/. Embryo/, exp. Morph. Vol. 31, 3, pp. 589-598, 1974
Printed in Great Britain
589
Further evidence that enterochromaffin cells are
not derived from the neural crest
By ANN ANDREW 1
From the Department of Anatomy, University of the Witwatersrand
SUMMARY
Recently, a previous finding that the enterochromaffin cells of chick embryos are not
derived from the neural crest has been contested, and so further evidence has been sought.
Presumptive gut, i.e. endoderm and adherent mesoderm, of embryos between the short headprocess stage and the 25-somite stage was grown on the chorio-allantoic membranes of host
embryos. Whether the presumptive gut was excised before or after the probable time of arrival
of neural crest cells in the gut, enterochromaffin cells occurred in the intestine in the grafts.
The presence or absence of enteric ganglia indicated the presence or absence, respectively, of
neural crest cells. Enterochromaffin cells were plentiful even if the donor had been at a stage
preceding that at which cells of the neural crest start to migrate, or preceding that at which
the crests themselves first appear. In a second experiment, presumptive gut of embryos at
10- to 21-somite stages was excised so as to exclude the portion underlying the somites.
Enteric ganglia were lacking in the intestine of these grafts, but enterochromaffin cells were
invariably present.
These experiments show that the precursors of enterochromaffin cells are present in the
more lateral part of the presumptive gut before the neural crest precursors of enteric ganglia
reach the region; and that they are present in the presumptive gut long before any crest cells
could have arrived there. This evidence supports the view that enterochromaffin cells are not
derived from the neural crest in chick embryos.
INTRODUCTION
Despite the chemical and biochemical similarities between enterochromaffin
cells, pigment cells and cells of the suprarenal medulla, it was shown some years
ago that enterochromaffin cells of chick embryos are not derived from the same
embryonic source as these other cell types, i.e. the neural crest (Andrew, 1963).
Recently, however, Pearse and his co-workers have become increasingly persuaded of the likelihood that endocrine cells of the gastro-intestinal tract (these
include enterochromaffin cells) do originate from the neural crest (Pearse, 1969;
Pearse & Polak, 1971a, b; Polak, Rost & Pearse, 1971).
Further evidence has therefore been sought. An experiment was designed to
find out whether or not progenitors of enterochromaffin cells are present in the
presumptive gut of chick embryos before neural crest cells migrate into it.
A second experiment which provides confirmatory evidence is also reported.
1
Author's address: Department of Anatomy, University of the Witwatersrand Medical
School, Hospital Street, Johannesburg, South Africa.
590
A. ANDREW
MATERIALS AND METHODS
Enteric ganglion cells are generally accepted to be neural crest derivatives in
chick embryos (Hammond & Yntema, 1947; Yntema & Hammond, 1952, 1954,
1955) and so the time at which they arrive in the presumptive gut indicates the
time of arrival of neural crest cells. Progenitors of enteric ganglion cells reach
the gut between the 10- and the 12-somite stages (Andrew, 1964), attained at
about 36 to 40 hours of incubation. This is the earliest reported time for this
event (cf. van Campenhout, 1931, 1932; Yntema & Hammond, 1954; Le
Douarin & Teillet, 1973). However, it is conceivable that other crest cells enter
the gut wall even earlier. Therefore, in some cases presumptive gut was excised
from donors considerably younger than the 10-somite stage. As a form of
control, donors of more than 12 somites were used to provide presumptive gut
already containing neuroblast progenitors.
It was noticed that if that part of the presumptive gut underlying the somites
was excluded from explants from donors of 10 or more somites, enteric ganglion
cells were lacking in the differentiated intestine. In a second experiment, explants were therefore designed in this way: the aim was to find out whether or
not progenitors of enterochromaffin cells would be present in the absence of
neuroblasts.
Black Australorp and other embryos served as donors in the first experiment,
and only Black Australorps in the second.
Delimitation of explants
(a) Experiment 1. Ectoderm and adherent mesoderm were peeled off the
endoderm and adherent mesoderm of blastoderms by the method adopted
previously (Andrew, 1964). The notochord is tightly applied to the endoderm,
and so the endoderm below it was excluded from the explants, as was the midline strip under the primitive node and primitive streak. Figure 1A illustrates the
limits of the explant in a young donor. Delimitation was similar at older stages;
however, once the anterior intestinal portal had developed, most of the invaginated part of the foregut was included in the explant, as illustrated in Fig. 1B.
