/ . Emhryol. exp. Morph. Vol. 29, 2, pp. 439-451, 1973
439
Printed in Great Britain
Effect of a proline analogue,
azetidine-2-carboxylic acid, on the morphogenesis
in vitro of mouse embryonic lung
By T. ALESCIO 1
From the Institute of Human Anatomy, Second Medical Faculty,
University of Naples
SUMMARY
A proline analogue, azetidine-2-carboxylic acid, which has been reported to selectively
depress collagen biosynthesis, affects lung morphogenesis in vitro by determining differential
modifications of the rate of epithelial growth and ramification activity.
Low concentrations of the analogue induce a sharp decline of the number of newly formed
terminal buds, leaving the rate of global growth of the epithelial tree almost unaffected. This
effect is reversed by increasing doses which appear more effective on global epithelial growth
than on budding. DNA and protein content at the end of the treatment are unequally reduced.
Protein concentration is more depressed with consequent rise of the DNA/protein ratio.
These results show that epithelial budding is very sensitive to the treatment with the
analogue, and draw attention to collagen biosynthesis as one of the possible mechanisms
by which the mesenchyme controls the spatial organization of the epithelial tree. This type
of control appears relatively independent on the actual rate of epithelial cell replication,
since budding is much more sensitive to the action of the drug.
INTRODUCTION
A number of reports in the last few years (Bernfield & Wessells, 1970) have
emphasized the importance of collagenase-sensitive materials in the morphogenesis of several embryonic organs whose developmental mechanics are known
to be based on the so called 'epithelio-mesenchymal interaction' (Fleischmajer
& Billingham, 1968). Most of that work is based on the effect of a collagenase
treatment of the epithelial component of the rudiments previously isolated by
trypsinization and cultured in a transfilter system in the presence of mesenchyme
(Grobstein, 1967; Wessells & Cohen, 1968). Since this procedure involves
drastic modifications on the normal developmental conditions, the observed
morphogenesis has been said to be 'reminiscent' (Wessells & Cohen, 1968) of
the normal developmental pattern. Consequently, when a molecular marker of
morphogenesis and/or differentiation is not available, morphological evaluation
of the observed effect is often questionable. In particular, when the involved
developmental event is epithelial budding it is difficult to assess what is morpho1
Author's address: Istituto di Anatomiaumana, NuovoPoliclinico, Cappella dei Cangiani
80131, Napoli, Italy.
440
T. ALESCIO
genetically 'normal' budding and what represents mere distortion of the
epithelial profile due to the removal of its investing mesenchyme.
Moreover, the presence of enzymic activities other than collagenolitic that are
detectable in several collagenase preparations, raises the possibility that 'the
dependence of morphogenesis on collagenase-susceptible materials does not
necessarily implicate collagen' (Bernfield & Wessells, 1970).
It is therefore of obvious interest to use an entirely different approach to the
problem of the morphogenetic significance of collagen biosynthesis, such as the
one recently indicated by the work of Lane, Dehm & Prockop (1971) and Lane,
Parkes & Prockop (1971).
They have shown that the proline analogue azetidine-2-carboxylic acid inhibits
collagen accumulation in growing chick embryos down to a level of approximately 20 % of the untreated controls. Therefore a morphogenetic effect of the
proline analogue on the developmental pattern would directly draw attention to
collagen, and other proline- and hydroxyproline-rich protein species.
The experiments reported in the current paper deal with the effect of azetidine2-carboxylic acid on the morphogenesis in vitro of embryonic mouse lungs. This
effect seems not to be entirely attributable to the depressed collagen biosynthesis,
since other accompanying modifications, probably not dependent on collagen
depletion, are present in the epithelial compartment of the rudiments when
treated with the analogue.
MATERIALS AND METHODS
The rationale of the experiments has been as follows: we assume after
Wessells (1970) that oriented collagen bundles act as a physical barrier to the
budding movement, thus stabilizing the epithelial structure; the sequence of
morphogenetic events should therefore be as depicted in Fig. 1 A, control proper:
the oriented collagen deposition selects along the epithelial surface a number of
special sites which are left open for budding movement, and the final result is the
production of a given number of terminal buds, accompanied by and strictly
correlated to an increase of the epithelial surface area; this represents what may
be defined as morphogenetic growth.
