Ultrastructural features of type II alveolar epithelial cells in early embryonic mouse lung.код для вставкиСкачать
THE ANATOMICAL RECORD 221:846-853 (1988) Ultrastructural Features of Type II Alveolar Epithelial Cells in Early Embryonic Mouse Lung ANK A.W. TEN HAVE-OPBROEK, JACQUELINE A. DUBBELDAM, AND CAROLINE J.M. O'ITO-VERBERNE Department of Anatomy and Embryology, Faculty of Medicine, University of Leiden, 2300 RC Leiden, The Netherlands ABSTRACT Immunofluorescence studies of type I1 alveolar epithelial cells indicate that they first appear in the pseudoglandular period of mouse lung development (around day 14.2).They are the only cell type to line the prospective pulmonary acinus at this time. The ultrastructural characteristics of this cell are defined by investigating embryos aged 13-16 days with transmission and scanning electron microscopy. Early embryonic type I1 cells appear as low-columnar or cuboid cells having large, approximately round nuclei and distinct ultrastructural features, including a welldeveloped Golgi apparatus with many associatedvesicles, multivesicular bodies, dense bodies, and large apical and basal glycogen fields. These fields represent a distinctive property of the cell. Frequently, they show compartmentalization due to the presence of membrane systems, and association with dense bodies of various sizes. Light microscope studies of lung development in the mouse (Ten Have-Opbroek, 1975, 19791, the rat (OttoVerberne and Ten Have-Opbroek, 1987a), and humans (Otto-Verberne and Ten Have-Opbroek, 198713) have made use of an antiserum that specifically recognizes a cell-specific antigen of the type I1 alveolar epithelial cell, presumably a surfactant-associated protein (Van Hemert et al., 1986; Otto-Verberne and Ten HaveOpbroek, 1987b), in the adult lung. These immunological studies first detect the type I1 cell in the embryonic lung in the pseudoglandular period, i.e., in the mouse on day 14.2, in the rat on day 16, and in humans around week 11 after conception. The presence of the antigen in embryonic type I1 cells-as well as in type I1 cells of alveolar and papillary mouse lung tumors (Rehm et al., 1988)-indicates that a fundamental cell property must be concerned here. Apart from the property of the unique cell marker, the early embryonic type I1 cell also shows distinct morphological features of the mature cell: an approximately cuboid shape and a large, more or less round nucleus; but it does not contain yet the characteristic multiamellar bodies. Therefore, this differentiating cell, which is present throughout lung development and presumably also in the mature lung (Ten Have-Opbroek, 1981), has been called the precursor cell of the type I1 alveolar epithelial cell (Ten Have-Opbroek, 1979). Initially, only cells of this type line the prospective respiratory portion of the lung and thus its unit, the pulmonary acinus; in later gestational stages this kind of cell gives rise to mature type I1 alveolar epithelial cells or to type I alveolar epithelial cells in this lining (Ten Have-Opbroek,1979; Otto-Verberneand Ten Have-Opbroek, 1987a, 1987b). Both the alveolar type and the bronchial type of epithelium originate from primordial epithelial cells, which line the original branching tubular system, the primordial system of the developing mouse lung. The primordial epithelial cells are present between the appearance of the lung anlage 0 1988 ALAN R. LISS, INC. around day 9.5 and approximately day 14.2, and in places also at later times (Ten Have-Opbroek, 1981; see also Otto-Verberne and Ten Have-Opbroek, 1987a). The present study was performed to define the ultrastructural characteristics of the early embryonic type I1 cell. For this purpose, lungs of embryos of the appropriate age (13-16 days) and belonging to the same inbred Swiss-type mouse strain (CPB-S) as used in the earlier studies were investigated by transmission (TEM) and scanning electron microscopy (SEMI, with special attention given to the cell type in the lining of the most peripheral branches of the tubular system present. TEM and SEM studies on the development of the respiratory portion of the respiratory tract or the type I1 cell of the alveolar epithelium have been performed in several species, including the mouse (Campiche