This figure also shows the position of the cranial limiting incision. The explants
included all of the presumptive gut area as mapped by Rosenquist (1971, fig. 5 c),
except for the most rostral end of the foregut. Also when the donors were older,
the region excised was split lengthwise along the midline, so providing two
explants each large enough to be grown successfully.
Donors ranged from the late primitive streak stage to the 25-somite stage,
and were well distributed among intervening stages.
(b) Experiment 2. Explants for this experiment were excised as above, except
that the median delimiting incisions were made at the level of the lateral borders
of the somites in the older embryos, or just slightly medial to this in the younger
Derivation of enterochromqffin cells
591
Fig. 1. Diagrams illustrating the delimitation of explants of presumptive gut of chick
embryos. The dotted lines show the outline of the endoderm and adherent mesoderm
excised after the ectoderm and adherent mesoderm had been stripped off. (A)Expt. 1.
Head-process stage donor. (B) Expt. 2. 12-somite stage donor.
embryos (Fig. IB). Donors were well distributed between the 10- and the
21-somite stages.
An attempt was made to roll up each explant and pinch the ends together to
form a tube with the endoderm on the inside. The ends sometimes separated
during grafting, however.
Culture of explants
Explants were grafted to the chorio-allantoic membranes of hosts (White
Leghorn or a Rock-Cornish cross) incubated for nine days, as described elsewhere (Andrew, 1963). After eight days' sojourn, the grafts were retrieved,
subdivided if large, and cultured for a further eight days in a second series of
hosts. As the donors had been incubated for one and a half to two days, the final
37
EMB
31
592
A. ANDREW
age of the grafts was 17^-18 days. At this age, both enteric ganglion cells and
enterochromaffin cells are numerous and well differentiated in normal embryos
(see Andrew, 1963, 1964).
Survival of grafts
Of 147 explants cultured, 59 were represented by the grafts retrieved from the
second hosts. The figure was low, due mainly to the poor survival of explants
prepared from donors younger than the early somite stages.
Histological methods
Retrieved grafts were fixed in 10 % formalin containing 2 % calcium acetate.
Paraffin sections were cut serially at 5 /im. A control section of the duodenum
of a normal 16-day Black Australorp embryo was mounted on each slide.
Sections on alternate slides were impregnated by modified Bodian protargol and
Masson-Fontana methods as before (Andrew, 1963). These procedures demonstrate nerve cells as well as the argyrophilia and argentaffinity of enterochromaffin cells. Impregnation and staining of both cell types in the control sections
provided evidence of the efficacy of the methods.
RESULTS
Well-differentiated intestine was present in the retrieved subdivisions of 41 out
of 59 original explants. Gizzard was found in five of these and in one other
graft. The criteria defined previously (Andrew, 1964) (with the exception of the
presence of enterochromaffin cells) were applied when deciding whether
intestine or gizzard was differentiated adequately for any progenitors of enterochromaffin cells and neurones present, to have become readily distinguishable.
Only gut meeting these requirements is analysed in Table 1. This degree of
differentiation is illustrated in Figs. 2 and 4.
Table 1 shows that if the presumptive gut was excised at or after the 10-somite
stage, enteric ganglion cells were present in the gut of all grafts except one. This
one had been prepared from a 12-somite donor, i.e. one of the youngest embryos
in this group. In the other grafts enteric ganglia were not numerous, but were
easily identifiable. On the other hand, enteric ganglia were absent from the gut
of grafts excised before the 10-somite stage. Enterochromaffin cells were
nevertheless present in the intestine and gizzard of all grafts, whether they had
been excised before or after the 10-somite stage. Enterochromaffin cells in the
intestine of a graft prepared from a 2-somite donor are illustrated in Fig. 3.
The results of the second experiment (Table 1) confirm the observation that
enteric ganglia are lacking if the presumptive gut beneath the somites is excluded
from explants (see p. 590). In these grafts, too, enterochromaffin cells were
invariably present (Fig. 5).
Derivation of enterochromaffin cells
593
200 /<m
4 -..-:•*>-- .
issmmm
s
Figs. 2 to 5. Sections through chorio-allantoic grafts of presumptive gut cultured
until 17J-18 days old. Figs. 2 and 4 illustrate the degree of differentiation in
intestine (V = villous folds; M = muscle coat). Figs. 3 and 5 demonstrate the
presence of enterochromaffin cells (EC).
