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Further investigation into the source of pancreatic endocrine cell types
GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
er Investigation
Department
of Anatomy,
University
44,
279-287
(198i)
into the Sour-c Cell Types
of the
Witwatersrand
Accepted
Medical
September
School,
Johannesburg
200/,
Sczzth
.4frica
4, 1980
In a previous study it was shown that pancreatic endocrine cells present in 3%day chick embryos are not derived from trunk levels of neurectoderm but the types of islet cell present in the operated embryos were not distinguished. In order to do this, quail neural tcbe containing neural crest was isotopically and isochronically transplanted to trunk levels of chick hosts at 4- to 22-somite stages and the embryos were sacrificed at 9 days of incubation. A, B, and D cells were identified by electron microscopy and by immunocytochemistry: none of them contained the quail nuclear marker. It is concluded ?hat these cell types are not derived from the levels of trunk neurectoderm transplanted. No conclusion can be drawn concerning PP cells which were very few in number.
It has been proposed that all APUD cells (cells which take up and decarboxylate amine precursors) arise from the neural crest (Pearse, 1969) or neurectoderm (Pearse ert al., 1972). This idea has been uncritically accepted by many and is prevalent in the literature, although Pearse (1977) as come to believe evidence to the Some of this evidence concerns pancreatic endocrine cells. Pictet et at. (1976) have shown that B cells in rat pancreas are not derived from the neural crest. Andrew (1976a) demonstrated that cells present in the pancreas of %-day chick embryos and showing the DOPA-provoked formaldehyde-induced fluorescence charcells, were not derived Is of the neurectoderm. did not show which types of pancreatic endocrine cells were represented among the APUD cells in these very young embryos. It cannot be assumed that every type was present: conceivably cells of one type or more could be of crest origin d could arrive in the pancreas after 3% days of incubation; or the arrival of one e or more from the neural crest could ve been retarded in operated embryos. In order to draw a valid conclusion concerning the origin of pancreatic endocrine
cells, it was therefore ~rn~orta~~ to establish whether any type does in from trunk levels of the neu this pm-pose operate incubated for a longer perio creatic endocrine cell types an experiment is reported here. Evidence that pancreatic islet ceils in chick embryos do not originate at vagal (caudal ~~~dbra~~) levels of the ~~~~a~crest has been presented by taine et ak. (1977). ~~bseq~e~tly we that at least A and cells were certainly not from any levels of ~~~~~~ and probably not from m drew and Kramer, 1979). It thus only remained for us to amplify the earlie Andrew (1976a) concerning Frlonk the neurectoderm. (i) Microsurgical fechnique. The experimental design was based on Le Douarin’s (1971) discovery that quail cells have exceptionally large Feulgen-positive nucleoii which allow them to be easily distinguished from chick cells. The details of the operation were as described previously (Andrew, 197Ba; Andrew and Kramer, 1979). Lengths of neural tube incorporating neura? crest were transplanted isotopically from Japanese quail embryos IQ Black Australorp chick hosts at the same stage of development. Grafts were 3- to 7-somi:e 279 0016-6480/81/070279-09$01.0010 Corwight @ 1981 by Academic Press. lot. All rights of reproduc:ion m any form reserved.
280
KRAMER
AND
lengths of neural tube and crest at the levels of somites 4 to 22. The levels selected for transplantation at any particular stage were determined on the basis of previous work (Andrew, 1963). They were those at which the crests had not yet formed as such, or at which no migration from the crests proper had yet taken place. Where a graft extended into the postsomitic region, the future somite level of its caudal end was estimated. The tissue excised from the quails was treated in 0.1% trypsin (Gibco) in calcium- and magnesium-free chick Ringer’s solution for 15 min at 4” to assist in separation of the neural tube from the surrounding tissue. The grafts were then transplanted into prepared sites in chick embryos at 4- to 22-somite stages. Operated embryos were sacrificed at 9 days of incubation. (ii) Electron microscopy. Pancreata of five of the operated embryos were fixed in Karnovsky’s fixative (4% paraformaldehyde and 0.5% glutaraldehyde in Millonig’s phosphate buffer at pH 7.4) for 2 hr at 4”. They were postfixed in 1% osmium tetroxide for I hr at 4” and embedded in araldite. Each block was completely sectioned and all usable sections were stained with uranyl acetate and lead citrate. Pancreas from a normal quail embryo of 9 days’ incubation was similarly prepared for electron microsCOPY. (iii) Immunocytochemistry.
The pancreata of three of the operated embryos were fixed in Bouin’s fluid (which is suitable for immunocytochemistry). embedded in paraplast and sectioned serially at 5 pm. The serial sections were divided into short ribbons which were mounted on several series of six slides for each block. Alternate slides were stained with hematoxylin and eosin to enable identification of the nuclear marker. The Feulgen method was not used because Bouin’s fluid is an inappropriate fixative for this procedure. However, hematoxylin demonstrates quail nucleoli adequately (Fontaine et al., 1977). The intervening slides were stained by an indirect immunoenzyme technique to demonstrate glucagon (in A cells), insulin (in B cells), and somatostatin (in D cells). Rabbit antiserum to ovalbumin-conjugated pancreatic glucagon, used at 1:500, was a gift from Professor K. Buchanan (Queen’s University, Belfast); guinea pig antiserum to porcine insulin, used at 1:3000, was prepared by Dr. P. H. Wright (Indiana School of Medicine, Indianapolis) and donated by Professor L. Orci (Institut d’Histologie et Embryologic, Geneve); and rabbit antiserum to synthetic cyclic SRIF conjugated to human serum albumin, used at 1:250, was kindly provided by Professor M. P. Dubois (Institut National de la Recherche Agronomique, Nouzilly). The latter antiserum was routinely absorbed with glucagon (20 &ml) as otherwise it stains A cells when used at this dilution. All antisera were diluted with 0.005 i\/l Tris-saline containing 1% nonimmune swine serum.
