We have performed experiments showing that the epithelial tree of embryonic rudiments, when cultured in vitro in the presence of hydrocortisone, sharply increases its glycogen content. The distribution of the polysaccharide remains very similar to that observed in the absence of the hormone.

The increased glycogen deposition in response to the hormonal treatment requires the presence of homologous (bronchial) mesenchyme, as shown by the complete inability of the epithelium to accumulate glycogen when experimentally associated with metanephrogenic mesenchyme.

The increased deposition of glycogen in the epithelial tree does not result in an enhanced rate of budding activity.

Recent investigations (Le Douarin, 1968; Morris & Moscona, 1970) have demonstrated that the existence of the proper interrelations among cells and cell groups is essential to the appearance of specific products in embryonic cells. In this context we have shown (Alescio & Dani, 1971) that in the epithelium of developing mouse lungs the presence of glycogen is dependent on the nature of the surrounding mesenchyme, and strictly associated, both topographically and chronologically, with the budding activity; hence a possible morphogenetic significance of glycogen in the developing lung was indicated.

The purpose of the current paper is to show first that substances (glucocorticoids) which are especially potent in affecting carbohydrate metabolism become almost totally ineffective when their target cells have lost the proper associations. Second, the data also show that an increased amount of glycogen in the embryonic lungs does not necessarily result in an enhanced rate of the epithelial morphogenetic growth.

The plan of the research has been as follows :

  1. Glycogen and protein determinations in 11-, 12- and 13-day lung rudiments developing in vivo.

  2. Glycogen and protein determinations in lung rudiments explanted at the 11th day of gestation and cultured in vitro for 24 and 48 h with and without hydrocortisone (1 μg/ml of culture medium).

  3. Effect of hydrocortisone-induced glycogen accumulation on the rate of morphogenetic growth, measured as increase of the epithelial surface area.

  4. Effect of the heterologous association of the main left bronchus with metanephrogenic mesenchyme on glycogen accumulation in the presence of hydrocortisone.

Points (1) and (2) are intended to give a quantitative estimate of glycogen content, relative to the total amount of protein, during the normal and in vitro development, and of the modifications ensuing from hydrocortisone treatment; point (3) should answer the question whether a modified glycogen content may affect the rate of the epithelial morphogenetic growth, while point (4) is meant to inform on the requirements for tissue interactions for the expression of the steroid effect.

Finally, in a collateral experimental series the epithelial trees of 11-day rudiments have been completely separated from mesenchyme and cultured in isolation in the presence of hydrocortisone, to show the direct effect of the hormone on the epithelial glycogen.

First-generation mouse hybrids from ♀C57BL × ♂ BALB/c have been used. The discovery of the copulation plug, looked for each 12 h, was taken as the beginning of the pregnancy. Lung rudiments have been cultured in a hanging drop at the surface of a clot made up with equal volumes of chicken plasma and 9-day chick embryo extract. Hydrocortisone (hydrocortisone alcohol, Mann Research Laboratory) was added to the culture medium, when required, at the final concentration of 1 μg/ml of culture medium. The grafting of the 11-day metanephrogenic mesenchyme on the left main bronchus, and successive reassociation with freshly dissected 11-day bronchial mesenchyme was performed as described previously (Alescio & Dani, 1971). Epithelial morphogenetic growth was measured as relative increment of the epithelial surface area, as in earlier experiments (Alescio & Colombo Piperno, 1967; Alescio & di Michele, 1968).

The complete separation of the epithelial tree from its mesenchymal mantle has been obtained by mild trypsinization as follows: 5min in calcium-magnesium-free Tyrode solution (CMF), followed by a 5 min treatment with 2% trypsin (Trypsin 1:250, Difco Laboratory) in CMF at 37 °C; the loosened mesenchyme has been then removed by cataract knives in 20 % horse serum containing DNase, 20 μg/ml. Glycogen was determined by the anthrone reagent after digesting the tissue in boiling 30 % KOH ; protein was determined by the method of Lowry et al. (1951), following ultrasound disintegration. The histochemical localization of glycogen was obtained using the periodic acid-Schiff (PAS) sequence after McManus (Pearse, 1968), with and without diastase digestion, as previously indicated (Alescio & Dani, 1971).

The embryonic stage between the 11th and the 13th day of development in vivo, morphogenetically characterized by intensive budding activity (Alescio, 1968), as expected, shows a marked increase in the amount of protein per rudiment (Fig. 1). This is representative of its intense growth rate, and is associated with an even more marked rise in the amount of glycogen (Fig. 2). In consequence, the specific concentration of glycogen (as expressed by the ratio: amount of glycogen/amount of protein) is not kept constant but rather increases with age (Fig. 3). The above sharp rise in protein and glycogen content is definitely but evenly depressed under conditions of growth in culture (Figs. 1, 2), hence the proportionality between the two is maintained and the specific concentration of glycogen remains very close to that of normal development (Fig. 3).