When the proline analogue is added to the culture medium, a remarkable
decline of collagen synthesis should ensue, as expected from the results by Lane
et ah (1971).
Then the expected morphogenetic result is represented in Fig. 1B, experiment
proper. The scarcity of oriented collagen bundles will prevent the formation of
definite budding sites, hence generalized enlargement of the epithelial surface
area and absence of new buds (i.e. non-morphogenetic growth) will be observed.
This result would ultimately demonstrate dissociation and relative independence
of the two developmental components, budding activity and global epithelial
growth.
This can be quantified by comparing the number of newly formed terminal
Azetidine-2-carboxylic acid and lung morphogenesis
441
A. Controls
Oriented collagen
Area = 2
deposition
Buds = 6
Arca=l
Buds = 0
Collagen deposition
inhibited
Area = 2
Buds = 0
B. Azetidine-2-carboxylic acid
Fig. 1. Plan of the experiments and expected results.
buds to the size of the epithelial surface area, and by estimating the degree of
correlation between the two, which is expected to decline, if the above assumption would prove correct, in the presence of the proline analogue.
Lung rudiments from 11-day first-generation mouse hybrids (? C57BLx^
BALB/c) were cultured in hanging drop at the surface of a plasma clot composed
by equal volumes of chicken plasma and 9-day chick embryo extract.
Azetidine-2-carboxylic acid (Calbiochem, Los Angeles) was introduced in the
culture medium (40, 60, and 80 /^g/ml). For measurements of morphogenetic and
non-morphogenetic growth all lung rudiments were drawn by camera lucida
immediately and after 21 and 45 h in culture. The number of terminal buds and
the measure of the epithelial surface area have been determined on the drawings
after the procedures previously described (Alescio & Colombo Piperno, 1967;
Alescio & di Michele, 1968). The data are reported in the form of total number of
buds produced in culture (i.e. number of buds at 21 and 45 h minus number of
buds at zero time), and of net increase of the epithelial area in culture (i.e. size
of the epithelial area, in arbitrary planimetric units, at 21 and 45 h minus size
of the epithelial area at zero time) in order to use the same arithmetical treatment
for both variables.
Histological examinations were carried out on Carnoy-fixed rudiments,
serially sectioned at 5 /<m and stained by the periodic acid-Schiff procedure after
McManus (Pearse, 1968).
442
T. A L E S C I O
Fig. 2. Lung rudiments explanted at the 1 lth day of gestation and cultured for 45 h.
(a) controls; (b)-(d) azetidine-2-carboxylic acid (/*g/ml) 40, 60, 80 respectively.
Photographs taken in the living state, x 40.
Azetidine-2-carboxyUc acid and lung morphogenesis
443
Oh
45 h
Control
40
Azetidine-2-carboxylic acid
60
80 //g ml
Fig. 3. Camera lucida drawings representing part of the main right bronchus of
the same rudiments as in Fig. 2.
Other rudiments were used for DNA and protein measurements respectively
after the method of Ceriotti (1952) as modified by Gwatkin & Biggers (1963),
and after the method of Lowry, Rosebrough, Farr & Randall, (1951).
RESULTS
The developmental pattern in vitro of the 11-day lung rudiments and the
effect of azetidine-2-carboxylic acid is exemplified in Fig. 2a-d. After a 45-h
treatment with 40 //g/ml of the analogue, the rudiments (Fig. 2b) tend to produce
less but larger terminal buds as compared with the untreated controls. The
branching activity is even more depressed with increasing doses of the analogue
(Fig. 2c, d) as the terminal portions of the epithelial tree are somewhat enlarged
with very little further branching. Simultaneously, however, the overall growth
of the epithelial tree is also clearly reduced with increasing doses as the rudiments
are much smaller in epithelial size as compared to the controls (Fig. 2a).
