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An Immunohistochemical and Morphometric Analysis of Insulin, Insulin-like Growth Factor I, Glucagon, Somatostatin, and PP in the Development of the Gastro-entero-pancreatic System of Xenopus laevis

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An Immunohistochemical and Morphometric Analysis of Insulin, Insulin-like Growth Factor I, Glucagon, Somatostatin, and PP in the Development of the Gastro-entero-pancreatic System of Xenopus laevis
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  An Immunohistochemical and Morphometric Analysisof Insulin, Insulin-like Growth Factor I, Glucagon,Somatostatin, and PP in the Developmentof the Gastro-entero-pancreatic System of   Xenopus laevis C. Maake,* W. Hanke,† and M. Reinecke* ,1 *  Institute of Anatomy, Division of Neuroendocrinology, University of Zu¨rich, Zu¨rich, Switzerland; and  †  Departmentof Zoology II, University of Karlsruhe, Karlsruhe, Federal Republic of Germany  Accepted January 5, 1998 The ontogeny of the classical islet hormones insulin(INS), glucagon (GLUC), somatostatin (SOM), and pan-creatic polypeptide (PP) as well as insulin-like growthfactor I (IGF-I) in the gastro-entero-pancreatic (GEP)system of   Xenopus laevis  (stages 41–66) was studiedusing double immunofluorescence and morphometricanalysis. As early as stage 41, clustered INS-immunoreac-tive (-IR) and isolated GLUC-IR cells occurred in thepancreas. The first SOM-IR cells appeared at stage 43,followed by PP-IR cells at stage 46. About 79%of the PPimmunoreactivity was confined to a subpopulation of theGLUC-IRcells. Both the GLUC/PP-IRcells and the PP-IRcells were located in a distinct area of the pancreas. Thefirst islets occurred in premetamorphosis (around stage50) and comprised mainly INS-IR and GLUC-IR cells.The majority of SOM-IR, PP-IR, and GLUC/PP-IR cellswas dispersed. The numbers of hormone cells remainedquite constant until the end of prometamorphosis (stage58). Around stages 60–62, the islets were partly disinte-grated and the numbers of islet cells slightly decreased.At stage 63, the cell number began to increase andreached the levels typical for the adult around stage 66.After metamorphic climax, the islets were reformed. Inthe gastrointestinal tract, transient INS-IRcells occurredprior to the adaptation of the gastrointestinal tract tofeeding (stages 41–44) and during metamorphosis whenthere is remodeling of the gastrointestinal tract (stages60–63). Therefore, INS released from the transientmucosal INS-IR cells may be involved in the temporaryproliferation of mucosal epithelial cells. The firstGLUC-IR and SOM-IR cells were seen at stage 41. PP-IRcells followed at stage 46. In contrast to the islets,GLUC-IRand PP-IRcells constituted different cell popu-lations. Around stage 46, the first IGF-I immunoreac-tions appeared in the GEP-system. In pancreas, IGF-Iimmunoreactivity was found in the GLUC/PP-IR cells(85–99%) but was absent from INS-IR, GLUC-IR, andSOM-IRcells. The IGF-I-IRgastro-entero-endocrine cells,however, seemed to contain none of the classical islethormones.   1998 Academic Press Development in anuran amphibians is associatedwith change in the larval herbivorous diet. To adaptthe organism to terrestrial life and the mainly carnivo-rous nutrition of the adult, numerous organs andtissues are modified during the complex process of ‘‘metamorphosis’’ (see Frieden and Just, 1970; DoddandDodd,1976).Theseprocesseswhichinvolveexten-sivecelldeathand/orcellproliferationanddifferentia-tion are certainly controlled by thyroid hormones and 1 To whom correspondence should be addressed at Institute of Anatomy, Division of Neuroendocrinology, University of Zu¨rich,Winterthurerstrasse 190, CH-8057 Zu¨rich, Switzerland. Fax: 0041 1635.57.02. E-mail: reinecke@anatomie.unizh.ch. General and Comparative Endocrinology  110,  182–195 (1998) Article No. GC987064 182 0016-6480/98 $25.00Copyright  1998 by Academic PressAll rights of reproduction in any form reserved.  