GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
26, 485-495 (1975)
APUD Cells in the Endocrine Pancreas of Chick Embryos
and the Intestine
ANN ANDREW Department of Anatomy, Medical School, Hospital Street, Johannesburg. 2001. South Africa Accepted March 19. 1975 Formaldehyde-induced fluorescence has demonstrated biogenic amine-storing (APUD) cells in the duodenum of chick embryos from day 13 or 14 of incubation. These are definitive intestinal endocrine cells. If DOPA was administered to the embryos, a few cells were fluorescent only 1 day sooner. Following DOPA-treatment, fluorescence revealed cells with biogenic amine-synthesizing ability (also APUD cells) scattered in the gut groove at 16. to 1S-somite stages, concentrated at the site of imminent evagination of the dorsal pancreatic bud later on, then localized within the bud in groups, and later grouped and scattered in the pancreas. In the absence of exogenous DOPA, their fluorescence was evident by day 13 or 14 only. It seems probable that the APUD cells scattered in the gut at early stages are the precursors of islet cells of one or more than one type.
Cells which have the ability to store a biogenic amine, or to synthesize one from an amine precursor such as DOPA, have been grouped together by Pearse (1966a) as APUD cells (APUD = Amine Precursor Uptake and Decarboxylation). The presence of biogenic amine in the cells may be detected by the formaldehyde-induced fluorescence method. Many of the cell types concerned secrete polypeptide hormones. Following administration of DOPA to pregnant mice, Pearse and Polak (1971a, b) found cells showing formaldehyde-induced fluorescence in 7- to g-day embryos. Judging from the position of these cells between the epidermis and the neural tube, they took them to be neural crest cells. Subsequently APUD cells were found “crossing” the mesenchyme to the pharynx and then within the pharynx, in the fourth pharyngeal pouch in particular. The fluorescent cells in the latter situation eventually became thyroid C cells (Pearse and Carvalheira, 1967). Other APUD cells assumed to be derived from the neural crest, were described invading the 485 Copyright All rights
@ 1975 by Academic Press, Inc. of reproduction in any form reserved.
dorsal pancreas, and the presumptive stomach and duodenum (Pearse and Polak, 1971 b). On this basis these authors concluded that at least some endocrine polypeptide cells of the gastrointestinal tract are derivatives of the neural crest. When applying the same methods to White Leghorn chick embryos, Polak, Rost, and Pearse (197 1) could find no APUD cells before the 36-somite stage, The first such cells appeared at the level of somites 6-12 “in the region of the neural crest.” These cells were held to become adrenal medullary cells. However, none were described en route to the foregut. The present author has previously investigated the possibility that enterochromaffin cells of the intestine are neural crest derivatives (Andrew, 1963, 1974), for which purpose Black Australorp chick embryos were used. Consequently, in embryos of this breed, a search was instigated for APUD cells arising from the neural crest and invading the gastrointestinal tract. None were found so engaged, but the presence of APUD cells in the intestine and the dorsal pancreatic bud was
486
ANN
ANDREW
revealed at unexpectedly early stages. The location of these APUD cells at successive stages provoked speculation concerning the situation of presumptive islet cells before the pancreatic buds form. METHODS Black Australorp embryos were processed at the following stages: early somite stage, 16-18 somites, 19-22, 23-25, 26-28, 29-32, and 34-38 somites (3 days); 3V4-4 days, 5. 6, 7, 9, 11, 12. 13, 14. and 16 days of incubation. At each stage or group of stages listed, some eggs were injected with DL-DOPA (B.D.H.) dissolved in chick Ringer’s solution, others with an equal volume of Ringer’s solution. Embryos of up to 7 days incubation received 150 yg DOPA in 150 ~1 Ringer’s solution; older embryos were treated with higher doses, calculated on the basis of their weights (according to Romanoff, 1960, p. 147) and the dose usually used for adult mice (100 mg/kg - see for example, Pearse and Polak, 1971a). The higher doses were administered in 0.25 ml (or 0.5 ml in the case of 16-day embryos), in order to dissolve the larger amounts of DOPA more easily. In general, eggs were incubated horizontally. At the time of treatment, the air space was punctured and a window made in the shell directly over the embryo. On removal of the underlying shell membrane, the contents of the egg displaced air from the air space, and the embryo dropped away from the window. DOPA solution or Ringer’s solution was injected directly onto the embryo or the chorioallantoic membrane and the window was sealed with cellotape. Embryos of less than 2 days of incubation tend to die if windowed in this way. Therefore eggs to be treated at earlier stages were incubated on end, and the DOPA solution or Ringer’s solution was injected into the air space through a small window, subsequently sealed. After reincubation for 1 hr, embryos were retrieved and dissected free of their extraembryonic membranes. Embryos of up to and including the 38-somite stage, were left intact; from embryos of 3Y4 to 16 days of incubation, a block of tissue including duodenum and pancreas was excised. One hundred and twenty-six embryos were treated as above. In addition, a further 20 were used in an attempt to see whether any accumulation of biogenic amine could be provoked in APUD cells of embryos at some of the younger stages (16-18 somites, 26-30 somites, and 3Y4 days). The monoamine-oxidase inhibitor, nialamide, was used: biogenic amines are normally broken down by this enzyme. Larson et al. (1966) treated mice with 100 mg/kg body wt, 1 hr before administering DOPA. This was the same dose as that of DOPA. Therefore 150 pg of nialamide (in
150 ~1 Ringer’s solution) was administered to some chick embryos; half of these received 150 kg DOPA I hr later. Other embryos received no nialamide: of these, some received DOPA and some did not. After incubation for a further hour, the eggs were opened and the intact embryos or pieces containing duodenum and pancreas were processed as below. The procedure adopted for demonstrating formaldehyde-induced fluorescence of biogenic amines was based on the methods used by Falck (1962). Emnko (I 967). Corrodi and Jonsson (1967). Polak et at. (1971), and Pearse and Polak (1971a). Tissues were washed in Ringer’s solution and quenched in melting isopentane cooled in liquid nitrogen. They were freeze-dried for 18-2 1 hr at -40” and I Oe3 Torr in an Edwards-Pearse EPD 2 tissue-dryer. The dried tissues were heated to 45-50” before admittance of air to the drying chamber, and then transferred to preheated vessels, either containing paraformaldehyde powder or empty. Some of the DOPAtreated embryos and all those not so treated. were subjected to formaldehyde vapour (generated by paraformaldehyde previously equilibrated over sulphuric acid to a humidity of 50%) at 80” for I hr (tissue from older embryos) or 3 hr (young embryos). Other DOPA-treated tissues were not subjected to formaldehyde vapour, but were kept at the same temperature for the same period in a separate oven uncontaminated by formaldehyde. Tissues were vacuum-embedded in paraplast (mp 58”). Serial sections were cut at IO pm and dry-mounted straight from the knife on specially cleaned, albumenized slides, and subsequently left to drain in an oven at 60” overnight. They were stored over CaCl, in a dessicator in a black plastic bag, in the refrigerator. Slides were dipped in xylol and cover-slipped. They were examined on a Reichert Fluorpan microscope equipped with an HBO 50 mercury burner. under blue bright ground illumination, with a 6 BG 12/h exciter filter and appropriate absorption filter. Photographs were taken with Kodak Tri-X Pan film (ASA 400) at an exposure of 10 sec.
