/ . Embryol. exp. Morph. Vol. 60, pp. 235-243, 1980
Printed in Great Britain © Company of Biologists Limited 1980
235
Induction of supernumerary tracheal
buds and the stimulation of DNA synthesis in the
embryonic chick lung and trachea by
epidermal growth factor
By GEOFFREY V. GOLDIN 1 AND LYNNE A. OPPERMAN 1
From the Department of Zoology, University of the
Witwatersrand, Johannesburg
SUMMARY
Epidermal growth factor (EGF) has been found to stimulale DNA synthesis in both the
trachea and bronchial tree of 5-day-old chick embryo lung rudiments in organ culture. After
20 h culture in the presence of 10 ng/ml EGF, the incorporation of tritiated thymidine into
DNA is stimulated two- to three-fold following a 2 h labelling period, as revealed by scintillation counting. Autoradiographic data indicate that this stimulation is most marked in the
epithelial tissue component of both the trachea and bronchial tree. Supernumerary 'lung'
buds have been induced in the normally unbranched tracheal epithelium by agarose pellets
containing EGF, such buds having been previously induced only by grafting a variety of
mesenchymal tissues alongside the tracheal epithelium. Since EGF has been shown to be a
potent stimulator of tracheal DNA synthetic activity it is suggested that the induction of
supernumerary buds by the EGF-agarose pellets is achieved through a localized stimulation
of cell proliferation in the tracheal epithelium. These data would further suggest that the
induction of supernumerary tracheal buds by various mesenchymal tissues is similarly due to
a localized increase in mitotic activity resulting from the action of some mitotic stimulator
substance(s) emanating from the inducing mesenchymal tissue. This conclusion may be
extended to include normal bud formation which occurs during branching morphogenesis in
everal developing organ systems.
INTRODUCTION
During the development of epithelial-mesenchymal organs such as lung,
mammary gland and salivary glands, both epithelial growth and morphogenesis
are dependent upon the presence of the mesenchymal tissue component
(Grobstein, 1953; Alescio, Cassini & Ladu, 1963). Although it has been demonstrated that continuing morphogenesis of lung and salivary gland epithelia is
dependent upon growth through cell proliferation (Alescio & diMichele, 1968;
Spooner & Wessells, 1972; Goldin & Wessells, 1979), there is no direct evidence
to suggest that the specific pattern of branching is a consequence of differential
1
Authors' address: Department of Zoology, University of the Witwatersrand, 1 Jan Smuts
Avenue, Johannesburg 2001, South Africa.
236
G. V. GOLDIN AND L. A. OPPERMAN
mitotic activity influenced by the mesenchymal tissue component. It is, however,
well established that the formation of the characteristic epithelial morphology
(pattern of morphogenesis) may be determined by the mesenchyme (Kratochwil,
1969; Lawson, 1972, 1974; Ball, 1974) and that the mesenchyme also influences
the epithelial growth rate (Alescio & Colombo Piperno, 1967; Alescio &
diMichele, 1968; Lawson, 1974). In addition, the mesenchymal factor (Ronzio
& Rutter, 1973) which can replace the mesenchymal tissue requirement for
pancreatic epithelial differentiation, purportedly stimulates DNA synthetic
activity and cell proliferation in isolated pancreatic epithelial rudiments.
Despite considerable data for a mitotic stimulatory role of mesenchyme, the
question as to whether cell proliferation, influenced by the mesenchyme, plays a
role in the formation of the characteristic branching of epithelia has not been
extensively studied. The only direct knowledge we know of which points to this
as a possible mechanism for morphogenesis has been provided by Bernfield,
Banerjee & Cohn (1972) and Bernfield, Cohn & Banerjee (1973) who reported a
greater concentration of proliferating cells at the distal ends of morphogenetically
active sites in the salivary gland epithelium. In a recent study, Goldin & Wessells
(1979) compared thymidine labelling in the induced supernumerary tracheal
buds with that in the adjacent tracheal epithelium whence the buds were derived.
Even after 3 days' culture, the branching buds showed a sustained high level of
labelling, while cell proliferation in the tracheal epithelium of the same explants
was markedly reduced. Although these data suggest that the bronchial mesenchyme (used to induce the supernumerary buds in the trachea) acts as a mitotic
stimulator, the evidence is not conclusive as to whether the initial event in bud
formation is a localized increase in mitotic activity in the responding epithelium.
We have approached this problem by employing agarose pellets impregnated
with epidermal growth factor (EGF) as the inducing agent for tracheal buds in
the same way as mesenchymal tissue has been used previously.