et al., 1963; Hitchcock OHare and Sheridan, 1970; Wang et al., 1973; Williams and Mason, 1977; Fukuda et al., 1983; Hilfer, 1983; Dilly, 1984). However, most of these studies did not start a t comparably early ages or follow the development at comparably small intervals. MATERIALS AND METHODS In the present as in the earlier studies in the mouse (Ten Have-Opbroek, 1975,1979,1981,1986; Van Hemert et al., 1986), we used an inbred Swiss-type mouse strain (CPB-S) (Centraal ProefdierenbedrijfTNO, Zeist, The Netherlands) with a gestation time of about 19 days, calculated from conception. The embryos used for TEM and SEM were obtained from female mice with a timed pregnancy, aged about 4 months and weighing 23-30 gm. The mothers usually carried 8 to 12 embryos having diverging weights. Since the weight of an embryo is a more sensitive indicator for the developmental stage than the actual age in days after conception, the weight is used as parameter in all of our studies. The Received November 24, 1986; accepted December 1, 1987. ULTRASTRUCTURE OF EMBRYONIC TYPE I1 CELL embryos were selected on the basis of their weight. The location of the weight of a mouse embryo on a growth curve (based on the relationship between weights and the corresponding ages) provides us with the mean corresponding age, which we call the “developmental age” (Goedbloed, 1976). The embryos used are indicated in the text by their developmental age and total body weight. In the mouse, the left lung consists of one large lobe, whereas the right lung has four lobes. The lung specimens for this study were taken from the left lung lobe; the histological picture was always compared with that of specimens taken from the right lung lobes. In these specimens, 10-15 peripheral lung areas were viewed per embryo. Three to five embryos from two mothers were investigated for each of the age groups indicated in Table 1. Transmission Electron Microscopy The pregnant mice were anesthetized with chloroform and killed by cervical dislocation. The embryos were removed from the uterus, weighed, and fixed by immersion in 2% paraformaldehyde and 1.25%glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for about 30 min at 4°C. The lungs were removed by thoracotomy, fured by immersion in the same furative for 24 hr at 4”C, and then postfixed in 1%osmium tetroxide in 0.1 M cacodylate buffer (pH 7.4) for 2 hr at room temperature. After dehydration via a graded alcohol series (70-loo%, each step lasting 10-20 min), the tissue was transferred to a mixture of propylene oxide and Epon (1:l) for 90 min, stored overnight in Epon in an exsiccator under low vacuum for better penetration of Epon into the lung tissue, and then embedded in fresh Epon. In embryos aged between 13.0 and 14.0 days, the complete heart-lung block was embedded; from day 14.0 onward, the right and left lungs were embedded separately. Sections (1 pm) stained with toluidine blue were used for orientation. Ultrathin sections (ca. 90 nm) were mounted on R 100 grids (Veco,The Netherlands), stained with aqueous uranyl acetate 7% (Pease, 1964) for 20 min and alkaline lead citrate (Reynolds, 1963) for 10 min, and examined with a Philips 300 electron microscope at 80 kV. Scanning Electron Microscopy system of the lung of an embryo aged 13.6 days (embryonic weight 200 mg), consisted of primordial cells with a more or less oval nucleus and many microvilli on the surface (Fig. 1). Usually no basal lamina was present along the most distal branches (not illustrated). Glycogen occurred diffusely scattered throughout the cytoplasm, both as single particles and aggregates (Fig. 2). Occasionally, a glycogen accumulation contained a whorl of membranes (see Figs. 5 and 6). The endoplasmic reticulum (Fig. 2) showed cisternae that were slightly distended and filled with a fine, homogeneous, slightly electron-dense material. The supranuclear Golgi apparatus (Fig. 2) was composed of curved, stacked saccules; a few associated small vesicles were located in the concavity of the stacks. Adjacent cells displayed distinct junctional complexes a t their apical borders. The primordial epithelium in peripheral areas of the lungs of embryos aged 13.0-13.7 days (embryonicweights 145-215 mg) showed a comparable ultrastructural picture (Table 1). On