An Electron Microscopic Study on the Type I Pneumocyte in the Cat: Differentiation ANTHONY R. MERCURI02 A N D JOHANNES A. G. RHODIN3 Department of Anatomy, N e w York Medical College, Valkalla, N e w York 10595

ABSTRACT This investigation describes the differentiation of the type I pneumocyte from undifferentiated pulmonary epithelium. Cells lining subpleural alveolar septa were photographed from serial sections with the electron microscope and a three dimensional representation of each cell was obtained by transferring the contours of the cell membranes from micrographs to transparent plastic sheets which were then spaced to scale and stacked. The results of this study indicate that: (1) the only reliable criterion for differentiating between type I and type I1 cells is the commencement of attenuation of the type I cell; ( 2 ) differentiation of the type I cell occurs via the formation of one or more cytoplasmic attenuations that eventually fuse peripherally, thereby surrounding the unattenuated cell soma; ( 3 ) with respect to individual cells, blood-air barriers tend to form in distal areas of the attenuating cytoplasm before proximal areas; ( 4 ) both type I and type I1 pneumocytes retain certain characteristics that reveal their common origin.

In the latter half of the nineteenth century there were two schools of thought regarding the existence of a n epithelium lining the respiratory portion of the lung. One school, supported by Chrzonszczewsky (1863, 1866), maintained that the epithelium was continuous; the other school, supported by Kolliker ( 1 8 8 l ) , maintained that the epithelium was made up of a combination of non-nucleated plates (type I cells) and clusters of small nucleated cells (type I1 cells). Miller ('37), utilizing pathological tissues demonstrating interstitial edema, convincingly demonstrated the existence of a continuous epithelium which not only had been swollen, rendering it within resolution of the light microscope, but also had been lifted from the alveolar wall, demonstrating its continuity. Any doubts remaining concerning the existence of a n epithelium were finally laid to rest by Low ( ' 5 2 ) , whose electron micrographs of rat lung clearly demonstrated that the epithelium was indeed continuous. Bremer ('04), i n his studies on the pouch young of the opossum, was the first to describe the transformation of the type I cell from its cuboidal form to its squamous form with its attenuations extending across the capillaries. Since the time of Low's publication many electron microscopic investiAM. J. AN AT.,^^^: 255-272.

gations have been carried out on the developing lung. Of those investigators who have published work on the developing alveolar lining, most have concentrated on the type I1 cell because of its probable role in the formation of surfactant (Campiche et al., '63; Balis and Conen, '64; Kikkawa et al., '65, '72; Sun, '66; Sorokin, '66; Banks and Epling, '71; Noack, '71; Schneeberger, '72 ) . Some investigators have ventured beyond a cursory description of the developing type I cell (Woodside and Dalton, '58; Leeson and Leeson, '64; Suzuki, '66; Kikkawa et al., '68; O'Hare and Sheridan, '70). However, none has described the threedimensional shape or size of the cell at various stages of development. It is the purpose of this study to report on observations made on the differentiation of the type I cell in the cat Felis domestica by utilizing electron micrographs of serial Accepted February 27, '76. 1 Supported by USPHS General Research Grant FR05398 and the Health Research Council of the CltY of New York U-1508. 2 Portion of a thesis submitted in partial fulfillment of the requirements for the Doctor of Philosophy degree a t New York Medical College. Current address: Department of Anatomy, New York University College of Dentistry, 421 First Avenue, New York, New York 10010. 3 Current address: Chairman, Department of Anatomy, University of Michigan, A n n Arbor, Michlgan 48104.

