Fish Physiology and Biochemistry vol. 7 nos 1-4 pp 125-131 (1989) Kugler Publications, Amsterdam/Berkeley
Morphogenesis and distribution of the endocrine pancreas in adult lampreys John H. Youson and W. Mark Elliott Department of Zoology and Scarborough Campus, University of Toronto, West Hill, Ontario MIC IA4, Canada Keywords: adult lamprey, endocrine pancreas, morphogenesis, distribution, immunocytochemistry, somatostatin, insulin
Abstract The endocrine pancreas of larval lampreys appears as islets of cells isolated in the submucosa and those both continuous with, and within, the gut epithelium at the intestinal-oesophageal-bile duct junction. The islets, and occasionally follicles, are composed of only insulin-secreting (B) cells. During metamorphosis, the bile duct either completely degenerates or its epithelium transforms into a caudal endocrine pancreas while a cranial pancreas appears as a specialization and expansion of the larval pancreas. Immunocytochemistry and histochemistry demonstrates that there is a wide variation in the distribution of the pancreatic tissue in adults of lamprey species, and this variation may result from interspecific differences in morphogenetic events at metamorphosis. Despite species variability in its distribution, the endocrine pancreatic tissue in all adult lampreys is composed of equal numbers of B cells and somatostatin-secreting (D) cells, but there are no glucagonsecreting (A) cells. Immunocytochemistry reveals that B and D cells of the caudal pancreas differentiate from cells of the larval bile duct during metamorphosis of the sea lamprey, Petromyzon marines.
Introduction Lampreys are extant representatives of the ancient jawless fishes, the Agnatha, and have received considerable attention during studies on the evolution of organ systems of vertebrates (Youson 1985). They also have a complex life cycle, involving a protracted larval period (3-5 years), a phase of metamorphosis, a juvenile period, and a period of sexual maturity which culminates with spawning and death. In nonparasitic species, juveniles enter sexual maturation immediately after metamorphosis without ever feeding, whereas adults of parasitic species feed and grow (Hardisty and Potter 1971). Due to the complexity and length of the life cycle, lampreys have provided unique opportunities to investigate early and late stages of ontogeny of organs
(Youson 1985). In recent years it has become quite clear that there is some diversity in organ structure among adults of lamprey species (Youson and Beamish 1986) and, in some cases, this may be the result of variation in morphogenetic events occurring during metamorphosis. In the present study, we use immuno-histochemistry and -cytochemistry to report the variable distribution of endocrine pancreatic tissue of adults of lamprey species and to explain how the tissue develops during metamorphosis.
Materials and methods Larval lampreys of Petromyzon marinus were obtained from streams in New Brunswick, Canada
126 and some were kept in laboratory aquaria with suitable substrate until they began metamorphosis. Between July and November the transforming animals were staged 1 to 7 according to the criteria of Youson and Potter (1979). Juvenile adults of P. marinus were obtained from Lake Washademoak, New Brunswick. Juvenile adults of species Lampetra richardsoni,L. richardsonivariety marifuga, L. macrostoma, L. tridentata, and L. ayresi were obtained from Dr. R.J. Beamish, Pacific Biological Station, Fisheries and Oceans, Government of Canada, Nanaimo, British Columbia (Youson et al. 1988). For light microscopic immunohistochemistry, all animals were first anaesthetized and decapitated, and were then placed in Bouin's fluid for 24 h before storage in 70°7 ethanol. A portion of the body between the last branchiopore and the tip of the liver was embedded in paraffin, serially sectioned, and stained with periodic acid-Schiff (PAS) and counterstains or with antisera against either synthetic somatostatin 14, lamprey somatostatin34 (Andrews et al., 1988), or bovine insulin using the peroxidase anti-peroxidase method (Elliott and Youson 1986). For routine electron microscopy, pancreatic and presumptive pancreatic tissues from P. marinus were double-fixed in glutaraldehyde and osmium tetroxide in phosphate buffer, embedded in Epon-araldite, and thin sections were stained with uranyl acetate and lead citrate. For immunocytochemistry, the tissues were fixed only in glutaraldehyde, embedded in Lowicryl K4M, and thin sections were treated with the above antisera and antisera to mammalian glucagon using the protein A-gold method (Elliott and Youson 1988). Data on larvae and adults of Geotria australiswere obtained from the studies of Hilliard et al. (1985) and Hilliard and Potter (1988).
Results Larvae The endocrine pancreatic tissue of ammocoetes of all lamprey species appears as islets or small follicles of epithelial cells in the submucosal connective tissue near the junction of the oesophagus with
b
a Al BD--
0- O-
Al-
D
C
d
CR >_
CR D
D
,-CD
O
Al
e CR
D
g
CR x 0o
h
D
CR
D _
D
D _
C~ U
Al
,-
I
CD
.:D -~ ......