days 13.9, 14.0, and 14.1 (embryonic weights 240, 250, and 264 mg), the primordial epithelial cells constituting the lining of the end-pieces of the primordial system were usually arranged in a single layer and looked somewhat different (see Table 1). The shape of the cells was columnar or low-columnar, and the nucleus was oval or approximately round. The cells bore a small number of microvilli. Glycogen aggregates were no longer detectable. An occasional cell contained a few dense inclusion bodies, or a Golgi apparatus with a large number of associated vesicles in the cytoplasm. In general, however, the fine structure of the cells resembled that seen in the earlier stages (Table 1). Type II Cell Precursor and Pulmonary Acinus On days 14.2, 14.3, and 14.4 (embryonic weights 279, 303, and 345 mg), the appearance of the end-pieces of the tubular system of the lung was often quite different (Fig. 3, Table 1). These end-pieces now represented the prospective respiratory units or pulmonary acini (Ten Have-Opbroek, 1979, 1981). At present, the term pulmonary minus is normally used as a collective term for structures situated distal to the terminal bronchiole, especially in work dealing with systemic histological descriptions of the respiratory tract (see review by Ten Have-Opbroek, 1986). Some textbooks (Gray’s Anatomy, 1973; Bloom and Fawcett, 1975; Gardner et al., 1975) use the termprimary lobule to describe this functional respiratory unit, frequently in a more regional context-that is, as subunit of the macroscopically visible larger lung lobule that is supplied by a preterminal bronchiole (also called secondary lobule). However, be- Lungs were obtained as described above. After 24 hr of immersion furation in the same fixative as used for TEM, the lung lobes were cut in half and the tissue was postfxed for 2 hr in 1% osmium tetroxide in 0.1 M cacodylate buffer (pH 7.4) a t room temperature. The specimens were then passed through a graded alcohol series (70-loo%, each step lasting 30 min), and transferred to a [email protected] for critical-point-drying with liquid carbon dioxide. The dried specimens were mounted on aluminum stubs with conductive carbon cement (Carbon Leit-C), coated with gold in a Polaron Sputter Coater, and examined in a Cambridge S 180 scanning electron microscope at BL C 15 kV. DB RESULTS Primordial Epithelium 847 G GA M N P The pseudostratified (or sometimes simple) columnar epitheiium that lined the distal part of the primordial s Abbreviations Basal lamina Capillary Dense body Glycogen Golgi apparatus Mesenchyme Nucleus Pleura Saccule 848 A.A.W. TEN HAVE-OPBROEK ET AL. Figs. 1, 2. "EM pictures of left lung lobe at 13.6 days (embryonic weight: 200 mg). Fig. 1. End-piece of the original branching tubular system, the primordid system of the lung. The pseudostratified columnar epithelium of the primordial tubule is studded with microvilli. The shape of the nuclei is usually oval. Glycogen occurs diffusely scattered throughout the cytoplasm. Boxed area is shown enlarged in Figure 2. X 1,430. cause this primary lobule also comprises all associated structures of the respiratory unit (blood vessels, lymphatics, nerves, connective tissue), this term is in fact not applicable in systemic histology. For this reason, probably, the term has been deleted from the text in recent editions of some textbooks (Fawcett, 1986; ORahilly, 1986). In the adult lung, the pulmonary acinus is composed of its definitive components, namely, the alveolated respiratory bronchiole, the alveolar duct, and the alveolar sac (reviewby Ten Have-Opbroek, 1986). The respiratory bronchiole, however, is not always present in mammalian species (review by Tyler, 1983). It is evident that this definition of the adult pulmonary acinus presents a main problem. It implies that the mature respiratory unit (unlike the developing equivalence, see below) is not defined as a separate histological entity. For the respiratory bronchiole contains not only alveolar epithelium, as is characteristic of the respiratory unit, bct also bronchial epithelium in its lining. The epithelial pattern of this tubule has generally been assumed to be of a mixed bronchial-alveolar Fig. 2. The cytoplasm of a primordial epithelial cell contains cisternae of smooth and