RESULTS
Embryos and tissues treated with DOPA but not subjected to formaldehyde vapour, served as controls demonstrating background fluorescence and autofluorescence. The notochordal sheath was autofluorescent, though before the 30-somite stage not always so in the caudal end of the embryo. Red blood cells were usually, but not invariably, autofluorescent; punctate yellow autofluorescence occurred in
APUD
CELLS
IN CHICK
parts of the mesonephric and metanephric tubules. Following DOPA administration and vapour fixation, specific fluorescence was apparent at 4 days incubation, in sympathetic ganglia and preaortic ganglia, and also in small numbers of cells medial to the mesonephros -presumably presumptive adrenal medullary cells. By 7 days incubafluorescent cells were tion, intensely present in the adrenal gland. Later on, fluorescence in all these structures was no longer dependent on exogenous DOPA. At no stage examined, whether DOPA had been administered or not, were cells showing specific fluorescence observed in the neural crest, invading the gastrointestinal tract or pancreas, or at sites en route from the crest to the tract. However, fluorescent cells were encountered within the presumptive intestine and dorsal pancreatic bud as shown in Tables 1 and 2. In the epithelium of the gut groove (i.e., the endodermal groove which will later close and become the intestine) of embryos as young as the 16-somite stage, cells scattered along a distance of more than seven somites’ length showed the ability to take up exogenous DOPA and decarboxylate it to dopamine. The cells TABLE
1
FORMALDEHYDE-INDUCED
FLUORESCENCE
APUD
CELLS IN THE OF CHICK EMBRYOS
DOPA administered
Stage” 16-18s 19-28s 29-32s 33-38s
fve +ve
No DOPA administered
or -veb +ve or
-ve -VE!
-ve
-ve
13d
few +ve -ve very few +ve few +ve
14d
+ve
+ve
16d
+ve
+ve
3$lld 12d
(3d)
IN
INTESTINAL EPITHELIUM DURING DEVELOPMENT
very
-ve -ve -ve few
+ve
u s = somites; d = days of incubation. * +ve = specific fluorescence; -ve = no fluorescent cells.
487
EMBRYOS TABLE
FORMALDEHYDE-INDUCED
APUD
CELLS
FLUORESCENCE IN
DEVELOPING
Stage
2
THE
PANCREAS
CHICK
IN OF
EMBRYOS”
DOPA administered
No
DOPA
administered
28-38s
+ve
- ve
3+13d 13-16d
+ve +ve
-ve +ve
‘I Abbreviations
as in Table
1.
were not very brightly fluorescent, and were not present in every embryo of the 16- to 18-somite group. In all embryos of 19-26 somites (Figs. 1 and 2) similar but more brightly fluorescent cells were distributed in the dorsal and lateral walls of the gut groove, from the anterior intestinal portal caudally. By the 24-somite stage, small groups were observed as well as single cells (Fig. 2). The endoderm in this region was considerably thickened. In 27-somite stage embryos, fluorescent APUD cells were more concentrated, most of them lying in the dorsal wall of the gut at the anterior intestinal portal (Fig. 3). Once the dorsal pancreatic diverticulum had begun to form (at or soon after the 27somite stage), a few scattered fluorescent cells were present in the roof of the (still open) gut immediately caudal to the diverticulum. The number of cells in this position subsequently dwindled, one or two being found occasionally in 3-day specimens. In the duodenum of 12- and 13-d&y DOPA-treated embryos, fluorescenr cells were again observed in the epithelium (Table 1). but these were very few. and their fluorescence not very hright. In the 14- and 16-day specimens, fluorescent cells were increasingly numerous and the fluorescence bright (Fig. 8). In embryos not treated with DOPA. no epithelial cells of the intestine up to 3 days of incubation, nor of the duodenum thereafter, showed fluorescence until day 13, when a very few were rather weakly
488
ANN
ANDREW
1. Scattered fluorescent cells in the gut groove of a 19-somite embryo. Longitudinal section. 2. Single and grouped fluorescent cells in the roof and lateral walls of the gut groove in a 24-somite embryo. Transverse section. FIG. 3. Fluorescent cells in the gut in the region of the anterior intestinal portal, where the dorsal pancreatic bud will form. Transverse section of 27-somite embryo. FIG. 4. Fluorescent cells, mostly in groups, at the periphery of the dorsal pancreatic bud of a 36-somite embryo. Transverse section. FIG. FIG.
FIGS. l-8. Cells showing specific formaldehyde-induced fluorescence in chick embryos treated with DOPA. (Magnification constant.) L-lumen of gut; A-dorsal aorta; DP-dorsal pancreatic bud.
APUD
CELLS
IN
fluorescent. At 14 days some were seen, but they were fewer and duller than when DOPA was administered; by 16 days, however, cells appeared equally numerous and equally brightly fluorescent. In the dorsal pancreatic bud of embryos treated with DOPA, brightly fluorescent cells were evident from the start of its evagination at the 27-somite stage (Table 2). These cells were present singly or in small groups within the diverticulum. Soon (by the 31- to 33-somite stage) small groups of fluorescent cells formed buds at the periphery of the advancing diverticulum. As the sprouts lengthened and branched during successive stages up to 14 days of incubation, the fluorescent cells remained at their tips (Figs. 4 and 5). The fluorescent material tended to lie basally in the cells. Later on-by 7 and 16 days, for example (Figs. 6 and 7) -numerous fluorescent cells were distributed singly and in groups (which were doubtless islets) in the parenchyma of the pancreas, and their cytoplasm was more uniformly fluorescent. As shown in Table 2, if no DOPA was administered, fluorescence was lacking in the pancreas until day 13 or 14. In this study, the various lobes of the pancreas were not distinguished. Fluorescent cells were absent from the ventral pancreatic buds while these were recognisable as distinct entities (4-5 days of incubation). Administration of nialamide to embryos of stages 17-20 somites, 26-30 somites, and 3314 days of incubation resulted in no obvious increase in the intensity of fluorescence in APUD cells in the presumptive intestine or the pancreas of DOPA-treated specimens. No definite fluorescence was provoked by nialamide in those embryos which had received no DOPA. DISCUSSION
In certain respects the present findings confirm for Black Australorp embryos, observations made by Polak et ul. (197 1) on
CHICK
EMBRYOS
489
APUD cells in DOPA-treated White Leghorn embryos: no cells in the neural crests were fluorescent: nor were any APUD cells seen invading the foregut. The fluorescent cells mentioned as likely to become incorporated in the adrenal medulla were doubtless those also seen by Polak et al. However, Polak et al. found no APUD cells at all in chick embryos before the 36somite stage, and made no mention of APUD cells in the presumptive intestine or in the dorsal pancreatic bud even after this time: in the present investigation, cells showing specific fluorescence after DOPA administration were evident in these sites well before the 36-somite stage and also in the differentiating pancreas and the duodenum later on. The reason why Polak et al. found no APUD cells at the early stages may lie in the different breed of their embryos or in the different method of DOPA administration used. They treated embryos removed from the eggs: in the present study embryos were treated in ova. Furthermore, paraformaldehyde equilibrated to a low humidity was used here. Fluorescence is not as intense as with higher humidities, but the fluorescent product is less likely to diffuse away. Polak et al. do not specify conditions of humidity. The appearance of fluorescent cells in the duodenal epithelium at and after 13 days of incubation was expected since Enemar et al. (1965) and Pentila and Mustakallio (1968) had demonstrated them first at 14 days (in the absence of exogenous DOPA). In the present study, administration of DOPA produced fluorescence only a day earlier-and then in only few cells. The above authors identified the fluorescent cells as enterochromaffin by silver impregnation. Other endocrine cell types in the intestine of mammals (ECL, G and D cells -nomenclature according to Solcia et al., 1973) are held to give the APUD reaction (Solcia et al., 1969; Hakan-
ANN
ANDREW
FIG. 5. Fluorescent cells in the growing buds sprouting from the dorsal pancreatic diverticulum in a 4-day embryo. FIG. 6. Scattered single and grouped cells in the pancreatic parenchyma of a 7-day embryo. FIG. 7. Scattered fluorescent cells in the pancreatic parenchyma of a 16-day embryo. FIG. 8. Fluorescent cells in the epithelium of the duodenum of a 16-day embryo. Transverse section.