Since the mitogenic nature of EGF has been established in only a few embryonic
systems, namely epidermis (Cohen, 1965), cornea (Hollenberg & Cuatrecasas,
1973; Cohen & Savage, 1974) and differentiated embryonal carcinoma cells
(Rees, Adamson & Graham, 1979), this study has also been directed at demonstrating EGF stimulation of DNA synthetic activity in the embryonic lung
system, which demonstration is pertinent to the interpretation of the results
obtained in the bud induction experiments.*
Our results show that the incorporation of tritiated thymidine into DNA in
both the trachea and bronchial tree is stimulated two-fold by EGF, and that
EGF-agarose pellets positioned alongside the tracheal epithelium elicit supernumerary buds in the 5-day-old embryonic chick trachea. Since EGF is a potent
stimulator of tracheal DNA synthetic activity it is suggested that the induction
of the supernumerary buds by the agarose pellets impregnated with EGF is
achieved through a localized stimulation of tracheal epithelial cell proliferation.
* See note added at proof stage on page 243.
EGF role in lung growth and morphogenesis
237
MATERIALS AND METHODS
Explants: Lung systems were dissected from 5-day-old hybrid chicken
embryos (Cornish females x White Rock males) in Hank's balanced salt solution.
For labelling studies, intact explants (trachea and bronchial tree) were placed
on top of Millipore culture platforms and cultured on either standard culture
medium or standard culture medium augmented with EGF for 20 h prior to
adding [3H]thymidine to the medium. In the bud induction experiments, a piece
of tracheal mesenchyme was removed surgically from the epithelium and the
explant transferred to a Millipore filter assembly. Once in position, the 'mesenchyme-free' wall of the trachea was gently scraped with the tip of an iridectomy
knife and a pellet of agarose (control) or EGF-agarose placed alongside the
denuded tracheal epithelium. In several explants, pieces of bronchial mesenchyme from 5-day-old chick embryo lungs were grafted alongside the denuded
tracheal epithelium to establish the responsiveness of the chick trachea to the
induction of supernumerary buds. The explants were then cultured for 24-48 h
on standard culture medium.
Cultures'. Both intact explants for labelling studies and those prepared for
the induction of tracheal buds were cultured on the upper surface of Millipore
filter culture platforms placed over liquid culture medium. The standard culture
medium was Eagle's MEM (Hank's salt) supplemented with 10% horse serum
and 10% 9-day-old chick embryo extract. Each millilitre of medium contained
100 units Penicillin, 100 units Streptomycin and 0-25 fig Fungizone. All the
media constituents and antibiotics were obtained from the Grand Island Biological Company, Glasgow. The epidermal growth factor (mouse EGF) was
kindly donated by Dr Stanley Cohen and was added to the Standard culture
medium to yield a concentration of ca. 10 ng/ml. Cultures were incubated in
a high humidity, 37 °C incubator under a gas phase of 5 % CO2 in air.
Agarose pellets'. Electrophoretic grade agarose (Merck) was added to 0-1 ml
Eagle's MEM (Hank's salt) containing 100 ng EGF, heated to 50 °C and then
cooled to room temperature. As a control, agarose was also added to Eagle's
MEM (Hank's salt) containing no EGF. Small pellets of EGF-agarose and of
agarose were obtained from the resulting solids and were used for the bud
induction experiments. On the basis of pellet size, it was estimated that the
EGF-agarose pellets contained 2-5 ng EGF.
Labelling: [Methyl-3H]thymidine (Radiochemical Centre, Amersham,
England; specific activity 25 Ci/mM) was added to the culture medium at a
concentration of 10 /iCi/ml after 20 h culture and the explants were labelled for
2h.
Liquid scintillation counting: The trachea (including primary bronchii) of
labelled explants was separated from the bronchial tree by a cross-cut and
each sample then transferred to a pre-weighed fibre glass disc (weighed to
the nearest microgram on a Mettler BE 22 electromicrobalance). Following
l6
EMB 60
238
G. V. GOLDIN AND L. A. OPPERMAN
Table 1: DNA synthetic activity in the trachea and bronchial tree of 5-day-old
embryonic chick lung explants cultured in the presence or absence of epidermal
growth factor. Explants labelled for 2 h with [methyl-3H]thymidine following
20 h culture
Mean cpm per trachea (10//g)
± standard deviation
Mean cpm per bronchial tree (15 fig)
± standard deviation
Standard culture
medium
Standard culture
medium + 10 ng/ml EGF
1679± 543 (n = 14)
4362± 1211 (n = 16)*
3591 ±771 (n = 14)
*P = < 0-01.
6428 ± 1158 (n = 16)*
treatment with 0-2 N - P C A (2 ml, 1 h, 5 °C), the specimens were washed in methanol and ether. Thedried specimens were then re-weighed to the nearest microgram.
