J. Embryol. exp. Morph. 82, 131-145 (1984)
Printed in Great Britain © The Company of Biologists Limited 1984
The distribution of endocrine cell progenitors in the
gut of chick embryos
ByB. B. RAWDON 1 , BEVERLEY KRAMER 1 AND
ANN ANDREW 2
1
Department of General Anatomy, School of Dentistry, University of the
Witwatersrand, 1 Jan Smuts Avenue, Milner Park, Johannesburg 2001,
South Africa
2
Department of Anatomy, Medical School, University of the Witwatersrand,
York Road, Parktown, Johannesburg 2193, South Africa
SUMMARY
The aim of this experiment was to find out whether or not, at early stages of development,
progenitors of the various types of gut endocrine cells are localized to one or more specific
regions of the gastrointestinal tract. Transverse strips of blastoderm two to four somites in
length were excised between the levels of somites 5 and 27 in chick embryos at 5- to 24-somite
stages and were cultured as chorioallantoic grafts. The distribution of endocrine cells in the
grafts revealed confined localization of progenitor cells only in the case of insulinimmunoreactive cells. The progenitors of cells with somatostatin-, pancreatic polypeptide-,
glucagon-, secretin-, gastrin/CCK-, motilin-, neurotensin- and serotonin-like immunoreactivity were distributed along the length of the presumptive gut at the time of explantation;
indeed, in many cases they were more widespread than are their differentiated progeny in
normal gut of the same age. This finding indicates that conditions in grafts must differ from
those that operate in the intact embryo. Also it may explain the occurrence of ectopic gut or
pancreatic endocrine cells in tumours of the digestive tract.
INTRODUCTION
The last decade has seen the discovery of an ever increasing number of types
of gastrointestinal endocrine cells containing as wide a variety of regulatory
peptides. Experimental evidence has shown that these cells are not neural crest
derivatives as had been proposed, but are endodermal in origin (Andrew,
Kramer & Rawdon, 1982). Nothing, however, is known about the mechanism
of their differentiation. An advantage for investigations of this problem, is that
in their differentiated state, the endocrine cell types are readily detected by
immunocytochemical methods. A disadvantage is that the cells are scattered in
the gastrointestinal tract. Were their progenitors gathered together at one place
at one time, the action of factors determining the path of their differentiation
would be easier to conceive of and to analyse.
Hence the experiment reported here was designed to find out whether or not,
at an early stage, progenitors of endocrine cells are localized in a particular
132
B. B. RAWDON, B. KRAMER AND A. ANDREW
region or regions of the presumptive gut. If so, many of them must subsequently
spread to other parts of the tract. The possibility of such localization was suggested
by the concentration of endocrine cells in the duodenum and pyloric region of
chicks (Rawdon & Andrew, 1981) and by the fact that the endocrine-rich dorsal
pancreatic bud originates here. Alumets et al. (1978) have expressed a similar
idea: they remark that soon after pancreatic polypeptide cells differentiate in the
developing chick pancreas, it appears that cells of this type spread cranially and
caudally from the duodenum. Furthermore, it seems that progenitors of some
types of pancreatic endocrine cells congregate at the site of evagination of the
dorsal pancreatic bud, subsequently dispersing through the gland (Drews,
Kussather & Usadel, 1969; Andrew, 1975).
Since progenitors of gut endocrine cells are not recognizable by any morphological features at early stages of chick development (Kramer & Andrew,
1983), their presence is detectable only by the demonstration of their differentiated progeny at a later time. Therefore blastoderm from different levels of
young chick embryos was isolated and cultured long enough for gut endocrine
cells to have differentiated. If the hypothesis were correct i.e. if progenitors of
a given type were localized to a specific region, the differentiated endocrine cells
would be restricted to grafts isolated from the relevant levels.
MATERIALS AND METHODS
The experiment was performed on Black Australorp chick blastoderms at 8to 24-somite stages (stages 8 to 16, Hamburger & Hamilton, 1951).