APUD
CELLS
IN
son et al., 1969; Dawson, 1970; Pearse et al., 1970). Pearse (197 1) considers that L (EG) and S cells are also members of the APUD series. Quite unexpected was the demonstration of the ability of cells in the gut at a very early stage (16-somite stage, i.e., about 48 hr of incubation), to produce biogenic amine from administered precursor. There is, however, other evidence that the processes involved do occur in chick embryos at such early stages. That DOPA is taken up by the embryo by the first day of incubation is indicated by Ignarro and Shideman (1968a). The conversion of DOPA to dopamine occurs already on the second day (ibid.; Burack and Badge, 1964). That this dopamine is indeed synthesized intraembryonically is shown by its absence from the yolk of the fertilized egg (Ignarro and Shideman, 1968b). Biogenic amines are broken down by monoamine oxidase: this enzyme has been detected at 3 days of incubation by Ignarro and Shideman (1968~) and Castellano et al. (1974). The occurrence of fluorescent cells in the dorsal pancreatic bud from the time of inception of its formation was also unexpected. A considerable period elapsed before this fluorescence became independent of DOPA administration (day 13): a similar time-lag occurs in the endocrine pancrease of the guinea pig embryo (Cegrell and Falck, 1968) and the mouse embryo (Pearse et al., 1973). During the early stages of development (up to 33/4 days), under the conditions of the present study, not even the administration of nialamide provoked fluorescence in the pancreas or the gut groove in the absence of treatment with DOPA. Nor was there any increase in fluorescence if the embryos received exogenous DOPA as well. The reason is probably that monoamine oxidase is absent, or present in insufficient quantity to break down any biogenic amine produced: Castellano et al. (1974) have been unable
CHICK
EMBRYOS
491
to detect the enzyme in the pancreas before 13 days of incubation. This maneuver has thus not thrown light on the question of whether any biogenic amines are normally produced at these early stages. In this connection, the role of biogenic amines synthesized by APUD cells in the gut and pancreas is of interest. Unfortunately the functions of these amines are not yet clear in adults, let alone in embryos. The only suggestion mooted is that they play a part in hormone production or release (Cegrell, 1968b; Falck and Owman, 1968; Slanina and Tjalve, 197 I ). Quickel et a/. ( 1971) have suggested that the endogenous biogenic amines of the pancreas, serotonin and dopamine, inhibit secretory activity of B islet cells -a view favoured by Hahn von Dorsche and Krause (1973). Thus any early production of biogenic amine in the endocrine pancreas might be related to synthesis of pancreatic hormones. That these hormones are made early in development is known. In the rat, glucagon and insulin are produced as soon as, or soon after, the dorsal pancreatic bud forms (Rutter et al., 1968; Wessels, 1968; Clark and Rutter, 1972; Pictet and Rutter, 1972; Rall et al., 1973). In chick embryos, glucagon has been demonstrated at the 30somite stage, i.e., soon after the appearance of the dorsal pancreatic bud (Beaupain and Dieterlen-Lievre, 1974) and insulin a little later, at the 40- to 43-somite stage (Dieterlen-Lievre and Beaupain, 1974). As in higher vertebrates. it seems that in the fowl, glucagon or a glucagonlike hormone is synthesized by cells corresponding to A, cells (Mikami and ‘One, 1962) and insulin or an insulin-like hormone by B cells (Thommes, 1960: Mikami and Mutoh, 1971; Green and Benzo, 1973). In chick embryos, A (probably A,) cells have been identified in the dorsal pancreatic bud at 3 or 3.5 days (i.e., soon after its evagination) (Dieterlen-Lievre, 1963: Przbylski, 1967) and B cells at the same
492
ANN
time (Przbylski, 1967) or at 4 days of incubation (Dieterlen-Lievre, 1965). The identity of the APUD cells in the dorsal pancreatic bud in chick embryos has yet to be established. In the duck, both A, and A, cells show formaldehyde-induced fluorescence (Falck and Hellman, 1963); in the pigeon, A, and some A, cells (Trandaburu, 1972) do so. (No amine precursor was administered in these studies.) It does not necessarily follow that fowl APUD cells are A cells: in different species of other vertebrate groups, different islet cell types give this reaction-in certain newts A, cells, in frogs B, and in reptiles none (Trandaburu, 1972), in some mammals A cells, in others B, and in others both A and B cells are APUD (Cegrell, 1967a; 1968a, c, d; Cegrell et al., 1968; Legg, 1968). Dieterlen-Lievre (1970) has shown by isolating and culturing dorsal and presumptive ventral pancreatic buds separately, that both can give rise to A and-B cells. It is therefore strange that APUD cells were confined to the dorsal bud in the present study-a finding more in line with the previously held view that the dorsal bud becomes most of the splenic lobe, in which the majority of A islets form (DieterlenLievre, 1963). The presence of APUD cells in the walls of the gut groove from the 16-somite stage until the evagination of the dorsal pancreatic bud was entirely unexpected. If they are the precursors of the definitive APUD cells of the intestine, recognized from 13 or 14 days of incubation, they must temporarily lose their biogenic aminesynthesizing ability. Another possibility is that they are not directly related developmentally to any definitive APUD cells and have a merely transitory existence. Most attractive, however, is the possibility that these cells become pancreatic islet cells of one or more than one type. Certainly, shortly before the dorsal pancreatic bud evaginates, the APUD cells are concen-
ANDREW
