Immunocytochemical localization of pancreatic endocrine cells in frog embryos and young larvae

GENERAL

AND

COMPARATIVE

ENDOCRINOLOGY

lmmunocytochemical

Localization of Pancreatic Endocrine Frog Embryos and Young Larvae HUE-LEE

Department

45, 204-211 (1981)

of Anatomy,

University

CHENG

Cells in

KAUNG

of Minnesota,

Minneapolis,

Minnesota

5545.5

Accepted February 18, 1981 The pancreatic endocrine cells are localized immunocytochemically in the leopard frog, during early development. Insulin containing cells are first detectable at stage 22 when the embryo is about 8 mm long and the pancreatic Anlage is first formed. Glucagon and pancreatic polypeptide containing cells are first identified at stage 24, whereas somatostatin containing cells are not present until the animals reach larval stage II, shortly after animals start feeding. As is reported for the adult, glucagon and pancreatic polypeptide are present in the same cells during early development. These various endocrine cells are scattered in the pancreas during the early developmental periods. They are arranged into adult type of islets when animals are of stage V in age.

Rana pipiens,

The development of the endocrine pancreas has been studied extensively in mammals (Hard, 1944; Pictet and Rutter, 1972; Like and Orci, 1972) and birds (Przybylski, 1967; Dieterlen-Lievre and Beaupain, 1976; Andrew, 1977). However, little work has been done on the development of pancreatic endocrine cells in amphibians. In the early literature, Hirata (1934) and Janes (1938) described the presence of two or more endocrine cell types in the larval pancreases of several anurans. More recently, Frye ( 1964) demonstrated pseudoisocyanine staining beta cells in the pancreases of Rana clamitans larvae. No other cell type was mentioned in his work. Beaumont ( 1968)) using electron microscopy, showed that alpha and beta cells were present in the larval pancreases of Alytes, Discoglossus, and Pelobates and that delta cells did not appear until the end of metamorphosis. Using immunocytochemistry, Kaung and Elde (1980) demonstrated in the pancreas of adult Rana pipens the presence of insulin containing cells, somatostatin containing cells, and cells containing both glucagon and pancreatic polypeptide. In this paper immunocytochemical lo-

calization of various endocrine cell types in pancreases of Rana pipiens embryos and young larvae is presented. Observations are made on the initial appearances of various cell types, their morphology, and their topographical distribution during the early development of Rana pipens. MATERIALS

204 0016-6480/81/100204-08!$01.00/0 Copyright @ 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.

AND METHODS

larvae were obtained from eggs artificially ovulated and fertilized (Rugh, 1934). Animals were kept in aerated tap water at 20”. They were fed canned spinach. The staging of the embryos was done according to Shumway (1940) and that of the larvae, according to Tayler and Kollros (1946). For this study, two to three animals each of embryonic stages 20, 22, 24, 25, and larval stages I, II, III, and V were used. At stage 20, the embryo is hatched from jelly coat. At stage 22 the pancreatic Anlage first appears and the animal begins feeding at stage I. During early larval stages the development of the animal is characterized by increase in size of hindlimb buds. Bouin-fixed whole animals (stages 20 through II) or dissected pancreases (stages III and V) were prepared as 4-p serial paraffin sections. At intervals of every six sections, four consecutive sections were stained with the following four antisera by the peroxidase-antiperoxidase method of Sternberger (1974). The antisera used were rabbit antiserum to anglerfish insulin, guinea pig antiserum to synthetic somatostatin (both are gifts from Dr. R. Elde, University of Minnesota, Minneapolis, Minn.), rabbit antiserum to porcine Rana