Transverse strips between two and four somites in length (Fig. 1) were excised
from levels (or presumptive levels) of somites 5 to 27 in Ringer's solution containing antibiotics (100 i.u. penicillin, 50//g garomycin and 100 fig neomycin per
ml). These strips included all three germ layers and extended only slightly
beyond the lateral confines of the embryo. The dorsal portions of the neural tube
and somites were removed from the explants when it became evident that the
grafts were rather large on retrieval. The lateral edges of the grafts were pinched
together prior to chorioallantoic grafting. Nine days later the grafts were
retrieved, subdivided if large, and regrafted to a second series of hosts. After a
further nine days of incubation the grafts were retrieved and fixed. On final
retrieval the grafts had attained a total incubation age of 20 to 21 days. By this
time endocrine cells are known to have differentiated in the gut of intact Black
Australorp embryos (Andrew, 1976a,b; Rawdon & Andrew, 1981).
The retrieved grafts were fixed with recrystallized parabenzoquinone (PBQ)
in one of two ways. Some grafts were snap-frozen in melting isopentane and then
freeze dried overnight at - 4 0 °C and 10~3 torr. This tissue was fixed in PBQ
vapour for 3 h at 60 °C (see Pearse & Polak, 1975) and embedded under vacuum
in a 1:1 epon: araldite mixture which was polymerized at 60 °C for 48 h. The
blocks were sectioned at 1 /im. Once gut had been located, the required number
Progenitors of gut endocrine cells
133
I
Fig. 1. Diagrams showing delimitation of a transverse strip of blastoderm two
somites wide from a donor chick embryo, and the explant, from which the dorsal
portions of the neural tube and somites have been removed.
of serial sections was cut and mounted. In order to locate gut more easily, the
remainder of the grafts was fixed in liquid PBQ for 1 to H h at 37 °C (as recommended by Bu'Lock, Vaillant, Dockray & Bu'Lock, 1982) and embedded in
paraplast. These grafts were completely serially sectioned at 5/im.
The indirect immunoenzyme technique was applied as previously described
(see Rawdon & Andrew, 1979). A list of the antisera used appears in Table 1.
The antisera to serotonin and secretin stained cells by the routine procedure in
the resin, but not in the paraplast, sections. However after pretreatment of the
latter sections for 30min at 37 °C with 0-05 % trypsin (Gibco) following Giddings, Griffin & Maclver (1982), secretin-like, but not serotonin-like, immunoreactivity was revealed.
The antiserum to avian pancreatic polypeptide (APP) was routinely absorbed
with 20/ig of insulin per ml diluted antiserum (see Rawdon & Andrew, 1979).
The antiserum L48, raised to the COOH-terminal octapeptide of cholecystokinin (CCK), is specific for the COOH-terminal pentapeptide (Dockray, 1979).
Since the latter is common to CCK and gastrin, the anti-serum reveals both
peptides.
Control procedures were applied to sections of each graft. These consisted of
replacement of the primary and secondary antisera with diluent alone, substitution of the primary antiserum by non-immune rabbit or guinea pig serum at
tDockray(1979)
$ Yanaihara et al. (1980).
Neurotensin
(fragment 8-13)
Bombesin (synthetic
anuran)
Serotonin
Pancreatic polypeptide
(natural avian)
Glucagon (porcine
pancreatic)
Secretin (natural porcine)
Cholecystokinin
(CCK8-II)
Motilin (synthetic
porcine)
1:100
1:1000/1:2000
—
t COOH-terminal
pentapeptide
X whole molecule
COOH-terminal
—
53
L48
1106
R94
—
fttaTA
1:500
? COOH-terminal
NH2-terminal
1)B31
2)YY59
We gratefully acknowledge these gifts.
1:16000
1:8000
1:16000
1:800
—
—
-8-11-8-76
1:8000
1:8000
Somatostatin (synthetic
cyclic ovine)
—
1:1000/1:2000
1:100
1:400/1:600
1:2000
1:1000
1:1000
—
1:8000
1:2000
1:2000
1:8000
Code
Insulin (natural porcine.)
