Fish Physiology and Biochemistry vol. 8 no. 5 pp 389-397 (1990) Kugler Publications, Amsterdam/Berkeley
Morphogenesis of somatostatin- and insulin-secreting cells in the lamprey endocrine pancreas John H. Youson and Richard Cheung Department of Zoology and Scarborough Campus, University of Toronto, West Hill, Ontario MIC IA4, Canada Keywords: morphogenesis, endocrine pancreas, lamprey, life cycle, immunohistochemistry
Abstract The development of the endocrine pancreatic tissue during the entire life cycle of lampreys was provided through a review of previous literature and through a description of new, preliminary immunohistochemical data on a number of species. There seems to be no firm conclusions on the method of development of the endocrine pancreas during embyrogenesis, but observations of 79-day old larvae suggest that somatostatinpositive cells are confined to the epithelium of the alimentary canal, which is also the site at which insulinimmunoreactive islets form. This type of cellular distribution and morphogenesis at the oesophago-intestinal junction continues throughout the larval period. In northern hemisphere species, a caudal pancreatic mass forms at metamorphosis from the transformed epithelium of the bile duct. Evidence is presented to suggest that the process of bile duct degeneration and transformation is highly synchronized and that interference with the morphogenetic event will alter the normal distribution of pancreatic islets in the adult. The position of the bile duct in larvae of southern hemisphere species results in no caudal pancreas in adults. The adult cranial pancreas of all lamprey species, forms from remnants of the larval islets and through proliferation of new islets from the epithelium of the developing intestinal diverticulum. In holarctic lampreys, the cranial and caudal masses are connected by a discontinuous intermediate cord of islets and all three regions contain equal numbers of somatostatin- and insulin-immunoreactive cells. Following metamorphosis, growth of the caudal pancreas occurs through cellular proliferation from within but the cranial pancreas continues to recruit islets from the diverticular epithelium through the upstream-migrant (prespawning) period.
Introduction There are three families of extant lampreys: Petromyzontidae, represented by thirty-six species in the northern hemisphere (holarctic lampreys); Geotriidae, with a single species in the southern hemisphere; Mordaciidae, with three species also in the southern hemisphere (Potter 1980, 1986). The lam-
prey life cycle provides an opportunity to study many events of vertebrate ontogeny as the organism passes through seven intervals of development: gametogenesis, embryogenesis, a larval period, metamorphosis, a juvenile period (with growth in parasitic species), sexual maturation, and senescence (Youson 1985). There are many organ systems which undergo radical changes at specific pe-
Addressforcorrespondence:J.H. Youson, Division of Life Sciences, Scarborough Campus, University of Toronto, West Hill, Ontario MIC 1A4, Canada
390 riods of the life cycle, and these include elements of the endocrine system. Two of the most dramatic changes in the endocrine system are seen at metamorphosis in the thyroid gland and the endocrine pancreas (Youson 1980), both of which are derived from endoderm and appear relatively early in embryogenesis. However, in addition, the endocrine pancreas undergoes a continuous development throughout the larva period in order to replace islets lost through degeneration (Barrington 1972). For the most part, members of the three families of lampreys undergo similar developmental changes during the life cycle (Youson 1985). However, the endocrine pancreatic tissue in adults of most holarctic lampreys is divided into distinct cranial and caudal portions (Epple and Brinn 1986, 1987), which although developing via different morphogenetic pathways, both contain insulin- and somatostatin-immunoreactive cells (Van Noorden et al. 1972; Elliott and Youson 1986, 1988, 1989). In contrast, adults of both Geotriidae and Mordaciidae have only a single cranial pancreas (islet organ) which, on the basis of histochemical observations, is thought to contain both cell types (Hilliard et al. 1985). Although somatostatin cells are not visible in the pancreas of larvae, cells immunoreactive with somatostatin antisera are located in the intestine (Van Noorden et al. 1972) and it is possible that their presence at this latter site may have some relevance to subsequent development of the definitive endocrine pancreas. In the present study, we review the development of endocrine pancreatic tissue in the larval period and during metamorphosis and provide preliminary data on our investigations into the ontogeny of somatostatin- and insulin-immunoreactive cells in this tissue during embryogenesis and adult life.