trated at its site of origin; once it evaginates they are increasingly confined to it. Less easy to explain is the incorporation into the dorsal bud, of cells distributed previously over a considerable extent of the gut groove. However, it is not inconceivable that this whole extent becomes included in the diverticulum. For one thing, the duodenum arises at the levels of somites 8-15 (Le Douarin, 1961); since the pancreatic bud arises from the future duodenum it is possible that much of the gut roof of the 16-somite embryo (from the anterior intestinal portal-located at about the level of somite 5 -to approximately the level of somites 12, 13, or even 14) could be incorporated into the dorsal bud. Other evidence which supports this suggestion is that Wessels and Cohen (1967) and Wessels and Evans (1968) found that evagination of the diverticulum in the mouse is due to changes in cell shape and to cell movements rather than to proliferation. This implies the incorporation of a considerable extent of the roof of the gut groove into the evaginating bud. Very much in line with the present observations on the presence and distribution of APUD cells in the early chick gut, are the findings of Drews et ul. (1969). They describe cholinesterase-rich cells scattered in the gut endoderm at the 16-somite stage. The grouping and concentration of such cells in the dorsal pancreatic diverticulum when this formed, the formation of complexes of the cells at the periphery of the diverticulum and their subsequent identification as islet cells, indicate that these are the very cells which show the APUD ability. Indeed, cholinesterase activity is a characteristic of many APUD cells (Pearse, 1966a). However, the enzyme activity of the cells disappears on the fifth day of incubation whereas the biogenic amine-producing ability persists. Drews et al. do not hesitate to assume that the scattered cholinesterase-rich cells of the 16somite stage become islet cells. Observa-
APUD
CELLS
IN
tions of cells showing similar features (such as cholinesterase or APUD reactions) at successive developmental stages is highly suggestive but not conclusive evidence that they belong to the same cell line, however. Before the evagination of the pancreatic diverticulum, Wessels and Evans (1968) and Pictet et al. (1972) found no ultrastructural evidence of endocrine cells in the mouse and rat. Pearse et al. (1973), however, describe cells with pleomorphic granules in the presumptive pancreatic region of the mouse embryo before evagination occurs. They maintain that these cells differentiate into the various types of islet cells. Thus they suppose that, in the mouse, “primitive endocrine cells” in the intestine become islet cells; Pearse and Polak (1971 b), however, did not find APUD cells in the intestine of the mouse prior to formation of the pancreatic diverticulum. The hypothetical picture proposed by Pearse et al. (1973), of scattered “primitive endocrine cells” in the intestine, corresponds to the belief that phylogenetically and ontogenetically islet cells have arisen from specialized cells in the intestinal epithelium (Steiner et al., 1969; Falkmer, 1972; Epple and Lewis, 1973). If the APUD cells described in the gut groove of 16-somite embryos in the present study, are indeed future islet cells, then the existence of this hypothetical stage (see Epple and Lewis, Fig. 1D) is confirmed. Whether or not some or all such cells, rather than being endodermal in origin, are derivatives of the neural crest as Pearse and Polak (197 1 b) and Pearse et al. (1973) believe, is another question. No evidence for or against this contention came to light in the present study.
493
CHICK EMBRYOS
Levitan, and Miss D. Croft for technical assistance. A grant from the South African Medical Research Council for the purchase of the freeze-dryer is acknowledged with gratitude.
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Hikanson, R., Lilja, B. and Owman, C. (1969). Cellular localization of histamine and monoamines in the gastric mucosa of man. Histochemie 18, 74-86. Ignarro, L. J. and Shideman, F. E. (1968a). Appearance and concentration of catecholamines and their biosynthesis in the embryonic and developing chick. J. Pharmacol. Exp. Ther. 159, 38-48. Ignarro, L. J. and Shideman, F. E. (1968b). Norepinephrine and epinephrine in the embryo and embryonic heart of the chick: uptake and subcellular distribution. J. Phurmacol. Exp. Ther. 159,49-58.
Ignarro, L. J. and Shideman, F. E. (1968~). CatecholO-methyl transferase and monoamine oxidase activities in the heart and liver of the embryonic and developing chick. J. Pharmucol. Exp. Ther. 159, 29-37.
Larson, B., Owman, C., and Sundler, F. ( 1966). Monoaminergic mechanisms in parafollicular cells of the mouse thyroid gland. Endocrinology 78, 1109-1114. Le Douarin, N. (1961). Radiodestructions partielles chez I’embryon de poulet aux stades jeunes et localisation des Cbauches digestives. J. Embryol. Exp. Morphol. 9, 1-8. Legg, P. G. (1968). Fluorescence studies on structures and endocrine cells in the pancreas of the cat. Z. Zellforsch. Mikrosk. Anat. 88, 487-495. Mikami, S. I. and Mutoh, K. (1971). Light and electron microscope studies of the pancreatic islet cells in the chick under normal and experimental conditions. Z. Zellforsch. Mikrosk. Anut. 116, 205-227. Mikami, S. I. and Ono, K. (1962). Glucagon deficiency induced by extirpation of alpha islets of the fowl pancreas. Endocrinology 71, 464-473. Pearse, A. G. E. (1966). Common cytochemical properties of cells producing polypeptide hormones, with particular reference to caicitonin and the thyroid C cells. Vet. Rec. 79, 587-590. Pearse, A. G. E. (1971). The endocrine polypeptide cells of the APUD series. (Structural and functional correlations) Mem. Sot. EndocrinoL 19, 543-556. Pearse, A. G. E. and Carvalheira, A. F. (1967). Cytochemical evidence for an ultimobranchial origin of rodent thyroid C cells. Nature (London) 214, 929-930. Pearse, A. G. E., Couling. I., Weavers, B., and Friesen, S. (1970). The endocrine polypeptide cells of the human stomach, duodenum and jejunum. Gut 11, 649-658. Pearse, A. G. E. and Polak, J. M. (197 la). Cy-
APUD
CELLS
IN
tochemical evidence for the neural crest origin of mammalian ultimobranchial C cells. Histochemie 27, 96-102. Pear-se, A. G. E. and Polak, J. M. (1971 b). Neural crest origin of the endocrine polypeptide (APUD) cells of the gastrointestinal tract and pancreas. Cur 12, 783-788. Pearse, A. G. E., Polak, J. M., and Heath, C. M. ( 1973). Development, differentiation and derivation of the endocrine polypeptide cells of the mouse pancreas. Immunofluorescence, cytochemical and ultrastructural Diabetologicu 9, 120-129. Pentila, A. and Mustakallio, A. (1968). Monoamine oxidase activity as related to the 5hydroxytryptamine stores in the chicken duodenum during development. Acta Histochem. 31, l-13. Pictet, R. L., Clark, W. R., Williams, R. H., and Rutter, W. J. (1972). An ultrastructural analysis of the developing embryonic pancreas. Develop. Biol.
29, 436-467.