Antiserum raised to
PBQ liquid
dilution
PBQ vapour
Regional
specificity
Table 1. Antisera used
* J. De Mey, Beerse
Immuno Nuclear Corporatin
* P. Emson, Cambridge
K. D. Buchanan, Belfast
* J. M. Polak, London
* G. J. Dockray, Liverpool
* W. G. Forssmann, Heidelberg
(Prepared by N. Yanaihara,
Shizuoka-Shi)
Milab
* J. R. Kimmel, Kansas City
* M. P. Dubois, Nouzilly
* L. Orci, Geneva
(prepared by P. H. Wright,
Indianapolis
Source
3
Z
w
o
Progenitors of gut endocrine cells
135
Table 2. Antigens used for absorption purposes
Antigen
Concentration
(^g/ml diluted antiserum)
Source
Insulin (natural porcine)
Somatostatin (synthetic)
Pancreatic polypeptide (avian
APP-II-117)
Glucagon (natural bovine/
porcine)
Secretin (synthetic porcine)
COOH-terminal pentapeptide
gastrin/CCK
Motilin (M5(ii), natural
porcine)
Neurotensin (synthetic
bovine)
Bombesin (synthetic
amphibian)
Serotonin
20
20
10
* Lilly Research Laboratories
Sigma Chemical Co.
* J. R. Kimmel, Kansas City
20
Research Plus Laboratories
20
20
* Hoffman-La Roche & Co.
Research Plus Laboratories
20
* J. C. Brown, Vancouver
40
Sigma Chemical Co.
20
Sigma Chemical Co.
500
Sigma Chemical Co.
' We gratefully acknowledge these gifts.
appropriate dilutions, or by primary antiserum absorbed with its antigen for 16
to 20 h at 4°C. Each antiserum absorbed with each of the other peptides listed
in Table 2 was tested on the gut of newly-hatched chicks. As a further control,
a section of chick gut known to contain endocrine cells of the relevant type was
included with each batch of graft sections to be stained with a particular antiserum.
Each antiserum was applied to gut in sections of the grafts at regular intervals
until at least ten sections had been stained or a minimum of ten positive cells per
graft had been revealed by that antiserum. This procedure was applied to every
subdivision of an original graft; the data from such subdivisions were combined
for analysis.
RESULTS
Of a total of 134 grafts prepared, 74 survived passage through two hosts. Of
these, 28 (38%) exhibited well-differentiated gut containing endocrine cells.
The removal of the dorsal portions of the neural tube and somites from 18 of
these explants did not affect the results of the experiment.
Simple tubular glands, similar to those in the pyloric region of newly hatched
chicks, were found in three grafts from somite levels 5 to 11. Pancreas was
identified in two grafts isolated from the levels of somites 5 to 8 and 10 to 13. The
remaining grafts exhibited villi with an epithelium of intestinal type (Fig. 3A)
containing variable numbers of goblet cells; it was not possible on the basis of
136
B. B. RAWDON, B . KRAMER AND A. ANDREW
Fig. 2A. A cell with neurotensin-like immunoreactivity (arrow) in a graft from the
level of somites 5-8 of a 13-somite embryo.
Fig. 2B. An adjoining control section treated with neurotensin antiserum preabsorbed with neurotensin. Bar equals lO/xm.
histology to distinguish with certainty between small and large intestine in the
grafts.
Fixation in PBQ vapour proved suitable for all of the endocrine cell types
sought. With the exception of serotonin-immunoreactive cells, this was true also
of material fixed in liquid PBQ. However, tissue fixed in liquid PBQ and embedded in paraplast revealed fewer cell types than did comparable grafts fixed
in PBQ vapour and embedded in resin. This suggests that the vapour fixation
and/or resin embedding is the superior procedure for demonstration of gastrointestinal endocrine cells in chicks.
Fig. 3A. Gut in a graft from the level of somites 21-22 of a 20-somite embryo.
Simple columnar epithelium lines well-formed villi. Bar equals 30jum.
Fig. 3B. Four groups of cells with insulin-like immunoreactivity, in pancreatic tissue
of a graft from the level of somites 10-13 of a 15-somite embryo. Bar equals 20 /im.
Fig. 3C. A cell with glucagon-like immunoreactivity, in a graft from the level of
somites 5-8 of a 12-somite embryo.
Fig. 3D. A cell with pancreatic polypeptide-like immunoreactivity, in a graft from
the level of somites 5-8 of a 12-somite embryo.
Fig. 3E. A cell with somatostatin-like immunoreactivity, in a graft from the level of
somites 8-9 of a 22-somite embryo.
Fig. 3F. Cells with gastrin/CCK-like immunoreactivity in a graft from the level of
somites 8-9 of a 22-somite embryo. Bar in (C-F) equals lOjum.