Materials and methods Larval lampreys (ammocoetes) of Petromyzon marinus L. were obtained from several streams and rivers in New Brunswick, Canada where juvenile adults were taken from migrating alewives in Lake Washademoak, New Brunswick. Many of the ammocoetes began to transform in the laboratory
while they were housed in freshwater aquaria with river substrate, following the criteria of Youson and Potter (1979). These were classified as to stage 1-7 before their sacrifice. Juveniles were fed on rainbow trout (Salmo gairdneri) in freshwater aquaria. Upstream-migrant, landlocked P. marinus were captured in the Humber River, Toronto, Canada and did not feed. Both larvae and late transforming brook lampreys, Lampetra lamottenii, were caught by electro-fishing in Duffins Creek, Ontario, Canada. Eggs from upstream-migrants of P. marinus were fertilized in finger bowls with sperm from the same species, and the embryos were allowed to develop to varying stages (Piavis 1971) before fixation in toto in Bouin's fluid. After anaesthetization, all animals were killed by decapitation and either portions of the body trunk region (ammocoetes, metamorphosing stages, juveniles) or dissected pancreatic tissue and intestine (upstream migrants) were placed in Bouin's fluid for 24 h before storage in 70% ethanol. Tissue was embedded in paraffin using routine methods, serially sectioned, and stained with either periodic acidSchiff and counterstains, or with antisera for immunohistochemistry using the peroxidase antiperoxidase method (Elliott and Youson 1986). The antisera were prepared in rabbits against synthetic mammalian somatostatin-14, lamprey somatostatin-34 (Andrews et al. 1988) or lamprey insulin (Plisetskaya et al. 1988).
Results and discussion Embryo Both Kupffer (1893) and Brachet (1897) studied early development of the alimentary canal, liver, and pancreas in lampreys. However, they disagreed as to the method of formation and the time at which endocrine pancreatic islets first appear. In fact, it is not clear that they identified pancreatic islets at any stage of embryogenesis. Brachet (1897) correctly concluded that, unlike most other vertebrates (Jollie 1973), the pancreas of larval lampreys is not formed from dorsal and ventral diverticula. Instead, Brachet (1897) concluded that pancreatic is-
391 lets arise from proliferation of the epithelium of the digestive tube and specifically, the anterior intestine. It is clear that this viewpoint was influenced by the observations of Schneider (1879) who earlier had described the distribution of islets in large ammocoetes. We are in the process of re-examining the development of the pancreas during embryogenesis and early larval life using immunohistochemistry, and have already observed sections of 79-day old larvae treated with antisomatostatin (Fig. 1) and anti-lamprey insulin (Fig. 2) antisera. Islet cells are immunoreactive to anti-lamprey insulin at the oesophago-intestinal junction and are located both within the basal epithelium of the intestine and the surrounding submucosal connective tissue (Fig. 2). In addition, somatostatin-14, but not somatostatin34, cells are present in the early alimentary canal (Fig. 1). Although this is presently the earliest observation of insulin- and somatostatin-immunoreactive cells in lamprey development, it is our belief that these endocrine cells will be found much earlier and that the islets likely arise in embryogenesis in the manner described for later ammocoetes (Figs. 3, 4).