Al
,
Fig. 1. Diagrammatic representation of the distribution of the pancreatic tissue in islets (P) of larvae and in cranial (CR), caudal (CD), and intermediate (1) aggregates of juvenile adults of several species of lampreys. Also included are the anterior intestine (Al), bile duct (BD), diverticulum (D), liver (L), and oesophagus (0). a. Larva of Geotria australis; b. larva of Petromyzon marinus; c. adult G. australis; d. adult P. marinus; e. adult Lampetra richardsoni, Puntledge River; f. adult L. richardsoni, Morrison Creek; g. adult L. richardsoni variety marifuga; h. adult Lampetra tridentata;i. adult Lampetra macrostoma; j. adult Lampetra ayresi. (a., b., and c. are modified from Hilliard et al. 1985).
the anterior intestine (Fig. la, b). In holarctic species the pancreatic tissue is aggregated at this junction which is also the site where the extrahepatic common bile duct enters the alimentary canal. However, in ammocoetes of the Southern Hemisphere species, Geotriaaustralis,the bile duct is connected to one of the two large, anteriorly-directed diverticula and the pancreatic tissue is located be-
127 tween the diverticula and the oesophagus (Fig. la). Immuno-histochemistry and -cytochemistry of Petromyzon marinus shows only insulin-immunoreactive (B) cells in the larval islet tissue (Fig. 2) but somatostatin-immunoreactive (D) cells are present in the intestinal epithelium. During larval life there is a turnover of islet tissue with new B cell islets first appearing in the base of the intestinal epithelium and then moving into the submucosal area (Fig. 2).
Metamorphosis Both the alimentary canal and the hepato-biliary system of lampreys undergo a dramatic transformation during metamorphosis (Youson 1981a, b). The junction of the oesophagus with the anterior intestine, which in ammocoetes is located near the caudal tip of the liver, gradually moves to a position at the cranial end of the liver by the end of metamorphosis (Figs la-d). The prominent diverticula of ammocoetes of the Southern Hemisphere disappear and only a small diverticulum forms during metamorphosis. In contrast, during metamorphosis of Petromyzontidae a prominent intestinal diverticulum usually develops at the new oesophageal-anterior intestinal junction. The most significant change in the hepato-biliary system during lamprey metamorphosis is the loss of the entire biliary tree: extra- and intra-hepatic common bile ducts, bile ducts, bile ductules, and bile canaliculi. Thus, the liver loses its capacity for exocrine release of bile products. Keibel (1927) and Boenig (1928) demonstrated from observations of serial sections of metamorphosing Lampetra planeri that the extrahepatic common bile duct of larvae never completely disappears but instead the cells transform into pancreatic endocrine cells. Immunohistochemistry with antiinsulin and antisomatostatin shows that the extrahepatic, and part of the intrahepatic, common bile duct of ammocoetes of P. marinuscontribute to the formation of the caudal pancreas during the formation of this pancreatic mass at metamorphosis. B cells appear earlier than D cells in this transformation (Fig. 3). Electron microscopy reveals cells with some morphological features of bile duct cells with either
typical B-cell granules (Fig. 4) or D-cell granules. These redifferentiating endocrine cells show respective B- or D-cell immunoreactivity with immunocytochemical procedures (Fig. 5). The Southern Hemisphere species, Geotria australis, does not show the transformation of the biliary system into endocrine pancreatic tissue and consequently has no caudal pancreas in the adult (Hilliard et al. 1985; Hilliard and Potter 1988). Immunohistochemistry used to follow the fate of the larval pancreatic tissue during metamorphosis in P. marinus, shows that the B-cell larval islet tissue accompanies the forward moving oesophageal-intestinal junction and, when the adult diverticulum begins to form, new islets (including D-cell islets) are added from the diverticular epithelium in the manner that is seen during larval life. The resulting pancreatic mass is termed the cranial pancreas.
Juvenile Routine light microscopy with PAS stain and immunohistochemistry with antisera to both somatostatin and insulin reveals differences in the distribution of the pancreatic tissue within juveniles of most lamprey species (Fig. Ic-j). There are also intraspecific differences in distribution of the endocrine pancreas between individuals from different populations of the nonparasitic lamprey, L. richardsoni, and between L. richardsoni and a parasitic variety marifuga which coexist in the same stream. In P. marinus, L. macrostoma, L. tridentata, L. ayresi, and L. richardsoni there are two major masses of pancreatic tissue, a cranial and caudal pancreas, and they are connected to a variable degree by randomly-distributed islets called the intermediate cord. G. australis lacks a caudal pancreas and most L. richardsonivar. marifuga have no cranial pancreas. The cranial pancreas is usually found enveloping the anterior end of the diverticulum but the length of the diverticulum and the volume of cranial pancreatic tissue is remarkably species specific. However, there seems to be no relationship between the size of the diverticulum and the amount of cranial pancreas. For instance,
128
Fig. 2. Insulin immunoreactivity in cells within the base of intestinal epithelium (arrowhead) and in several positions in the submucosal connective tissue (arrows) of larval P. marinus, P, pigment. x 280. Fig. 3. Insulin immunoreactivity in some cells (arrowheads) of the extrahepatic common bile duct (BD) at stage 4 of metamorphosis in P. marinus. L, liver. x 285. Fig. 4. Electron micrograph of a cell from the extrahepatic common bile duct at stage 4 of metamorphosis in P. marinus showing aggregates of intermediate filaments (F) and some granules (arrowheads). x 22,000.