various growth factors, e.g., prolactin and growthhormone (GH) (Kikuyama  et al.,  1993). Particularly, thedigestive tract is completely modified during thetransition from herbivorous to carnivorous diet.Morphological aspects of the larval, metamorphos-ing, and mature structures of the gastro-entero-pancreatic (GEP) system have been described for someanuran species. In  Xenopus laevis,  the dorsal anlage of the pancreas forms around stages 34–35 (Nieuwkoopand Faber, 1975), followed by the differentiation of thetwo ventral pancreatic anlagen around stages 37–38.The three rudiments fuse around stage 40 and themean absolute weight of the pancreas steadily in-creases, although its weight relative to total bodyweight decreases, as shown for  Rana pipiens  (Race  et al., 1966) and  R. catesbeiana  (Hulsebus and Farrar, 1985).During spontaneous and induced metamorphosis thepancreas regresses (Blatt  et al.,  1969; Bollin  et al.,  1973)so that pancreas weight decreases in absolute termsand relative to body weight (Race  et al.,  1966; Hulsebusand Farrar, 1985) diminishing to about 20% of itssrcinal size. Furthermore, around stages 59 to 61 in Xenopus  there are signs of pancreatic histiolysis andcell death (Frieden and Just, 1970; Nieuwkoop andFaber, 1975; Dodd and Dodd, 1976; Leone  et al.,  1976).The remaining rudiment regenerates and differentiatesinto the adult pancreas (Bollin  et al.,  1973; Nieuwkoopand Faber, 1975).However, remodeling of the larval GEP systemlikely involves tissue morphology and biochemicalchanges (Shi and Ishizuya-Oka, 1996). There is evi-dence for a reorganisation of hormone-producing cells(Leone  et al.,  1976). Because the gastric region, theintestinal wall, and the pancreas are the distributionsites of the GEP hormone system, especially of theclassicalislethormonesinsulin(INS),glucagon(GLUC),somatostatin (SOM), and pancreatic polypeptide (PP),these hormones may be especially involved.Insulin-like growth factor I (IGF-I) may play a role inthe development of   Xenopus  (De Pablo  et al.,  1990;Perfetti et al., 1994),asitislikelyinmammals(seeJonesand Clemmons, 1995) and birds (see De Pablo  et al., 1990, 1993; Reinecke and Collet, 1998). Most investiga-tions on the IGF system in  Xenopus  development havefocused on oocytes and early embryogenesis but littleis known about the role of IGF-I in postembryonicdevelopment (Perfetti  et al.,  1994; Reinecke and Collet,1998). Few studies have addressed IGF-I at the proteinlevel (Pancak-Roessler and Lee, 1990) or its cellularlocalization (Reinecke  et al.,  1995).Thus, a developmental investigation has been car-ried out on the GEP system of   X. laevis  from stage 41 tostage 66. The study using double immunofluorescenceaimed (1) to localize the classical islet hormones insulin,glucagon, somatostatin, and pancreatic polypeptide inthe developing and metamorphosing gastro-entero-pancreatic system; (2) to investigate the potentialcolocalisation of these hormones during developmentand metamorphosis; and (3) to identify the potentialIGF-I production sites in the gastrointestinal tract andpancreas during  Xenopus  development.Amorphomet-ric analysis was also performed. MATERIAL AND METHODS Specimens.  