Progenitors of gut endocrine cells
137
Control procedures for immunocytochemistry gave satisfactory results (Fig. 2)
with one exception: in a small number of grafts, cells stained weakly with the
antiserum to bombesin but such staining was not quenched by preabsorption of
V
\
vt
B
Fig. 3
L
15
0
0
0
0
0
0
52
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
insulin
137
160
30
170
77
25
50
65
17
27
37
0
7
24
6
20
12
2
58
25
6
7
0
5
11
14
1
12
12
somatostatin
6
88
17
5
43
2
60
44
18
24
17
0
1
19
0
1
13
0
4
4
0
0
1
8
2
0
0
7
7
polypeptide
paiiuictiiit
213
48
33
17
72
23
30
43
30
26
44
0
1
105
0
19
60
0
14
95
0
4
6
37
0
0
0
24
26
glucagon
0
2
20
0
30
7
47
5
6
6
17
0
0
1
0
0
0
0
0
1
0
0
0
2
0
1
0
0
0
secretin
0
6
0
630
0
1
0
1
0
2
2
0
0
2
0
1
9
7
32
10
0
11
5
9
5
0
3
1
8
gastrin/CCK
0
9
7
5
33
1
2
0
2
38
28
4
1
32
0
3
13
0
5
8
0
21
0
11
4
3
1
7
12
motilin
Mean number of cells per 10 sections showing immunoreactivity for
$ Subdivisions of the same graft; in all other cases (*) data from subdivisions are poolec1.
t Serotonin-like immunoreactivity not demonstrable in liquid PBQ-wax blocks.
24-27
20-21
21-22
22-25
23-26
19-20
19-22
15
22
22
21
20
15*
19
21
16*
20*
17
23
20
20
21*
20
20*
23
22
24
20*
23*
12
21*
8-11
10-12
10-13
11-12
13-14
14-15
14-17
15-16
16-17
16-19
17-18
18-19
18-21
\
22* f
8- 9
r
8
12
5- 8
1 3
Donor stage
in somites
Somite
levels
Table 3. Numbers of endocrine cells in grafts from different somite levels
0
57
45
33
297
6
33
20
33
50
123
31
8
17
10
45
40
27
60
23
45
87
12
27
12
88
17
103
39
243
0
1
180
—
33
—
48
36
140
—
21
27
—
53
225
—
—
270
—
—
35
208
—
50
—
60
59
—t
neurotensin serotonin
u
w
M
u
>
>
w
w
Cd
o
w
w
Of
t—
Progenitors of gut endocrine cells
139
Table 4: Frequency of endocrine cell types in grafts from
different somite levels
Frequency of endocrine cells in grafts showing immunoreactivity for:
0 cells/10 sections
16-40 cells/10 sections
1-4
41-99
5-15
100^
"•"Subdivisions of the same graft; in all other cases ( * ) data from subdivisions are pooled.
ASerotonin-like immunoreactivity not demonstrable in liquid PBQ-wax blocks.
140
B. B. RAWDON, B. KRAMER AND A. ANDREW
the antiserum with its antigen. It was therefore considered to be non-specific.
Although this antiserum is known to stain cells specifically in the proventriculus
and gizzard (Rawdon & Andrew, 1981), it does stain cells in the intestine nonspecifically (personal observation).
In nine cases, grafts were subdivided before re-grafting into the second host.
With one exception (see later), analysis of the subdivisions of each of these grafts
showed good agreement with respect to the types of endocrine cells present.
Cells with insulin-, somatostatin-, APP-, glucagon-, secretin-, gastrin/CCK-,
motilin-, neurotensin- and serotonin-like immunoreactivity were demonstrated in
grafts (Figs 2A, 3B to.F). No cells were found to stain specifically for bombesin.
The distribution of endocrine cells in grafts is shown in Tables 3 and 4. The number
and variety of endocrine cell types differed from one graft to another. The tables
show the relationship of these differences to the somite levels explanted.