This is more clearly shown in the diagrams reproduced in Fig. 3, where the
epithelial profile of the distal part of the main right bronchus (caudally to the
origin of the infra-cardiac branch) is represented.
We have therefore some evidence that the developmental pattern of the
epithelial tree tends to shift, under the effect of the analogue, from morphogenetic
growth (prominent budding activity) to non-morphogenetic growth (global enlargement of the epithelial structures with fewer new branches). This effect appears
most perceivable when 40 //g/ml of the analogue are used, while with increasing
doses non-morphogenetic growth also appears increasingly affected (Fig. 3).
The above result is quantified by the data reported in Figs. 4 and 5, where the
average number of newly formed terminal buds and the average increase of the
epithelial surface area in the presence of different doses of azetidine-2-carboxylic
acid are plotted as a function of time in culture. They show that the general
trend of the response of both budding activity and global growth to the analogue
is similar, in the sense that both these developmental parameters undergo an
444
T. ALESCIO
1
i
1
1
1
•
100 -
)0
90 -8-0 -
/
70 o. 60
/
--
50
/
40
j
/
-
•c
30 -
20 10 1
12
24
Time in culture (li)
1
36
1
4S
Fig. 4. Average number of terminal buds produced in culture as a function of time.
0: controls; A-C: azetidine-2-carboxylic acid Og/ml) 40, 60, 80 respectively.
Uncertainties are standard errors.
inhibitory effect directly dependent on time in the presence of the analogue and
its concentration. However the magnitude and the temporal pattern of the
observed inhibitory effect show that budding activity is more sensitive to the
analogue, being more depressed than global growth at 40//g/ml; the last in
turn, when the analogue concentration becomes adequate, is affected more
precociously than budding activity. The above information is summarized by
the data in Table 1, where budding activity and global growth at 21 and 45 h
under differing analogue concentrations are reported as percentage of controls;
they show that budding activity possesses a lower threshold of sensitivity to the
analogue, but the events required for the expression of this effect demand a
longer time interval than those necessary to establish a reduced rate of global
growth at higher concentrations.
The interdependence of budding activity and global growth may be considered
Azetidine-2-carboxylic acid and Jung morphogenesis
445
10 12
24
36
Time in culture (h)
Fig. 5. Average net increase of the epithelial surface area in culture as a function
of time, in arbitrary planimetric units. 0: controls; A-C: azetidine-2-carboxylic acid
(//g/ml) 40, 60, 80 respectively. Uncertainties are standard errors.
next, by plotting the average number of newly formed terminal buds after 45 h
in culture as a function of the corresponding measures of surface area (Fig. 6).
The resulting curve shows, as already implicated by the data in Fig. 4 and 5, a
clearly diphasic trend with a definite change of slope dependent on analogue
concentration. This provides further evidence that a dose of 40 /*g/ml of the
analogue determines a marked inhibition of budding accompanied by relatively
little depression of global growth; the effect is reversed at higher analogue
concentrations, which appear to have maximal inhibitory effect on global growth.
Independent fluctuations of budding and growth are therefore definitely substantiated.
This relative independence of the two variables brought about by the experimental treatment may then be quantitatively assessed by estimating the degree
of statistical correlation between the two. This has been done using the pooled
29
E M B 29
446
T. ALESCIO
Table 1. Effect of azetidine-2-carboxylic acid on budding activity
and global growth
45 h
21 h
A
Azetidine-2-carboxylic acid (/tg/ml)
Newly formed terminal buds
(% of control)
Increase of epithelial surface area
(% of control)
4
60
80
40
60
64-58
66-66
62-50
5612
38-78
35-71
69-90
65-28
4002
77-75
42-48
28-71
Azctidine-2-carboxylic acid (/m ml)
<
40
80
Controls
_
20
30
40
50
60
70
80
90
Average increase of epithelial surface area
Fig. 6. Average number of newly made terminal buds after 45 h in culture as a
function of the corresponding average increase of the epithelial surface area, in
controls and in the presence of increasing analogue concentrations.
data at both time points, 21 and 45 h, to obtain a more complete evaluation of
the dependence on time of the involved fluctuations. The estimated values of the
correlation coefficient (r) are plotted as a function of dose in Fig. 7. The highly
significant (P < 0-01) statistical correlation existing in normal development
(controls) is strongly depressed under the effect of the analogue, and falls below
the level of statistical significance when its concentration is of 60 /<g/ml or more.
