nerves, involving cholinergic and serotonergic nerves, are indicated. The relative impor- tance of the different systems vary between teleost species.

It is suggested that, in evolution, tachykinins had no effect on the intestinal motility in the most primitive state and subse-

quently adopted an endocrine action on the smooth muscle to arrive at the most ad- vanced state, wherein hormonal and neuro- nal mechanisms integrate direct and indirect effects on the smooth muscle.

ACKNOWLEDGMENTS

We thank professor August0 Abe, University of Rio Claro, S.P., Brazil, and Dr. Eduardo Bicudo, Univer- sity of Sao Paulo, Brazil, for the opportunity to work on lungfish at the University of Rio Claro. We also thank Mr. S. Rodstrom, Smogen for supplying the ray, Dr. Hans Lundberg. Umel for supplying the lamprey, Mrs. Christina Hagstrom for help with immunohistochemistry, and Mrs. Birgitta Vallander and Ms. Inger Holmqvist for help with the figures and photography. The kind gifts of antisera from Professor J. Fumess, Flinders University, South Australia and Dot. A.-C. Jonsson. Goteborg are gratefully acknowledged. This study was supported by grants from the Swedish Nat- ural Science Research Council, the Hierta-Retzius Foundation, the Erna and Victor Hasselblad Founda- tion, and the Helge Ax:son Johnson Foundation

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