Fig. 2. Expt. 1: neural crest excluded. Donor: 5 somites (Formalin; Bodian
protargol).
Fig. 3. Expt. 1: neural crest excluded. Donor: 2 somites (Formalin; Fontana).
Figs. 4 and 5. Expt. 2: prospective enteric ganglion cells excluded. Donor: \1\
somites (Formalin; Fontana).
37-2
594
A. ANDREW
Table 1. The occurrence of enterochromaffin cells and enteric ganglion cells
in chorio-allantoic grafts of presumptive gut of chick embryos
No. of grafts
showing presence of
Experiment
Explant delimited:
Donor stage
1
After crest arrival
Before crest arrival
2
Lateral to somites
10 to 25 s*
Short head process
to 9 s
10 to 21 s
* s = pairs of somites.
No. of
grafts
Enteric
Enteroganglion chromaffin
cells
cells
8
16
7
0
8
16
17
0
17
In almost all the intestine in grafts resulting from both experiments, enterochromaffin cells were numerous - often they were obviously more plentiful
than in a comparable extent of intestinal epithelium of intact 16- or 17-day
embryos. Both the reaction for argyrophilia and that for argentaflfinity gave
positive results on the sections of intestine. In the gizzard, only argyrophil
cells were present, and these were few: this is the situation in normal
embryos.
Pigment cells, being neural crest derivatives, might have been expected in
grafts excised after the arrival of neural crest cells. Yet, although most of the
donors used in the two experiments belonged to a highly pigmented breed, with
one exception no pigment cells were found. The exception was a graft prepared
in experiment 2 from an 11-somite donor. The median delimiting incision was
near the lateral border of the somites as in Fig. 1B. Pigment cells were extremely
numerous, lying even between the muscle layers and the lamina propria of the
intestine. Clearly, neural crest cells had accidentally been included in this
explant. It is surprising that enteric ganglia were not present too. This is an
interesting observation, but possible explanations are not relevant here.
DISCUSSION
The first experiment was based on the premise that neural crest cells do not
reach the presumptive gut before the 10-somite stage. The presence of enteric
ganglion cells in the gut of grafts which had been excised at or after this stage
confirmed the presence of neural crest cells in all cases except one. That the
numbers of enteric ganglia were small is because nothing like the full complement of prospective neuroblasts could have reached the presumptive gut by
even the oldest donor stage used (just under two days' incubation): they begin
to arrive in some parts of the digestive tract only at five days (Le Douarin &
Teillet, 1973). The occurrence of enterochromaffin cells in these grafts showed
Derivation of enterochromaffin cells
595
that enterochromaffin cells can differentiate in chorio-allantoic grafts of
presumptive gut. Enteric ganglia were lacking, but enterochromaffin cells were
plentiful if the explants had been prepared from donors younger than the
10-somite stage. Therefore, progenitors of enterochromaffin cells are present in
the presumptive gut before the prospective neuroblasts arrive there from the
neural crest. It is conceivable that other neural crest cells arrive even earlier than
do prospective neuroblasts. They could not, however, arrive before the time at
which the migration of cells away from the neural crest begins. This happens at
the 8-, or perhaps the 7-, somite stage (see Holmdahl, 1928; Romanoff, 1960;
Andrew, 1963). Therefore only exceptionally could any crest cells reach the
presumptive gut before the 7-somite stage. Eight of the grafts analysed in
Expt. 1 were from donors having fewer than 8 pairs of somites. In fact, all eight
donors were younger than the 6-somite stage, the stage at which the neural
crests make their very first appearance (Holmdahl, 1928). The differentiation of
enterochromaffin cells in all these explants therefore provides good evidence
that enterochromaffin cells are not derived from the neural crest.
The prospective neuroblasts which have reached the presumptive gut by
stages at and after the 10-somite stage can be excluded from explants by
excluding that part of the gut underlying the somites: this is demonstrated by
the lack of ganglion cells in the grafts of Expt. 2. That enterochromaffin cells
nevertheless occur in these grafts is consistent with the conclusion of the first
experiment.
Here again, one wonders whether other neural crest cells might arrive in the
gut before those destined to become neuroblasts. In the author's experience
(Andrew, 1969, 1970), it is usual that if neural crest cells are included in chorioallantoic grafts, enteric ganglion cells appear. (Definite exceptions number 4 out
of 50 grafts in the work cited; one exception has been noted in the present
study, p. 594). If crest cells are excluded, enteric ganglia are invariably absent.