ANDREW
Sections were dewaxed and reacted with 10% swine serum for 10 min before the incubation in the specific antiserum for 22 hr at 4”. During the latter incubation precautions were taken to prevent evaporation of the antiserum (Rawdon, 1978). The second antiserum, horseradish peroxidase-conjugated antiserum, either swine anti-rabbit IgG (Dakopatts) or rabbit anti-guinea pig IgG (Miles) was used at 1:20 for 30 min at room temperature. The horseradish peroxidase was located histochemically by treating sections with a solution of 3,3-diaminobenzidine tetrahydrochloride (DAB) containing hydrogen peroxide (Graham and Kamovsky, 1966). Control procedures consisted of replacement of the first and second antisera with diluent alone, substitution of normal rabbit serum for the first antiserum, and absorption of the antihormone serum with its antigen (glucagon: Lilly; porcine insulin: a gift from Lilly Research Laboratories; somatostatin: Sigma) at 20 Kg/ml diluted antiserum. (iv) Light microscopy. The grafted region of spinal cord and adherent tissue were excised from all operated embryos, fixed in 10% Formalin containing 2% calcium acetate, and embedded in paraplast. Serial 5-pm sections were stained with the Feulgen method for DNA (Pearse, 1960). Pancreas of a normal quail at the same age as the operated embryos was fixed in Bouin’s fixative and sections were stained with hematoxylin and eosin.
RESULTS
Of 22 operated embryos, 10 survived. As a rule the grafts appeared to have healed in position and developed well. The development of the pancreas appeared normal; however, sometimes the gland was a little small. The most important criterion for successful grafting was the appearance of numerous quail cells with large Feulgenpositive nucleoli in spinal ganglia (Fig. l), sympathetic ganglia, and neurilemmal (Schwann) cells of the chick hosts. This was evidence for the normal formation and migration of quail neural crest cells into host chick tissue. In this way 8 of the 10 grafted embryos were judged reliable for further analysis. The number of times each level of neural tube had been transplanted in successfully operated embryos is shown in Table 1. (I) Electron microscopy. A and D cells were identified in all five successfully operated embryos studied by electron micros-
ORIGIN
PANCREATIC
ENDOCRINE
281
CELLS
FIG. 1. Quail nucleoli in the spinal cord and an adjacent spinal ganglion in an operated embryo. Neural tube from a 12-somite Quail donor was transplanted to somite levels 14 to 21 of a L4-somite chick host. Formal acetate, Feulgen.
cells were identified in four. ion was based on the typical features described for islet cells in chick eterlen-Lievre (1963, 1965), , Macerollo (1977), and AnKramer (1979). A granules have round, electron-dense profiles with loosely fitting membranes (Fig. 2). The granules vary in size in embryonic tissue. B cells were plentiful but not as numerous as A ells, and are distinguished by electronense bars enclosed in spherical mem3). Numerous D cells were enThey have round granules which vary in electron density and have
were by no means as uumerous as the
that there are elongate as well as round granule profiles; electrou density are somew than granules (Andrew au 1980). No cells ide~t~~ab~e electro ically as A, , or PI? showed the iias nucleolus of graph of au islet from au operated ern-
TABLE SUCCESSFCLTRANSPLANTATIOF~
OFNEURAL
1
TUBE
ANDNEURALCRESTATVAR!OUS~,EVE~S
Somite level
No. of times transplanted
Somite level
No. of times transplanted
Somite ievel
4 5 6 7 8 9 10
I 1 1 1 1 2 3
I1 32 13 14 15 16
5 5 6 6 5 4
17 18 19 20 2: 22
5C times transplanted
No.
3 2 1 1 I 1
282
KRAMER
FIG.
received FIG.
received FIG.
received FIG.
embryo 9-somite
AND
ANDREW
2. Electron micrograph of a pancreatic A cell with a chick nucleus, in a chick embryo which a graft of quail neural tube and crest at the levels of somites 6 to 8 at the 6-somite stage. 3. Electron micrograph of a pancreatic B cell with a chick nucleus, in a chick embryo which a graft of quail neural tube and crest at the levels of somites 6 to 8 at the 6-somite stage. 4. Electron micrograph of a pancreatic D cell with a chick nucleus, in a chick embryo which a graft of quail neural tube and crest at the levels of somites 11 to 15 at the 1 I-somite stage. 5. Electron micrograph of a pancreatic polypeptide (PP) cell with a chick nucleus, in a chick which received a graft of quail neural tube and crest at the levels of somites 10 to 14 at the stage.
