The development of the chick tertiary bronchus

J. Anat. (1972), 113, 3, pp. 325-340 325With 16 figuresPrinted in Great Britain

The development of the chick tertiary bronchusII. The origin of the surface lining system

ALED W. JONES* AND CAROLYN J. P. RADNORPathology Department, Manchester University

(Accepted 9 October 1972)

INTRODUCTION

The structure of the pulmonary surface lining ofbirds differs from that ofmammals.In the avian lung there is a uniform thin trilaminar surface layer (Tyler & Pangborn,1964) while the surface lining of the mammalian lung is a duplex structure of varyingwidth, consisting of a hypophase covered by a thin smooth surface layer (Weibel &Gil, 1968). The mammalian structure is considered to represent surfactant to whichthe osmiophilic inclusion body (OIB) of the granular pneumocyte (type II cell) con-tributes material (Gil & Weibel, 1969).Although it is agreed that surfactant is present in mammals, the surfactant status

of birds is controversial. Pattle & Hopkinson (1963) claim that it is present, whilstMiller & Bondurant (1961) and Klaus et al. (1962) were unable to demonstrate any.Thus, although OIBs are present in the avian lung, their role in surfactant productionis uncertain, as also is their relationship to the development of the characteristictrilaminar lining layer (Tyler & Pangborn, 1964; Lambson & Cohn, 1968; Petrik &Reidel, 1968 a, b; Akester, 1970).The purpose of this study is to trace the development of the distinctive avian

trilaminar layer in the embryonic lung and to elucidate the relationship between thisand the OIB, in order to comprehend more fully the nature and function of thetrilaminar system.

MATERIALS AND METHODS

These have been described in the previous paper (Jones & Radnor, 1973).

OBSERVATIONS

Respiration commenced when the air cell of the egg was pierced by the beak of thechick. The expansion of the gas exchange area of the tertiary bronchus was a gradualprocess, with progressive increase in diameter and unequal expansion of contiguoustertiary bronchi.

Before breathing started, the air capillary zone was compact, the air capillariesbeing relatively unexpanded, with plump lining cells containing plentiful cytoplasm.The blood capillaries were expanded and the interstitium was cellular (Fig. 1). The

* Present address: P.O. Box 7072, Pathology Department, Makerere University, Kampala, Uganda.

A. W. JONES AND C. J. P. RADNOR

Fig. 1. Part of an unexpanded tertiary bronchus of a 21 day unhatched chick showing thelumen (1), the atria (a) and the infundibula (i). The bronchus was fixed by intra-tracheal glutar-aldehyde infusion, thus producing some artefactual expansion of the air capillaries of thecellular unexpanded air capillary zone (acz). 1 ,um thick Araldite-embedded section. Toluidineblue. x 750.

subsequent decrease in cellularity of the air capillary zone was due to the expansionof the air capillaries with attenuation of the cytoplasm of the lining cells andcompression of the interstitial cells (Fig. 2).The overall architecture of the atrial area did not alter greatly following the com-

mencement of breathing. The most significant change was the appearance of largequantities of trilaminar material in the atrium, either lying loose in the lumen orclosely applied to the atrial lining cells (Fig. 3). This material consisted of lamellaewhich were either closely apposed or loosely adherent. The thickness of each lamellawas between 8-8 and 13-1 nm and consisted of two external osmiophilic bandsbounding a central osmiophobic area (Fig. 4).

In the 'neonatal' period the greatest amount of the trilaminar material was manu-factured in the atrial area but it was also produced in the air capillary area. In theatria it was formed in atrial lining cells not containing OJBs and it was produced here

326

Development of chick tertiary bronchus. II

Fig. 2. Part of an expanded tertiary bronchus in a hatched 1 day old chick, showing the smoothmuscle bundles (sm) and the atria (a). There is complete expansion of the air capillaries (ac).The tertiary bronchus is bounded externally by a thin connective tissue septum (s). 1 ,tm thickAraldite-embedded section. Toluidine blue. x 750.