Fig. 1.

Amount of protein, expressed as μg per lung rudiment, as a function of developmental age. HC = rudiments cultured in the presence of hydrocortisone. Uncertainties are standard errors of the average of 3 to 8 determinations.

Fig. 1.

Amount of protein, expressed as μg per lung rudiment, as a function of developmental age. HC = rudiments cultured in the presence of hydrocortisone. Uncertainties are standard errors of the average of 3 to 8 determinations.

Fig. 2.

Amount of glycogen, expressed as μg per lung rudiment, as a function of developmental age. HC = rudiments cultured in the presence of hydrocortisone. Uncertainties are standard errors of the average of 3 to 8 determinations.

Fig. 2.

Amount of glycogen, expressed as μg per lung rudiment, as a function of developmental age. HC = rudiments cultured in the presence of hydrocortisone. Uncertainties are standard errors of the average of 3 to 8 determinations.

Fig. 3.

Specific concentration of glycogen expressed as units of glycogen per unit of protein, as a function of developmental age. HC = rudiments cultured in the presence of hydrocortisone. Uncertainties are standard errors of the average of 3 to 8 ratios.

Fig. 3.

Specific concentration of glycogen expressed as units of glycogen per unit of protein, as a function of developmental age. HC = rudiments cultured in the presence of hydrocortisone. Uncertainties are standard errors of the average of 3 to 8 ratios.

The effect of hydrocortisone added to the culture medium at a final concentration of 1 μg/ml consists in a moderate yet statistically significant (P < 0 ·05) increase in the amount of protein per lung rudiment (Fig. 1) while the glycogen content goes up strikingly to more than twice the value reached in culture in the absence of the steroid (Fig. 2); consequently the specific concentration of glycogen is clearly above the level of both the in vivo and in vitro development (Fig. 3). The increased amount of glycogen follows closely the characteristic distribution pattern of this polysaccharide in developing lungs, as shown in Figs. 4 and 5, where it appears predominantly localized in the terminal buds.

Fig. 4.

Eleven-day lung rudiment cultured for 2 days in the presence of hydrocortisone. Presence of large quantities of glycogen in the epithelial tree, with preferential localization in the terminal buds. PAS-haematoxylin, × 65.

Fig. 4.

Eleven-day lung rudiment cultured for 2 days in the presence of hydrocortisone. Presence of large quantities of glycogen in the epithelial tree, with preferential localization in the terminal buds. PAS-haematoxylin, × 65.

Fig. 5.

Higher magnification of the same rudiment as in Fig. 4. PAS-haematoxylin, ×240.

Fig. 5.

Higher magnification of the same rudiment as in Fig. 4. PAS-haematoxylin, ×240.

The rate of epithelial growth of the hydrocortisone-treated rudiments, as determined by measurement of the epithelial surface area, is not significantly higher than that of rudiments cultured in the absence of the steroid, as also implied by the above mentioned divergent increase of protein and glycogen with consequent rise of the polysaccharide specific concentration. Therefore the increase of glycogen does not suffice, in itself, to bring about a clear-cut enhancement of budding rate.

The next question to be examined is whether the above sharp increase in glycogen concentration, resulting from hydrocortisone treatment, is dependent on the existance of the proper cell associations. We have previously shown (Alescio & Dani, 1971) that when the bronchial epithelium is associated with metanephrogenic mesenchyme, budding activity ceases immediately and completely, and no PAS-positive, diastase-sensitive material can be traced in the epithelial cells. When this experiment is performed in the presence of the steroid, introduced in the culture medium at the final concentration of 1 μg/ml, the bronchial epithelium associated with metanephrogenic mesenchyme becomes totally insensitive to the presence of the hormone. This is shown in Figs. 6 and 7, where the main left bronchus surrounded by metanephrogenic mesenchyme shows no progress of bronchial arborization and also complete absence of PAS-positive material, in spite of the presence of the steroid; this is in sharp contrast with the controlatéral bronchial branches from the main right bronchus, where the normal tissue association was maintained (Fig. 7).

Fig. 6.

Eleven-day lung rudiment cultured for 2 days in the presence of hydrocortisone after association of the main left bronchus (arrows) with metanephrogenic mesenchyme. Absence of epithelial glycogen as a result of the heterologous association. PAS-haematoxylin, × 70.

Fig. 6.

Eleven-day lung rudiment cultured for 2 days in the presence of hydrocortisone after association of the main left bronchus (arrows) with metanephrogenic mesenchyme. Absence of epithelial glycogen as a result of the heterologous association. PAS-haematoxylin, × 70.

Fig. 7.