ORIGIN
PANCREATIC
ENDOCRINE
CELLS
283
FIG. 6. Electron micrograph of an A islet in a 9-day operated embryo shows the lack of quaii nucleoli. The chick host received a graft of quail neural tube and crest at the levels of somites 1 I to !‘7 at the 9-somite stage. FIG. 7. Electron micrograph of an islet from a normal quail embryo of9 days of incubaticn. Note the very large distinctive quail nucleoli. Compare with the chick nuclei in Fig. 6.
ryo an iliustrating the absence of quail nucleoli (Fig. 6) may be compared with one of an islet from normal quail pancreas showing typical large quail nucleoli (Fig. 7). (2) Immunocytochemistry. Glucagonand somatostatin-immunoreactive (A and D) cells were identified in the pancreata of all three successfully operated embryos studied by immunocytochemistry. Insulinimmunoreactive (B) cells were identified in two of these embryos. (It is possible that part 0 e pancreas was not retrieved from the th embryo.) Somatostatin-immunoreactive cells occurred peripherally in islets (Fig. 8) and scattered in the exocrine parenchyma. ~~s~Ii~-imm~noreactive cells made up (Fig. 10). Glucagonall (B) islets
immunoreactive cells were mainly confine to larger (A) islets; occasional cells were found in the exocrine ~are~cbyma~ A cent sections stained with he
quail cells (see Figs. 9, 1 comparison, an islet fr embryo, showing typi
satisfactory
results.
FIG. 8. An immunocytochemical preparation demonstrating the presence of somatostatin in pancreatic endocrine cells of a chick embryo which had received a graft of quail neural tube and crest at the levels of somites 10 to 16 at the IO-somite stage. FIG. 9. A section adjoining that shown in Fig. 8 demonstrating the lack of quail nucleoli in the somatostatin cells. Bouin; hematoxylin and eosin. FIG. 10. An immunocytochemical preparation demonstrating the presence of insulin in pancreatic endocrine cells of a chick embryo which had received a graft of quail neural tube and crest at the levels of somites 14 to 21 at the 12-somite stage. FIG. 11. A section adjoining that shown in Fig. 10. The lack of quail nucleoli is evident in the insulin cells. Bouin; hematoxylin and eosin. 284
ORIGIN
PANCREATIC
ENDOCRINE
CELLS
preparation demonstrating the presence of glucagon in pancreatic FIG. 12. An immunocytochemical endocrine cells of a chick embryo which had received a graft of quail neural tube and crest at the levels of somites 10 to 16 at the IO-somite stage. FIG. 13. A section adjoining that shown in Fig. 12 demonstrating the lack of quail nucleoli in the glucagon cells. Bouin: hematoxylin and eosin.
FIG. 14. Islet tissue from a normal 9-day quail embryo showing the evident large nucleoli typical of quail cells. Bouin: hematoxylin and eosin.
286
KRAMER
AND
microscopy and by immunocytochemistry corresponded to the situation in normal chick embryos at the same stage of development (see Andrew, 1976b). DISCUSSION
In this experiment it was vital that migration of neural crest cells from the host neural tube should not have occurred in the operated region before grafting of the donor tissue took place. With this in mind, the criteria for the selection of levels for transplantation were strictly adhered to and have been shown to be sound (Andrew, 1963). Likewise it is important to be sure that quail neural crest cells migrate normally in chick tissue, This was confirmed by the presence of large numbers of quail cells in the spinal ganglia, sympathetic ganglia, and neurilemmal (Schwann) cells of the chick hosts. It has been repeatedly shown by Le Douarin and her co-workers that isotopic quail to chick grafts are prolific in the formation of migratory neural crest cells (Le Douarin and Teillet, 1970, 1973, 1974; Le Lievre and Le Douarin, 1975). The most likely level of origin of pancreatic islet cells was that at which the duodenum develops, i.e., the level of somites 8 to 15 (Le Douarin, 1961). The number of times neural tube containing neural crest at each of these somite levels was transplanted and the number of successfully operated embryos justify the conclusions drawn. A, B, D, and PP cells were identified by electron microscopy in the operated chicks. Confirmation of the presence of A, B, and D cells was given by the immunocytochemical analysis. PP cells were sought immunocytochemically but not found, no doubt due to the fact that only few are present at 9 days of incubation, the earliest stage at which Alumets et al. (1978) have identified PP cells in the pancreas of chick embryos. For this reason no definite conclusion was drawn concerning PP cells. The complete absence of the quail nuclear marker from A, B, and D cells indi-
ANDREW
cated their origin from a source other than the neurectoderm of the most likely trunk levels. It is therefore now possible to conclude that these specific pancreatic endocrine cell types are not derived from these trunk levels of the neurectoderm. With the proposal that neural crest cells might arise from a ventral neural ridge (Takor Takor and Pearse, 1975) the possibility arose that this might be a source of pancreatic endocrine cells. However, we could find no evidence for a ventral neural ridge or related neural crest cells (Levy ef al., 1980). Taking the present results together with others (Andrew, 1976a; Fontaine et al., 1977; Andrew and Kramer, 1979), it has thus been shown that pancreatic (APUD) cells are not derived from rhombencephalic, probably not from mesencephalic, and not from trunk levels of the neurectoderm. The specific pancreatic endocrine cell types embraced by this conclusion are at least A and B cells for cephalic levels and A, B, and D cells for the most likely trunk levels. After combining mesoderm and endoderm from early chick and quail embryos, we have observed that, in the few grafts in which pancreas differentiated, the nuclear features of A and B cells were in accordance with an endodermal origin (unpublished results and see Andrew, 1978). The most likely source of these pancreatic endocrine cells therefore remains the endoderm. ACKNOWLEDGMENTS The authors would like to thank the Medical Research Council of South Africa and the Council and Senate Research Committees of the University of the Witwatersrand, Johannesburg, for supporting this study. We are grateful to Mr. W. Tadiello, Mrs. G. Holmes, and Mrs. J. Layzell for skilled technical assistance. Our thanks are also due to Professor P. V. Tobias, Head of the Department of Anatomy, for his continued interest and encouragement.