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gestation. Due to the tedious and time-consuming nature of three-dimensional reconstruction, a single ceIl was reconstructed MATERIALS A N D METHODS for each developmental stage. Outlines of Fetuses of 28, 32, 41 and 47 days’ ges- the type I cells were traced from microtation (term = 63 days) were obtained graphs to clear plastic acrylic sheets onefrom cats anesthetized by injection of sixteenth inch thick (Acrylite, Cyanamid, Nembutal (sodium pentobarbital) into the Wallingford, Connecticut), The sections cephalic vein. Age was estimated, inter- were then cut from the plastic sheet using polated, or extrapolated from crown-rump- a router fitted with a one-sixteenth inch length measurements (Ackerman, ’67). veining bit. Spacers (plastic rod) cut to Samples were taken from the costal sur- scale were cemented with ethylene dichloface of the left diaphragmatic lobe. Sam- ride between the plastic sections. Drawings ples were selected from the same lobe to were then made from the models. eliminate a possible source of error i n OBSERVATIONS chronology because the upper lobes probably mature before the lower lobes (Howatt Undifferentiated epithelium et al., ’65; Brumley et al., ’67; Ablow and Gestation: 32 days Orzalesi, ’71; Blackburn et al., ’72). The Extracellular morphology. At this stage tissues were fixed in 1.5% glutaraldehyde for one hour and post-osmicated in 1% of gestation the lung was in the glandular osmium tetroxide for one hour. Both fixa- (pseudoglandular) stage of development. tives were buffered at pH 7.4 in 0.1 M The epithelium consisted of tall columnar s-collidine (Polysciences Inc., Warrington, cells resting on a basal lamina. Large inPa. ). Phosphate-buffered osmium was not tercellular spaces were frequently seen beused because fixing lung tissue in glutaral- tween the non-interdigitating cell memdehyde followed by osmium tetroxide in branes. Some microvilli were present in the phosphate buffer results in the appearance apical portions of the cells. Tight junctions of “spherical electron dense granules . . . were seen between the apical portions of as artifacts of the alveolar surface” (Gil adjacent cells. Intracellular morphology. The ovoid nuand Weibel, ’68; OHare and Braunshweig, ’75). Specimens were dehydrated in etha- clei containing prominent nucleoli were located basally. Virtually all of the chronol and embedded in Epon. Sections 0.5 pm thick were stained with matin was euchromatic. The cytoplasm toluidine blue-0 for light microscopy. Spec- abounded with electron-lucent areas from imens containing subpleural alveolar septa which glycogen probably had been lost durwere then serially sectioned for electron ing fixation. Most of the remaining cytomicroscopy using a diamond knife. The plasm was filled with polyribosomes. An respiratory portions of the lung develop occasion a1 mitochondrion, thick-walled vesin the hilar regions before the peripheral icle, centriole or segment of rough endoregions (Kikkawa et al., ’68). The error plasmic reticulum was observed. introduced due to the different portions of the lung being at different stages of de- Gestation: 41 days Extracellular morphology. At this stage velopment was eliminated by examining alveolar septa in the same area (sub- of gestation the epithelium in the proximal portions of the lung was in the canalicular pleural). Ribbons of sections were picked u p on stage of development. However, the cells of formvar-coated grids with a 1 x 2 m m the proliferating terminal buds remained hole and doubly stained with a saturated undifferentiated. Although the cells of the aqueous solution of uranyl acetate followed terminal buds remained undifferentiated by Karnovsky’s “A’ lead monoxide (Kar- they differed morphologically from cells of novsky, ’61). Grids were examined in a the 32-day fetus. Siemens Elmiskop 1A. The cellular height of the epithelium was Models of entire type I cells were con- reduced to low columnar or high cuboidal. structed for fetuses of 41 and 47 days’ The model of one of these undifferentiated

sections to reconstruct three-dimensional plastic models.