Higher magnification of the same rudiment as in Fig. 6 to show the striking difference between the main left bronchus (arrows) associated with metanephrogenic mesenchyme and buds from the main right bronchus where bronchial mesenchyme is present. PAS-haematoxylin, × 150.

Fig. 7.

Higher magnification of the same rudiment as in Fig. 6 to show the striking difference between the main left bronchus (arrows) associated with metanephrogenic mesenchyme and buds from the main right bronchus where bronchial mesenchyme is present. PAS-haematoxylin, × 150.

This is only a provisional effect of the heterologous association, as demonstrated by the subsequent reassociation of the morphogenetically quiescent main left bronchus with bronchial mesenchyme (Figs. 8, 9) : this immediately makes the bronchial epithelium highly responsive to the steroid, as shown by the intense deposition of glycogen in the epithelial cells and resumption of budding activity.

Fig. 8.

Eleven-day lung rudiment cultured for 2 days in the presence of hydrocortisone after association of the main left bronchus with metanephrogenic mesenchyme; the metanephrogenic mesenchyme has then been removed and bronchial mesenchyme reassociated for two additional days in culture in the presence of the steroid. Reappearance of the epithelial glycogen in the main left bronchus. PAS-haematoxylin, ×70.

Fig. 8.

Eleven-day lung rudiment cultured for 2 days in the presence of hydrocortisone after association of the main left bronchus with metanephrogenic mesenchyme; the metanephrogenic mesenchyme has then been removed and bronchial mesenchyme reassociated for two additional days in culture in the presence of the steroid. Reappearance of the epithelial glycogen in the main left bronchus. PAS-haematoxylin, ×70.

Fig. 9.

Higher magnification of the same rudiment as in Fig. 8. PAS-haematoxylin, ×150.

Fig. 9.

Higher magnification of the same rudiment as in Fig. 8. PAS-haematoxylin, ×150.

The above results, suggesting a dependence of the epithelial responsiveness to the steroid on the nature of the surrounding mesenchyme, give rise to the question of whether the homologous mesenchyme is specifically required or whether the heterologous mesenchyme may actively inhibit or somehow suppress the epithelial response. Therefore the ability of the isolated epithelium to accumulate glycogen in the presence of the steroid was tested. The mesenchyme-deprived epithelial tree cultured in hanging drop for 2 days shows progressive disorganization and spreading of the epithelial cells from tissue-like organization, and absence of glycogen stores. The presence of hydrocortisone in the culture medium does not modify the above situation.

We have shown in a previous paper (Alescio & Dani, 1971) that in the epithelial tree of developing lungs the ability of the terminal buds to accumulate glycogen is dependent on the nature of the mesenchymal environment, and that the presence of glycogen is associated, with impressive constancy, to the budding movement of the epithelial cells.

The main features of the present results are that (1) the responsiveness of the epithelial cells to substances (corticosteroids) which are also specifically able to interfere with glycogen metabolism in other embryonic organs (Greengard & Dewey, 1970) is dependent on the surrounding mesenchyme; and (2) a remarkable increase in glycogen stores does not suffice, per se, to determine an increased rate of budding.

As far as point (1) is concerned, it is noteworthy first that the sharp increase in the specific concentration of glycogen following hydrocortisone treatment tends to conform with the normal pattern of glycogen distribution, so that the final effect takes the appearance of some kind of magnification of the normal behaviour of glycogen deposition.

The results of the experimental association with heterologous (metanephrogenic) mesenchyme show that the epithelial metabolic activities involved in glycogen deposition, which are normally dependent on the availability of the homologous mesenchyme (Le Douarin, 1968; Alescio & Dani, 1971), cannot be elicited even by a specific and potent activator (hydrocortisone) in the absence of external signals provided by tissue interactions. In this context Morris & Moscona (1970) have recently shown that the existence of the proper organization at the cellular level is required for the hormonal induction of glutamine synthetase in the embryonic neural retina. They have also shown that inducibility is irreversibly lost after cell dissociation, as result of metabolism-dependent processes following the persistent separation of the cells from tissue-like associations. Our findings stress the idea that, also at the level of tissue organization, similar interacting phenomena are actively operating since an overall metabolic process, namely glycogen metabolism, appears highly dependent on tissue interactions. Moreover, the degree of such dependence is so stringent as to suppress completely the epithelial response to the hormone. This fact is noteworthy in the evaluation of the developmental significance and specificity levels of tissue interactions in developing lungs (Wessells, 1970), since it now seems likely that mesenchymal ‘information’ is required for the maintenance of normal metabolic patterns. This is also stressed by the fact that isolated pulmonary epithelium appears insensitive to hydrocortisone, suggesting that the condition ‘absence of mesenchyme’ is possibly equivalent, from this point of view, to the condition of ‘heterologous association’. Should this idea prove correct, not inhibition from metanephrogenic mesenchyme but rather active involvement of bronchial mesenchyme would explain the described results.