Pictet, R. and Rutter, W. J. (1972). Development of the embryonic pancreas. In “Handbook of Physiology” (D. F. Steiner and N. Freinkel, eds.), Sect. 7, Vol. 1, pp. 25-66. American Physiological Society, Washington D.C. Polak, J. M., Rost, F. W. D., and Pearse, A. G. E. ( 197 1). Fluorogenic amine tracing of neural crest derivatives forming the adrenal medulla. Gen. Camp. Endocrinol. 16, 132- 136. Przbylski, R. J. (1967). Cytodifferentiation of the chick pancreas. I. Ultrastructure of the islet cells and the initiation of granule formation. Gen. Camp. Endocrinol. 8, 115- 128. Quickel, K. E., Feldman, J. M., and Lebovitz, H. E. (1971). Inhibition of insulin secretion by serotonin and dopamine: species variation. Endocrinology 89, 129% 1302. Rall, L. B., Pictet, R., Williams, R. H., and Rutter, W. J. (1973). The early differentiation of glucagon-producing cells in the embryonic pancreas. Proc.
Nat.
Acad.
Sci. USA
70, 3478-3482.
Romanoff, A. (1960). “The Avian Embryo: Structural and Functional Development,” p. 147. MacMillan, New York. Rutter, W. J., Kemp, J. D., Bradshaw, W. S., Clark,
CHICK
495
EMBRYOS
W. R., Ronzio, R. A., and Sanders, R. G. (1968). Regulation of specific protein synthesis in cytodifferentiation. J. Cell. Physiol. 72, (Suppl. 1), 2-18. Slanina, P. and Tjiilve, H. (1971). Uptake of $Hatropine in pancreatic islets and the parafollicular cells of the thyroid in mice shown by microautoradiography. Acra Physiol. Stand. 84, 254-260. Solcia, E., Pearse, A. G. E., Grube, D., Kobayashi, S., Bussolati, G., Creutzfeldt, W., and Gepts, W. (1973). Revised Wiesbaden classification of gut endocrine cells. Rend. Gastroenterol. 5, 13- 16. Solcia, E., Vassallo, G., and Capella, C. (1969). Cytology and cytochemistry of hormone producing cells of the upper gastrointestinal tract. In “Origin, Chemistry, Physiology and Pathophysiology of the Gastrointestinal Hormones” (W. Creutzfeldt, ed.), International Symposium, Wiesbaden, pp. 3-29. Schatthauer, Stuttgart and New York. Steiner, D. F., Clark, J. L., Nolan, C., Rubenstein, A. H., Margoliash, E., Aten, B., and Oyer, P. E. (1969). Proinsulin and the biosynthesis of insulin. Rec. Progr.
Horm.
Res. 25, 207-272.
Thommes, R. C. (1960). A histochemical study of insulin in the chick embryo pancreas. Growth 24, 69-80. Trandaburu, T. (1972). Comparative observations on adrenergic innervation and monoamine content in endocrine pancreas of some amphibians, reptiles and birds. Endokrinologie 59, 260-264. Wessels, N. K. (1968). Problems in the analysis of determination, mitosis and differentiation. In “Epithelio-Mesenchymal Interactions” (R. Fleischmajer and R. E. Billingham, eds.), 18th Hahnemann Symposium, pp. 132- 15 1. Williams and Wilkins, Baltimore. Wessels, N. K. and Cohen, J. H. (1967). Early pancreas organogenesis: morphogenesis, tissue interactions and mass effects. Develop. Biol. 15, 237-270. Wessels, N. K. and Evans. J. (1968). Ultrastructural studies of early morphogenesis and cytodifferentiation in the embryonic mammalian pancreas. Develop. Biol. 17, 413-446.
AND
COMPARATIVE
ENDOCRINOLOGY
26, 485-495 (1975)
APUD Cells in the Endocrine Pancreas of Chick Embryos
and the Intestine
ANN ANDREW Department of Anatomy, Medical School, Hospital Street, Johannesburg. 2001. South Africa Accepted March 19. 1975 Formaldehyde-induced fluorescence has demonstrated biogenic amine-storing (APUD) cells in the duodenum of chick embryos from day 13 or 14 of incubation. These are definitive intestinal endocrine cells. If DOPA was administered to the embryos, a few cells were fluorescent only 1 day sooner. Following DOPA-treatment, fluorescence revealed cells with biogenic amine-synthesizing ability (also APUD cells) scattered in the gut groove at 16. to 1S-somite stages, concentrated at the site of imminent evagination of the dorsal pancreatic bud later on, then localized within the bud in groups, and later grouped and scattered in the pancreas. In the absence of exogenous DOPA, their fluorescence was evident by day 13 or 14 only. It seems probable that the APUD cells scattered in the gut at early stages are the precursors of islet cells of one or more than one type.
Cells which have the ability to store a biogenic amine, or to synthesize one from an amine precursor such as DOPA, have been grouped together by Pearse (1966a) as APUD cells (APUD = Amine Precursor Uptake and Decarboxylation). The presence of biogenic amine in the cells may be detected by the formaldehyde-induced fluorescence method. Many of the cell types concerned secrete polypeptide hormones. Following administration of DOPA to pregnant mice, Pearse and Polak (1971a, b) found cells showing formaldehyde-induced fluorescence in 7- to g-day embryos. Judging from the position of these cells between the epidermis and the neural tube, they took them to be neural crest cells. Subsequently APUD cells were found “crossing” the mesenchyme to the pharynx and then within the pharynx, in the fourth pharyngeal pouch in particular. The fluorescent cells in the latter situation eventually became thyroid C cells (Pearse and Carvalheira, 1967). Other APUD cells assumed to be derived from the neural crest, were described invading the 485 Copyright All rights
@ 1975 by Academic Press, Inc. of reproduction in any form reserved.