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cells measured 12 x 14 x 8 pm (fig. l ) . 4 old of differentiation contained osmioIn overall dimensions the cell would have philic lamellar bodies. The reconstructed been classified as cuboidal, but most of the cell contained four of these lamellar bodies, cellular volume was confined to dimen- all displaying concentric lamellae (fig. 5). sions of 12 X 7 X 4 pm. These dimensions Generally, there was a gradation in numwould have classified the cell as low colum- ber of lamellar bodies, decreasing i n numnar. The discrepancy in dimensions was ber in cells toward the more proximal portions of the lung. due to the cell having a broad base. In the reconstructed cell some of the The basal lamina consisted of a proximal electron-lucent zone and a distal electron- cisternae of a n elongate Golgi complex condense zone (fig. 2 ) . The basal surface of tained a fine granular material. A smaller the cells conformed to the almost spherical poorly developed Golgi complex had a curvature of the terminal bud, the bases multivesicular body associated with it. Ribosomes were either associated with of the cells each overlying a small portion of the spherical basal lamina. This spheri- endoplasmic reticulum or formed polycal shape was interrupted where the basal ribosomes, the latter being more prevalent. lamina entered the space between the A n occasional dense-core body or centriole bases of adjacent cells (fig. 2 ) . All of the was seen (figs. 4, 5). cellular surfaces, including the basal surDifferentiation faces, demonstrated irregularities, most of Gestation: 47 days which took the form of microvillous projections (fig. 2). The only reliable criterion for distinTight junctions were present between guishing between a differentiating type I apical portions of adjacent cells (fig. 3 ) . cell and a differentiating type I1 cell was The tight junction surrounding the apical the commencement of attenuation. Two portion of the cell from which the model other criteria aided in distinguishing bewas constructed formed an irregular circu- tween the two cell types. One, cells having lar pattern. large numbers of lamellar bodies, displayA capillary, located beneath the basal ing parallel stacked plates, probably diflamina of the reconstructed cell, was sep- ferentiated into type I1 cells. This type of arated from it by an interstitial space. lamellar body was rarely seen in type I cells. The other criterion was the amount (fig. 4). Intracellular morphologv. The nucleus of glycogen present in the cell. The larger of the reconstructed cell-was centrally lo- the amount of glycogen in the cell the more cated in the basal aspect of the cell. Its likely it was to be destined to differentiate shape was roughly that of a truncated into a type I1 cell. tetrahedron not unlike the overall shape Extracellular morphology. At this stage of the cell. A prominent nucleolus was of gestation the epithelium subjacent to the located centrally and a smaller nucleolus pleura was also in the canalicular stage of was situated basally near the nuclear mem- development, with the cells in the process brane. Most of the chromation was euchro- of attenuation. Some areas appeared unmatic but some evidence of heterochroma- differentiated but serial sections revealed tin was observed, predominantly subjacent attenuations in deeper sections. I n overall to the nuclear membrane (figs. 3, 4). dimensions the reconstructed cell at this Areas of lost glycogen persisted in some stage of gestation was cuboidal, measuring of these undifferentiated cells while other 9.5 x 9 x 9 pm (fig. 6 ) . However, a seccells had retained some of the glycogen tion through the superficial portion rethat was fixed in situ (fig . 3 ) . vealed the cell's squamous shape (fig. 7 ) , Ovoid, elongate or irregular mitochon- a section bisecting the nucleus demondria were ubiquitous. A moderate number strated the cuboidal shape (fig. 8 ) ,whereas of mitochondria1 cristae formed incomplete a section through the deeper end revealed transverse septa. Approximately 28 mitochondria were counted in the reconstructed 4 H e i g h t (basal lamina .to lumen) x w i d t h . (at a right angle t o the helght m the plane of sectlon) x cell. thickness (dimension perpendicular to the plane of Virtually all of these cells on the thresh- section).

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the cell's columnar shape (fig. 9). The almost semicircular attentuated portion of the reconstructed cell originated from the upper third of the cell soma (fig. 6). Instead of appearing in their usual location near the apex of the cell, tight junctions can be seen nearer the base of the reconstructed cell (fig. 9). Numerous microvilli can be seen projecting from the cell surface (fig. 9). Unlike the 41-day stage, the basal lamina on which the reconstructed cell rested was highly irregular in shape, reflecting invasion by capillaries. Compare the basal lamina in figure 3 with the basal lamina in figure 7. A nascent blood-air barrier, 0.74 pm thick, was present in the attenated area of the reconstructed cell. The interstitial space had not been completely eliminated by the fused basal laminae of the type I cell and the endothelial cell (fig. 7). However, sections through the center of the cell revealed fused basal laminae before commencement of attenuation in this region (fig. 8). Presumably, it is arbitrary whether attenuation or fusion of basal laminae occurs first in the formation of the barrier. The endothelium was not fully attenuated at this early stage (figs. 7, 8). Intracellular morphology. The eggshaped, centrally located nucleus of the reconstructed cell had a few basal grooves. The single nucleolus present was in the central portion of the nucleus. The ratio of euchromatin to heterochromatin was about the same as that of the 41-day fetus. Compare figures 3 and 9. Glycogen was found throughout the reconstructed cell in discrete masses containing one or more lamellar bodies. The mitochondria, numbering over 60 in the reconstructed cell, were elongate or irregular in shape with sparse transverse plate-like cristae. The lamellar bodies, about 30 in number in the reconstructed cell, varied in appearance, size and electron-density. Most consisted of concentric lamellae, some had irregularly shaped lamellae and others possessed an electron-dense amorphous material (figs. 8, 9, 10). Two small Golgi complexes were located in the apical region of the reconstructed cell. One contained a finely granular mate-

rial in one of the cisternae; the other had dilated cisternae (fig. 11). The cytoplasm of the reconstructed cell contained a moderate amount of rough endoplasmic reticulum, the remaining cytoplasm being filled with polyribosomes. A number of thick-walled vesicles were observed, mostly confined to the basal portion of the reconstructed cell (fig. 11). Three multivesicular bodies were observed in the reconstructed cell, two with electron-lucent matrices (fig. 9 ) and one with an electrondense matrix (fig. 10). A centriole was located in the apical region of the cell. DISCUSSION