Insulin-like immunoreactivity occurred in only two grafts and in both instances
was restricted to pancreatic tissue separate from the gut epithelium. Secretinimmunoreactive cells were found in less than fifty percent of grafts and were
confined largely to explants from somite levels 5 to 15 with sporadic occurrence
more caudally. Cells showing serotonin- and neurotensin-like immunoreactivity
were the most numerous endocrine cell types found and occurred along with
somatostatin-immunoreactive cells in nearly all grafts; glucagon-, APP-, motilinand gastrin/CCK-immunoreactive cells were not as numerous, or present in as
many grafts, as the former three cell types. Cells showing APP-, somatostatinand glucagon-like immunoreactivity showed a decrease in number in grafts from
more caudal levels; with the exception of one graft from somite levels 8 to 9, the
reverse appeared to be true of gastrin/CCK immunoreactive cells. In one subdivision of this particular graft, cells with gastrin/CCK-like immunoreactivity
were very numerous in a pylorus-like region. No such cells were found in the
other subdivision of the same graft, but here the gut had the features of small
intestine. Cells with secretin-like immunoreactivity were present in the latter but
not in the former subdivision.
DISCUSSION
In interpreting the results of this experiment, the existence of recognizable
endocrine cell types in grafts was used as evidence for the presence of their
progenitor cells in the presumptive gut. Thus even a single cell of a given type
was proof of the existence of a progenitor in the explant. On the other hand,
failure to demonstrate any members at all of a particular cell type in a graft, was
only an indication that progenitors of that type were absent from the level
isolated. Seldom was more than one section in ten stained with a given antiserum: therefore if there were only a few cells of a type in a graft they might have
gone undetected. This limitation was mitigated by the fact that any given somite
level was represented in more than one graft.
Progenitors of gut endocrine cells
141
Restriction of progenitors to a confined region was indicated for cells with
insulin-like immunoreactivity. These cells were found only in the two grafts in
which pancreas differentiated. This finding is in fact not surprising: the levels of
the explants appear to be those from which the dorsal pancreatic bud normally
arises (see later and Drews, Kussather & Usadel, 1969; Andrew, 1975, 1984).
There is no reason to believe that the absence of demonstrable bombesin cells
from the grafts was attributable to technical failure. From the observations made
by Le Douarin (1964) it seems likely that the proventriculus develops from the
levels of the third and succeeding one or perhaps two somites, the gizzard from
the level of somite 5 and possibly the levels of somites 3 to 7. Although the most
cranial level included in the grafts in the present experiment was that of somite
5, recognizable proventriculus or gizzard were not detected. Hence it is conceivable that progenitors of bombesin cells are restricted to a more cranial level than
those tested: since in chicks at hatching bombesin cells are found only in the
stomach, evidence for extent of their progenitors to more caudal levels, although
possible, would not be expected.
Gastrin and CCK are not distinguished from one another by the COOHterminal-specific antiserum used in this study. Probably the very numerous cells
staining in one of the grafts with pyloric-type differentiation were gastrin cells;
at least the majority of sparse immunoreactive cells in intestine in a number of
grafts could have been CCK cells. Hence the results may mask separate populations of gastrin and CCK cell progenitors, each confined to different levels. On
the other hand separate progenitors may not exist: Larsson & J^rgensen (1978),
working on the duodenum of rats and of human foetuses, suggested that certain
cells may change from gastrin to CCK production during development.
The majority of cell types tested for were found in grafts from all levels. Thus
progenitors for cells with somatostatin-, pancreatic polypeptide-, glucagon-,
secretin-, motilin-, neurotensin- and serotonin-like immunoreactivity were not
confined to any region of the endoderm at the time of excision. However some
cell types diminished in frequency in grafts derived from successively more
caudal levels (notably secretin-, and also pancreatic polypeptide-, somatostatinand glucagon-immunoreactive cells), but their progenitors were obviously
present and widely distributed throughout the length of the early gut.
In general then, it seems that with a few possible exceptions, the progenitors
of endocrine cell types were not restricted to any region(s) of the gut at the stage
of excision of the explants. Where there did appear to be confinement of
progenitors of insulin cells, it is of course possible that in other regions their
progenitors might be present but unable to differentiate in grafts. It is apparent
that insulin cells do not normally differentiate in the gut of higher vertebrates the only instance known to us relates to the discovery of isolated cells with
insulin-like immunoreactivity in a specimen of small intestine from a 14-week
human foetus by Warson and Gepts (pers. commun.).