pipiens

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present in the same cells because of the small amount of their cytoplasm. The pancreases of stages I and II larvae are much more differentiated (Figs. 4- 10). Exocrine cells are large and polygonal shaped whereas endocrine cells are much smaller OBSERVATIONS with oval or angular shapes. Pigment granules are still present in some cells, but Pancreatic Anlage can be first identified in Rana pipiens embryos at or shortly be- not always in endocrine cells. Insulin conpresent in fore stage 22 when the embryo is about 8 taining cells are predominantly mm in length. At this stage, the pancreas is clusters of two to five cells and contain intensely stained cytoplasm (Fig. 4). Pancrefilled with yolk platelets and pigment and glucagon containing granules; cell boundaries are indistinct and atic polypeptide only a few nuclei are recognizable in the cells appear as single cells with occasional section (Fig. 1). Immunocytochemical small clusters of two cells (Figs. 6 and 7). staining with the four antisera showed that Comparison of adjacent serial sections of only insulin immunoreactive cells can be stage II animals showed that many cells and found in a pancreas of this age (Fig. 1). This contain both pancreatic polypeptide immunocytochemical reactivity to insulin is glucagon (Figs. 6 and 7 arrows). Somatoabsorbable with anglerfish insulin. These statin containing cells are first present at cells are mostly spherical, oval, or angular stage II. These cells are small and are oval and contain little cytoplasm. They appear or spindle shaped (Fig. 5). They are only singly or in groups of three or four cells. found as single cells at this stage. When Many of the insulin containing cells also the adjacent serial sections stained with different antisera are compared (Figs. 8, 9, contain large numbers of pigment granules. and 10) one observes that as late as stage However, there is no definitive correlation III different cell types are scattered in the between the presence of insulin immunoreactivity and heavy pigment gran- pancreas and are not arranged into islets, ules. the cellular topography typical of the adult In stages 24 and 25 embryos, the cells and (Kaung and Elde, 1980). In the stage V animals, insulin containing their nuclei are much more well defined in the pancreas. Occasional yolk platelets are cells assume a more cuboidal or low columstill present. Pigment granules can be found nar shape and form larger clusters. They form the core of the islet with glucagonin many cells. Morphologically, endocrine pancreatic polypeptide containing and cells are not distinguishable from exocrine cells without immunocytochemical stain- somatostatin containing cells located at the ing. Immunostaining of the stage 24 em- periphery (Figs, 11, 12, and 13). This is the bryonic pancreas demonstrated that, in ad- typical islet cell arrangement found in the dition to insulin containing cells, glucagon adult pancreas (Kaung and Elde, 1980). and pancreatic polypeptide containing cells Single cells of any type are also present. are first identified (Figs. 2 and 3). These DISCUSSION cells, like insulin containing cells, are spherical, oval, or angular shaped and With immunocytochemistry, cells concontain scarce cytoplasm. They may ap- taining peptides similar to angler-fish insupear singly or in clusters of two or three lin, porcine glucagon, bovine pancreatic cells. At this stage it was difficult to ascer- polypeptide, and synthetic somatostatin are tain from adjacent serial sections whether identified in Rana pipiens embryonic and pancreatic polypeptide and glucagon are larval pancreases. As early as stage II it can

glucagon (gift from Dr. R. McEvoy, Mount Sinai School of Medicine, N.Y.), and rabbit antiserum to bovine pancreatic polypeptide (gift from Dr. R. Chance, Eli Lilly Corp., Indianapolis, Ind.). For specificity controls, antisera were absorbed with their own specific antigens or other antigens before staining.

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be demonstrated that the glucagon immunoreactivity and pancreatic polypeptide immunoreactivity are present in the same cells. The dual presence of these two hormones were previously reported for the adult frog pancreas (Kaung and Elde , 1980). It is very likely that the presence of these hormones in the same cells in this species occurs when glucagon and pancreatic polypeptide immunoreactivities are first identified at stage 24. Very recently bovine pancreatic polypeptide immunoreactivity and immunoreactivity of glicentin, a glucagon-like immunoreactant isolated from porcine intestine, are reported to be present in same cells in human rectal carcinoids (Fiocca et al., 1980) and in cat colon (Ravazzola and Orci, 1980). These observations shed light on the nature of the content of glucagonpancreatic polypeptide cells in Rana pipiens. They further support a close evolutionary relationship between these two hormones. As in the higher animals, the endocrine cells of Rana pipiens embryos appear concomitant with or shortly after the formation of pancreatic Anlage. In the Rana pipiens embryos, this happens at stage 22 when the embryo is about 8 mm in length. However, the order of appearance of the various endocrine cell types is different from what has been reported for mammals and birds. Electron microscopic studies showed that in human and rat (Like and Orci, 1972; Pictet and Rutter, 1972), alpha cells are the first endocrine cell type to appear during development, delta cells follow, and beta cells appear last. In the chicken embryos (Przybylski, 1967; Dieterlen Lievre and Beaupain, 1976; Andrew, 1977), alpha and beta cells appear at about the same time, with alpha cells appearing slightly earlier. Delta cells appear later than alpha and beta cells. In the present immunocytochemical study of frog embryonic and larval pancreases, the cells containing insulin immunoreactivity are first identified during ontogeny at stage 22, glucagon and pan-