Lamellar bodies Lamellar bodies, found predominantly in type I1 pneumocytes, are believed to contain precursors of, or the definitive, surface-active material lining lung alveoli (Buckingham and Avery, '62; Pattle and Thomas, '61; Campiche et al., '63; Kikkawa et al., '65, '68; Kikkawa and Spitzer, '69; Finley et al., '68; Page-Roberts, '72). According to Blackburn et al. ('72) different morphological appearances of granules within type I1 cells represent a maturational sequence. The granular component synthesized by rough endoplasmic reticulum may form a multivesicular body in which lamellae appear in flat stacks; sub. sequently the lamellae curve and at maturity appear as parallel stacks or as concentric whorls, neither of which display any remaining granular component. Balis and Conen ('64), in their study of lamellar bodies in developing human lung, often observed both lamellar bodies and multivesicular bodies in close proximity to Golgi complexes, suggesting that the Golgi complex is involved in the metabolism of lamellar bodies. Apparently mature lamellar bodies assume different configurations in different species (Kikkawa and Spitzer, '69) and in the same species with different fixatives (Campiche, '60). The lamellar body containing parallel stacks of lamellae is the predominant configuration in the cat. In the present investigation lamellar bodies were observed in type 11, type I and undifferentiated cells of the cat. While lamellar bodies found in type I cells are less numerous and convincing, a study by Kik-

DIFFERENTIATING TYPE I PNEUMOCYTE

kawa and Kaibara (’72), utilizing osmiumethanol treatment to enhance osmiophilia of membranes, clearly demonstrated the existence of lamellar bodies in type I cells. This technique revealed osmiophilic bars previously unreported in type I1 cells. In addition, lamellar stacks were observed in Golgi complexes, multivesicular bodies and vesicles. This was the first report of type I cells containing lamellar bodies in adult lung. However, in developing rat lung epithelium, Balis and Conen (’64) reported that lamellar bodies were “seldom” seen in type I cells. Suzuki (’66) reported that lamellar bodies had decreased in number in type I cells of the 21-day rat fetus compared to earlier stages.

Microbodies Microbodies (peroxisomes ) , discovered by one of us (Rhodin, ’54), have been identified morphologically and cytochemically in type I1 cells by Schneeberger (’72). According to Schneeberger, microbodies in type I1 cells “may play an indirect role in surf actant synthesis.” The dense-core vesicles (dense-core bodies, granules) found in type I cells in the present study were morphologically identical to the microbodies of Schneeberger’s study. Kikkawa et al. (’68) noticed that dense-core bodies are seen in type I cells in earlier stages of their differentiation but disappear completely in later stages. Microvilli Whether, in fact, the microvilli of the differentiating type I cell were less numerous than those of the type I1 cell was not investigated in this study of the cat. The reconstructed differentiating type I cell (47 days) displayed a two-fold increase in microvilli over the undifferentiated cell. Differentiation Differentiation of the pulmonary epithelium into type I and type I1 cells commenced with the attenuation of cuboidal pulmonary epithelial cells. Attenuation was the only characteristic that could have been utilized to identify a differentiating type I cell. Kikkawa et al. (’68) arrived at the same conclusion in their study of fetal rabbits. Balis and Conen (’64) reported findine attenuations in tvae I1 cells in deD

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veloping human lung, On the other hand, observing lamellar bodies in a fetal alveolar epithelial cell is not a criterion for identifying the cell as type 11, because undifferentiated and differentiating type I cells also contain lamellar bodies. We suggest, therefore, that the attenuations described by Balis and Conen (’64) were those of a differentiating type I cell and not a type I1 cell. As definitive as the differentiation between type I and type I1 pneumocytes may be, both cell types retain certain characteristics that reveal their common origin; for example, microvilli are present in both cell types, suspected precursors of lamellar bodies are found in both cell types, both cell types are capable of phagocytosis (Suzuki et al., ’72), lamellar bodies have been observed in type I pneumocytes, phylogenetically, adult amphibian type I pneumocytes contain lamellar bodies morphologically identical to those of type I1 pneumocytes (Okada et al., ’61) and both type I and type I1 cells may be capable of modulating to cuboidal shapes after exposure to noxious gases (Macklin, ’39) or in pathological conditions (Bell, ’43; Geever et al., ’43). ACKNOWLEDGMENTS