REFERENCES Alumets, J., H&kanson, R., and Sundler, Distribution, ontogeny and ultrastructure
F. (1978). of pan-
ORIGIN
PANCREATIC
creatic polypeptide (PP) cells in the pancreas and gut of the chicken. Cell Tissue Res. 194,377-386. Andrew, A. (1963). A study of the developmental relationship between enterochromaftin cells and the neural crest. J. Embryo/. Exp. Morphol. 11, 307-324. Andrew, A. (1976a). An experimental investigation into the possible neural crest origin of pancreatic APUD (islet) cells. J. Embryo/. Exp. Morphol. 35, 517-593. Andrew, A. (1976b). Pancreatic APUD cells in older chick embryos with special reference to their identity. Histochemistry 49, 213-219. Andrew, A. (1978). The embryonic origin of pancreatic islet (APUD) cells in the chick. In “Molecular Endocrinology” (I. MacIntyre and M. Szelke, eds.), pp. 355-359. ElsevieriNorth-Holland, Amsterdam/New York. Andrew, A., and Kramer, B. (1979). An experimental investigation into the possible origin of pancreatic islet cells from rhombencephalic neurectoderm. J. Embryo!.
Exp.
Morphol.
52, 23-38.
Andrew, A., and Rawdon, B. B. (1980). The ultrastructnral identity of pancreatic polypeptide sells in chicks. Histochemistry 65, 261-267. Dieterlen-Lievre, F. (1963). Demonstration de l’activite precoce des cellules A du pancreas chez l’embryon de poulet. C.R. Seance. Acad. Sci. Paris
256,
1597-
1599.
Dieterlen-Lievre. F. (1965). Etude morphologique et experimentale de la differentiation du pancreas chez l’embryon de poulet. Bull. Biol. France Belg. 99,3-116. Fontaine. J.. Le Lievre, C., and Le Douarin. N. M. (1977). What is the developmental fate of the neural crest cells which migrate into the pancreas in the avian efnbrya? Gen. Comp. Endocrinol. 33, 394-404.
Graham. R. C.. and Karnovsky. M. J. (1966). The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem. 14, 291-302. Le Douarin, N. M. (1961). Radiodestructions partielies chez l’embryon de poulet aux stades jeunes en localisation des ebauches digestives. J. Embvol. E.xp.
Morphoi.
9, 1-8.
Le Douarin, N. M. (1971). Caracteristiques ultrastructurales du noyau interphasique chez la caille et chez ie poulet et utilisation de cellules de caille comme ‘marqueurs biologiques’ en embryologie experimentale. Ann. Embryol. Morphol.
4, 125-135.
Le Douarin, N. M., and Teillet, M.-A. (1970). Sur quelques aspects de la migration des cellules neurales chez l’embryon de poulet ttudiee par la
ENDOCRINE
CELLS
284
methode des greffes heterospeci~q~es de tube nerveux. C.R. Senilce. Sot. Biol. 164, 390-397. Le Douarin, N. M., and Teillet, M.-A. (3973). The migration of neural crest celis to the wall of the digestive tract in avian embryos. J. Embryo!. E,xl;. Morphol.
30, 31-48.
Le Douarin, N. M., and Teillet, M.-A. (1974). Experimental analysis of the migration and differentiation of neuroblasts of the autonomic nervous system and of neurectodermal mesenchymal derivatives. using a biological cell marking recbnique. Develop. Morphol. 41, 162-184. Le L!evre, C. S.. and Le Douarin, N. M. (1975). Mesenchymal derivatives of the neural crest: Analysis of chimaeric quail and chick embryos. J. Embryol.
Exp.
Morphol.
34, 125-154.
Levy, N.. Andrew, A., Rawdon, B. E., and Kramer, B. (1980). Is there a ventral neural ridge? Implications for the origin ofadenohypophyseal and other APUD cells. J. Embryo/. Exp. Morpho!~ 57, 71-7a.
Maceroho, P. (1977). Pancreatic islet cells in very young chick embryos with special reference to D cells. Gen. Comp. Endocrinol. 33. 139-146. Machino, M. (1966). Electron microscopic observations of pancreatic islet cells of the early chick embryo. Nailcre (London) 210, 853-854. Pearse, A. 6. E. (1960). “Histochemistry. Theoretical and Applied,” 2nd ed., p. 822. Churchill, London. Pearse, A. G. E. (1969). The cytochemistry and ultrastructure of po~ypeptide-hormo~~e producing cells of the APUD series and the embryologic, physiologic and pathologic implications of the concept. J. Histochem. Cytochem. 6’7, 303--313. Pearse, A. 6. E. (1977). The APUD concept and irs implications: Related endocrine peptides in brain, intestine. pitaitary, placenta and anuran cutaneous glands. Med. Biol. 55, 115-125. Pearse, A. G. E.. Polak. .I. M., and Bussoiatl, 6, (1972). The neural crest origin of gastro-intestinal and pancreatic endocrine polypeptide cells and their distinction by sequential immunoflaorescence. Folia Hisiochem. Cytochem. Krako,!, 10, 115-120. Pictet, R. L., Rail, L. B., Phelps. P., and Rutter, (1976). The neural crest and the origin of the insulin-producing and other gastrointestinal hormone-producing cells. Science 191, Y9i -i93. Rawdon, B. B. (1978). Preventing evaporation of antiserum during indirect immunocyiochemical staining. Stain Technol. 53, 289-290. Takor Takor, T., and Pearse, A. 6. E. ;1915). Neuroectodermal origin of avian hypothalamobypopbyseal complex: The role of the ventral neural ridge. J. Embryol. Exp. Morphol. 34, 311-325.