both in the 'neonatal' lungs and in adult cockerel lungs. In the air capillaries, lami-nated material was formed in some of the epithelial lining cells, the greatest amountbeing produced during the first day, after which production decreased. In the maturebird, none was identified in this site. The trilaminar material was derived fromhitherto undescribed inclusion bodies, named avian inclusion bodies, which wereproduced in the non-granular pneumocytes of the atrium and in certain cells in theair capillaries. The majority of these bodies were usually oval or spherical (Fig. 5),and were composed of regular trilaminar lamellae. They differed in size from theOIB and a comparison between their surface areas is shown in Fig. 6. The totalthickness of the lamellae varied between 8-8 and 13-1 nm, approximately corres-ponding to the thickness of the extruded material. Prior to discharge, the compactoval or spherical avian inclusion body partly unfurled inside the cell to give a varietyof shapes, the most common being an elongated or torpedo-like body (Fig. 5).Further unwinding of the lamellae took place near the free cell surface so that a

327


Fig. 3. Extruded trilaminar material in the atrial area of a tertiary bronchus; it is closely appliedto the atrial lining cells and intimately mixed with the long cytoplasmic processes of some ofthese lining cells (arrows). Part of the smooth muscle bundle of a 'club' is shown (sm). Theextruded material partly overlies three atrial lining cells (a, b and c). Cell 'a' contains a partlyunfurled avian inclusion body showing prominent laminations. Cell 'b' contains a homo-geneous avian inclusion body with no laminations, whilst in cell 'c' the inclusion is partlylaminated. 1 day old chick. x 15000.

tangle of trilaminar material was found lying loose in the superficial cell cytoplasm(Fig. 7). Release of this laminated material either in the compact form or as a loosetangle was accompanied by partial disintegration of the superficial part of the cell(Fig. 8), suggestive of an apocrine type of secretion. Release was an untidy processin which unfurled or partially unfurled lamellae plus tongues or villi of cytoplasmwere discharged into the atrial lumen (Fig. 9). There was some difficulty in distin-guishing cell boundaries under these circumstances, as much of the dischargedlamellar material, with its long interleaved cytoplasmic processes and attached cyto-plasm from its cell of origin, came to overlie and compress the neighbouring granularpneumocytes.A similar discharge process could be seen in some air capillary lining cells situated

in the angles between two blood capillaries (Fig. 10), with intracytoplasmic unfurlingof the avian type inclusion bodies (Fig. 11). This process was seen during the first

328


Fig. 4. Extruded trilaminar material closely apposed to, and overlying and partly compressingvilli (v) of atrial lining cells (a). The lumen of the tertiary bronchus is seen (tb). 1 day old chick.x 100000.

Fig. 5. An atrial cell containing two typical avian inclusion bodies showing a trilaminarpattern. Inclusion body 'a' is compact, whilst inclusion 'b' has partly unfurled. 3 day old chick.x 50000.

329

A. W. JONES AND C. J. RADNOR

100 _

50 _

Lul IB

2 15 | H L l Avian0

C

0

-o10Ez

5

1 2 3 4Surface area (/rM2)

Fig. 6. Comparison of the surface areas of sectioned avian inclusion bodies and OIBs.

week after hatching, but it was during the first day that this was most obvious; nosuch changes were seen in the mature cockerel.During the first day, the surface of the air capillaries became covered by a single

layer of trilaminar material (Fig. 12); although discontinuities were seen, these wereconsidered to be artefacts. In general, only a single trilaminar covering was presentbut occasionally, in the infundibular area, two or three such lamellae could beidentified (Fig. 13). The scanty amount in the air capillaries contrasted strongly withthat in the atria, where large masses of material were found.No complex series of avian inclusion body precursors was identified early in

embryonic life. However, in the atrial cells and air capillary lining cells it was possibleto find homogeneous, osmiophilic structures of a predominantly oval or circularshape (Fig. 3, cell 'b') although bizarre shapes could also be seen (Figs. 14, 16). Thishomogeneous material changed into a lamellar structure and bodies in transitionbetween the homogeneous and lamellar states were identified (Fig. 3, cell 'c').No avian inclusion bodies were identified in the air capillary lining cells in the

mature cockerel lung. This is as would be expected, as these cells functioned as amechanical barrier only and, in the mature animal, did not have the necessaryapparatus to manufacture inclusion bodies.The typical form of discharge of the lining material has already been described

but a different pattern was also noted, in which the unwound trilaminar materialinside the cell was bounded on either side by a membrane (Fig. 15) prior to partialcell disintegration and discharge of the material.

330

Development of chick tertiary bronchus. II 331

Fig. 7. Unfurled trilaminar material lying loose in the superficial part of thecytoplasm of an atrial cell. 3 day old chick. x 37 500.