Perusal of the results obtained from our grafts suggested that in general, rather
142
B. B. RAWDON, B. KRAMER AND A. ANDREW
Dorsal pancreas
Normal gut
derived from
relevant somite
levels
Duodenum
Pylorus
Ileum
Rectum
Gizzard
Proventriculus
Insulin
Somatostatin
Pancreatic
polypeptide
Glucagon
Secretin
Gastrin/CCK
Motilin
Neurotensin
Bombesin
Serotonin
Somite levels
of explants
5-8
Possible
Definite
8-15
extent of
presumptive
gut region
16-23
24-27
Normal gut
Normal gut
and grafts
Grafts
Fig. 4. Distribution of progenitors of gut endocrine cells in embryos and of
endocrine cells in chicks at hatching.
than demonstrating concentration of progenitors, we had revealed a more
widespread distribution than is indicated by the types present in normal gut of
the same age. To make this comparison, we used available data on the
distribution of gut endocrine cells in chicks at hatching: the data were mostly
immunocytochemical (Rawdon & Andrew, 1981), but were solely ultrastructural for enterochromaffin (serotonin-containing) cells (Andrew, 197'6a,b). We
related these in Fig. 4 to data provided by Le Douarin (1964) and reinforced by
Sumiya (1976) on the prospective fate of the endoderm at various somite levels
of 9- to 27-somite chick embryos. In this way levels isolated in the present
experiment could be correlated with the regions of the gastrointestinal tract into
which they normally develop. The pyloric region of chicken gut having been
recognized only recently (Larsson et al. 1974), it was not referred to in the above
reports. Consideration of Le Douarin's findings suggests to us that it arises from
Progenitors
of gut endocrine cells
143
the vicinity of somites 7 and 8. According to observations recorded by Le
Douarin (1964) and by Sumiya (1976), presumptive dorsal pancreatic bud tissue
lies between the levels of somites 9 and 13, possibly extending as far cranially as
somite 6. Available evidence of Le Douarin & Bussonnet (1966) suggests that
presumptive ventral pancreatic bud tissue extends even further cranially.
Therefore all four pancreatic endocrine cell types could be expected in grafts
isolated from these levels. In chick embryos the dorsal pancreatic bud has been
shown by Manning (1984) to be the source of pancreatic insulin, glucagon and
somatostatin cells whereas pancreatic polypeptide cells are derived from the
ventral bud.
It is known that much of the presumptive fore- and mid-gut shifts caudally
during development and thus is situated opposite more cranial somites at early
than at later stages (see for example, Noden, 1983). Le Douarin (1964) for
instance, noted such a shift between 9- to 15-somite stages on one hand and four
or five days of incubation on the other. Therefore -the presumptive fate of
endoderm excised at the level of the same somites in embryos of different ages
would vary. The difference is probably small in the embryos used in the present
experiment - the most advanced were only about a day and a half older than the
youngest. However, in order to minimize this source of error in compiling Fig.
4, grafts from more cranial levels of the older donors were excluded.
As shown above, completely accurate equation of somite levels with prospective regions of the digestive tract was not possible in all cases. Nevertheless it is
clear from Fig. 4 that (with the possible exception of bombesin cells) all the cell
types present in any given region in chicks at hatching, were also present in the
corresponding grafts. Surprisingly, however, it was apparent that additional cell
types had differentiated in grafts from certain levels, especially the more caudal
ones. Thus although cells with secretin-like immunoreactivity are present in the
hatched chick only in the duodenum, they occurred in grafts from levels giving
rise to more cranial and more caudal regions of the gut; cells with somatostatin-,
pancreatic polypeptide-, glucagon-, gastrin/CCK- and motilin-like immunoreactivity were present in grafts from levels giving rise to hindgut, and in the case
of motilin-immunoreactive cells, also to caudal small intestine: yet these cell
types have not been found in the respective parts of the gut in chicks at hatching.
The above discrepancies might conceivably result from a difference in the rate
of differentiation between grafts and normal gut but it is to be hoped that any
retardation or acceleration in the grafts was minimal. Certainly on retrieval the
grafts had reached the same incubation age as the chicks with which they have
been compared.