It would be of obvious biological interest to clarify the mechanism of the mesenchymal dependence of the epithelial response to the steroid; at the moment we cannot establish with the available data whether the site of the primary hormonal effect resides in the mesenchymal or in the epithelial compartment of the rudiment, and therefore the two hypotheses are in principle both acceptable. However, since increased glycogen stores expressed in the epithelium are the final effect of the steroid, the evidence is that there must be, in any case, a flow of influences from mesenchyme to epithelium. The binding of the steroid to the epithelial cell receptors or any of the subsequent steps preceding the expression of the steroid effects, as recently elucidated in rat thymus cells (Mosher, Young & Munck, 1971), may represent the mesenchyme-dependent steps in our experimental system.

Our second point indicates that an increase in glycogen stores does not suffice, in itself, to enhance significantly the rate of morphogenetic growth. Clearly this fails to prove that the presence of the polysaccharide is unrelated to budding activity, and we must therefore conclude that we are still lacking the demonstration of a causal relationship between the two, beyond their coincidental occurrence. An answer to the question of the possible morphogenetic significance of glycogen in terminal buds might perhaps require an experimental system where glycogen could be cleared from budding epithelium while preserving the normal association with the morphogenetically active bronchial mesenchyme.

Aumento da idrocortisone della deposizione di glicógeno e sua dipendenza dalle interazioni tessutali nelpolmone embrionale di topo coltivato in vitro

Gli esperimenti eseguiti indicano che 1’abbozzo epiteliale del polmone embrionale, coltivato in vitro in presenza di idrocortisone, aumenta fortemente il proprio contenuto in glicógeno. La sua distribuzione rimane simile a quella osservata in assenza dell’ormone.

L’aumento della deposizione di glicógeno in risposta al trattamento ormonale richiede la presenza di mesenchima omologo (bronchiale), come è dimostrato dalla completa incapacità dell’epitelio ad accumulare glicógeno quando sia sperimentalmente associate a mesenchima metanefrogeno.

L’aumento della deposizione di glicógeno nell’albero epiteliale non provoca un aumento del tasso di gemmazione.

This research has been performed with a contribution from the Italian C.N.R.

Alescio
,
T.
(
1968
).
L’azione del mesenchima sulla morfogenesi epiteliale nello sviluppo in vitro del polmone embrionale di topo
.
Arch. ital. Anat. Embriol.
73
,
295
319
.
Alescio
,
T.
&
Colombo Piperno
,
E.
(
1967
).
A quantitative assessment of mesenchymal contribution to epithelial growth rate in mouse embryonic lung developing in vitro.
J. Embryol. exp. Morph.
17
,
213
227
.
Alescio
,
T.
&
Dani
,
A. M.
(
1971
).
The influence of mesenchyme on the epithelial glycogen and budding activity in mouse embryonic lung developing in vitro.
J. Embryol. exp. Morph.
25
,
131
140
.
Alescio
,
T.
&
Di Michele
,
M.
(
1968
).
Relationship of epithelial growth to mitotic rate in mouse embryonic lung developing in vitro.
J. Embryol. exp. Morph.
19
,
227
237
.
Greengard
,
O.
&
Dewey
,
H. K.
(
1970
).
The premature deposition of glycogen in livers of fetal rats injected with hydrocortisone or glucagon
.
Devi Biol.
21
,
452
461
.
Le Douarin
,
N.
(
1968
).
Synthèse du glycogène dans les hépatocytes en voie de différentiation : rôle des mésenchymes homologue et hétérologue
.
Devl Biol.
17
,
101
104
.
Lowry
,
O. H.
,
Rosebrough
,
N. J.
,
Farr
,
A. L.
&
Randall
,
R. J.
(
1951
).
Protein measurement with the Folin phenol reagent
.
J. biol. Chem.
193
,
265
275
.
Morris
,
J. E.
&
Moscona
,
A. A.
(
1970
).
Induction of glutamine synthetase in embryonic retina: its dependence on cell interactions
.
Science, N. Y.
167
,
1736
.
Mosher
,
K. M.
,
Young
,
D. A.
&
Munck
,
A.
(
1971
).
Evidence for irreversible, actinomycin D-sensitive, and temperature-sensitive steps following binding of cortisol to glucorticoid receptors and preceding effects on glucose metabolism in rat thymus cells
.
J. biol. Chem.
246
,
654
659
.
Pearse
,
A. E. G.
(
1968
).
Histochemistry, Theoretical and Applied.
London
:
Churchill
.
Wessells
,
N. F.
(
1970
).
Mammalian lung development: interactions in formation and morphogenesis of tracheal buds
.
J. exp. Zool
.
175
,
455
466
.