KAUNG

creatic polypeptide immunoreactivities are present later at stage 24, and the somatostatin immunoreactivity is identified last at stage II. Although frequent section samples through whole pancreases of at least two animals were examined for each age group, it is possible that glucagon, pancreatic polypeptide, or somatostatin containing cells were present in stage 22 animals and were not observed. On the other hand, it is possible that the immunocytochemical method is sensitive enough to detect cellular immunoreactivities which may not be present in granule form. If this is the case, then the early presence of insulin in amphibians in the present investigation is consistent with the observation that insulin was found in rat pancreas very early by radioimmunoassay (Pictet and Rutter, 1972). Electron microscopic studies on these embryonic pancreases will further characterize the present immunohistochemical observations. Our observations, however, do indicate the order of frequency of the islet cell types in the early stages of the developing pancreas in this species. Preliminary quantitation of various cell types of stage II larval pancreas (unpublished) using a linear scanner developed by Carpenter and Lazarow (1962) confirms this observation. The quantitative data showed that insulin containing cells are the most predominant, glucagon-pancreatic polypeptide containing cells are the second most frequent cell type and the somatostatin containing cells are least frequent. It is interesting to note that the adult type of topographical organization of various islet cell types is not observed until the animal reaches stage IV or V (Figs. 11, 12, 13). At stage II or III the various islet cell types are still randomly distributed without an organized pattern or arrangement (Figs. 8, 9, 10). How the typical adult islet cell organization is achieved during development is unknown. With the exception of Frye’s study (1964) which showed that Rana tadpoles do not

FIG. 1. A section of pancreas of a stage 22 embryo. Note the abundance of yolk platelets and pigment granules. The section was stained with antiserum to anglerfish insulin. Arrows point to the insulin containing cells. Other dark-colored cells in the micrograph are cells containing pigment granules. Some insulin containing cells also contain pigment granules. x625. FIG. 2. A section of pancreas of a stage 24 embryo. Cells are more differentiated compared to those of stage 22 embryos. The section was stained with antiserum to porcine glucagon. Arrows point to the glucagon containing cells. Other dark-colored cells are cells containing pigment granules. x 625. FIG. 3. A section of a stage 24 pancreas stained with antiserum to bovine pancreatic polypeptide. Arrows show pancreatic polypeptide containing cells. Pigment granules are present in these pancreatic polypeptide containing cells as well as in other cells. x625. 207

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FIG. 4. A section of a stage II larval pancreas stained with antiserum to anglerfish insulin. Note the well-differentiated exocrine cells and the darkly stained insulin containing cells in small clusters (arrows). Pigment granules are still present in some cells including some insulin containing cells. x 625. FIG. 5. A section of a stage II larval pancreas, stained with antisomatostatin serum. Somatostatin containing cell (arrow) is always seen as single cell at this stage. x 625. FIGS. 6 AND 7. Two adjacent sections of the pancreas of a stage II animal stained with antiporcine glucagon serum (Fig. 6) and antibovine pancreatic polypeptide serum (Fig. 7). Note antiglucagon serum and antipancreatic polypeptide serum stain same cells (arrows). FIG. 6. Stage II pancreas stained with antiporcine glucagon serum. ~375. FIG. 7. Stage II pancreas stained with antibovine pancreatic polypeptide serum. Section is adjacent to the one shown in Fig. 6. x 375.

FIGS. 8- 10. Low-power view of consecutive sections of stage II pancreas illustrating the random distribution of insulin containing (Fig. 8), glucagon containing (Fig. 9), and somatostatin containing (Fig. 10) cells in the pancreas. No organization of these three cell types into islets is apparent. L, liver; S, stomach; I, intestine. FIG. 8. Section of pancreas of stage II larva stained with antianglerfish insulin serum. (Arrows point to insulin containing cells.) X 95. FIG. 9. Section of pancreas of stage II larva adjacent to the one shown in Fig. 8. Section was stained with antiporcine glucagon serum. (Arrows indicate glucagon containing cells.) x95. FIG. 10. Section of pancreas of stage II larva adjacent to that shown in Fig. 9 and stained with antisomatostatin serum. (Arrows indicates somatostatin containing cell.) x 95. 209