129 G. australis has a small diverticulum but a large cranial mass or islet organ (Hilliard et al. 1985) while var. marifuga also has a limited diverticulum and no cranial mass. The islets of the intermediate cord are best visualized through immunohistochemistry and, although general patterns of distribution are visualized, the patterns are not sufficiently consistent to be considered as species specific. Several examples of a continuous thick cord of endocrine pancreatic cells between the cranial and caudal pancreas has only been noted in L. richardsoni which coexist in a stream with L. richardsoni var. marifuga (Fig. If). The caudal pancreas is located approximately at the mid-point of the liver where the hepatic portal vein and hepatic artery enter the liver. The pancreatic tissue is positioned within submucosal connective tissue which connects the liver to the typhlosole of the anterior intestine. The extent to which the pancreatic tissue is positioned with respect to either the liver or the typhlosole is variable and seems to be species specific. Thus, L. richardsoni(Fig. le) shows the caudal pancreas within a deep invagination of the liver, whereas in L. tridentata(Fig. lh) the endocrine tissue is mainly confined to the typhlosole. In others, such as P. marinus the caudal pancreas is in an intermediate position between the liver and the typhlosole (Fig. ld). The caudal pancreas of var. marifuga is distinctly different in many respects from that of other juvenile lampreys. The typhlosole of the variety commences much more posteriorly than in other lampreys and, although the anterior portion of the caudal pancreas is found in the typhlosole, most of the caudal pancreas is in an intrahepatic position with several partially connected branches of pancreatic tissue and many smaller intrahepatic cords or islets (Fig. Ig). Bile ductules are occasionally located near the antiinsulinand antisomatostatin-immunoreactive intrahepatic cords (Fig. 6).
All regions of endocrine pancreatic tissue in all juvenile lampreys examined show immunostaining with antiinsulin and antisomatostatin. B cells and D cells or P cells (Brinn and Epple 1976) are present in approximately equal numbers in the cranial pancreas of G. australis, in the caudal pancreas of var. marifuga (Fig. 6), and in the cranial and caudal pancreas of all other species (Fig. 7). The use of the designation P cell (Brinn and Epple 1976) is in recognition of the peculiar histochemical staining properties of the lamprey somatostatin-secreting cells and the fact that they may not be homologous to D cells of higher vertebrates. A third cell type of distinctive fine structure, but of unknown nature, is recognized in both P. marinusand L. ayresi (Elliott and Youson 1988; Youson et al. 1988). An argyrophilic cell is present in G. australis (Hilliard et al. 1985). The endocrine pancreas of juvenile lampreys is not immunoreactive with antisera to mammalian glucagon.
Discussion The distribution of the endocrine pancreas in lampreys at all stages of the life cycle is directly related to the morphogenesis of the tissue. Variability in distribution of endocrine pancreatic tissue between species may result from morphogenetic and anatomical differences. The distribution of islets in larvae of Geotriidae, Mordaciiae, and Petromyzontidae is fundamentally the same, despite the fact that the bile duct, which is believed to provide the source of new islets (Barrington 1972), enters the alimentary canal at entirely different positions in representatives of each of these families. The common feature among larvae of these families is that the islets are aggregated at or near the junction of the oesophagus with the anterior intestine. This feature may be explained by the fact that, although the bile duct
Fig. 5. Gold particles showing insulin immunoreactivity in granules (arrowheads) of a cell from the extrahepatic common bile duct at stage 4 of metamorphosis in P. marinus. F, intermediate filaments. x42,000. Fig. 6. Immunoreactivity (dark staining) of the caudal pancreas (CD) and intrahepatic islets (arrowheads) of L. richardsoni var. marifuga with antisomatostatin-14. Al, anterior intestine; L, liver; P, pigment. x 115. Fig. 7. Immunoreactivity (dark staining) of the caudal pancreas (CD) of juvenile P. marinus with antisomatostatin-34. Al, anterior intestine; L, liver. x 100.