The probability that the experiment has revealed a real difference between the
cell types appearing in grafts and in normal gut developed from the same somite
levels is an exciting one. It may be deduced that in normal gut, progenitors for
some cell types are present in certain regions (the hind gut in particular), but do
not fulfill their potentialities. This implies that in grafts the conditions must differ
144
B. B. RAWDON, B. KRAMER AND A. ANDREW
from those in normal developing gut. An analysis of the differences should prove
significant for an understanding of the factors affecting the differentiation of
these cells.
The presence in a given region, of endocrine cell progenitors which do not
normally differentiate there might explain the occurrence of ectopic endocrine
tumours in that part of the human gastrointestinal tract. Even where there is no
evidence for progenitors in grafts or normal gut from certain regions, precursors
may nevertheless exist and may become neoplastic. Insulin cells for instance
have been found in tumours of the hind gut by Alumets, Hakanson & Sundler
(1981).
Another point of interest in relation to oncognesis concerns stem cells in
mature gut. Whereas restricted localization of progenitors of endocrine cells
would imply that these cells arise from separate stem cells, the present findings
leave open the possibility that the endocrine cells are produced by the same stem
cells as other epithelial cells. The latter view was put forward by Cheng &
Leblond (1974a,b) and has been supported by Bjerknes & Cheng (1981a, fr),
Inokuchi, Fujimoto & Kawai (1983) and Stein & Morris (1984a, b).
The present experiment has revealed little evidence for localization of
progenitors of endocrine cells in the digestive tract of early embryos. Nevertheless, the demonstration in certain regions, of progenitors which normally fail to
develop further may provide unexpected clues to the control of differentiation
of gut endocrine cell types.
The authors wish to thank sincerely the donors of antisera and antigens indicated in Tables
1 and 2. We acknowledge with gratitude grants from the South African Medical Research
Council and the Research Committee of the Council of the University of the Witwatersrand,
Johannesburg. We should also like to thank our research assistants and departmental technicians for their skilled technical assistance.
REFERENCES
ALUMETS, J., ALM, P., FALKMER, S., HAKANSON, R., LJUNGBERG, O., MARTENSSON, H.,
SUNDLER, F. & TIBBLIN, S. (1981). Immunohistochemical evidence of peptide hormones in
endocrine tumors of the rectum. Cancer 48, 2409-2415.
& SUNDLER, F. (1981). Distribution, ontogeny and ultrastructure
of pancreatic polypeptide (PP) cells in the pancreas and gut of the chicken. Cell Tissue Res.
194, 377-386.
ANDREW, A. (1975). APUD cells in the endocrine pancreas and the intestine of chick embryos. Gen. comp. Endocr. 26, 485-495.
ANDREW, A. (1976a). Intestinal endocrine cells of chicks around the time of hatching. Cell
Tissue Res. 172,541-551.
ANDREW, A. (19766). Endocrine cells of the stomach of chicks around the time of hatching.
Cell Tissue Res. 172, 553-561.
ANDREW, A. (1984). The development of the gastro-entero-pancreatic neuroendocrine system in birds. In Evolution and Tumour Pathology of the Neuroendocrine System. Fernstrom
Foundation Series, vol. 4. Amsterdam: Elsevier (in press).
ANDREW, A., KRAMER, B. & RAWDON, B. B. (1982). The embryonic origin of endocrine cells
of the gastrointestinal tract. Gen. comp. Endocr. 47, 249-265.
ALUMETS, J., HAKANSON, R.
Progenitors of gut endocrine cells
145
M. & CHENG, H. (1981a). The stem-cell zone of the small intestinal epithelium. I.
Evidence from Paneth cells in the adult mouse. Am. J. Anat. 160, 51-63.
BJERKNES, M. & CHENG, H. (19816). The stem cell zone of the small intestinal epithelium. II.
Evidence from Paneth cells in the newborn mouse. Am. J. Anat. 160, 65-75.
BU'LOCK, A. J., VAILLANT, C , DOCKRAY, G. J. &BU'LOCK, J. D. (1982). A rational approach
to the fixation of peptidergic nerve cell bodies in the gut using parabenzoquinone. Histochemistry 74, 49-55.
CHENG, H. & LEBLOND, C. P. (1974a). Origin, differentiation and renewal of the four main
epithelial cell types in the mouse small intestine. III. Entero-endocrine cells. Am. J. Anat.