The tissues were solubilized with 0-5 ml Soluene 350 (Packard & Co. Inc.) for
12 h at room temperature and thereafter 8 ml scintillation fluid (100 ml Permafluor III per litre toluene) was added to each vial. The samples were counted for
10 min and the cpm for each sample were expressed per 10 /tg for tracheal
samples and per 15 fig for the bronchial trees.
Autoradiography: Labelled explants were fixed in Bouin's fluid for 1 h and
following dehydration through an ethanol series and embedding in Paraplast,
5 /im thick serial sections were cut. The mounted sections were hydrated and
coated with Kodak NTB 2 nuclear emulsion (diluted 1:1 by volume, with 2 %
glycerol) by the dipping technique. After 5 days' exposure in the dark at 5 °C,
the slides were developed with D 19 for 3 min at 18 °C, fixed with acid fixer for
5 min and washed in running water for 30 min. The sections were then stained
through the emulsion with Mayer's haemotoxylin for 2 min.
RESULTS
3
Incorporation of [methyl- H]thymidine into DNA: DNA synthetic activity, as
measured by scintillation counting following 2 h labelling with tritiated thymidine, was compared in the lung system (trachea and bronchial tree) of 5-dayold chick embryo explants cultured for 20 h on standard culture medium augmented with 10 ng/ml EGF to control lung explants cultured on standard
culture medium. As reflected in Table 1, the presence of EGF in the culture
medium stimulates DNA synthesis in both the trachea and bronchial tree of
lung explants. The extent of this stimulation is in the order of two- to three-fold
and clearly indicates that EGF is a potent mitogen for the embryonic lung
system at nanogram concentrations comparable to those employed in other
studies (Hollenberg & Cuatrecasas, 1973, 1975; Cohen & Savage, 1974).
While the scintillation counting data include the incorporation of the isotope
239
EGF role in lung growth and morphogenesis
Fig. 1. Autoradiographs showing 5-day-old embryonic chick lung explants cultured
on standard medium (control) (A) and standard medium augmented with 10 ng/ml
EGF (B) for 20 h prior to a 2 h labelling period with [methyl-3H]thymidine. DNA
synthetic activity is stimulated by EGF and this stimulation appears to be most
marked in the epithelial tissue component of both the trachea and bronchial tree.
The thymidinc-labelling is non-uniformly distributed in the lung epithelium in both
theEGF-treated and control explants. A considerably lower number of labelled cells
occur in the interbud regions compared to the highlabellingin the morphogenetically
active bud tips, x 60. T, trachea; Br, primary bronchii.
into both the epithelial and mesenchymal tissue components of trachea and
bronchial tree, autoradiographic evidence (Fig. 1) shows that the EGF stimulation of DNA synthesis occurs in both these tissue components, but that the
stimulation is most marked in the epithelial tissue component. The autoradiographs also reveal a non-uniform distribution of thymidine-labelled cells in the
bronchial (lung) epithelium of both the EGF-treated and control explants.
Tn the morphogenetically inactive epithelial regions between the lung buds the
level of labelling is considerably lower (and in places absent) while at the growing
tips of the morphogenetically active buds the level of labelling is high. This
observation in the chick lung system compares with the distribution of thymidine-labelled cells in the salivary gland epithelium reported by Bernfield et al.
(1972, 1973).
Supernumerary trachea! bud induction: Supernumerary buds have been induced
16-2
240
G. V. GOLDIN AND L. A. OPPERMAN
Table 2. The induction of supernumerary tracheal buds in the 5-dayold embryonic chick lung system
Type of inducer
Bronchial mesenchyme
(5-day-old chick lung)
EGF-agarose pellets
Agarose pellets
Number of
explants
Buds induced
No buds
induced
.10
0
11
15
10
21
15
*».
Fig. 2. Supernumerary bud (arrow) induced in the 5-day-old embryonic chick trachea
by a pellet of agarose impregnated with EGF. Living explant after 48 h culture, x 40.
Br, primary bronchii.
in the trachea of the 5-day-old embryonic chick lung explants by grafting
bronchial mesenchyme alongside the tracheal epithelium or by placing agarose
pellets containing EGF (EGF-agarose) alongside the tracheal epithelium stripped of tracheal mesenchyme (Table 2; Fig. 2). As a control, agarose pellets were
also employed as the inducing agent and as is evident in Table 2, were not able
to elicit tracheal buds.