Complete statistical dissociation of budding activity and global epithelial growth
is therefore gradually achieved with increasing doses.
The histological examination has failed to show noticeable structural modifications related to the presence of the analogue. The histological structure,
Azetidine-2-carboxylic acid and lung morphogenesis
1
1
1
1
447
1
0-7 -
-
P<0-0l
0-6
-
COCM
0-5 -
cor iclaticm
o
o
0-4 -
\
0-3 —
N.S.\
\
P<005 -
\
\
0-2 -
0
Degrees of freed o m = 18
01
1
1
1
1
1
0
20
40
60
80
Azelidine-2-carbo.\ylic acid (//g.'ml)
Fig. 7. Estimated values of r, correlation coefficient, of number of newly made
terminal buds to corresponding net increase of epithelial surface area, as a function
of the analogue concentration. Data at 21 and 45 h have been pooled. Transverse
dashed lines indicate the levels of significance of r. N.S. = not significant.
Table 2. Effect of azetidine-2-carboxylic acid on DNA and
protein content
Controls
DNA (/ig/rudiment)
Protein (/<g/rudiment)
Ratio (DNA/protein)
0-7850
16-52
00475
Azetidine-2-carboxylic
acid (60 /tg/ml)
Control (%)
0-6000
11-53
00520
76-43
69-79
109-47
including the peculiar glycogen distribution (Alescio &Dani, 1971), is apparently
unchanged (Fig. 8 a-d), and no appreciable indications of noteworthy cell
degeneration are present over the entire dose range used. Finally, some hints on
the molecular mechanisms of the described results may be derived from DNA
and protein measurements, performed on samples of randomly pooled lung
rudiments from several litters, and cultured for 45 h in the absence and in the
presence of 60/^g/ml of azetidine-2-carboxylic acid. Both DNA and protein
content per lung rudiment is reduced in the presence of the analogue (Table 2).
However, protein concentration is more affected than DNA, with consequent
rise of the ratio DNA/protein per lung rudiment. The interpretation of the
above result must, however, consider that it is based on the assumption that the
29-2
448
T. ALESCIO
(C)
Fig. 8. Histological sections of a control culture (a), and of a rudiment treated with
40/tg/ml of the analogue, after 45 h in culture (b). PAS-haematoxylin. x 190.
(c, d) Higher magnifications of the same rudiments as in (a) and (6) respectively.
PAS-haematoxylin. x 390.
Azetidine-2-carboxylic acid and lung morphogenesis
449
average content of DNA and protein per lung rudiment is the same in the control
and experimental groups at explanation, so that the observed final modifications are representative of modified accumulation rates due to the experimental
treatment.
DISCUSSION
This work has been based on the assumption that two distinct developmental
components are operating on the epithelial compartment in lung morphogenesis.
The first one is increase in size of the epithelial tree, mostly but probably not
entirely due (Alescio & di Michele, 1968) to cell replication, particularly intense
in lung morphogenesis (Wessells, 1970). Its function is to provide materials for
growth, which in turn are rendered morphogenetically significant by the second
developmental component, that is budding movement. The two processes must
be closely integrated for the normal course of development.
We have asked whether it is possible to experimentally dissociate the two
developmental components, and the answer is positive but only to a limited
extent.
The first morphogenetically noticeable effect of low doses of the proline
analogue azetidine-2-carboxylic acid, which is known to inhibit collagen accumulation (Lane, Dehm & Prockop, 1971), is in fact a reduced branching
activity, leading to partial uncoupling of growth and ramification, with prevailing
non-morphogenetic growth. The rudiment continues to grow as the epithelial
structures are enlarged, but less new branches are produced because of the
reduced number of bifurcation points.