rough endoplasmic reticulum, which may be slightly distended (arrowheads),as well as a supranuclear Golgi apparatus with a few associatedvesicles. Glycogen is present as single particles or aggregates. x 9,600. type (review by Ten Have-Opbroek, 1986). To the contrary, however, ultrastructural studies of the adult mouse lung (Ten Have-Opbroek, 1986) have shown that, in this species, the respiratory bronchiole portion of the respiratory tract consists of two sharply demarcated parts-a proximal bronchial part (columnar epithelium) and a distal (very probably) alveolar part (cuboid and squamous epithelium). On this basis we have proposed a different classification for this type of tubule (Ten Have-Opbroek, 19861, which proposes that the proximal part should be assigned to the bronchial system, and the distal part to the respiratory system. This means that the proximal part must be called terminal bronchiole, whereas the constituents of the distal part (in analogy with the other components of the respiratory unit) can be called alveolar tubules (tubuli alveolares). These conclusions may also hold for other mammals possessing a respiratory bronchiole, in view of existing homology (reviewby Ten Have-Opbroek, 1986) and comparable development (see below). Introduction of a classification of the respiratory bronchiole in this 849 ULTRASTRUCTURE OF EMBRYONIC TYPE I1 CELL TABLE 1. Ultrastructural characteristics of mouse lung epithelia in peripheral areas around the time of pulmonary acinus formation Interval (developmental age in days) Primordial cell 13.0-13.7 13.9-14.1 Cell arrangement Pseudostratified Col. Oval Cell shape’ Nucleus shape Structures2 Microvilli Endoplasmic reticulum Mitochondria Golgi apparatus Multivesicular bodies Glycogen pattern Disseminated3 Fields4 Membrane systems associated with glycogen fields Dense inclusion bodies + + + * - 14.2-14.4 Type I1 cell precursor 14.7-14.8 15.0-16.1 Simple Simple Simple Simple (Low) col. Ovalhound Low col./cub. Round Cub./low col. Round Cub./(low col.) Round + + + + + + + + + + + + + + + 2 + + + - + 2 - + + + + + - - ? + + + + ‘Col. = columnar; Cub. = cuboid. ‘Presence, graded as: distinct + , not distinct f , absent - . 3As single particles or aggregates throughout the cytoplasm. ‘Large fields in the apical or basal cell region. sense leads t o a new definition of the adult pulmonary acinus, which posits that its components are the alveolar tubule, the alveolar duct, and the alveolar sac. As shown below, this approach certainly benefits the utility of the pulmonary acinus as a separate functional unit in lung research. All of the components of the adult pulmonary acinus according to the new definition originate from one kind of basic structure in the developing prenatal and postnatal lung, that is, a tubule (or sprout) lined by approximately cuboid epithelium (Ten Have-Opbroek, 1979, 1981; Otto-Verberne and Ten Have-Opbroek, 1987a,b). This kind of tubule has long been considered to belong to the prospective bronchial part of the respiratory tract, because of the presence of a light microscopically distinct epithelium and the absence of proximity between epithelium and capillary system (review by Ten HaveOpbroek, 1981).Our studies, on the contrary, have clearly shown that this epithelium consists of type I1 cells or their precursor cells, which means that the tubule belongs to the future pulmonary acinus (Ten HaveOpbroek, 1979, 1981; Otto-Verberne and Ten HaveOpbroek, 1987a,b). Therefore, this basic precursor structure for the pulmonary acinus has been called the acinar tubule (Ten Have-Opbroek, 1979). In the light of these results, the term bronchial tree (frequently used in embryonic lung research to indicate the complex of tubules with a light microscopically distinct epithelium in the developing lung) could better be replaced by the neutral term “respiratory tract.” In the developing lung, the prospective pulmonary acinus is always clearly demarcated from the prospective bronchial part of the respiratory tract (criteria; approximately cuboid, and later also squamous, epithelium; presence of the specific type I1 cell marker versus columnar epithelium; absence of this marker). Initially, the pulmonary acinus consists only of an increasing number of branching acinar tubules. In