Fig. 8. An avian inclusion body in the process of being discharged. Part of the smooth musclebundle of an atrial 'club' is seen (sm) partly covered by three atrial lining cells (a, b and c).Cells 'b' and 'c' contain mitochondria with dense matrices. Cell 'a' is partly disrupted as alarge, partly unfurled, avian inclusion body is discharged from it. This partly overlies theadjacent cell (cell 'b'). The lamellar osmiophilic material is admixed with cytoplasmic villi(arrow) and in the adjacent areas the osmiophilic material has a lamellar appearance. 3 dayold chick. x 16875.

DISCUSSION

Surface lining systemThe present study has confirmed the presence of a trilaminar lining in the newly

hatched chick lung (Petrik & Reidel, 1968a) and in the mature bird using conven-tional fixation and staining methods. The width of the extruded material measuresbetween 8-6 and 12-5 nm, which is in agreement with previous studies in the chick,goose, pigeon and sparrow: 7-5 nm (2'5-2-5-2-5 nm), Tyler & Pangborn (1964);

AWW 4%1w


Fig. 9. A partly disrupted non-granular pneumocyte containing unfurling avian inclusionbodies, and overlying an atrial granular pneumocyte containing 5 mature OIBs. The avianinclusion bodies have a laminated appearance. The superficial cell boundary of the granularpneumocyte is indicated by the white arrow heads. 1 day old chick. x 17500.

8-12 nm, Lambson & Cohn (1968); 13 nm (5-3-5 nm), Petrik & Reidel (1968a);15 nm (5-5-5 nm), Petrik & Reidel (1968b) and 10 nm (3 3-3 3-3 3 nm), Akester(1970). These size differences are most likely to be the result of technical problemsand difficulties in defining the edges of the material, but species variations cannotdefinitely be ruled out.The appearance of trilaminar material in the atria and the air capillaries is related

to the onset of breathing and it is not seen prior to this. This is in contrast with theobservation of Petrik & Reidel (1969 a), who claimed to have identified such a liningat day 19 of incubation. It is possible that these authors failed to realize that in thosespecimens claimed not to be air breathing, the air cell of the egg had already beenpierced and air breathing had commenced, although the shell was still intact.The mode of production of the lining has previously been in doubt. The OIB

(Tyler & Pangborn, 1964; Akester, 1970), the endoplasmic reticulum of the granularpneumocyte (Lambson & Cohn, 1968) and extracellular 'disintegrating osmiophilicmasses' (Petrik & Reidel, 1968a) have all been considered possible sources of thematerial. This study has shown that it is derived from special inclusion bodies calledavian inclusion bodies produced by the non-granular pneumocyte lining cells of theatrium in both newly hatched and mature lungs and, during the early post-hatchingperiod only, in some of the air capillary lining cells. The avian inclusion body differsin many respects from the OIB (Table 1), the most significant of these being the timeof its first appearance, the mode of development and the method of discharge from

332

'U "4M,oO-T-. 7-73- -..

Ap.. + .....1,-;oO\ - ..


Fig. 10. Part of an air capillary lining cell of a 1 day old chick containing partly unfurledtrilaminar material which is derived from an irregularly shaped avian inclusion body. Aninterstitial cell (i) is seen adjacent to the air capillary lining cell. x 18 750.

the cell. The similar periodicity of the intra-inclusion and extruded laminationsaffords strong support for the site of origin.The atrial lining cells that produce avian inclusion bodies are distinct from granular

pneumocytes. During early embryonic life, however, it is not possible to predict byany cytological characteristic or anatomical position which cell is destined to pro-duce them. No complex sequence of precursor bodies has been identified, as in thecase of the OIB, as the only precursors recognized prior to hatching have been large,sometimes irregular but predominantly circular, homogeneous, osmiophilic bodies.The transformation of the relatively homogeneous bodies to the laminated form inthe embryo appears initially to be related to the exposure to air.