The authors wish to thank Miss Mary Lorenc for the drawings of the reconstructed cells. LITERATURE CITED Ablow, R. C., and M. M. Orzalesi 1971 Localized roentgenographic pattern of hyaline membrane disease. Am. J. Roentgenol. Radium Ther. Nucl. Med., 112: 23-27. Ackerman, G. A. 1967 Developmental relationship between the appearance of lymphocytes and lymphopoietic activity in the thymus and lymph nodes of the fetal cat. Anat. Rec., 158: 387400. Balis, J. U., and P. E. Conen 1964 The role of alveolar inclusion bodies i n developing lung. Lab. Invest., 13: 1215-1229. Banks, W. J., and G. P. Epling 1971 Differentiation and origin of the type I1 pneumocyte: A n ultrastructural study. Acta anat., 78: 604620. Bell, E. T. 1943 Hyperplasia of the pulmonary alveolar epithelium i n disease. Am. J. Path., 19: 901-911. Blackburn, W. R., H. Travers and D. M. Potter 1972 The role of the pituitary-adrenal-thyroid axes i n lung differentiation. Lab. Invest., 26: 306-3 18. Bremer. J. L. 1904 On the lung- of the OPOSsum. Am. J. Anat., 3 : 67-74.

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Brumley, G. W., V. Chernick, W. A. Hodson, E. Normand, A. Fenner and M. E. Avery 1967 Correlation of mechanical stability, morphology, pulmonary surfactant, and phospholipid content in developing lamb lung. J. Clin. Invest., 46: 863-873. Buckingham, S., and M. E. Avery 1962 Time of appearance of lung surfactant i n the foetal mouse. Nature, 193: 688-689. Campiche, M. 1960 Les inclusions lamellaires des cellules alveolaires dans le poumon du Raton. J. Ultrastruct. Res., 3: 302-312. Campiche, M., A. Gautier, E. I. Hernandez and A. Reymond 1963 An electron microscope study of the fetal development of human lung. Pediatrics, 32: 976-994. Chrzonszczewsky, N. 1863 Beber das Epithel der Lungenblaschen (SIC) der Saugethiere; Wiirzburger med. Zeitschr. Bd. 4 cited by W.S. Miller. In: The Lung. Thomas, Springfield, 1937,pp. 56-57. - 1866 Zur Lehre von dem Lungenepithel. Arch f . pathol. Anat. u. Physiol. Bd 35. Cited by W. S. Miller. In: The Lung. Thomas, Springfield, 1937,PP. 56-57. Finley, T. M., S. A. Pratt, A. J. Ladman, L. Brewer and M. B. McKay 1968 Morphological and lipid analysis of the alveolar lining material in dog lung J. Lipid. Res., 9: 357-365. Geever, E. F., K. T. Neubuerger, C. L. Davis 1943 The pulmonary alveolar lining under various pathologic conditions in man and animals. Am. J. Path., 19: 913-937. Gil, J., and E. R. Weibel 1968 The role of buffers in lung fixation with glutaraldehyde and osmium tetroxide. J. Ultrastruct. Res., 25: 331348. Howatt, W. F., M. E. Avery, P. W. Humphreys, I. C. Norman, L. Reid and L. B. Strang 1965 Factors affecting pulmonary surface properties i n foetal lambs. Clin. Sc., 29: 239-248. Karnovsky, M. J., 1961 Simple methods for “staining with lead” at high pH in electron microscopy. J. Biophys. Biochem. Cytol., 11: 729-732. Kikkawa, Y., and M. Kaibara 1972 The distribution of osmiophilic lamellae within the alveolar and bronchiolar walls of the mammalian lungs as revealed by osmium-ethanol treatment. Am. J. Anat., 134: 203-220. Kikkawa, Y., E. K. Motoyama and C. D. Cook 1965 The ultrastructure of the lungs of lambs Am. J. Path., 47: 877-904 . Kikkawa, Y., E. K. Motoyama and L. Gluck 1968 Study of the lungs of fetal and newborn rabbits. Am. J. Path., 52: 177-210. Kikkawa, Y., and R. Spitzer 1969 Inclusion bodies of type I1 alveolar cells: species differences and morphogenesis. Anat. Rec., 163: 525542. Kolliker, A. 1881 Zur Kenntnis des Baues der Lunge des Menschen; Verhandl. dl. phys.-med. Gesell zu Wiirzburg., cited by W. S. Miller: In: The Lung. Thomas, Springfield, 1937, pp. 5659,61.