Tadpoles were staged according toNieuwkoop and Faber (1975). Table 1 gives the num- bers of the animals investigated. Fixation was carriedout by whole-body immersion in Bouin’s solutionwithout acetic acid for 3–5 h at room temperature(18°C). Adult frogs ( n  4) were decapitated, andpancreas and gastrointestinal tract were rapidly dis-sectedandfixedinthesamesolutionasabovefor3–5hat room temperature. Thereafter, all specimens weredehydrated in an ascending series of ethanol androutinely embedded in paraplast. Serial sections werecut at 4 µm and collected onto gelatin-coated slides.  Antibodies and peptides.  For the immunohisto-chemical detection of IGF-I, three different antisera(codes 116, 117, K37) raised in rabbits were used. Theantisera have been shown to be specific for IGF-I (Zapf  et al.,  1981; Hansson  et al.,  1988; Reinecke  et al.,  1992)and used in several previous phylogenetic studies(Reinecke  et al.,  1992, 1993a,b, 1995; Berwert  et al., 1995). For the detection of the classical islet hormonesthe following antibodies were used: guinea pig anti-porcine insulin (DAKO, Glostrup, Denmark), mouseanti-porcine glucagon (Sigma, Buchs, Switzerland),rabbit anti-porcine glucagon (DAKO), rat anti-somato-statin (Eugene Tech, Ridgefield Park, NJ), and rabbitanti-bovine pancreatic polypeptide (Bio-Science, Em-menbru¨cke, Switzerland). Criteria of specificity have Classical Islet Hormones and IGF-I in  Xenopus  183 Copyright  1998 by Academic PressAll rights of reproduction in any form reserved.   been described previously (Maake and Reinecke, 1993;Berwert  et al.,  1995). For the detection of the primaryantiserathefollowingsecondaryantibodieswereused: biotinylatedsheepanti-mouseIgG(Amersham,Zu¨rich,Switzerland), biotinylated goat anti-rat IgG (Bio-Science), and biotinylated goat anti-guinea pig IgG(Bio-Science). Visualisation was achieved with fluores-cein-isothiocyanate (FITC)-coupled swine anti-rabbitIgG (DAKO) and streptavidin Texas Red (Amersham).Preabsorption of the primary antisera was carried outusing recombinant human (h)IGF-I (Bachem, Buben-dorf, Switzerland), bovine (b)INS, bGLUC (Sigma),SOM (Sigma), and hPP (Peninsula, Heidelberg, Ger-many).  Immunohistochemical technique.  For the detec-tion and localisation of the classical islet hormones andIGF-I the double immunofluorescence technique wasused on serial sections. Deparaffinized and rehydratedsections were blocked with 0.1 M phosphate-bufferedsaline (PBS), pH 7.4, containing 2% bovine serumalbumin (BSA) and 2% normal goat serum (DAKO) for30 min at room temperature. For the investigation of the classical islet hormones two consecutive sectionswere incubated overnight at 4°C with guinea piganti-INS (1:8000) and rat anti-SOM (1:800), respec-tively. After buffer wash, biotinylated anti-guinea pigIgG (1:100) or biotinylated anti-rat IgG (1:100) wasapplied for 30 min at room temperature followed byvisualisation with streptavidin Texas Red (1:100) for 30min at room temperature. The same sections wereincubated with a second set of primary antibodiesovernight at 4°C, i.e., rabbit anti-GLUC (1:300) andrabbit anti-PP (1:600), respectively. Visualisation wasobtained with FITC-labeled anti-rabbit IgG (1:50) for30 min at room temperature. For the analysis of coexistence of the classical islet hormones and IGF-I,single sections were incubated with guinea pig anti-INS, mouse anti-GLUC (1:5000), or rat anti-SOM over-night at 4°C. Bound antibodies were visualised byincubation with biotinylated anti-species IgG followed by streptavidin Texas Red. The same sections wereincubated with one of the rabbit IGF-I antisera (116,1:300; 117, 1:150; K37, 1:100) overnight at 4°C followed by detection with FITC-conjugated anti-rabbit IgG. TodetermineapossiblelocalisationofIGF-Iimmunoreac-tivity in PP immunoreactive (-IR) cells, a comparisonwas made between consecutive sections incubatedwith one of the rabbit IGF-I antisera and with