Luzatti & Husson (1962) studied the electron microscopical configuration ofmolecules in phospholipid-water systems and showed that, if the water content is30 % or more, no lamellar pattern is seen. The appearance of laminations in the avianinclusion body could be due to a similar relative change in phospholipid-water con-centration. It has further been suggested (Stoeckenius, 1962; Chapman & Fluck,1966) that, in the lamellar phase of a 3-7-4-4 nm periodicity, phospholipid and waterare arranged in parallel stacks of lamellae, the lipid molecules being arranged insymmetrical bimolecular leaflets with polar hydrophilic groups in contact with waterand the long hydrophobic hydrocarbon chains pointing away from the water phasetowards the centre of the leaflet. Osmium tetroxide reacts with the hydrophilic polargroups of the phospholipid and thus the centre osmiophobic area corresponds to the

333


L Fig. 11. The contents of the air capillary lining cell seen in Fig. 10 shown in greater detail. Theunfurling material is mostly non-lamellar, but in a few places the characteristic lamellar appear-ance can be distinguished (arrows). x 26250.

b

-.1

e

Fig. 12. The surface of an air capillary cell covered by a single layer of trilaminar material(arrow). The air capillary lining cell (arrow 'e') is extremely attenuated. It rests on a thinbasement membrane (arrow 'b') which separates it from a blood capillary, the cytoplasm (c)and nucleus (n) of which is shown. 4 day old chick. x 75000.

334

Development of chick tertiary bronchus. I3

.'N

e

A ,

c

Fig. 13. Tnfundibular area of the air capillary zone. Several layers of trilaminar material areseen. The thin air capillary lining cell (arrow 'e') is separated from the blood capillary (c) by aninterstitial cell (i). 3 day old chick. x 50000.

hydrocarbon chains (Hayes, Lindgren & Gofman, 1963; Chapman & Fluck, 1966).The width of the avian lining layer is between 8-6 and 12-5 nm, which is clearlyoutside the range of a pure water-phospholipid system (Chapman & Fluck, 1966)and is more akin to that of the unit membrane. Stoeckenius (1962) has postulatedthat the water phase may contain protein; the increased width of the avian liningsystem over a pure phospholipid-water system could therefore be due to proteinmolecules attached in some way to the polar group of the phospholipid. Thus, thelining could be regarded as a lipoprotein, and this seems to concur with the view ofPetrik & Reidel (1968b).At the time of expansion of the air capillaries and during the first 7 days, two sources

appear to contribute trilaminar material: the atrial non-granular pneumocyte cellsand certain air-capillary lining cells. The time of expansion is the time of maximumneed for lining material, as prior to breathing the air capillary zone is unlined. Afterthe first day of hatching there is a decrease in the amount produced in the air capil-lary zone. This suggests that in the mature bird the material is produced only in theatrium, at some distance from where it is likely to be needed, as in the mature state

335

22 ANA II 3


tA, .X

Fig. 14. A bizarre-shaped avian inclu3ion body precursor in an air capiilary lining cell in a 21day embryo. No lamellae are yet apparent. The lumen (I) of the air capillary is shown. x 35000.

the air capillary cells do not seem to have the capacity to produce avian inclusionbodies.

Tyler & Pangborn (1964) noted that in atrial cells trilaminar material appeared todip down into the cell. A similar appearance has been seen in this study, and it is nowknown that this represents the end stage of discharge of an unfurled avian inclusionbody. However, this appearance has been observed by Petrik & Reidel (1968b) andLambson & Cohn (1968) in the capillary lining cells in mature animals. In the pre-sent study, no avian inclusion bodies have been seen in these cells in the mature bird,so the phenomenon described by the above authors must represent an alternativemode of production of trilaminar material in the adult air capillary zone, a featurewhich requires further study.During unfurling of the avian inclusion body, trilaminar material is usually found

lying loose in the cell cytoplasm. However, a slightly different mode of unfurling hasalso been noted in which, prior to discharge, the unfurled trilaminar material is seento be bounded on both sides by a cell membrane of normal thickness (Fig. 15). Asimilar appearance has been noted by Petrik & Reidel (1966a) in the atrial cells ofnewly hatched chicks. Most of the avian inclusions are membrane-bound circularor oval bodies, but some inclusions and precursor bodies have bizarre shapes (Fig.14, 16). If a trilaminar lamella were to be formed in one of the thin narrowed areas of

336


Fig. 15. An atrial lining cell containing trilaminar material which is bounded on both sides bycell membrane (arrows). 1 day old chick. x 120000.

an inclusion such as that shown in Fig. 16, then the appearance described abovewould be produced. It is thus considered that the two methods of unfurling aredependent on whether the laminations are formed in bulk in a compact spherical oroval body or in a bizarre shaped inclusion body.