The lower proportion of positive inductions (ca. 50 %) with EGF-agarose
when compared to supernumerary bud induction with bronchial mesenchyme
EGF role in lung growth and morphogenesis
241
(70-80%) in both the chick (this study) and mouse (Goldin & Wessells, 1979)
lung systems may be due to the non-tissue nature of this inducing agent. Previously, Wessells (1970) demonstrated that in unsuccessful inductions with
bronchial mesenchyme, tracheal mesenchyme cells had invaded the graft and had
formed a layer between the responding tracheal epithelium and the inducing
bronchial mesenchyme tissue. Clearly, the EGF-agarose pellet does not attach
to the Millipore filter substratum and presumably would be less likely to block
or hinder the migration of tracheal mesenchyme cells, to the site of induction the 'naked' tracheal epithelium. In support of this notion, we have observed
that more positive cases of induction have resulted when large expanses of
tracheal epithelium were cleared of investing mesenchyme.
The supernumerary tracheal bud, induced with EGF-agarose, shown in Fig. 2
represents the maximum level of growth and complexity recorded in these
experiments thus far. In two cases, even after 4 days in culture, no hint of branching or of continued growth has been recorded. While no material has yet been
examined histologically, a mesenchymal layer, presumably tracheal in origin,
is present at the distal tip of the bud after 48 h in culture. This could well explain the failure of the bud to elongate further, a situation comparable to that of
grafting tracheal mesenchyme to the growing tip of a bronchus (Wessells,
1970).
DISCUSSION
Our results have shown that epidermal growth factor, at nanogram concentrations, stimulates DNA synthetic activity (cell proliferation) two-fold in the
5-day-old embryonic chick trachea and bronchial tree. In addition to the epidermis (Cohen, 1965) and cornea (Hollenberg & Cuatrecasas, 1973; Cohen &
Savage, 1974), the lung system represents a further embryonic organ that is
responsive to the mitogenic action of EGF. However, this does not provide
evidence to suggest that EGF plays a role in regulating embryonic growth and
development - it remains to be shown that embryonic organs produce this
growth factor (Carpenter & Cohen, 1978). Recent evidence which supports this
possibility has been provided by Catterton et al. (1979) who found that EGF
accelerates foetal rabbit lung differentiation and maturation.
Since EGF has been demonstrated to be a potent stimulator of tracheal DNA
synthetic activity, it seems likely that the induction of supernumerary tracheal
buds by the EGF-agarose pellets may be the result of a localized stimulation
of cell proliferation (by EGF) in the tracheal epithelium adjacent to the pellet.
Agarose alone is not able to elicit such a response, which previously has only
been achieved through grafting various mesenchymes alongside the normally
unbranched tracheal epithelium (Wessells, 1970; Goldin & Wessells, 1979).
When coupled with the earlier report (Goldin & Wessells, 1979) of a sustained
high level of cell proliferation in the induced tracheal buds and a concomitant
decline in cell proliferation in the tracheal epithelium whence the buds were
242
G. V. GOLDIN AND L. A. OPPERMAN
derived, this conclusion may be extended to include bud induction with mesenchyme. A similar localized stimulation in tracheal epithelial cell proliferation
may result from the action of some mitotic stimulator substance(s) emanating
from the inducing tissue. Certainly both EGF and the mesenchymal factor
(Ronzio & Rutter, 1973) could serve such a role, although as pointed out
previously, no developmental role for EGF has yet been established.
Finally, if supernumerary tracheal bud induction is analogous to normal bud
formation in the lung, then it may be suggested that localized differences in
cell proliferation, coupled with selective degradation of basal lamina glycosaminoglycans (Banerjee, Cohn & Bernfield, 1977) to facilitate the action of a
mitotic stimulator, may be the mechanism for branching morphogenesis in the
lung and other developing organs which undergo this form of organogenesis.
Our autoradiographs and those of Bernfield et al. (1972, 1973) suggest that
such localized differences in cell proliferation do exist in the epithelia of organs
that undergo branching morphogenesis.
We are grateful to Dr Norman K. Wessells for the stimulating discussions which led to the
formulation of this study and to Dr Barry Fabian for helpful suggestions during the preparation of the manuscript. We thank Miss W. Maier foi photographic assistance. This research
was supported by grants from The Atomic Energy Board, The Council for Scientific and
Industrial Research and The Mellor Research Fellowship.
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{Received 20 December 1979, revised 1 May 1980)
Note added at proof stage
Nex0, Hollenberg, Figueroa & Pratt (1980) have recorded an increase both in the
receptors for EGF and in a substance (presumably EGF) that can occupy the receptor
during foetal mouse development.
E., HOLLENBERG, M. D., FIGUEROA, A. & PRATT, R. M. (1980). Detection of epidermal growth factor-urogastrone and its receptor during fetal mouse development.
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NEX0,