dorsal pancreas, and the presumptive stomach and duodenum (Pearse and Polak, 1971 b). On this basis these authors concluded that at least some endocrine polypeptide cells of the gastrointestinal tract are derivatives of the neural crest. When applying the same methods to White Leghorn chick embryos, Polak, Rost, and Pearse (197 1) could find no APUD cells before the 36-somite stage, The first such cells appeared at the level of somites 6-12 “in the region of the neural crest.” These cells were held to become adrenal medullary cells. However, none were described en route to the foregut. The present author has previously investigated the possibility that enterochromaffin cells of the intestine are neural crest derivatives (Andrew, 1963, 1974), for which purpose Black Australorp chick embryos were used. Consequently, in embryos of this breed, a search was instigated for APUD cells arising from the neural crest and invading the gastrointestinal tract. None were found so engaged, but the presence of APUD cells in the intestine and the dorsal pancreatic bud was
486
ANN
ANDREW
revealed at unexpectedly early stages. The location of these APUD cells at successive stages provoked speculation concerning the situation of presumptive islet cells before the pancreatic buds form. METHODS Black Australorp embryos were processed at the following stages: early somite stage, 16-18 somites, 19-22, 23-25, 26-28, 29-32, and 34-38 somites (3 days); 3V4-4 days, 5. 6, 7, 9, 11, 12. 13, 14. and 16 days of incubation. At each stage or group of stages listed, some eggs were injected with DL-DOPA (B.D.H.) dissolved in chick Ringer’s solution, others with an equal volume of Ringer’s solution. Embryos of up to 7 days incubation received 150 yg DOPA in 150 ~1 Ringer’s solution; older embryos were treated with higher doses, calculated on the basis of their weights (according to Romanoff, 1960, p. 147) and the dose usually used for adult mice (100 mg/kg - see for example, Pearse and Polak, 1971a). The higher doses were administered in 0.25 ml (or 0.5 ml in the case of 16-day embryos), in order to dissolve the larger amounts of DOPA more easily. In general, eggs were incubated horizontally. At the time of treatment, the air space was punctured and a window made in the shell directly over the embryo. On removal of the underlying shell membrane, the contents of the egg displaced air from the air space, and the embryo dropped away from the window. DOPA solution or Ringer’s solution was injected directly onto the embryo or the chorioallantoic membrane and the window was sealed with cellotape. Embryos of less than 2 days of incubation tend to die if windowed in this way. Therefore eggs to be treated at earlier stages were incubated on end, and the DOPA solution or Ringer’s solution was injected into the air space through a small window, subsequently sealed. After reincubation for 1 hr, embryos were retrieved and dissected free of their extraembryonic membranes. Embryos of up to and including the 38-somite stage, were left intact; from embryos of 3Y4 to 16 days of incubation, a block of tissue including duodenum and pancreas was excised. One hundred and twenty-six embryos were treated as above. In addition, a further 20 were used in an attempt to see whether any accumulation of biogenic amine could be provoked in APUD cells of embryos at some of the younger stages (16-18 somites, 26-30 somites, and 3Y4 days). The monoamine-oxidase inhibitor, nialamide, was used: biogenic amines are normally broken down by this enzyme. Larson et al. (1966) treated mice with 100 mg/kg body wt, 1 hr before administering DOPA. This was the same dose as that of DOPA. Therefore 150 pg of nialamide (in
150 ~1 Ringer’s solution) was administered to some chick embryos; half of these received 150 kg DOPA I hr later. Other embryos received no nialamide: of these, some received DOPA and some did not. After incubation for a further hour, the eggs were opened and the intact embryos or pieces containing duodenum and pancreas were processed as below. The procedure adopted for demonstrating formaldehyde-induced fluorescence of biogenic amines was based on the methods used by Falck (1962). Emnko (I 967). Corrodi and Jonsson (1967). Polak et at. (1971), and Pearse and Polak (1971a). Tissues were washed in Ringer’s solution and quenched in melting isopentane cooled in liquid nitrogen. They were freeze-dried for 18-2 1 hr at -40” and I Oe3 Torr in an Edwards-Pearse EPD 2 tissue-dryer. The dried tissues were heated to 45-50” before admittance of air to the drying chamber, and then transferred to preheated vessels, either containing paraformaldehyde powder or empty. Some of the DOPAtreated embryos and all those not so treated. were subjected to formaldehyde vapour (generated by paraformaldehyde previously equilibrated over sulphuric acid to a humidity of 50%) at 80” for I hr (tissue from older embryos) or 3 hr (young embryos). Other DOPA-treated tissues were not subjected to formaldehyde vapour, but were kept at the same temperature for the same period in a separate oven uncontaminated by formaldehyde. Tissues were vacuum-embedded in paraplast (mp 58”). Serial sections were cut at IO pm and dry-mounted straight from the knife on specially cleaned, albumenized slides, and subsequently left to drain in an oven at 60” overnight. They were stored over CaCl, in a dessicator in a black plastic bag, in the refrigerator. Slides were dipped in xylol and cover-slipped. They were examined on a Reichert Fluorpan microscope equipped with an HBO 50 mercury burner. under blue bright ground illumination, with a 6 BG 12/h exciter filter and appropriate absorption filter. Photographs were taken with Kodak Tri-X Pan film (ASA 400) at an exposure of 10 sec.
RESULTS
Embryos and tissues treated with DOPA but not subjected to formaldehyde vapour, served as controls demonstrating background fluorescence and autofluorescence. The notochordal sheath was autofluorescent, though before the 30-somite stage not always so in the caudal end of the embryo. Red blood cells were usually, but not invariably, autofluorescent; punctate yellow autofluorescence occurred in
APUD
CELLS
IN CHICK
parts of the mesonephric and metanephric tubules. Following DOPA administration and vapour fixation, specific fluorescence was apparent at 4 days incubation, in sympathetic ganglia and preaortic ganglia, and also in small numbers of cells medial to the mesonephros -presumably presumptive adrenal medullary cells. By 7 days incubafluorescent cells were tion, intensely present in the adrenal gland. Later on, fluorescence in all these structures was no longer dependent on exogenous DOPA. At no stage examined, whether DOPA had been administered or not, were cells showing specific fluorescence observed in the neural crest, invading the gastrointestinal tract or pancreas, or at sites en route from the crest to the tract. However, fluorescent cells were encountered within the presumptive intestine and dorsal pancreatic bud as shown in Tables 1 and 2. In the epithelium of the gut groove (i.e., the endodermal groove which will later close and become the intestine) of embryos as young as the 16-somite stage, cells scattered along a distance of more than seven somites’ length showed the ability to take up exogenous DOPA and decarboxylate it to dopamine. The cells TABLE
1
FORMALDEHYDE-INDUCED
FLUORESCENCE
APUD
CELLS IN THE OF CHICK EMBRYOS
DOPA administered
Stage” 16-18s 19-28s 29-32s 33-38s
fve +ve
No DOPA administered
or -veb +ve or
-ve -VE!
-ve
-ve
13d
few +ve -ve very few +ve few +ve
14d
+ve
+ve
16d
+ve
+ve
3$lld 12d
(3d)
IN
INTESTINAL EPITHELIUM DURING DEVELOPMENT
very
-ve -ve -ve few
+ve
u s = somites; d = days of incubation. * +ve = specific fluorescence; -ve = no fluorescent cells.