Comparison between mammalian and avian surface liningThe duplex mammalian alveolar lining requires special fixation methods for its

demonstration (Weibel & Gil, 1968; Gil & Weibel, 1969). The reason why it cannotbe demonstrated by ordinary ultrastructural fixation methods may be that thesmooth, superficial, osmiophilic layer is composed of phospholipid molecules, thehydrophobic hydrocarbon chain end of which is directed towards the gas phase whilstthe polar group is embedded in the hypophase. Conventional aqueous fixatives dis-rupt this layer, which is washed away before fixation can take place, or artefactualmicelles are formed at the surface (Gil & Weibel, 1969). In contrast, the avian liningis demonstrated by conventional techniques, probably because it has a differentmolecular configuration. Whilst it is considered that it has a phospholipid com-ponent, the hydrophobic carbon chains may be directed towards the centre of the

22-2

337


4A

.~ ~ ~ ~~ ~ ~ ~ ~ ~~~~~~~~~~~~A

Fig. 16. A bizarre-shaped inclusion body precursor with no laminations. If a trilaminar

lamination were to develop in the narrow area (arrow), an appearance similar to that seen in

Fig. 15 would be produced. 21 day old embryo. x 50000.

Table 1. Differences between the osmiophilic inclusion

body and the avian inclusion body

OIB Avian

Time of appearance 17 days At commencement of breathingPrecursor body MVB/dense body Large osmiophilic inclusionDiameter 0-2-1-2 ,m 0-2-2-9 ItmSurface area 01-1-5 ,um2 0-28X-5 Aum2Site of production Atrial granular pneumocytes Non-granular pneumocyte atrial lining

cells + (air capillary area*)Laminations Irregular Even and trilaminarLamination periodicity 4-65-3 nm 8-8-13-1 nmMethod of release Merocrine ApocrineSecretion product Myelin bodies Trilaminar lining

* During the first week after hatching only.

338

Development of chick tertiary bronchus. IIbimolecular leaflet, leaving the hydrophilic polar groups and the postulated attachedprotein groups directed towards the air phase. Under these circumstances, contactwith aqueous fixatives would not produce disruption.

If the periodicity of the rat OIB laminations (3-8-5 1 nm) and the micelles pro-duced at the surface of the rat hypophase (3 8-5-1 nm) (Weibel & Gil, 1968), theperiodicity of in vitro phospholipid-water systems (3 7-4 4 nm) (Chapman & Fluck,1966) and the periodicity of avian OIB laminations (44-5 3 nm) (Jones & Radnor,1972) are compared, it is seen that they all fall within an approximately similarrange, suggesting that they all consist of a phospholipid-water system. Thus, theosmiophilic surface lining of the mammalian duplex layer is probably pure phos-pholipid, whilst it is suggested that the avian layer is phospholipid plus protein.

Pattle (1958) has postulated that the avian lung requires surfactant and that theavian lining is present in the situation where, theoretically, there is a need for it.Weibel & Gil (1968) have said that any surface lining must be smooth, to iron outsurface irregularities of the underlying cells; the avian lining manages to do thiswithout the need of a hypophase as the air capillaries have a predominantly smoothoutline. Thus, there is much circumstantial evidence to suggest that the avian lininglayer has a surfactant function. However, current opinion (Clements, 1970; Kikkawa,Motayama & Gluck, 1968) is in favour of the OIB as being the site of surfactantproduction in the mammalian lung, although the present study has shown that theOIB appears to contribute no morphological component to the avian surface lining.If one accepts this reasoning, the function of the OIB in the avian lung becomeshighly problematical.

It is evident that further studies evaluating the surfactant status of birds arerequired. Sequential surfactant studies of the chick embryo and newly hatched chickalong similar lines to that of Buckingham & Avery (1962) in the mouse might behelpful, for it might then be possible to correlate the appearance of surfactantactivity with either the appearance of the OIB at 17 days or with the avian inclusionbody at the time of hatching and the commencement of air breathing.

SUMMARY

The distinctive avian trilaminar lining is derived from a hitherto undescribedorganelle named the avian inclusion body which appears at the time of hatching,being produced in the non-granular pneumocyte type of atrial lining cell and someair capillary lining cells.A precursor body consisting of uniformly osmiophilic material was identified in the

cytoplasm, and this transformed into a laminated avian inclusion body followinghatching; the type and width of its laminations corresponded with the extruded tri-laminar lining.A variable degree of unfurling of the avian inclusion body within the cell was

followed by partial disintegration of the superficial part of the cell cytoplasm andrelease of the trilaminar material into the atria or air capillaries. Production ofavian inclusion bodies was seen in the atrium both in the newly hatched and maturelung, but in the air capillary cells production was limited to the first week of life onlyand here it was most prominent during the first day.