Leeson, T. S., and C. R. Leeson 1964 A light and electron microscope study of developing respiratory tissue in the rat. J. Anat. (London), 98: 183-193. Low, F. N. 1952 Electron microscopy of the rat lung. Anat. Rec., 113: 437-450. Macklin, C. C. 1939 Sections showing a continuous layer of cuboidal epithelium on the alveolar walls of the cat’s lung after exposure to the fumes of osmium tetroxide. Anat. Rec., 73 suppl. 2: p. 71. Miller, W. S. 1937 The Lung. Charles C Thomas, Springfield, pp. 60-62. Noack, W. 1971 Das electronenmikroskopische Bild des Lungenepithels von Rattenembryonen vom Tag 16 bis zur Geburt. Acta. anat., 79: 445-465. O’Hare, K. H., and R. J. Braunsweig 1975 The effects of various fixative-buffer combinations o n lung fine structure. Anat. Rec., 181: 545560. O’Hare, K. H., and M. N. Sheridan 1970 Electron microscopic observations on the morphogenesis of the albino rat lung with special reference to pulmonary epithelial cells. Am. J. Anat., 127: 181-206. Okada, Y.S., Ishoko, S. Daido, J. Kim and S. Ikeda 1961 Comparative morphology of the lung with special reference to the alveolar epithelial cells: I. Lung of the amphibia. Acta. Tuberc. Jap., 11: 7/63-16/72. Page-Roberts, B. A. 1972 Preparation and partial characterization of a lamellar body fraction from rat lung. Biochem. Biophys. Acta., 260: 334-338. Pattle, R. E.,and L. C. Thomas 1961 Lipoprotein composition of the film lining the lung. Nature, 189: 844. Rhodin, J. 1954 Correlation of ultrastructural organization and function in normal and experimentally changed proximal convoluted tubule cells of the mouse kidney. Thesis, Karolinska Institutet, Stockholm. Schneeberger, E. E. 1972 A comparative cytochemical study of microbodies (peroxisomes) i n great alveolar cells of rodents, rabbit and monkey. J. Histochem. Cytochem., 20: 180-191. Sorokin, S. P. 1966 A morphologic and cytochemical study on the great alveolar cell. J. Histochem. Cytochem., 14: 884-897. Sun, C. M. 1966 Lattice structures and osmiophilic bodies i n the developing respiratory tissue of rats. J. Ultrastruct. Res., 15: 380-388. Suzuki, Y. 1966 The structural differentiation of the alveolar lining cells: I. Electron microscopic studies on the prospective alveolar epithelium i n the lung tissue of rat embryos. Okaj. Fol. Anat. Jap., 42: 114-147. Suzuki, Y., J. Churg and T. Ono 1972 Phagocytic activity of the alveolar epithelial cells i n pulmonary asbestosis. Am. J. Pathol., 69: 373388. Woodside, G. L., and A. J. Dalton 1958 The ultrastructure of lung tissue from newborn and embryo mice. J. Ultrastruct. Res., 2: 28-54.

PLATES

PLATE 1 EXPLANATION OF FIGURE

1 Drawing from a plastic reconstruction of a cell from the undifferentiated pulmonary epithelium of 41-day-old fetus. Base of cell is toward bottom of page. Potential alveolar space is toward top of page above tight junction (TJ). This cellular orientation, with base toward bottom and apex toward top of page, is maintained throughout the figures. Nucleus ( N ) Capillary (CL).

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PLATE 1

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PLATE 2 EXPLANATION OF FIGURES

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2

Basal portions of several undifferentiated cells from a terminal bud of a 41-day fetal lung. Basal lamina (arrows) can be seen entering space formed between bases of adjacent cells. Microvillous projections (MV). ~ 8 , 4 0 0 .