later stages of prenatal and postnatal lung development, it also contains structures derived from these tubules. These derivative structures are, depending on their size and shape, called dilatated tubules, saccules, or pouches (Ten Have-Opbroek, 1979,1981; Otto-Verberne and Ten Have-Opbroek, 1987a,b). The nomenclature used for the components of the adult pulmonary acinus is not applicable to the developing pulmonary acinus (see below). The type I cells in the lining of the derivative structures derive from the type I1 cells (or precursors) present (Ten Have-Opbroek, 1979,1981; Otto-Verberne and Ten Have-Opbroek, 1987a,b). Contrary to the general opinion, we believe that the derivative structures occurring in these later stages of lung development are not identical to the similarly sized and shaped components of the pulmonary acinus of the mature lung. They must be transitional structures, because the developing pulmonary acinus has proved to grow by budding of acinar tubules in a mode that is, in principle, not restricted to any particular pattern (Ten HaveOpbroek, 1981).The primitive pulmonary acinus formed by the derivative structures is, therefore, subject to gross remodeling during lung development. Final alveolarization of the maturing pulmonary acinus is also due to budding of acinar tubules or sprouts along its lining (Ten Have-Opbroek, 1981). Formation of these buds along the complete lining may result in the presence of only alveolar ducts and sacs, a picture observed in some 850 A.A.W.TEN HAVE-OPBROEK ET AL. mammalian species (see above). The definitive pulmonary acinus is present in mammals when growth has stopped, that is, after puberty (Ten Have-Opbroek, 1981). In the stages under investigation, the pulmonary acini contained only acinar tubules. Striking ultrastructural characteristics of the lining epithelium, that is, type I1 cell precursors (Fig. 3, Table l), include the shape of the cells (low-columnar, sometimes approximately cuboid), the shape of the nuclei (large, approximately round), and the presence of large glycogen fields in the apical and basal parts of the cells.The apical cell surface was covered with microvilli. As can be seen in Figure 4, the cytoplasm may contain a well-developed Golgi apparatus surrounded by many small vesicles and some larger vacuoles, both of light density. Multivesicular bodies, dense bodies, and profiles of sometimes variably distended smooth and rough endoplasmic reticulum were also present. The apical and basal glycogen fields frequently displayed a homogeneous organization, but sometimes compartments could be seen, apparently formed by membrane systems in these fields (Fig. 5; cf. Fig. 6). Some fields also showed a close relationship with dense bodies, sometimes lying nearby or even inside them and frequently in apparently glycogen-free areas. The dense bodies varied in size as well as in shape (round to oblong), and sometimes displayed a membrane-bound, centric or paracentric core of light density; they occurred alone or grouped in smaller or larger clusters. Cells with these striking characteristics were the only type lining the prospective pulmonary acini. On days 14.7 and 14.8 (embryonic weights 374 and 385 mg), the type I1 cell precursors lining the prospective pulmonary acini showed a picture more or less similar to that on days 14.2-14.4 (Table 1). The glycogen fields now frequently showed compartmentalization by distinct membranes forming loops in these areas (Fig. 6). Clefts seen along these membranes within the glycogen fields might be interpreted as indicating the presence of lumens but could also be due to artifacts. Investigation of lungs of older embryos (15.0, 15.4, 15.9, 16.0, and 16.1 days old, weighing 409, 475, 560, 572, and 598 mg, respectively) yielded similar histological findings except that type I1 cell precursors lining the prospective pulmonary acini had become approximately cuboid (Table 1). Scanning electron microscopy (Fig. 7a,b) showed sharp transitions from the approximately cuboid type I1 cell precursors of the prospective respiratory portion to the columnar epithelium of the prospective bronchial portion of the respiratory tract. These abrupt transitions were first observed on day 14.7. This SEM approach (Fig. 7a,b) also gave