AND
COMPARATIVE
ENDOCRINOLOGY
er Investigation
Department
of Anatomy,
University
44,
279-287
(198i)
into the Sour-c Cell Types
of the
Witwatersrand
Accepted
Medical
September
School,
Johannesburg
200/,
Sczzth
.4frica
4, 1980
In a previous study it was shown that pancreatic endocrine cells present in 3%day chick embryos are not derived from trunk levels of neurectoderm but the types of islet cell present in the operated embryos were not distinguished. In order to do this, quail neural tcbe containing neural crest was isotopically and isochronically transplanted to trunk levels of chick hosts at 4- to 22-somite stages and the embryos were sacrificed at 9 days of incubation. A, B, and D cells were identified by electron microscopy and by immunocytochemistry: none of them contained the quail nuclear marker. It is concluded ?hat these cell types are not derived from the levels of trunk neurectoderm transplanted. No conclusion can be drawn concerning PP cells which were very few in number.
It has been proposed that all APUD cells (cells which take up and decarboxylate amine precursors) arise from the neural crest (Pearse, 1969) or neurectoderm (Pearse ert al., 1972). This idea has been uncritically accepted by many and is prevalent in the literature, although Pearse (1977) as come to believe evidence to the Some of this evidence concerns pancreatic endocrine cells. Pictet et at. (1976) have shown that B cells in rat pancreas are not derived from the neural crest. Andrew (1976a) demonstrated that cells present in the pancreas of %-day chick embryos and showing the DOPA-provoked formaldehyde-induced fluorescence charcells, were not derived Is of the neurectoderm. did not show which types of pancreatic endocrine cells were represented among the APUD cells in these very young embryos. It cannot be assumed that every type was present: conceivably cells of one type or more could be of crest origin d could arrive in the pancreas after 3% days of incubation; or the arrival of one e or more from the neural crest could ve been retarded in operated embryos. In order to draw a valid conclusion concerning the origin of pancreatic endocrine
cells, it was therefore ~rn~orta~~ to establish whether any type does in from trunk levels of the neu this pm-pose operate incubated for a longer perio creatic endocrine cell types an experiment is reported here. Evidence that pancreatic islet ceils in chick embryos do not originate at vagal (caudal ~~~dbra~~) levels of the ~~~~a~crest has been presented by taine et ak. (1977). ~~bseq~e~tly we that at least A and cells were certainly not from any levels of ~~~~~~ and probably not from m drew and Kramer, 1979). It thus only remained for us to amplify the earlie Andrew (1976a) concerning Frlonk the neurectoderm. (i) Microsurgical fechnique. The experimental design was based on Le Douarin’s (1971) discovery that quail cells have exceptionally large Feulgen-positive nucleoii which allow them to be easily distinguished from chick cells. The details of the operation were as described previously (Andrew, 197Ba; Andrew and Kramer, 1979). Lengths of neural tube incorporating neura? crest were transplanted isotopically from Japanese quail embryos IQ Black Australorp chick hosts at the same stage of development. Grafts were 3- to 7-somi:e 279 0016-6480/81/070279-09$01.0010 Corwight @ 1981 by Academic Press. lot. All rights of reproduc:ion m any form reserved.
280
KRAMER
AND
lengths of neural tube and crest at the levels of somites 4 to 22. The levels selected for transplantation at any particular stage were determined on the basis of previous work (Andrew, 1963). They were those at which the crests had not yet formed as such, or at which no migration from the crests proper had yet taken place. Where a graft extended into the postsomitic region, the future somite level of its caudal end was estimated. The tissue excised from the quails was treated in 0.1% trypsin (Gibco) in calcium- and magnesium-free chick Ringer’s solution for 15 min at 4” to assist in separation of the neural tube from the surrounding tissue. The grafts were then transplanted into prepared sites in chick embryos at 4- to 22-somite stages. Operated embryos were sacrificed at 9 days of incubation. (ii) Electron microscopy. Pancreata of five of the operated embryos were fixed in Karnovsky’s fixative (4% paraformaldehyde and 0.5% glutaraldehyde in Millonig’s phosphate buffer at pH 7.4) for 2 hr at 4”. They were postfixed in 1% osmium tetroxide for I hr at 4” and embedded in araldite. Each block was completely sectioned and all usable sections were stained with uranyl acetate and lead citrate. Pancreas from a normal quail embryo of 9 days’ incubation was similarly prepared for electron microsCOPY. (iii) Immunocytochemistry.