The mechanism of this effect may well be depressed collagen biosynthesis, and
this would confirm the morphogenetic importance attributed to oriented collagen
deposition (Wessells, 1970). Since collagen is mostly of mesenchymal origin
(Bernfield, 1970), the mesenchyme must be regarded as the site of this effect.
However, when the analogue concentration increases the epithelial growth
undergoes more powerful and more precocious depression than budding activity.
The difference between relative inhibition of growth and relative inhibition of
budding is maximal after 21 h, and then decreases after 45 h of treatment
(Table 1). The anticipated involvement of growth is a particularly relevant point,
showing that the effect on global growth is probably not a consequence
of the prevented budding.
We may therefore assume two rather independent mechanisms of action for
the proline analogue. Part of the available analogue may be immediately and
preferentially incorporated in precollagen polypeptides, thereby inhibiting
collagen accumulation and finally preventing further budding activity; this effect
may demand relatively longer time for perceivable final expression. When the
concentration of the analogue is sufficiently high, a residual fraction, not involved
in collagen metabolism, may become available for interfering in other, possibly
also protein biosynthetic pathways, which found a more precocious final
450
T. ALESCIO
expression in a reduced rate of cell replication; hence the noticed reduction of the
DNA content per rudiment. Lane, Dehm & Prockop (1971) have shown, in fact,
that the inhibitory effect on collagen biosynthesis is not entirely specific, since
the net accumulation of non-collagen proteins is also somewhat reduced. A final
conclusion on this particular point awaits, however, measurements of extent of
the inhibition of collagen synthesis in this particular system, and a more detailed
study not only of the modifications of protein and DNA content under the effect
of the drug, but also of their rates of synthesis in the presence of the drug. Anyhow
whatever the mechanism, it clearly entails loss of correlation of global growth
to budding activity, directly dependent on analogue concentration, and opens
a new possibility for further study of the reciprocal role of cell replication and
budding movement in lung morphogenesis.
This work has been limited, at the moment, to whole lung rudiments, so that
the observed results may well be a composite of mesenchymal and epithelial
effects variously combined to give the described final pattern. Nothing therefore
can be said up to now about the relevance of this experimental system for further
understanding of the nature of the epithelio-mesenchymal interaction, besides
that partial inhibition of one specific mesenchymal function such as collagen
biosynthesis has morphogenetic consequences on the spatial organization of the
epithelial tree. Further helpful information may come from knowledge of
individual responses of epithelium and mesenchyme to the analogue, and of
possible modifications of patterns of macromolecular synthesis in both tissues.
RIASSUNTO
Effetto di un analogo delta prolina sulla morfogenesi in vitro
del polmone embrionale di topo
Un analogo della prolina, azetidine-2-carboxylic acid, che e stato dimostrato capace di
ridurre in maniera specifica la biosintesi del collagene, influenza la morfogenesi del polmone
in vitro determinando modificazioni differenziali del tasso di accrescimento epiteliale e della
attivita di ramificazione.
Basse concentrazioni di analogo determinano una netta riduzione del numero di gemme
terminali prodotte, lasciando relativamente indenne il tasso di crescita complessiva dell'albero
epiteliale. Tale effetto e capovolto sotto l'influsso di dosi piu elevate, che risultano piu attive
sulla crescita che sulla ramificazione. II contenuto di DNA e proteine al termine del trattamento e ridotto in maniera diversa, con maggiore riduzione del contenuto proteico e
conseguente aumento del valore del rapporto DNA/proteine.
Questi risultati mostrano che la gemmazione epiteliale e molto sensibile al trattamento
con l'analogo, e richiamano l'attenzione sulla biosintesi del collagene come uno dei possibili
meccanismi con cui il mesenchima controlla l'organizzazione spaziale dell'albero epiteliale.
Questo tipo di controllo risulta relativamente indipendente dal tasso effettivo di replicazione
delle cellule epiteliali, perche la gemmazione e molto piu sensibile alPazione della sostanza.
This research has been performed with a contribution from the Italian C.N.R.
Azetidine-2-carboxylic acid and Jung morphogenesis
451
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