rabbitanti-PP, respectively, followed by visualisation withFITC-labeled anti-rabbit IgG.The specificity of the reactions obtained was testedusing the following controls: (1) Replacement of theprimary antiserum by nonimmune serum; (2) preab-sorption of the primary antiserum with hIGF-1, bINS, bGLUC, hPP, and SOM (0.4, 4, 40, 400 µg peptide/mldiluted antiserum), respectively; and (3) change of theorder of the double immunofluorescence method.As apositive control, sections of rat pancreas (Maake andReinecke, 1993) were processed in parallel.PhotomicrographsweretakenwithaZeissAxiophot(Zeiss, Zu¨rich, Switzerland). For photography, thefluorochromes were visualised with fluorescence mod-ules for FITC (BP 450–490 nm, FT 510, LP 515–565 nm)and for Texas Red (BP 546, FT 580, LP 590 nm).Examination of coexistence was mainly carried outusing a FITC/Texas Red module (BP 485/20 546/12,FT 500/560, LP 515–530/580–630). TABLE 1 Number of Animals Investigated from Different Stages (Days of Development of   Xenopus  by Rearing at 22°C and Feeding ad Libitum )Stage Day Number41 3.3 342 3.5 343 3.7 344 4 245 4.5 346 5 447 7 348 8 349 12 250 15 351 18 252 21 453 24 454 26 355 32 356 38 457 41 358 44 359 45 260 46 261 48 462 49 363 51 364 53 365 56 266 58 2 184  Maake, Hanke, and Reinecke Copyright  1998 by Academic PressAll rights of reproduction in any form reserved.  Determination of cell number.  The different typesof endocrine cells in  Xenopus  pancreas were countedapplying the following protocol. Sections of the entirepancreas were photographed at magnification   25.Prints were mounted and the number of INS-IR,GLUC-IR, GLUC/PP-IR, PP-IR, SOM-IR, and GLUC/PP/IGF-I-IR cells per section determined. For everystage, two to four individuals were used for theevaluation, and, from every individual, 7–10 sectionsof the pancreas were analysed. The distance betweenthe sections evaluated amounted to about 20 µm. Bythe use of a MOPVideoplan (Kontron, Zu¨rich, Switzer-land) the surface of the pancreatic sections was mea-sured. For every stage and hormone investigated thecell density was calculated as average number of hormone-IR cells per 0.1 mm 2 pancreatic tissue. In thegastrointestinal tract, a semiquantitative grading of the hormone-IR cells was carried out (Berwert  et al., 1995). RESULTS General  Reactions obtained by the use of the antisera againstINS, GLUC, SOM, PP, and IGF-I were extinguished bypreabsorption with the respective antigen but per-sisted after preabsorption with the other peptides.Analysis of consecutive sections showed that thedifferent rabbit IGF-I-antisera used most likely reactedwith identical cells. Thus, controls and reaction pat-ternsindicatedthespecificityofthereactionsobtained.The stages of appearance and the time course duringdevelopment of the different subpopulations of classi-cal islet hormone cells and their numbers per 0.1 mm 2 of pancreatic section surface are given in Fig. 1. Figure2depictstheratiooftotalGLUC-IR,GLUC/PP-IR,andGLUC/PP/IGF-I-IR cells throughout development.Similarly, the time course of appearance of INS-IR,GLUC-IR, SOM-IR, PP-IR, and IGF-I-IR cells and asemiquantitative grading of their frequencies in thegastrointestinal tract are shown in Table 2. The criticalphasesofpancreaticdevelopment,suchaspremetamor-phosis, metamorphic climax, and end of metamorpho-sis, are illustrated in Figs. 4–6.  Appearance of Classical Islet Hormonesin the GEP System Pancreas.  