The pancreata of three of the operated embryos were fixed in Bouin’s fluid (which is suitable for immunocytochemistry). embedded in paraplast and sectioned serially at 5 pm. The serial sections were divided into short ribbons which were mounted on several series of six slides for each block. Alternate slides were stained with hematoxylin and eosin to enable identification of the nuclear marker. The Feulgen method was not used because Bouin’s fluid is an inappropriate fixative for this procedure. However, hematoxylin demonstrates quail nucleoli adequately (Fontaine et al., 1977). The intervening slides were stained by an indirect immunoenzyme technique to demonstrate glucagon (in A cells), insulin (in B cells), and somatostatin (in D cells). Rabbit antiserum to ovalbumin-conjugated pancreatic glucagon, used at 1:500, was a gift from Professor K. Buchanan (Queen’s University, Belfast); guinea pig antiserum to porcine insulin, used at 1:3000, was prepared by Dr. P. H. Wright (Indiana School of Medicine, Indianapolis) and donated by Professor L. Orci (Institut d’Histologie et Embryologic, Geneve); and rabbit antiserum to synthetic cyclic SRIF conjugated to human serum albumin, used at 1:250, was kindly provided by Professor M. P. Dubois (Institut National de la Recherche Agronomique, Nouzilly). The latter antiserum was routinely absorbed with glucagon (20 &ml) as otherwise it stains A cells when used at this dilution. All antisera were diluted with 0.005 i\/l Tris-saline containing 1% nonimmune swine serum.
ANDREW
Sections were dewaxed and reacted with 10% swine serum for 10 min before the incubation in the specific antiserum for 22 hr at 4”. During the latter incubation precautions were taken to prevent evaporation of the antiserum (Rawdon, 1978). The second antiserum, horseradish peroxidase-conjugated antiserum, either swine anti-rabbit IgG (Dakopatts) or rabbit anti-guinea pig IgG (Miles) was used at 1:20 for 30 min at room temperature. The horseradish peroxidase was located histochemically by treating sections with a solution of 3,3-diaminobenzidine tetrahydrochloride (DAB) containing hydrogen peroxide (Graham and Kamovsky, 1966). Control procedures consisted of replacement of the first and second antisera with diluent alone, substitution of normal rabbit serum for the first antiserum, and absorption of the antihormone serum with its antigen (glucagon: Lilly; porcine insulin: a gift from Lilly Research Laboratories; somatostatin: Sigma) at 20 Kg/ml diluted antiserum. (iv) Light microscopy. The grafted region of spinal cord and adherent tissue were excised from all operated embryos, fixed in 10% Formalin containing 2% calcium acetate, and embedded in paraplast. Serial 5-pm sections were stained with the Feulgen method for DNA (Pearse, 1960). Pancreas of a normal quail at the same age as the operated embryos was fixed in Bouin’s fixative and sections were stained with hematoxylin and eosin.
RESULTS
Of 22 operated embryos, 10 survived. As a rule the grafts appeared to have healed in position and developed well. The development of the pancreas appeared normal; however, sometimes the gland was a little small. The most important criterion for successful grafting was the appearance of numerous quail cells with large Feulgenpositive nucleoli in spinal ganglia (Fig. l), sympathetic ganglia, and neurilemmal (Schwann) cells of the chick hosts. This was evidence for the normal formation and migration of quail neural crest cells into host chick tissue. In this way 8 of the 10 grafted embryos were judged reliable for further analysis. The number of times each level of neural tube had been transplanted in successfully operated embryos is shown in Table 1. (I) Electron microscopy. A and D cells were identified in all five successfully operated embryos studied by electron micros-
ORIGIN
PANCREATIC
ENDOCRINE
281
CELLS
FIG. 1. Quail nucleoli in the spinal cord and an adjacent spinal ganglion in an operated embryo. Neural tube from a 12-somite Quail donor was transplanted to somite levels 14 to 21 of a L4-somite chick host. Formal acetate, Feulgen.
cells were identified in four. ion was based on the typical features described for islet cells in chick eterlen-Lievre (1963, 1965), , Macerollo (1977), and AnKramer (1979). A granules have round, electron-dense profiles with loosely fitting membranes (Fig. 2). The granules vary in size in embryonic tissue. B cells were plentiful but not as numerous as A ells, and are distinguished by electronense bars enclosed in spherical mem3). Numerous D cells were enThey have round granules which vary in electron density and have
were by no means as uumerous as the
that there are elongate as well as round granule profiles; electrou density are somew than granules (Andrew au 1980). No cells ide~t~~ab~e electro ically as A, , or PI? showed the iias nucleolus of graph of au islet from au operated ern-
TABLE SUCCESSFCLTRANSPLANTATIOF~
OFNEURAL
1
TUBE
ANDNEURALCRESTATVAR!OUS~,EVE~S
Somite level
No. of times transplanted
Somite level
No. of times transplanted
Somite ievel
4 5 6 7 8 9 10
I 1 1 1 1 2 3
I1 32 13 14 15 16
5 5 6 6 5 4
17 18 19 20 2: 22
5C times transplanted
No.
3 2 1 1 I 1
282
KRAMER
FIG.
received FIG.
received FIG.
received FIG.
embryo 9-somite
AND
ANDREW
2. Electron micrograph of a pancreatic A cell with a chick nucleus, in a chick embryo which a graft of quail neural tube and crest at the levels of somites 6 to 8 at the 6-somite stage. 3. Electron micrograph of a pancreatic B cell with a chick nucleus, in a chick embryo which a graft of quail neural tube and crest at the levels of somites 6 to 8 at the 6-somite stage. 4. Electron micrograph of a pancreatic D cell with a chick nucleus, in a chick embryo which a graft of quail neural tube and crest at the levels of somites 11 to 15 at the 1 I-somite stage. 5. Electron micrograph of a pancreatic polypeptide (PP) cell with a chick nucleus, in a chick which received a graft of quail neural tube and crest at the levels of somites 10 to 14 at the stage.