3

Undifferentiated epithelium from a terminal bud of 41-day fetal lung. Tight junctions (TJ and inset) are seen between the apical portions and large intercellular spaces ( x ) between the more basal portions of the epithelial cells. Golgi complex (Go) and lamellar bodies (LB and inset) are apparent. Areas of “washed out” glycogen ( G ) can be seen surrounded by areas of glycogen fixed i n situ. Basal lamina (BL) x 5,700. Insets x 12,000.

4

Basal portions of cells of undifferentiated epithelium from a 41-day fetal lung. Labelled mitochondria ( M ) are within the cell from which the reconstruction was made. In this section, the cell, roughly the shape of a parallelogram, is separated from a capillary ( C ) by a n interstitial space (x). A n adjacent cell contains dense-core vesicles (DCV and inset) and thick-walled vesicles ( V and inset). ~ 8 , 1 0 0 .

5

Apical portion of reconstructed cell from 41-day fetus displaying nucleus ( N ) , nucleolus ( N u ) and lamellar bodies (LB). Centriole ( C ) and cilium (Ci) are apparent i n adjacent cells. X 7,500. Inset x 12,000.

DIFFERENTIATING TYPE I PNEUMOCYTE Anthony R. Mercurio and Johannes A. G . Rhodin

PLATE 2

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PLATE 3 EXPLANATION O F F I G U R E

6

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Drawing made from a reconstructed differentiating type I pneumocyte from a fetus of 47 days’ gestation. Notice that the attenuation arises from the apical portion of the cell to approximate the capillary ( C ) passing below. The centrally located nucleus ( N ) and perinuclear cytoplasm are protruding into the lumen of the canaliculus above the tight junction ( T J ) . This cell was sectioned sequentially from right to left as depicted in the drawing, i n a vertical plane perpendicular to that of the drawing. The arrows indicate the three areas of the cell from which micrographs of the following three figures were made.

DIFFERENTIATING TYPE I PNEUMOCYTE Anthony R . Mercurio and Johannes A. G . Rhodin

PLATE 3

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PLATE 4 EXPLANATION

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O F FIGURES

7

Superficial section through the attenuation of the reconstructed type I cell from a 47-day fetal lung. The tight junction (unlabelled arrows) can be seen at both ends of the attenuated portion of cytoplasm. The attenuation and poorly defined interstitial space separate the capillary ( C ) from the potential alveolar space ( A S ) of a canaliculus. Endothelium ( E n ) , fused basal laminae (BL) of endothelial and epithelial cells. x 11,100.

8

Section through the center of the reconstructed differentiating type I cell from the lung of a 47-day fetus. Note that cell is cuboidal and its basal lamina (BL) is fused with the basal lamina of the capillary endothelial cell, i n anticipation of the impending attenuation in this area. Microvilli (MV) project into the potential alveolar space ( A S ) . Capillary lumen ( C ) . Cell apical region can be seen to form a tight junction ( T J ) with adjacent cell o n right. Cellular contents include: glycogen ( G ) , basally located thick-walled vesicles ( V ) , mature lamellar bodies (LB and lower inset) and immature lamellar bodies (ILB and upper inset). X 8,100. Insets X 12,000.

DIFFERENTIATING TYPE I PNEUMOCYTE Anthony R. Mercurio a n d J o h a n n e s A. G . Rhodin

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PLATE 5 EXPLANATION O F F I G U R E S

9

Section through deep end of the reconstructed type I cell from a lung of a 47-day old fetus. Note columnar shape of cell. Contents include multivesicular body (MVB and inset) with electron-lucent matrix and a lamellar body ( L B ) . Tight junction ( T J ) . Numerous microvilli are present. x 7,200.

10 Reconstructed cell of 47-day fetus containing multivesicular body with electron-dense matrix (MVB) and lamellar bodies i n different stages of maturation ( L B ) . x 12,000.

11

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Apex of reconstructed cell from lung of 47-day fetus. Two Golgi complexes (Go) thick-walled vesicles ( V ) and microvilli ( M V ) . x 12,000.

DIFFERENTIATING TYPE I PNEUMOCYTE Anthony R. Mercurio and Johannes A. G. Rhodin

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An electron microscopic study on the type I pneumocyte in the cat: differentiation.

An Electron Microscopic Study on the Type I Pneumocyte in the Cat: Differentiation ANTHONY R. MERCURI02 A N D JOHANNES A. G. RHODIN3 Department of Ana...
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