13 FIGS. 1 l- 13. Consecutive sections of portion of pancreas of a stage V animal. Sections in Figs. 11, 12, and 13 were stained with antisera to insulin, glucagon, and somatostatin, respectively. Note that the three cell types are arranged into an islet with insulin containing cells forming the center of the islet and glucagon and somatostatin containing cells surrounding the core of insulin containing cells. FIG. 11. Section of stage V larval pancreas stained with antianglerfish insulin serum. (Insulin containing cells are between arrows.) x 375. FIG. 12. Adjacent section of that shown in Fig. 11 stained with antiporcine glucagon serum. (Arrows point to glucagon containing cells.) X 375. FIG. 13. Adjacent section of that shown in Fig. 12 stained with antisomatostatin serum. (Arrows point to somatostatin containing cells.) x 375. 210

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regulate blood sugar until animal reaches stage V, there has been practically no work suggesting the functional significance of these various endocrine cells during the early stage of development of amphibians. Nor is there any indication whether the hormones are being secreted to the circulation. Biochemical and physiological studies are needed to understand the development of these cells in amphibians. ACKNOWLEDGMENTS The author thanks Drs. R. Elde, R. Chance and R. McEvoy for providing the antisera; Drs. D. Hamilton and A. Carpenter for reading the manuscript; Mr. B. Detloff for technical assistance. This research was supported by a grant from the American Diabetes Association, Inc., and Grant AM19868 from National Institutes of Health.

REFERENCES Andrew, A. (1977). Pancreatic D cells in very young chick embryos. Gen. Comp. Endocrinol. 31, 463-465. Beaumont, A. (1968). Les types cellulaires dan le pancreas endocrine des larves d’Amphibiens anoures. J. Microscop. 7, No. 4, 21a. Dieterlen-Lievre, F., and Beaupain, D. (1976). Immunocytological study of endocrine pancreas ontogeny in the chick embryo; normal development and pancreatic potentialities in the early splancknopleure. In “The Evolution of Pancreatic Islets”. (T. Grille, L. Leibson, and A. Epple, eds.), pp. 37-50. Pergamon, New York. Fiocca, R., Capella, C., Buffa, R., Fontana, R., Solcia, E., Hage, E., Chance, R. E., and Moody, A. J. ( 1980). Glucagon-, glicentin- and pancreatic polypeptide-like immunoreactivities in rectal carcinoids and related colorectal cells. Amer. J. Pathol.

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81-91.

Frye, B. E. (1964). Metamorphic changes in the blood sugar and the pancreatic islets of the frog, Rana clumitans. J. Exp. 2001. 155, 215-223.

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Hard, W. L. (1944). The origin and differentiation of the alpha and beta cells in the pancreatic islets of the rat. Amer. J. Anat. 75, 369-403. Hirata, K. (1934). On the histogenesis of the island of Langerhans in Rana juponica (Gunther). Science Reports of the Tohoku Imp. Univ. 4th Series 9, 159- 182. Janes, R. (1938). Studies on the amphibian digestive system III. The origin and development of pancreatic islands in certain species of aura. J. Morphol.

62, 375-391.

Kaung, H. C., and Elde, R. (1980). Distribution and morphometric quantitation of pancreatic endocrine cell types in the frog, Rana pipiens. Anat. Rec. 196, 173-181. Lazarow, A., and Carpenter, A. M. (1962). Component quantitation of tissue sections. I. Characterization of the instrument. J. Histochem. Cytochem. 10, 324- 328. Like, A., and Orci, L. (1972). Embryogenesis of the human pancreatic islets: A light and electron microscopic study. Diabetes 21, 511-534 (suppl.). Pictet R., and Rutter, W. J. (1972). Development of the embryonic endocrine pancreas. In “Endocrine Pancreas, Handbook of Physiology” Section 7, Vol. 1, (D. F. Steiner and N. Freinkel, eds.), pp. 25-66. Williams & Wilkins, Baltimore, Md. Przybylski, R. J. (1967). Cytodifferentiation of the chick pancreas I. Ultrastructure of the islet cells and the initiation of granule formation. Gen. Comp. Endocrinol. 8, 115- 128. Ravazzola, M., and Orci, L. (1980). A pancreatic polypeptide (PP)-like immunoreactant is present in the glicentin-containing cell of the cat intestine. Histochemistry 67, 221- 224. Rugh, R. (1934). Induced ovulation and artificial fertilization in the frog. Biol. Bull. 66, 22-24. Shumway, W. (1940). Stages in the normal development of Runa pipiens. Anat. Rec. 78, 139- 148. Sternberger, L. A. (1974). “Immunocytochemistry.” Prentice-Hall, Englewood Cliffs, N.J. Taylor, A. C., and Kollros, J. J. (1946). Stages in the normal development of Ranu pipiens larvae. Anat.

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94, 7-24.