Larva The difference in distribution of pancreatic islets in larvae of northern and southern hemisphere lampreys has been recently reviewed (Potter 1986) and is illustrated in Fig. 5. In larvae of holarctic lampreys, pancreatic islets are generally concentrated in the connective tissue at the junction of the anterior intestine with the oesophagus and the extrahepatic common bile duct (Figs. 3, 5). There is little, if any, intestinal diverticulum at this junction in northern hemisphere species (Youson 1981). The aggregations of epithelial cells at this site were described as a pancreatic endocrine tissue by Langerhans (1873) and occasionally are referred to as islets of Langerhans. In the southern hemisphere species, Geotria australis, islets are present in the connective tissue located between the oesophagus and the two forwardly-directed diverticula (Hilliard et al. 1985). The bile duct in this species empties
into the anterior region of the left diverticulum (Fig. 5). In another southern hemisphere species, Mordacia mordax, the pancreatic islets are located at the oesophageal-intestinal junction (Fig. 5) and the bile duct enters at the cranial end of a single, left diverticulum (Potter 1986). In ammocoetes of all sizes and in all species so far examined (Morris and Islam 1969; Hilliard et al. 1985; Elliott and Youson 1986; Yui et al. 1988), histological sections of the epithelium of the alimentary canal at the oesophago-intestinal junction always reveal clumps of cells both in the basal epithelium and protruding into the surrounding submucosal layers where they are associated with the pancreatic islets (Fig. 4). According to Barrington (1972), there are dorsal and ventral zones of similar form and these zones represent sites where islets and/or follicles are released into the surrounding connective tissue to replace those that have degenerated. In Lampetra planeri it appears that new follicles or islets arise from mainly the oesophageal epithelium but may also orginate from pre-existing follicles located within the connective tissue underlying the epithelium (Morris and Islam 1969). Islet renewal is relatively slow. Mitotic figures and DNA synthesis, as revealed through autoradiography after 3 H-thymidine injection, are rarely encountered (Elliott 1989). That the intraepithelial and submucosal follicles are pancreatic endocrine tissues has been confirmed in L. planeri by immunofluorescence with a heterologous antiserum (Van Noorden et al. 1972). Immuno-histochemical and -cytochemical observations on larvae of P. marinus with antisera against bovine insulin (Elliott and Youson 1986, 1987, 1988), together with our observations using a homologous insulin antiserum (Figs. 3, 4) indicate that the intestinal epithelium at its junction with the oesophageal epithelium is the source of islets and follicles. This also seems to be the case for L. lamottenii (J.H. Youson, unpublished observations) and Lampetra japonica (Yui et al. 1988). As in early larvae, somatostatin immunoreactivity of the pancreaticintestinal system of older larvae is found only in the epithelium of the intestine. However, it may be noteworthy that immunostaining with antisomatostatin-34 and -14 antisera was substantially reduced
Fig. 1. Somatostatin-14 immunoreactivity (arrowhead) in the intestinal epithelium of a 79-day old larval P. marinus. P, pronephric duct. x 369. Fig. 2. Insulin immunoreactivity in intraepithelial (arrowhead) and submucosal (arrows) pancreatic islets of a 79-day old larval P. marinus at the junction of the intestine (I) and oesophagus (0). x 369. Fig. 3. Pancreatic islets (arrows) located at the junction of the bile duct (BD), oesophagus (0), and anterior intestine (I) of larval P. marinus are immunoreactive with antisera directed against lamprey insulin. Note the positively stained groups of cells within the intestinal epithelium (arrowheads). x 90.
393 or absent in larva P. marines kept in captivity for any time after 6 months.
Metamorphosis When larval lampreys undergo metamorphosis there is a dramatic change in the biliary tree of the liver involving the complete regression of the intrahepatic gallbladder, the bile canaliculi, the bile ductules, and the bile ducts (Sidon and Youson 1983). There is an almost complete loss of the intrahepatic common bile duct, but the fate of the epithelium in the extrahepatic common bile duct shows familial variability. In the southern hemisphere species, G. australis and M. mordax, the extrahepatic portion of the common bile duct completely regresses along with the intrahepatic portion (Hilliard et al. 1985; Potter 1986; Hilliard and Potter 1988). It was originally demonstrated by Keibel (1927) and Boenig (1928) that the extrahepatic common bile duct of L. planeri becomes an endocrine pancreas