130 epithelium may contribute some islets, the majority originate within the epithelia of the oesophagus and the anterior intestine (Elliott and Youson 1986). The more widespread distribution of islets in Geotriidae can be accounted for by the contribution of the diverticular epithelium in islet formation (Hilliard et al. 1985). More recent evidence on adult lampreys is that there may be a wider species variation in distribution of endocrine pancreatic tissue than was formerly described (Barrington 1972; Youson 1981a; Potter 1986). Marked differences are noted between both Geotriidae and Mordaciidae and the more northerly, Petromyzontidae, but it is becoming apparent that there are subtle and sometimes pronounced differences in pancreatic distribution in members of the latter family. The lack of involvement of the bile duct in development of the pancreas in Geotriidae and Mordaciidae accounts for the absence of a caudal pancreas in adults (Hilliard et al. 1985; Hilliard and Potter 1988). There are subtle differences in the distribution of the cranial, intermediate, and caudal pancreatic tissues of P. marinus and the four Lampetra sp. of the west coast of Canada. This diversity may be the result of slight differences in the position of the larval bile duct and its contribution to the developing caudal pancreas and the extent to which the developing adult diverticulum provides islets for the enlarging cranial mass during metamorphosis. Immunohistochemical evidence on metamorphosing P. marinus indicates that enlargement of the cranial pancreas coincides with development of the diverticulum and that the islets arise from the epithelium of the latter in a manner similar to that seen in larval life (Elliott and Youson 1987). The distribution of the endocrine pancreas in adults of L. richardsoni var. marifuga primarily within a caudal mass is unique among the lampreys. It is not entirely clear why the distribution should be so different from that of other Lampetra sp. and, in particular, from that of L. richardsoni in the same stream. In turn, the latter is different from animals of the same species in another watershed. We strongly suspect that the situation in var. marifuga is a direct result of either reduced, different or delayed morphogenetic processes taking
place during metamorphosis in the variety compared to other Lampetra sp. The fact that the animal feeds while sexually mature is in itself highly unique among lampreys (Beamish and Withler 1986). The relationship between var. marifuga and L. richardsoniin a small stream on Vancouver Island, British Columbia is still a matter of debate (R.J. Beamish, personal communication) but the confusion is compounded by our evidence of diversity in distribution of pancreatic tissue in at least two populations of the latter species. Since L. richardsonihas such a wide dispersal along the west coast of Canada, it is possible that our investigation which is presently underway on other samples of this species will yield results which show a population specific distribution of pancreatic tissue. A consistent feature in all lamprey species is the presence of only insulin-secreting cells in the larval pancreas and the existence of approximately equal numbers of insulin- and somatostatin-secreting cells in the adult pancreas. Future research on the endocrine pancreas of lampreys should be directed towards the role of somatostatin, for clearly the significance of this hormone to adult life is emphasized by the number of cells within the pancreas. An explanation as to why, in most species, the common bile duct is sacrificed for the development of at least part of the pancreas may result from such an investigation.
Acknowledgements Supported by the Natural Sciences and Engineering Research Council of Canada.
References cited Andrews, P.C., Pollock, H.G., Elliott, W.M., Youson, J.H. and Plisetskaya, E.M. 1988. Isolation and characterization of a variant somatostatin-14 and two related somatostatins of 34 and 37 residues from lamprey (Petromyzon marinus) J. Biol. Chem. 263: 15809-15814. Barrington, E.J.W. 1972. The pancreas and intestine. In The Biology of Lampreys, vol. 2. pp. 135-169. Edited by M.W. Hardisty and I.C. Potter. Academic Press, London. Beamish, R.J. and Withler, R.E. 1986. A polymorphic population of lampreys that may produce parasitic and nonparasitic
131 varieties. In Indo-Pacific Fish Biology: Proc. Second Int. Conf. Indo-Pacific Fishes. pp. 31-49. Edited by T. Uyeno, R. Arai, T. Taniuchi and K. Matsuura. Ichthyological Society of Japan, Tokyo. Boenig, H. 1928. Studien zur Morphologie und Entwicklungsgeschichte des Pankreas beim Bachneunauge (Lampetra (Petromyzon) planeri). II. Tiel. Z. mikrosk. -anat. Forsch. 12: 537-594. Brinn, J.E. and Epple, A. 1976. New types of islets cells in a cyclostome, Petromyzon marinus. Cell Tiss. Res. 171: 317-330. Elliott, W.M. and Youson, J.H. 1986. Immunocytochemical localization of insulin and somatostatin in the endocrine pancreas of the sea lamprey, Petromyzon marinus L., at various stages of its life cycle. Cell Tiss. Res. 243: 629-634. Elliott, W.M. and Youson, J.H. 1987. Immunohistochemical observations of the endocrine pancreas during metamorphosis of the sea lamprey, Petromyzon marinus L. Cell Tiss. Res. 247: 351-357. Elliott, W.M. and Youson, J.H. 1988. Fine structure and immunocytochemistry of cells within the endocrine pancreas of larval and adult sea lampreys, Petromyzon marinus L. Am. J. Anat. 182: 73-83. Hardisty, M.W. and Potter, I.C. 1971. The general biology of adult lampreys. In The Biology of Lampreys. pp. 127-206. Edited by M.W. Hardisty and I.C. Potter. Academic Press, London. Hilliard, R.W. and Potter, I.C. 1988. Morphology of the exocrine pancreas of the Southern Hemisphere lamprey, Geotria australis Gray, and changes during metamorphosis. J. Morphol. 197: 33-52. Hilliard, R.W., Epple, A. and Potter, I.C. 1985. The morphology and histology of the endocrine pancreas of the Southern Hemisphere lamprey, Geotria australis Gray. J. Morphol. 184: 253-261.