Already at stage 41, INS and GLUCimmunoreactivities were present in the pancreas. In agiven section about 2–4 INS-IR cells formed smallislet-like clusters (Fig. 3a). The GLUC-IR cells weredistributed throughout the exocrine parenchyma (Fig.3c) and showed no relation to the INS-IR cell clusters.No colocalisation of INS and GLUC immunoreactivi-ties was detectable.Around stage 43, the first SOM-IR cells occurred inpancreas.TheSOM-IRcellswereinfrequentandconsti-tuted a cell population different from the INS-IR andGLUC-IR cells.At this stage, two or three small INS-IRclusters per section containing 4–7 cells were distrib-uted throughout the pancreatic tissue, in addition tosome scattered INS-IR and GLUC-IR cells.Cells exhibiting PP immunoreactivity were not ob-served before stages 45–46. In contrast to the other islethormones, PP-IR cells showed a local distributionrestricted to only one part of the pancreas. About 79%of the PP-IR cells constituted a subpopulation of thehomogeneously occurring GLUC-IR cells (Figs. 5b and5d).TheremainingportionexhibitedonlyPPimmuno-reactivity. Similarly, about 25% of the GLUC-IR cellsshowed no colocalisation with PP immunoreactivity(Fig. 1). Around stage 46, multiple individual INS-IRcells appeared alongside an increasing number of small INS-IR islets.From stage 46, when all classical islet hormonesoccurred in pancreas, until at about stage 58 theaverage number of endocrine cells per 0.1 mm 2 pancre-atic surface remained consistently low (Fig. 1) withonly slight variations. Around stage 50 the first islets,which consisted mainly of numerous INS-IR and someGLUC-IR cells, were observed.With the onset of metamorphic climax, the islets, inpart, started to disintegrate. Around stages 60–62, thenumber of all endocrine cells per 0.1 mm 2 sectionsurface exhibited a slight decrease but started toincrease at around stage 63. This increase in celldensity continued until around stage 66 (Fig. 1).At stage 66, the pancreas showed large restoredINS-IR clusters. The INS-IR cell accumulations nowconsisted of 20–40 cells per sectioned islet (Fig. 6a). Inaddition, there were many separate INS-IR cells.GLUC-IR cells occurred in large numbers (Fig. 1). The Classical Islet Hormones and IGF-I in  Xenopus  185 Copyright  1998 by Academic PressAll rights of reproduction in any form reserved.  GLUC-IR cells, in part, formed aggregates consistingof 3–4 cells. Some of the GLUC-IR cell clusters layadjacent to the INS-IR islets. The GLUC/PP-IR andPP-IR cells were limited to an area of the pancreas(Figs. 6b and 6d) whereas the SOM-IR cells werescattered throughout the exocrine parenchyma (Fig.6c). The relative cell frequencies and distribution pat-terns of the INS-IR, GLUC-IR, GLUC/PP-IR, PP-IR,and SOM-IR cells of stage 66 froglets were comparableto those of adult  Xenopus. Gastrointestinaltract.  Inthegastrointestinaltract,throughout development there were fewer endocrinecells in more distal parts of the gut compared to theproximal portions and stomach. Only very few, if any,endocrine cells were present in the hindgut. All typesof endocrine cells occurred in the mucosal epithelium.Transient INS-IR cells appeared in stomach fromstages 41 to 44 (Fig. 3b). Furthermore, between stage 60and stage 63 single INS-IR cells transiently occurred instomach and foregut.GLUC-IR cells were present in the gastric (Figs. 3cand 8a) and intestinal mucosa throughout develop-ment. The number of the GLUC-IR cells fluctuatedfrom low numbers at stage 41 to a first maximum atstage 46, followed by a decrease around stage 49 and aslight increase to a second maximum at stage 59 (Table2). Thereafter, only infrequent GLUC-IR cells werefound in the gastrointestinal tract. FIG. 1.  Number of all subpopulations of classical islet hormone containing cells per 0.1 mm 2 pancreatic tissue in  Xenopus  during development. 186  Maake, Hanke, and Reinecke Copyright  1998 by Academic PressAll rights of reproduction in any form reserved.
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