during metamorphosis. In a more recent study, immunohistochemistry was used to show that part of the intrahepatic portion and all of the extrahepatic common bile duct in P. marines transforms into a caudal endocrine pancreas (Elliott and Youson 1987). This transformation is accomplished through a dedifferentiation of bile duct cells and their redifferentiation into cells which eventually acquire immunoreactivity to antisera against either somatostatin or insulin (Elliott and Youson 1987; Elliott 1989; Youson and Elliott 1989). It is apparent that not all the small intrahepatic bile ducts degenerate to the same degree in every holarctic species and their epithelia do not undergo the same dedifferentiation and differentiation found in larger ducts (Youson et al. 1988). The result is that some species contain intrahepatic patches of islets (Youson et al. 1988; Youson and Elliott 1989). The integrity of bile ducts of all sizes at the time when pancreatic islet cell differentiation
occurs is undoubtedly a major factor in determining the final distribution of islets in the caudal pancreas. We have recently observed a situation in L. lamottenii which further illustrates the significance of the synchronization between bile duct transformation and endocrine cell differentiation with the eventual distribution of pancreatic tissue in adults of holarctic lampreys. In some individuals of L. lamottenii at late stage 7 or immediately postmetamorphic, there are still large and small intrahepatic bile ducts and many cells within their epithelia and in the surrounding connective tissue are immunoreactive to antiinsulin (Fig. 6). Other L. lamottenii at the same period of the life cycle showed no intrahepatic pancreatic islets. This intraspecific variation may be partially explained by the variable degree to which the larvae of this lamprey species were infected by nematodes prior to the beginning of metamorphosis, for they are a natural host to the nematode, Truttaedacnitis stelmioides. Larvae of this nematode reside in the bile ducts of ammocoetes of L. lamottenii and, during metamorphosis of the host, they migrate to the intestine where they will eventually reach sexual maturity (Pybus et al. 1978). Infected ammocoetes have more highly enlarged, and apparently many more, bile ducts and ductules than noninfected individuals (F. Eng and J.H. Youson, unpublished observations). It seems that during metamorphosis some of the larval nematodes become trapped in the bile ducts which respond by enlarging rather than regressing. The remaining bile ducts ultimately show immunoreactivity for the adult lamprey pancreatic hormones, insulin and somatostatin-34. We feel that this host-parasite relationship emphasizes that the dedifferentiation and redifferentiation processes of the bile duct epithelium during lamprey metamorphosis are highly synchronized. It also strongly suggests that deviation from a normal ontogenetic sequence results in phenotypic changes in members of the same population (i.e., alterations
Fig. 4. Higher magnification of a portion of a region of Figure 3 showing clumps of cells immunoreactive to insulin antiserum within the intestinal epithelium (arrowheads) and in the submucosa (arrow) of a larva. x 234.
in developmental canalization). The sensitivity of the events of bile duct transformation to external influences may explain the intraspecific variation in pancreatic distribution which has been noted in different populations of other brook lampreys (Youson and Elliott 1989; J.H. Youson and R.J. Beamish, unpublished observations). The fate of the larval pancreatic islets during metamorphosis of P. marinushas been recently followed using immunohistochemistry (Elliott and Youson 1987). These islets are present throughout metamorphosis and eventually form part of the cranial pancreas at the junction of the oesophagus and diverticulum of the anterior intestine on the left dorso-lateral surface of the pericardium. There is immunoreactivity to only antiinsulin through stages 1-5 of metamorphosis, but eventually the cells become immunoreactive to antisomatostatin14 antiserum. This immunoreactivity coincides with the appearance of the diverticulum and the new islets are added from the diverticular epithelium in the manner described during larval life.
Fig. 5. Diagrammatic presentation of the distribution of endocrine pancreatic tissue in the larva (a) and adult (b) of Geotria australis, the larva of Mordaciamordax (c) and the larva (d) and adult (e) of Petromyzon marinus. See the text for further description. Al, anterior intestine; BD, bile duct; CD, caudal pancreas; CR, cranial pancreas; D, diverticulum; I, intermediate pancreatic cord; L, liver; 0, oesophagus; P, pancreatic islets of larva; ID, left diverticulum; rD, right diverticulum.