ORIGIN
PANCREATIC
ENDOCRINE
CELLS
283
FIG. 6. Electron micrograph of an A islet in a 9-day operated embryo shows the lack of quaii nucleoli. The chick host received a graft of quail neural tube and crest at the levels of somites 1 I to !‘7 at the 9-somite stage. FIG. 7. Electron micrograph of an islet from a normal quail embryo of9 days of incubaticn. Note the very large distinctive quail nucleoli. Compare with the chick nuclei in Fig. 6.
ryo an iliustrating the absence of quail nucleoli (Fig. 6) may be compared with one of an islet from normal quail pancreas showing typical large quail nucleoli (Fig. 7). (2) Immunocytochemistry. Glucagonand somatostatin-immunoreactive (A and D) cells were identified in the pancreata of all three successfully operated embryos studied by immunocytochemistry. Insulinimmunoreactive (B) cells were identified in two of these embryos. (It is possible that part 0 e pancreas was not retrieved from the th embryo.) Somatostatin-immunoreactive cells occurred peripherally in islets (Fig. 8) and scattered in the exocrine parenchyma. ~~s~Ii~-imm~noreactive cells made up (Fig. 10). Glucagonall (B) islets
immunoreactive cells were mainly confine to larger (A) islets; occasional cells were found in the exocrine ~are~cbyma~ A cent sections stained with he
quail cells (see Figs. 9, 1 comparison, an islet fr embryo, showing typi
satisfactory
results.
FIG. 8. An immunocytochemical preparation demonstrating the presence of somatostatin in pancreatic endocrine cells of a chick embryo which had received a graft of quail neural tube and crest at the levels of somites 10 to 16 at the IO-somite stage. FIG. 9. A section adjoining that shown in Fig. 8 demonstrating the lack of quail nucleoli in the somatostatin cells. Bouin; hematoxylin and eosin. FIG. 10. An immunocytochemical preparation demonstrating the presence of insulin in pancreatic endocrine cells of a chick embryo which had received a graft of quail neural tube and crest at the levels of somites 14 to 21 at the 12-somite stage. FIG. 11. A section adjoining that shown in Fig. 10. The lack of quail nucleoli is evident in the insulin cells. Bouin; hematoxylin and eosin. 284
ORIGIN
PANCREATIC
ENDOCRINE
CELLS
preparation demonstrating the presence of glucagon in pancreatic FIG. 12. An immunocytochemical endocrine cells of a chick embryo which had received a graft of quail neural tube and crest at the levels of somites 10 to 16 at the IO-somite stage. FIG. 13. A section adjoining that shown in Fig. 12 demonstrating the lack of quail nucleoli in the glucagon cells. Bouin: hematoxylin and eosin.
FIG. 14. Islet tissue from a normal 9-day quail embryo showing the evident large nucleoli typical of quail cells. Bouin: hematoxylin and eosin.
286
KRAMER
AND
microscopy and by immunocytochemistry corresponded to the situation in normal chick embryos at the same stage of development (see Andrew, 1976b). DISCUSSION
In this experiment it was vital that migration of neural crest cells from the host neural tube should not have occurred in the operated region before grafting of the donor tissue took place. With this in mind, the criteria for the selection of levels for transplantation were strictly adhered to and have been shown to be sound (Andrew, 1963). Likewise it is important to be sure that quail neural crest cells migrate normally in chick tissue, This was confirmed by the presence of large numbers of quail cells in the spinal ganglia, sympathetic ganglia, and neurilemmal (Schwann) cells of the chick hosts. It has been repeatedly shown by Le Douarin and her co-workers that isotopic quail to chick grafts are prolific in the formation of migratory neural crest cells (Le Douarin and Teillet, 1970, 1973, 1974; Le Lievre and Le Douarin, 1975). The most likely level of origin of pancreatic islet cells was that at which the duodenum develops, i.e., the level of somites 8 to 15 (Le Douarin, 1961). The number of times neural tube containing neural crest at each of these somite levels was transplanted and the number of successfully operated embryos justify the conclusions drawn. A, B, D, and PP cells were identified by electron microscopy in the operated chicks. Confirmation of the presence of A, B, and D cells was given by the immunocytochemical analysis. PP cells were sought immunocytochemically but not found, no doubt due to the fact that only few are present at 9 days of incubation, the earliest stage at which Alumets et al. (1978) have identified PP cells in the pancreas of chick embryos. For this reason no definite conclusion was drawn concerning PP cells. The complete absence of the quail nuclear marker from A, B, and D cells indi-
ANDREW
cated their origin from a source other than the neurectoderm of the most likely trunk levels. It is therefore now possible to conclude that these specific pancreatic endocrine cell types are not derived from these trunk levels of the neurectoderm. With the proposal that neural crest cells might arise from a ventral neural ridge (Takor Takor and Pearse, 1975) the possibility arose that this might be a source of pancreatic endocrine cells. However, we could find no evidence for a ventral neural ridge or related neural crest cells (Levy ef al., 1980). Taking the present results together with others (Andrew, 1976a; Fontaine et al., 1977; Andrew and Kramer, 1979), it has thus been shown that pancreatic (APUD) cells are not derived from rhombencephalic, probably not from mesencephalic, and not from trunk levels of the neurectoderm. The specific pancreatic endocrine cell types embraced by this conclusion are at least A and B cells for cephalic levels and A, B, and D cells for the most likely trunk levels. After combining mesoderm and endoderm from early chick and quail embryos, we have observed that, in the few grafts in which pancreas differentiated, the nuclear features of A and B cells were in accordance with an endodermal origin (unpublished results and see Andrew, 1978). The most likely source of these pancreatic endocrine cells therefore remains the endoderm. ACKNOWLEDGMENTS The authors would like to thank the Medical Research Council of South Africa and the Council and Senate Research Committees of the University of the Witwatersrand, Johannesburg, for supporting this study. We are grateful to Mr. W. Tadiello, Mrs. G. Holmes, and Mrs. J. Layzell for skilled technical assistance. Our thanks are also due to Professor P. V. Tobias, Head of the Department of Anatomy, for his continued interest and encouragement.