487
EMBRYOS TABLE
FORMALDEHYDE-INDUCED
APUD
CELLS
FLUORESCENCE IN
DEVELOPING
Stage
2
THE
PANCREAS
CHICK
IN OF
EMBRYOS”
DOPA administered
No
DOPA
administered
28-38s
+ve
- ve
3+13d 13-16d
+ve +ve
-ve +ve
‘I Abbreviations
as in Table
1.
were not very brightly fluorescent, and were not present in every embryo of the 16- to 18-somite group. In all embryos of 19-26 somites (Figs. 1 and 2) similar but more brightly fluorescent cells were distributed in the dorsal and lateral walls of the gut groove, from the anterior intestinal portal caudally. By the 24-somite stage, small groups were observed as well as single cells (Fig. 2). The endoderm in this region was considerably thickened. In 27-somite stage embryos, fluorescent APUD cells were more concentrated, most of them lying in the dorsal wall of the gut at the anterior intestinal portal (Fig. 3). Once the dorsal pancreatic diverticulum had begun to form (at or soon after the 27somite stage), a few scattered fluorescent cells were present in the roof of the (still open) gut immediately caudal to the diverticulum. The number of cells in this position subsequently dwindled, one or two being found occasionally in 3-day specimens. In the duodenum of 12- and 13-d&y DOPA-treated embryos, fluorescenr cells were again observed in the epithelium (Table 1). but these were very few. and their fluorescence not very hright. In the 14- and 16-day specimens, fluorescent cells were increasingly numerous and the fluorescence bright (Fig. 8). In embryos not treated with DOPA. no epithelial cells of the intestine up to 3 days of incubation, nor of the duodenum thereafter, showed fluorescence until day 13, when a very few were rather weakly
488
ANN
ANDREW
1. Scattered fluorescent cells in the gut groove of a 19-somite embryo. Longitudinal section. 2. Single and grouped fluorescent cells in the roof and lateral walls of the gut groove in a 24-somite embryo. Transverse section. FIG. 3. Fluorescent cells in the gut in the region of the anterior intestinal portal, where the dorsal pancreatic bud will form. Transverse section of 27-somite embryo. FIG. 4. Fluorescent cells, mostly in groups, at the periphery of the dorsal pancreatic bud of a 36-somite embryo. Transverse section. FIG. FIG.
FIGS. l-8. Cells showing specific formaldehyde-induced fluorescence in chick embryos treated with DOPA. (Magnification constant.) L-lumen of gut; A-dorsal aorta; DP-dorsal pancreatic bud.
APUD
CELLS
IN
fluorescent. At 14 days some were seen, but they were fewer and duller than when DOPA was administered; by 16 days, however, cells appeared equally numerous and equally brightly fluorescent. In the dorsal pancreatic bud of embryos treated with DOPA, brightly fluorescent cells were evident from the start of its evagination at the 27-somite stage (Table 2). These cells were present singly or in small groups within the diverticulum. Soon (by the 31- to 33-somite stage) small groups of fluorescent cells formed buds at the periphery of the advancing diverticulum. As the sprouts lengthened and branched during successive stages up to 14 days of incubation, the fluorescent cells remained at their tips (Figs. 4 and 5). The fluorescent material tended to lie basally in the cells. Later on-by 7 and 16 days, for example (Figs. 6 and 7) -numerous fluorescent cells were distributed singly and in groups (which were doubtless islets) in the parenchyma of the pancreas, and their cytoplasm was more uniformly fluorescent. As shown in Table 2, if no DOPA was administered, fluorescence was lacking in the pancreas until day 13 or 14. In this study, the various lobes of the pancreas were not distinguished. Fluorescent cells were absent from the ventral pancreatic buds while these were recognisable as distinct entities (4-5 days of incubation). Administration of nialamide to embryos of stages 17-20 somites, 26-30 somites, and 3314 days of incubation resulted in no obvious increase in the intensity of fluorescence in APUD cells in the presumptive intestine or the pancreas of DOPA-treated specimens. No definite fluorescence was provoked by nialamide in those embryos which had received no DOPA. DISCUSSION
In certain respects the present findings confirm for Black Australorp embryos, observations made by Polak et ul. (197 1) on
CHICK
EMBRYOS
489
APUD cells in DOPA-treated White Leghorn embryos: no cells in the neural crests were fluorescent: nor were any APUD cells seen invading the foregut. The fluorescent cells mentioned as likely to become incorporated in the adrenal medulla were doubtless those also seen by Polak et al. However, Polak et al. found no APUD cells at all in chick embryos before the 36somite stage, and made no mention of APUD cells in the presumptive intestine or in the dorsal pancreatic bud even after this time: in the present investigation, cells showing specific fluorescence after DOPA administration were evident in these sites well before the 36-somite stage and also in the differentiating pancreas and the duodenum later on. The reason why Polak et al. found no APUD cells at the early stages may lie in the different breed of their embryos or in the different method of DOPA administration used. They treated embryos removed from the eggs: in the present study embryos were treated in ova. Furthermore, paraformaldehyde equilibrated to a low humidity was used here. Fluorescence is not as intense as with higher humidities, but the fluorescent product is less likely to diffuse away. Polak et al. do not specify conditions of humidity. The appearance of fluorescent cells in the duodenal epithelium at and after 13 days of incubation was expected since Enemar et al. (1965) and Pentila and Mustakallio (1968) had demonstrated them first at 14 days (in the absence of exogenous DOPA). In the present study, administration of DOPA produced fluorescence only a day earlier-and then in only few cells. The above authors identified the fluorescent cells as enterochromaffin by silver impregnation. Other endocrine cell types in the intestine of mammals (ECL, G and D cells -nomenclature according to Solcia et al., 1973) are held to give the APUD reaction (Solcia et al., 1969; Hakan-
ANN
ANDREW
FIG. 5. Fluorescent cells in the growing buds sprouting from the dorsal pancreatic diverticulum in a 4-day embryo. FIG. 6. Scattered single and grouped cells in the pancreatic parenchyma of a 7-day embryo. FIG. 7. Scattered fluorescent cells in the pancreatic parenchyma of a 16-day embryo. FIG. 8. Fluorescent cells in the epithelium of the duodenum of a 16-day embryo. Transverse section.