339

340 A. W. JONES AND C. J. P. RADNOR

The avian inclusion body differed from the OIB in the following respects: thetime of first appearance, site and mode of production, surface area, type of lamina-tion and mode of discharge from cells.The significance of the morphology of the avian body laminations was discussed

with reference to its possible chemical composition and compared with that of theOIB, together with its relation to surfactant production.

This work was supported by a grant from The British Heart Foundation.

REFERENCES

AKESTER, A. R. (1970). Osmiophilic bodies as the source of laminated membrane in the epithelial liningof the avian tertiary bronchi. Journal of Anatomy 107, 189.

BUCKINGHAM, S. & AVERY, M. E. (1962). The time of appearance of lung surfactant in the foetal mouse.Nature, London 193, 688-689.

CHAPMAN, D. & FLUCK, D. J. (1966). Physical studies of phospholipids. III. Electron microscopestudies of some pure fully saturated 2,3-diacyl-DL-phosphatidyl-ethanolamines and phosphatidyl-cholines. Journal of Cell Biology 30, 1-11.

CLEMENTS, J. A. (1970). Pulmonary surfactant. American Review of Respiratory Disease 101, 984-990.GIL, J. & WEIBEL, E. R. (1969). Improvements in the demonstration of lining layer of the lung alveoli by

electron microscopy. Respiration Physiology 8, 13-36.HAYES, T. L., LINDGREN, F. T. & GOFMAN, J. W. (1963). A quantitative determination of the osmium

tetroxide-lipoprotein interaction. Journal of Cell Biology 19, 251-255.JONES, A. W. & RADNOR, C. J. P. (1972). The development of the chick tertiary bronchus. I. General

development and the mode of production of the osmiophilic inclusion body. Journal of Anatomy 113,303-324

KIKKAWA, Y., MOTOYAMA, E. K. & GLUCK, L. (1968). Study of the lungs of fetal and newborn rabbits.Morphological, biochemical and surface physical development. American Journal of Pathology 52,177-210.

KLAUS, M., REISS, 0. K., TOOLEY, W. H., PIEL, C. & CLEMENTS, J. A. (1962). Alveolar epithelial cellmitochondria as the source of the surface-active lung lining. Science, New York 137, 750-751.

LAMBSON, R. 0. & COHN, J. E. (1968). Ultrastructure of the lung of the goose and its lining of surfacematerial. American Journal of Anatomy 122, 631-649.

LUZATTI, V. & HUSSON, F. (1962). The structure of the liquid-crystalline phases of lipid-water systems.Journal of Cell Biology 12, 207-219.

MILLER, D. A. & BONDURANT, S. (1961). Surface characteristics of vertebrate lung extracts. Journal ofApplied Physiology 16, 1075-1077.

PATTLE,R. E. (1958). Properties, function andoriginof the alveolar lining layer. Proceedings of the RoyalSociety (London) B 148, 217-240.

PATTLE, R. E. & HOPKINSON, D. A. W. (1963). Lung lining in bird, reptile and amphibian. Nature, London200, 894.

PETRIK, P. & REIDEL, B. (1968a). A continuous osmiophilic non-cellular membrane at the respiratorysurface of the lungs of fetal chickens and young chicks. Laboratory Investigation 18, 54-62.

PETRIK, P. & REIDEL, B. (1968 b). An osmiophilic bilaminar lining film at the respiratory surfaces of theavian lungs. Zeitschrift fur Zellforschung und mikroskopische Anatomie 88, 204-219.

STOECKENIUS, W. (1962). Some electron microscopical observations on liquid-crystalline phases inlipid-water systems. Journal of Cell Biology 12, 221-229.

TYLER, W. S. & PANGBORN, J. (1964). Laminated membrane surface and osmiophilic inclusions in avianlung epithelium. Journal of Cell Biology 20, 157-164.

WEIBEL, E. R. & GIL, J. (1968). Electron microscopic demonstration of an extracellular duplex lininglayer of alveoli. Respiration Physiology 4, 42-57.

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The development of the chick tertiary bronchus