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F. (1978). of pan-
ORIGIN
PANCREATIC
creatic polypeptide (PP) cells in the pancreas and gut of the chicken. Cell Tissue Res. 194,377-386. Andrew, A. (1963). A study of the developmental relationship between enterochromaftin cells and the neural crest. J. Embryo/. Exp. Morphol. 11, 307-324. Andrew, A. (1976a). An experimental investigation into the possible neural crest origin of pancreatic APUD (islet) cells. J. Embryo/. Exp. Morphol. 35, 517-593. Andrew, A. (1976b). Pancreatic APUD cells in older chick embryos with special reference to their identity. Histochemistry 49, 213-219. Andrew, A. (1978). The embryonic origin of pancreatic islet (APUD) cells in the chick. In “Molecular Endocrinology” (I. MacIntyre and M. Szelke, eds.), pp. 355-359. ElsevieriNorth-Holland, Amsterdam/New York. Andrew, A., and Kramer, B. (1979). An experimental investigation into the possible origin of pancreatic islet cells from rhombencephalic neurectoderm. J. Embryo!.
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Andrew, A., and Rawdon, B. B. (1980). The ultrastructnral identity of pancreatic polypeptide sells in chicks. Histochemistry 65, 261-267. Dieterlen-Lievre, F. (1963). Demonstration de l’activite precoce des cellules A du pancreas chez l’embryon de poulet. C.R. Seance. Acad. Sci. Paris
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Dieterlen-Lievre. F. (1965). Etude morphologique et experimentale de la differentiation du pancreas chez l’embryon de poulet. Bull. Biol. France Belg. 99,3-116. Fontaine. J.. Le Lievre, C., and Le Douarin. N. M. (1977). What is the developmental fate of the neural crest cells which migrate into the pancreas in the avian efnbrya? Gen. Comp. Endocrinol. 33, 394-404.
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Le Douarin, N. M. (1971). Caracteristiques ultrastructurales du noyau interphasique chez la caille et chez ie poulet et utilisation de cellules de caille comme ‘marqueurs biologiques’ en embryologie experimentale. Ann. Embryol. Morphol.
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Le Douarin, N. M., and Teillet, M.-A. (1974). Experimental analysis of the migration and differentiation of neuroblasts of the autonomic nervous system and of neurectodermal mesenchymal derivatives. using a biological cell marking recbnique. Develop. Morphol. 41, 162-184. Le L!evre, C. S.. and Le Douarin, N. M. (1975). Mesenchymal derivatives of the neural crest: Analysis of chimaeric quail and chick embryos. J. Embryol.
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Maceroho, P. (1977). Pancreatic islet cells in very young chick embryos with special reference to D cells. Gen. Comp. Endocrinol. 33. 139-146. Machino, M. (1966). Electron microscopic observations of pancreatic islet cells of the early chick embryo. Nailcre (London) 210, 853-854. Pearse, A. 6. E. (1960). “Histochemistry. Theoretical and Applied,” 2nd ed., p. 822. Churchill, London. Pearse, A. G. E. (1969). The cytochemistry and ultrastructure of po~ypeptide-hormo~~e producing cells of the APUD series and the embryologic, physiologic and pathologic implications of the concept. J. Histochem. Cytochem. 6’7, 303--313. Pearse, A. 6. E. (1977). The APUD concept and irs implications: Related endocrine peptides in brain, intestine. pitaitary, placenta and anuran cutaneous glands. Med. Biol. 55, 115-125. Pearse, A. G. E.. Polak. .I. M., and Bussoiatl, 6, (1972). The neural crest origin of gastro-intestinal and pancreatic endocrine polypeptide cells and their distinction by sequential immunoflaorescence. Folia Hisiochem. Cytochem. Krako,!, 10, 115-120. Pictet, R. L., Rail, L. B., Phelps. P., and Rutter, (1976). The neural crest and the origin of the insulin-producing and other gastrointestinal hormone-producing cells. Science 191, Y9i -i93. Rawdon, B. B. (1978). Preventing evaporation of antiserum during indirect immunocyiochemical staining. Stain Technol. 53, 289-290. Takor Takor, T., and Pearse, A. 6. E. ;1915). Neuroectodermal origin of avian hypothalamobypopbyseal complex: The role of the ventral neural ridge. J. Embryol. Exp. Morphol. 34, 311-325.