Keibel, F. 1927. Zur Entwicklungsgeschichte des Vorderdarmes und des Pankreas beim Bachneunauge (Lampetra (Petromyzon) planeri) und beim Flubneunauge (Lampetra (Petromyzon) fluviatilis). Z. mikrosk.-anat. Forsch 8: 408-476. Potter, I.C. 1986. The distinctive characters of Southern Hemisphere lampreys (Geotriidae and Mordaciidae). In IndoPacific Fish Biology: Proc. Second Int. Conf. Indo-Pacific Fishes. pp. 9-19. Edited by T. Uyeno, R. Arai, T. Taniuchi, and K. Matsuura. Ichthyological Society of Japan, Tokyo. Youson, J.H. 1981a. The alimentary canal. In The Biology of Lampreys, vol. 3. pp. 95-189. Edited by M.W. Hardisty and I.C. Potter. Academic Press, London. Youson, J.H. 1981b. The liver. In The Biology of Lampreys, vol. 3. pp. 263-332. Edited by M.W. Hardisty and I.C. Potter. Academic Press, London. Youson, J.H. 1985. Organ development and specialization in lamprey species. In Evolutionary Biology of Primitive Fishes. pp. 141-164. Edited by R.E. Foreman, A. Gorbman, J.M. Dodd, and R. Olsson. Plenum, New York. Youson, J.H. and Beamish, R.J. 1986. Morphological comparison of organs in several species of lampreys on the west coast of Canada. In Indo-Pacific Fish Biology: Proc. Second Int. Conf. Indo-Pacific Fishes. p. 926. Edited by T. Uyeno, R. Arai, T. Taniuchi, and K. Matsuura. Ichthyological Society of Japan, Tokyo. Youson, J.H. and Potter, I.C. 1979. A description of the stages in the metamorphosis of the anadromous sea lamprey, Petromyzon marinus L. Can. J. Zool. 57: 1808-1817. Youson, J.H., Elliott, W.M., Beamish, R.J. and Wang, D.W. 1988. A comparison of endocrine pancreatic tissue in adults of four species of lampreys in British Columbia: a morphological and immunohistochemical study. Gen. Comp. Endocrinol. 70: 247-261.
Morphogenesis and distribution of the endocrine pancreas in adult lampreys John H. Youson and W. Mark Elliott Department of Zoology and Scarborough Campus, University of Toronto, West Hill, Ontario MIC IA4, Canada Keywords: adult lamprey, endocrine pancreas, morphogenesis, distribution, immunocytochemistry, somatostatin, insulin
Abstract The endocrine pancreas of larval lampreys appears as islets of cells isolated in the submucosa and those both continuous with, and within, the gut epithelium at the intestinal-oesophageal-bile duct junction. The islets, and occasionally follicles, are composed of only insulin-secreting (B) cells. During metamorphosis, the bile duct either completely degenerates or its epithelium transforms into a caudal endocrine pancreas while a cranial pancreas appears as a specialization and expansion of the larval pancreas. Immunocytochemistry and histochemistry demonstrates that there is a wide variation in the distribution of the pancreatic tissue in adults of lamprey species, and this variation may result from interspecific differences in morphogenetic events at metamorphosis. Despite species variability in its distribution, the endocrine pancreatic tissue in all adult lampreys is composed of equal numbers of B cells and somatostatin-secreting (D) cells, but there are no glucagonsecreting (A) cells. Immunocytochemistry reveals that B and D cells of the caudal pancreas differentiate from cells of the larval bile duct during metamorphosis of the sea lamprey, Petromyzon marines.
Introduction Lampreys are extant representatives of the ancient jawless fishes, the Agnatha, and have received considerable attention during studies on the evolution of organ systems of vertebrates (Youson 1985). They also have a complex life cycle, involving a protracted larval period (3-5 years), a phase of metamorphosis, a juvenile period, and a period of sexual maturity which culminates with spawning and death. In nonparasitic species, juveniles enter sexual maturation immediately after metamorphosis without ever feeding, whereas adults of parasitic species feed and grow (Hardisty and Potter 1971). Due to the complexity and length of the life cycle, lampreys have provided unique opportunities to investigate early and late stages of ontogeny of organs
(Youson 1985). In recent years it has become quite clear that there is some diversity in organ structure among adults of lamprey species (Youson and Beamish 1986) and, in some cases, this may be the result of variation in morphogenetic events occurring during metamorphosis. In the present study, we use immuno-histochemistry and -cytochemistry to report the variable distribution of endocrine pancreatic tissue of adults of lamprey species and to explain how the tissue develops during metamorphosis.