141, 503-520.
CHENG, H. & LEBLOND, C. P. (19746). Origin, differentiation and renewal of the four main
epithelial cell types in the mouse small intestine. V. Unitarian theory of the origin of the four
epithelial cell types. Am. J. Anat. 141, 537-562.
DOCKRAY, G. (1979). Cholecystokinin-like peptides in avian brain and gut. Experientia 35,
628-629.
DREWS, U., KUSSATHER, E. &USADEL, K. H. (1969). Cholinesterase-Aktivitatbeider Friihentwicklung der Inselanlagen des Huhnerembryos. Hormone metabolism Res. 1, 14-18.
GIDDINGS, J., GRIFFIN, R. L. & MACIVER, A. G. (1982). Demonstration of immunoproteins
in araldite-embedded tissues. /. Clin. Path. 35, 111-114.
HAMBURGER, V. & HAMILTON, H. L. (1951). A series of normal stages in the development of
the chick embryo. /. Morph. 88, 49-92.
INOKUCHI, H., FUJIMOTO, S. & KAWAI, K. (1983). Cellular kinetics of gastrointestinal mucosa,
with special reference to gut endocrine cells. Arch. Histol. Jap. 46, 137-157.
KRAMER, B. & ANDREW, A. (1983). Are precursors of gut or pancreatic endocrine cells
morphologically detectable? IRCS Med. Sci. 11, 201-202.
LARSSON, L.-I. & J0RGENSEN, L. M. (1978). Ultrastructural and cytochemical studies on the
cytodifferentiation of duodenal endocrine cells. Cell Tissue Res. 194, 79-102.
LARSSON, L.-I., SUNDLER, F., HAKANSON, R., REHFELD, J. F. & STADIL, F. (1974).
Distribution and properties of gastrin cells in the gastrointestinal tract of chicken. Cell
Tissue Res. 154, 409-422.
LE DOUARIN, N. (1964). Etude experimental de l'organogenese du tube digestif et du foie
chez l'embryon de Poulet. Bull. Biol. Fr. Belg. 98, 543-673.
LE DOUARIN, N. & BUSSONNET, C. (1966). Determination precoce et role inducteur de
l'endoderm pharyngien chez l'embryon de poulet. C.r. hebd. Sianc. Acad. Sci., Paris 263,
1241-1243.
MANNING, D. M. (1984). The potential of the dorsal and ventral pancreatic buds in the avian
embryo to giveriseto endocrine cells. Ph.D. thesis, University of the Witwatersrand, Johannesburg.
NODEN, D. M. (1983). The embryonic origins of avian cephalic and cervical muscles and
associated connective tissues. Am. J. Anat. 168, 257-276.
PEARSE, A. G. E. & POLAK, J. M. (1975). Bifunctional reagents as vapour and liquid phase
fixatives for immunohistochemistry Histochem. J. 7, 179-186.
RAWDON, B. B. & ANDREW, A. (1979). An immunocytochemical study of the distribution of
pancreatic endocrine cells in chicks, with special reference to the relationship between
pancreatic polypeptide- and somatostatin-immunoreactive cells. Histochem. 59, 189-197.
RAWDON, B. B. & ANDREW, A. (1981). An immunocytochemical survey of endocrine cells in
the gastrointestinal tract of chicks at hatching. Cell Tissue Res. 220, 279-292.
STEIN, B. A. & MORRIS, J. F. (1984a). The development of the gastric antral epithelium in the
rat. I. Epithelial cell types (submitted to Cell Tissue Res.).
STEIN, B. A. & MORRIS, J. F. (19846). The development of the gastric antral epithelium in the
rat. II. Epithelial cell kinetics (submitted to Cell Tissue Res.).
SUMIYA, M. (1976). Localization of autodifferentiation potencies in the early endoderm of the
chick embryo. /. Fac. Sci. Univ. Tokyo Sect. IV, 13, 363-381.
BJERKNES,
YANAIHARA, N., YANAIHARA, C , NAGAI, K., SATO, H., SHIMIZU, F., YAMAGUCHI, K. & ABE,
K. (1980). Motilin-like immunoreactivity in porcine, canine, human and rat tissues.
Biomed. Res. 1, 76-83.
(Accepted 12 March 1984)