These findings apply to vagal and to trunk levels of the neural crest (Andrew,
1969, 1970) even though, according to Le Douarin & Teillet (1971, 1973), the
more cranial trunk levels do not give rise to enteric ganglia in intact embryos. This
means that crest cells which do not normally differentiate into enteric ganglia
can do so in chorio-allantoic grafts. It has long been held that the environment
at their final location plays a part in determining the fate of some neural crest
cells (see Balinsky, 1970; Willier, Weiss & Hamburger, 1956) though perhaps
not pigment cells (Stevens, 1954). In other words, it is possible that some crest
cells are still pluripotent on arrival at their destination. This accords with the
above idea that in the absence of the neuroblasts which normally become enteric
ganglion cells, other crest cells can and do differentiate in this way. Thus, in
general, the absence of enteric ganglia is evidence that neural crest cells are
absent.
The results of the present investigation clearly confirm the conclusion drawn
previously (Andrew, 1963) that enterochromaffin cells are not derived from the
596
A. ANDREW
neural crest. The earlier experiment was based on the culture of all three germ
layers of the blastoderm, pieces of which were excised so as to exclude the neural
crests as such from the experimental grafts, and so as to include them in controls.
Enterochromaffin cells were present in all well-differentiated gut of experimental
as well as control grafts.
Very recently, in an article concerned mainly with the origin of enteric ganglia,
Le Douarin & Teillet (1973) have reported pertinent observations. They grafted
quail neural crest to chick embryos at, and caudal to, rhombencephalic levels,
and could find no cells with characteristics of quail cells in the epithelium of the
digestive tract. The enterochromaffin cells seen were host cells. These authors
conclude that enterochromaffin cells do not arise from vagal or trunk levels of
the neural crest.
The connection between the above conclusions and Pearse's view must now
be considered. Enterochromaffin cells are among the cell types which Pearse
has grouped together as the APUD cell series, on the basis of common cytochemical, ultrastructural and functional similarities (see for instance Pearse.
1966«, 1969). ('APUD' stands for Amine Precursor-Uptake and Decarboxylation.) Other examples are pituitary corticotrophs and melanotrophs, melanocytes, pancreatic A and D cells, mast cells, suprarenal chromaffin cells and
thyroid C cells (Pearse, 1966«, b, 1969). Though aware of the diverse origins
ascribed to these cell types, Pearse (1966a, b, 1969) has suggested that they all
arise from the neural crest. Originally, his idea was based on the mechanism of
amine storage and the presence of cholinesterase, both common to the firstidentified members of the series. These features suggested to him a common
ancestor of neural, perhaps neural crest, origin (Pearse, 1966a).
More recently, Pearse and his co-workers have emphasized the likelihood that
some, probably all, the endocrine polypeptide (APUD) cells of the gastrointestinal tract are derived from the neural crest. This view is founded on the
claim that they have traced cells showing the APUD reaction, from the neural
crest to the suprarenal medulla of chick embryos (Polak et ah 1971) and to the
foregut (including stomach and duodenum) of mouse embryos (Pearse & Polak,
1971Z?); it was reinforced when they found that thyroid C cells originate in the
neural crest (Pearse & Polak, 1971 a).
The above studies provide the basis for Pearse's attractive hypothesis, but
morphological observations of cells in embryos at successive stages of development cannot constitute conclusive evidence that APUD cells of the gut are
derived from the neural crest. Even though they give the same reactions, the cells
in the suprarenal gland or in the gut wall at later stages, and the cells designated
as neural crest cells at the earlier stages, may not be the same cells. The latter
may be neural crest cells with some other destiny: prospective sympathetic
ganglion cells, for instance, acquire APUD characteristics (Cohen, 1969).
In any case, it has once again been demonstrated that one cell type belonging
to the endocrine APUD group in the gastro-intestinal tract, the enterochromaffin
Derivation of enterochromaffin cells
597
cell, is clearly not derived from the neural crest; the evidence of Pearse and his
co-workers cannot gainsay this conclusion.
The author would like to thank Professor P. V. Tobias for his continued interest and
encouragement; Mr R. J. Herman, Mrs N. G. de Maar and Mrs B. Levitan for proficient
technical assistance; Miss J. Walker for preparing the diagrams; and Mrs K. Copley for
typing the manuscript.
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{Received 12 August 1973)