APUD
CELLS
IN
son et al., 1969; Dawson, 1970; Pearse et al., 1970). Pearse (197 1) considers that L (EG) and S cells are also members of the APUD series. Quite unexpected was the demonstration of the ability of cells in the gut at a very early stage (16-somite stage, i.e., about 48 hr of incubation), to produce biogenic amine from administered precursor. There is, however, other evidence that the processes involved do occur in chick embryos at such early stages. That DOPA is taken up by the embryo by the first day of incubation is indicated by Ignarro and Shideman (1968a). The conversion of DOPA to dopamine occurs already on the second day (ibid.; Burack and Badge, 1964). That this dopamine is indeed synthesized intraembryonically is shown by its absence from the yolk of the fertilized egg (Ignarro and Shideman, 1968b). Biogenic amines are broken down by monoamine oxidase: this enzyme has been detected at 3 days of incubation by Ignarro and Shideman (1968~) and Castellano et al. (1974). The occurrence of fluorescent cells in the dorsal pancreatic bud from the time of inception of its formation was also unexpected. A considerable period elapsed before this fluorescence became independent of DOPA administration (day 13): a similar time-lag occurs in the endocrine pancrease of the guinea pig embryo (Cegrell and Falck, 1968) and the mouse embryo (Pearse et al., 1973). During the early stages of development (up to 33/4 days), under the conditions of the present study, not even the administration of nialamide provoked fluorescence in the pancreas or the gut groove in the absence of treatment with DOPA. Nor was there any increase in fluorescence if the embryos received exogenous DOPA as well. The reason is probably that monoamine oxidase is absent, or present in insufficient quantity to break down any biogenic amine produced: Castellano et al. (1974) have been unable
CHICK
EMBRYOS
491
to detect the enzyme in the pancreas before 13 days of incubation. This maneuver has thus not thrown light on the question of whether any biogenic amines are normally produced at these early stages. In this connection, the role of biogenic amines synthesized by APUD cells in the gut and pancreas is of interest. Unfortunately the functions of these amines are not yet clear in adults, let alone in embryos. The only suggestion mooted is that they play a part in hormone production or release (Cegrell, 1968b; Falck and Owman, 1968; Slanina and Tjalve, 197 I ). Quickel et a/. ( 1971) have suggested that the endogenous biogenic amines of the pancreas, serotonin and dopamine, inhibit secretory activity of B islet cells -a view favoured by Hahn von Dorsche and Krause (1973). Thus any early production of biogenic amine in the endocrine pancreas might be related to synthesis of pancreatic hormones. That these hormones are made early in development is known. In the rat, glucagon and insulin are produced as soon as, or soon after, the dorsal pancreatic bud forms (Rutter et al., 1968; Wessels, 1968; Clark and Rutter, 1972; Pictet and Rutter, 1972; Rall et al., 1973). In chick embryos, glucagon has been demonstrated at the 30somite stage, i.e., soon after the appearance of the dorsal pancreatic bud (Beaupain and Dieterlen-Lievre, 1974) and insulin a little later, at the 40- to 43-somite stage (Dieterlen-Lievre and Beaupain, 1974). As in higher vertebrates. it seems that in the fowl, glucagon or a glucagonlike hormone is synthesized by cells corresponding to A, cells (Mikami and ‘One, 1962) and insulin or an insulin-like hormone by B cells (Thommes, 1960: Mikami and Mutoh, 1971; Green and Benzo, 1973). In chick embryos, A (probably A,) cells have been identified in the dorsal pancreatic bud at 3 or 3.5 days (i.e., soon after its evagination) (Dieterlen-Lievre, 1963: Przbylski, 1967) and B cells at the same
492
ANN
time (Przbylski, 1967) or at 4 days of incubation (Dieterlen-Lievre, 1965). The identity of the APUD cells in the dorsal pancreatic bud in chick embryos has yet to be established. In the duck, both A, and A, cells show formaldehyde-induced fluorescence (Falck and Hellman, 1963); in the pigeon, A, and some A, cells (Trandaburu, 1972) do so. (No amine precursor was administered in these studies.) It does not necessarily follow that fowl APUD cells are A cells: in different species of other vertebrate groups, different islet cell types give this reaction-in certain newts A, cells, in frogs B, and in reptiles none (Trandaburu, 1972), in some mammals A cells, in others B, and in others both A and B cells are APUD (Cegrell, 1967a; 1968a, c, d; Cegrell et al., 1968; Legg, 1968). Dieterlen-Lievre (1970) has shown by isolating and culturing dorsal and presumptive ventral pancreatic buds separately, that both can give rise to A and-B cells. It is therefore strange that APUD cells were confined to the dorsal bud in the present study-a finding more in line with the previously held view that the dorsal bud becomes most of the splenic lobe, in which the majority of A islets form (DieterlenLievre, 1963). The presence of APUD cells in the walls of the gut groove from the 16-somite stage until the evagination of the dorsal pancreatic bud was entirely unexpected. If they are the precursors of the definitive APUD cells of the intestine, recognized from 13 or 14 days of incubation, they must temporarily lose their biogenic aminesynthesizing ability. Another possibility is that they are not directly related developmentally to any definitive APUD cells and have a merely transitory existence. Most attractive, however, is the possibility that these cells become pancreatic islet cells of one or more than one type. Certainly, shortly before the dorsal pancreatic bud evaginates, the APUD cells are concen-
ANDREW
trated at its site of origin; once it evaginates they are increasingly confined to it. Less easy to explain is the incorporation into the dorsal bud, of cells distributed previously over a considerable extent of the gut groove. However, it is not inconceivable that this whole extent becomes included in the diverticulum. For one thing, the duodenum arises at the levels of somites 8-15 (Le Douarin, 1961); since the pancreatic bud arises from the future duodenum it is possible that much of the gut roof of the 16-somite embryo (from the anterior intestinal portal-located at about the level of somite 5 -to approximately the level of somites 12, 13, or even 14) could be incorporated into the dorsal bud. Other evidence which supports this suggestion is that Wessels and Cohen (1967) and Wessels and Evans (1968) found that evagination of the diverticulum in the mouse is due to changes in cell shape and to cell movements rather than to proliferation. This implies the incorporation of a considerable extent of the roof of the gut groove into the evaginating bud. Very much in line with the present observations on the presence and distribution of APUD cells in the early chick gut, are the findings of Drews et ul. (1969). They describe cholinesterase-rich cells scattered in the gut endoderm at the 16-somite stage. The grouping and concentration of such cells in the dorsal pancreatic diverticulum when this formed, the formation of complexes of the cells at the periphery of the diverticulum and their subsequent identification as islet cells, indicate that these are the very cells which show the APUD ability. Indeed, cholinesterase activity is a characteristic of many APUD cells (Pearse, 1966a). However, the enzyme activity of the cells disappears on the fifth day of incubation whereas the biogenic amine-producing ability persists. Drews et al. do not hesitate to assume that the scattered cholinesterase-rich cells of the 16somite stage become islet cells. Observa-
APUD
CELLS
IN
tions of cells showing similar features (such as cholinesterase or APUD reactions) at successive developmental stages is highly suggestive but not conclusive evidence that they belong to the same cell line, however. Before the evagination of the pancreatic diverticulum, Wessels and Evans (1968) and Pictet et al. (1972) found no ultrastructural evidence of endocrine cells in the mouse and rat. Pearse et al. (1973), however, describe cells with pleomorphic granules in the presumptive pancreatic region of the mouse embryo before evagination occurs. They maintain that these cells differentiate into the various types of islet cells. Thus they suppose that, in the mouse, “primitive endocrine cells” in the intestine become islet cells; Pearse and Polak (1971 b), however, did not find APUD cells in the intestine of the mouse prior to formation of the pancreatic diverticulum. The hypothetical picture proposed by Pearse et al. (1973), of scattered “primitive endocrine cells” in the intestine, corresponds to the belief that phylogenetically and ontogenetically islet cells have arisen from specialized cells in the intestinal epithelium (Steiner et al., 1969; Falkmer, 1972; Epple and Lewis, 1973). If the APUD cells described in the gut groove of 16-somite embryos in the present study, are indeed future islet cells, then the existence of this hypothetical stage (see Epple and Lewis, Fig. 1D) is confirmed. Whether or not some or all such cells, rather than being endodermal in origin, are derivatives of the neural crest as Pearse and Polak (197 1 b) and Pearse et al. (1973) believe, is another question. No evidence for or against this contention came to light in the present study.
493
CHICK EMBRYOS
Levitan, and Miss D. Croft for technical assistance. A grant from the South African Medical Research Council for the purchase of the freeze-dryer is acknowledged with gratitude.
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