During the initial phase of the juvenile period of P. marinus, the pancreatic endocrine tissue shows strong reactivity to both antisomatostatin and antiinsulin in the pancreatic tissue. Immunostaining of adjacent sections shows that in both the caudal and cranial pancreas the same cells react with antisera directed against either somatostatin-34 or somatostatin-14 (Fig. 7). This finding is in contrast to that viewed in some other fishes, where a "big somatostatin" and somatostatin-14 reside in different cells (McDonald et al. 1987). However, it is interesting that in the intestines of both juveniles and upstream-migrant adults of P. marinus there is a more extensive distribution of cells immunoreactive to somatostatin-34 compared to stomatostatin-14 (Fig. 8). With only the few exceptions noted above, the caudal pancreas of most holarctic species occupies the former position of the intra- and extrahepatic common bile duct, but there is no sign of bile duct remnants or any extensive mitosis and DNA synthesis (Elliott 1989). Moreover, since there
Fig. 6. Cells (arrowheads) within bile ducts (BD) and within the liver parenchyma (L) are immunoreactive to insulin antiserum in an immediately postmetamorphic adult of Lampetra lamottenii. x 234. Fig. 7. Adjacent sections of the caudal pancreas of a juvenile P. marinus showing immunoreactivity in the same cells to both antisomatostatin-14 (a) and -34 (b). x 189. Fig. 8. Adjacent sections of the anterior intestine of a juvenile P. marinus showing that immunoreactivity to antisomatostatin-14 (a) is less extensive than to antisomatostatin-34 (b). x 369. Fig. 9. The cranial pancreatic islets (P) and those in the intestinal diverticular epithelium (arrowhead) and the submucosa (arrow) of upstream migrant P. marinus are immunoreactive with antisomatostatin-34 and indicate renewal of the pancreatic islets still occuring late in the lamprey life cycle. x 234.
396 is no renewal of the islet population from the intestinal epithelium, as has been noted for ammocoetes, it appears that growth of the caudal pancreas takes place relatively slowly within the tissue mass. Despite the apparent similarity in development of the caudal pancreas in holarctic lampreys during their metamorphosis (Boenig 1928; Elliott and Youson 1987), juveniles of the Petromyzontidae show interspecific variation in the distribution of the caudal pancreatic tissue within the submucosal connective tissue connecting the intestinal typhlosole and the liver (Youson et al. 1988; Youson and Elliott 1989). Observations of juveniles of different populations of Lampetra richardsoni,indicate that intraspecific variation in the distribution of endocrine pancreatic tissue also occurs in at least some holarctic lampreys (Youson et al. 1988; J.H. Youson and R.J. Beamish, unpublished observations). When immunoreactivity to antisomatostatin is compared in the caudal pancreas of juveniles and upstream migrants of P. marinus, reactivity is greatly reduced during the latter period. It is suspected that in the absence of a system for renewal of the islets after metamorphosis, the reduced immunoreactivity in upstream migrants reflects the depletion of somatostatin during this period in the life cycle. There is also a stronger reaction to the antisomatostatin-14 antiserum than to antisomatostatin-34 during the upstream migration. A discontinuous, intermediate cord of pancreatic tissue containing insulin and somatostatin cells is present between the caudal and cranial masses in holarctic lampreys (Youson et al. 1988) but the development and renewal of this minor pancreatic portion has not been investigated. In P. marinus, the proliferation of pancreatic islets from the epithelium of the intestinal diverticulum continues in adult life and assures that there is a potential source for renewal (repopulation) of islets for the cranial pancreas. In G. australis, islets proliferation occurs at a single point from the end of the adult diverticulum. Immunohistochemistry in P. marinus shows insulin and somatostatin cells in clumps within the diverticular epithelium, in the submucosal connective tissue, and within the cranial pancreas proper throughout adult life, including
during the upstream migration (Fig. 9). This contrast in immunoreactivity between cranial and caudal pancreatic masses in the upstream migration is no doubt related to the continued production of islets in the former, and the fact that there is no source for islet renewal in the latter. It is of interest that this renewal of the cranial pancreatic islets is still possible during the upstream migration even though the intestinal epithelium is undergoing atrophy (Youson 1981). It may be noteworthy that it has been demonstrated in G. australis that the oesophageal artery, which is the main pathway of blood for the cranial islet organ, is still patent during the upstream migration (Hilliard et al. 1985). In addition, electron microscopic observations of the diverticulum and anterior intestine of upstream migrant P. marinus, has shown that enteroendocrine cells, some of which are a possible source of pancreatic islet cells, retain their complete integrity after the loss of absorptive and secretory (zymogen) cells (J.H. Youson, unpublished observations).
Acknowledgements Supported by the Natural Sciences and Engineering Research Council of Canada.
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