Materials and methods Larval lampreys of Petromyzon marinus were obtained from streams in New Brunswick, Canada
126 and some were kept in laboratory aquaria with suitable substrate until they began metamorphosis. Between July and November the transforming animals were staged 1 to 7 according to the criteria of Youson and Potter (1979). Juvenile adults of P. marinus were obtained from Lake Washademoak, New Brunswick. Juvenile adults of species Lampetra richardsoni,L. richardsonivariety marifuga, L. macrostoma, L. tridentata, and L. ayresi were obtained from Dr. R.J. Beamish, Pacific Biological Station, Fisheries and Oceans, Government of Canada, Nanaimo, British Columbia (Youson et al. 1988). For light microscopic immunohistochemistry, all animals were first anaesthetized and decapitated, and were then placed in Bouin's fluid for 24 h before storage in 70°7 ethanol. A portion of the body between the last branchiopore and the tip of the liver was embedded in paraffin, serially sectioned, and stained with periodic acid-Schiff (PAS) and counterstains or with antisera against either synthetic somatostatin 14, lamprey somatostatin34 (Andrews et al., 1988), or bovine insulin using the peroxidase anti-peroxidase method (Elliott and Youson 1986). For routine electron microscopy, pancreatic and presumptive pancreatic tissues from P. marinus were double-fixed in glutaraldehyde and osmium tetroxide in phosphate buffer, embedded in Epon-araldite, and thin sections were stained with uranyl acetate and lead citrate. For immunocytochemistry, the tissues were fixed only in glutaraldehyde, embedded in Lowicryl K4M, and thin sections were treated with the above antisera and antisera to mammalian glucagon using the protein A-gold method (Elliott and Youson 1988). Data on larvae and adults of Geotria australiswere obtained from the studies of Hilliard et al. (1985) and Hilliard and Potter (1988).
Results Larvae The endocrine pancreatic tissue of ammocoetes of all lamprey species appears as islets or small follicles of epithelial cells in the submucosal connective tissue near the junction of the oesophagus with
b
a Al BD--
0- O-
Al-
D
C
d
CR >_
CR D
D
,-CD
O
Al
e CR
D
g
CR x 0o
h
D
CR
D _
D
D _
C~ U
Al
,-
I
CD
.:D -~ ......
Al
,
Fig. 1. Diagrammatic representation of the distribution of the pancreatic tissue in islets (P) of larvae and in cranial (CR), caudal (CD), and intermediate (1) aggregates of juvenile adults of several species of lampreys. Also included are the anterior intestine (Al), bile duct (BD), diverticulum (D), liver (L), and oesophagus (0). a. Larva of Geotria australis; b. larva of Petromyzon marinus; c. adult G. australis; d. adult P. marinus; e. adult Lampetra richardsoni, Puntledge River; f. adult L. richardsoni, Morrison Creek; g. adult L. richardsoni variety marifuga; h. adult Lampetra tridentata;i. adult Lampetra macrostoma; j. adult Lampetra ayresi. (a., b., and c. are modified from Hilliard et al. 1985).
the anterior intestine (Fig. la, b). In holarctic species the pancreatic tissue is aggregated at this junction which is also the site where the extrahepatic common bile duct enters the alimentary canal. However, in ammocoetes of the Southern Hemisphere species, Geotriaaustralis,the bile duct is connected to one of the two large, anteriorly-directed diverticula and the pancreatic tissue is located be-
127 tween the diverticula and the oesophagus (Fig. la). Immuno-histochemistry and -cytochemistry of Petromyzon marinus shows only insulin-immunoreactive (B) cells in the larval islet tissue (Fig. 2) but somatostatin-immunoreactive (D) cells are present in the intestinal epithelium. During larval life there is a turnover of islet tissue with new B cell islets first appearing in the base of the intestinal epithelium and then moving into the submucosal area (Fig. 2).
Metamorphosis Both the alimentary canal and the hepato-biliary system of lampreys undergo a dramatic transformation during metamorphosis (Youson 1981a, b). The junction of the oesophagus with the anterior intestine, which in ammocoetes is located near the caudal tip of the liver, gradually moves to a position at the cranial end of the liver by the end of metamorphosis (Figs la-d). The prominent diverticula of ammocoetes of the Southern Hemisphere disappear and only a small diverticulum forms during metamorphosis. In contrast, during metamorphosis of Petromyzontidae a prominent intestinal diverticulum usually develops at the new oesophageal-anterior intestinal junction. The most significant change in the hepato-biliary system during lamprey metamorphosis is the loss of the entire biliary tree: extra- and intra-hepatic common bile ducts, bile ducts, bile ductules, and bile canaliculi. Thus, the liver loses its capacity for exocrine release of bile products. Keibel (1927) and Boenig (1928) demonstrated from observations of serial sections of metamorphosing Lampetra planeri that the extrahepatic common bile duct of larvae never completely disappears but instead the cells transform into pancreatic endocrine cells. Immunohistochemistry with antiinsulin and antisomatostatin shows that the extrahepatic, and part of the intrahepatic, common bile duct of ammocoetes of P. marinuscontribute to the formation of the caudal pancreas during the formation of this pancreatic mass at metamorphosis. B cells appear earlier than D cells in this transformation (Fig. 3). Electron microscopy reveals cells with some morphological features of bile duct cells with either
typical B-cell granules (Fig. 4) or D-cell granules. These redifferentiating endocrine cells show respective B- or D-cell immunoreactivity with immunocytochemical procedures (Fig. 5). The Southern Hemisphere species, Geotria australis, does not show the transformation of the biliary system into endocrine pancreatic tissue and consequently has no caudal pancreas in the adult (Hilliard et al. 1985; Hilliard and Potter 1988). Immunohistochemistry used to follow the fate of the larval pancreatic tissue during metamorphosis in P. marinus, shows that the B-cell larval islet tissue accompanies the forward moving oesophageal-intestinal junction and, when the adult diverticulum begins to form, new islets (including D-cell islets) are added from the diverticular epithelium in the manner that is seen during larval life. The resulting pancreatic mass is termed the cranial pancreas.
Juvenile Routine light microscopy with PAS stain and immunohistochemistry with antisera to both somatostatin and insulin reveals differences in the distribution of the pancreatic tissue within juveniles of most lamprey species (Fig. Ic-j). There are also intraspecific differences in distribution of the endocrine pancreas between individuals from different populations of the nonparasitic lamprey, L. richardsoni, and between L. richardsoni and a parasitic variety marifuga which coexist in the same stream. In P. marinus, L. macrostoma, L. tridentata, L. ayresi, and L. richardsoni there are two major masses of pancreatic tissue, a cranial and caudal pancreas, and they are connected to a variable degree by randomly-distributed islets called the intermediate cord. G. australis lacks a caudal pancreas and most L. richardsonivar. marifuga have no cranial pancreas. The cranial pancreas is usually found enveloping the anterior end of the diverticulum but the length of the diverticulum and the volume of cranial pancreatic tissue is remarkably species specific. However, there seems to be no relationship between the size of the diverticulum and the amount of cranial pancreas. For instance,
128
Fig. 2. Insulin immunoreactivity in cells within the base of intestinal epithelium (arrowhead) and in several positions in the submucosal connective tissue (arrows) of larval P. marinus, P, pigment. x 280. Fig. 3. Insulin immunoreactivity in some cells (arrowheads) of the extrahepatic common bile duct (BD) at stage 4 of metamorphosis in P. marinus. L, liver. x 285. Fig. 4. Electron micrograph of a cell from the extrahepatic common bile duct at stage 4 of metamorphosis in P. marinus showing aggregates of intermediate filaments (F) and some granules (arrowheads). x 22,000.
129 G. australis has a small diverticulum but a large cranial mass or islet organ (Hilliard et al. 1985) while var. marifuga also has a limited diverticulum and no cranial mass. The islets of the intermediate cord are best visualized through immunohistochemistry and, although general patterns of distribution are visualized, the patterns are not sufficiently consistent to be considered as species specific. Several examples of a continuous thick cord of endocrine pancreatic cells between the cranial and caudal pancreas has only been noted in L. richardsoni which coexist in a stream with L. richardsoni var. marifuga (Fig. If). The caudal pancreas is located approximately at the mid-point of the liver where the hepatic portal vein and hepatic artery enter the liver. The pancreatic tissue is positioned within submucosal connective tissue which connects the liver to the typhlosole of the anterior intestine. The extent to which the pancreatic tissue is positioned with respect to either the liver or the typhlosole is variable and seems to be species specific. Thus, L. richardsoni(Fig. le) shows the caudal pancreas within a deep invagination of the liver, whereas in L. tridentata(Fig. lh) the endocrine tissue is mainly confined to the typhlosole. In others, such as P. marinus the caudal pancreas is in an intermediate position between the liver and the typhlosole (Fig. ld). The caudal pancreas of var. marifuga is distinctly different in many respects from that of other juvenile lampreys. The typhlosole of the variety commences much more posteriorly than in other lampreys and, although the anterior portion of the caudal pancreas is found in the typhlosole, most of the caudal pancreas is in an intrahepatic position with several partially connected branches of pancreatic tissue and many smaller intrahepatic cords or islets (Fig. Ig). Bile ductules are occasionally located near the antiinsulinand antisomatostatin-immunoreactive intrahepatic cords (Fig. 6).
All regions of endocrine pancreatic tissue in all juvenile lampreys examined show immunostaining with antiinsulin and antisomatostatin. B cells and D cells or P cells (Brinn and Epple 1976) are present in approximately equal numbers in the cranial pancreas of G. australis, in the caudal pancreas of var. marifuga (Fig. 6), and in the cranial and caudal pancreas of all other species (Fig. 7). The use of the designation P cell (Brinn and Epple 1976) is in recognition of the peculiar histochemical staining properties of the lamprey somatostatin-secreting cells and the fact that they may not be homologous to D cells of higher vertebrates. A third cell type of distinctive fine structure, but of unknown nature, is recognized in both P. marinusand L. ayresi (Elliott and Youson 1988; Youson et al. 1988). An argyrophilic cell is present in G. australis (Hilliard et al. 1985). The endocrine pancreas of juvenile lampreys is not immunoreactive with antisera to mammalian glucagon.
Discussion The distribution of the endocrine pancreas in lampreys at all stages of the life cycle is directly related to the morphogenesis of the tissue. Variability in distribution of endocrine pancreatic tissue between species may result from morphogenetic and anatomical differences. The distribution of islets in larvae of Geotriidae, Mordaciiae, and Petromyzontidae is fundamentally the same, despite the fact that the bile duct, which is believed to provide the source of new islets (Barrington 1972), enters the alimentary canal at entirely different positions in representatives of each of these families. The common feature among larvae of these families is that the islets are aggregated at or near the junction of the oesophagus with the anterior intestine. This feature may be explained by the fact that, although the bile duct
Fig. 5. Gold particles showing insulin immunoreactivity in granules (arrowheads) of a cell from the extrahepatic common bile duct at stage 4 of metamorphosis in P. marinus. F, intermediate filaments. x42,000. Fig. 6. Immunoreactivity (dark staining) of the caudal pancreas (CD) and intrahepatic islets (arrowheads) of L. richardsoni var. marifuga with antisomatostatin-14. Al, anterior intestine; L, liver; P, pigment. x 115. Fig. 7. Immunoreactivity (dark staining) of the caudal pancreas (CD) of juvenile P. marinus with antisomatostatin-34. Al, anterior intestine; L, liver. x 100.
130 epithelium may contribute some islets, the majority originate within the epithelia of the oesophagus and the anterior intestine (Elliott and Youson 1986). The more widespread distribution of islets in Geotriidae can be accounted for by the contribution of the diverticular epithelium in islet formation (Hilliard et al. 1985). More recent evidence on adult lampreys is that there may be a wider species variation in distribution of endocrine pancreatic tissue than was formerly described (Barrington 1972; Youson 1981a; Potter 1986). Marked differences are noted between both Geotriidae and Mordaciidae and the more northerly, Petromyzontidae, but it is becoming apparent that there are subtle and sometimes pronounced differences in pancreatic distribution in members of the latter family. The lack of involvement of the bile duct in development of the pancreas in Geotriidae and Mordaciidae accounts for the absence of a caudal pancreas in adults (Hilliard et al. 1985; Hilliard and Potter 1988). There are subtle differences in the distribution of the cranial, intermediate, and caudal pancreatic tissues of P. marinus and the four Lampetra sp. of the west coast of Canada. This diversity may be the result of slight differences in the position of the larval bile duct and its contribution to the developing caudal pancreas and the extent to which the developing adult diverticulum provides islets for the enlarging cranial mass during metamorphosis. Immunohistochemical evidence on metamorphosing P. marinus indicates that enlargement of the cranial pancreas coincides with development of the diverticulum and that the islets arise from the epithelium of the latter in a manner similar to that seen in larval life (Elliott and Youson 1987). The distribution of the endocrine pancreas in adults of L. richardsoni var. marifuga primarily within a caudal mass is unique among the lampreys. It is not entirely clear why the distribution should be so different from that of other Lampetra sp. and, in particular, from that of L. richardsoni in the same stream. In turn, the latter is different from animals of the same species in another watershed. We strongly suspect that the situation in var. marifuga is a direct result of either reduced, different or delayed morphogenetic processes taking
place during metamorphosis in the variety compared to other Lampetra sp. The fact that the animal feeds while sexually mature is in itself highly unique among lampreys (Beamish and Withler 1986). The relationship between var. marifuga and L. richardsoniin a small stream on Vancouver Island, British Columbia is still a matter of debate (R.J. Beamish, personal communication) but the confusion is compounded by our evidence of diversity in distribution of pancreatic tissue in at least two populations of the latter species. Since L. richardsonihas such a wide dispersal along the west coast of Canada, it is possible that our investigation which is presently underway on other samples of this species will yield results which show a population specific distribution of pancreatic tissue. A consistent feature in all lamprey species is the presence of only insulin-secreting cells in the larval pancreas and the existence of approximately equal numbers of insulin- and somatostatin-secreting cells in the adult pancreas. Future research on the endocrine pancreas of lampreys should be directed towards the role of somatostatin, for clearly the significance of this hormone to adult life is emphasized by the number of cells within the pancreas. An explanation as to why, in most species, the common bile duct is sacrificed for the development of at least part of the pancreas may result from such an investigation.
Acknowledgements Supported by the Natural Sciences and Engineering Research Council of Canada.
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