an impression of the dimension of the prospective pulmonary acinus at day 15.0: The short first-order branch (acinar tubule, see above) divided into two second-order branches (acinar tubules), each of which ended in a terminal saccule. DISCUSSION As mentioned in the Introduction, we have termed the original branching tubular system of the mammalian luGg, which origikates from the lung anlage, the primordial system (Ten Have-Opbroek, 1981; see also Otto-Verberne and Ten Have-Opbroek, 1987a). In the mouse, this system is present between about days 9.5 and 14.2. As shown by the present results, all primordial epithelial cells lining the end-pieces of the primordial system of the lungs of embryos aged 13.0-14.1 days (weights ca. 145-264 mg) appear similar (see Table 1). In contrast, the precursors of the type I1 cell-which occur from about day 14.2 (279 mg) onward and then line the end-pieces of the differentiating tubular system, i.e., the prospective pulmonary acini (Ten Have-Opbroek, l979,1981)-showed some distinct ultrastructural features. These cells have a low-columnar or approximately cuboid shape, a large, more or less round nucleus, and a cytoplasm displaying typical features of a differentiated cell (see Table 1).Some of the more striking features reported here, like the presence of a few multivesicular or dense bodies, cannot serve to conclusively demonstrate the type I1 cell precursor in conventional electron microscope studies. However, a quite distinctive feature of this cell type is the presence of large glycogen fields in the apical or basal cytoplasmic areas. These fields frequently show an intimate relationship with scrolled membrane systems within their area, and association with dense bodies of various sizes. On the basis of these relationships the type I1 cell precursor can be recognized easily. Therefore, we cannot agree with other investigators who found epithelial cells with similar morphological characteristics in peripheral areas of the developing lung and considered these cells to be undifferentiated or endodermal epithelial cells (Campiche et al., 1963; Kikkawa et al., 1968; Hitchcock 0 Hare and Sheridan, 1970; Wang et al., 1973; Williams and Mason, 1977; Hilfer, 1983; Fukuda et al., 1983) or to belong to primitive bronchiolar zones (Fukuda et al., 1983). We believe that these cells should be called type I1 cell precursors. Figs. 3-6. TEM pictures of left lung lobe on day 14.4 (Figs. 3, 41, day 14.3 (Fig. 5), and day 14.7 (Fig. 6). Embryonic weights: 345 mg (Figs. 3, 41, 303mg (Fig. 5) and 374 mg (Fig. 6). Fig. 3. End-piece of the differentiatingbranching tubular system (i.e., the prospective respiratory unit or pulmonary acinus). The simple epithelium consists of type I1 cell precursors. Marked features of the cell -seen especially at the bottom of the end-piece-are the shape of the nucleus (large, approximately round), the shape of the cell (low-columnar, sometimes approximately cuboid), and the presence of large apical and basal glycogen fields. Microvilli are present. Proximity to blood vessels is evident. Boxed area is shown enlarged in Figure 4. x 1,500. Fig. 4. The cytoplasm of a type I1 cell precursor contains a welldeveloped Golgi apparatus with a large number of associated vesicles, a single (or sometimesa few) multivesicularbody (arrow),or dense bodies (not shown). Glycogen fields may show a homogeneous organization at this stage. x 19,800. Fig. 5. Glycogen fields in the cytoplasm of the type I1 cell precursor may also show compartmentalization due to the presence of membranes winding through the area (arrowheads).Dense bodies of various sizes (arrows) lie near or even inside these fields, in an apparently glycogenfree area; these bodies sometimes have a membrane-bound, central or peripheral core of light density. There is a continuous basal lamina. x 14,100. Fig. 6. Glycogen field in a type I1 cell precursor showing a few compartments. The cleR between two membranes (star) suggests the presence of a lumen but may be an artifact. A few multivesicular bodies (arrows), profiles of smooth endoplasmic reticulum (arrowheads),and a dense body are SO present. ~20,300. ULTRASTRUCTURE OF EMBRYONIC TYPE I1 CELL 851 852 A.A.W.TEN HAVE-OPBROEK ET AL. Fig. 7a,b. Successive SEM pictures of left lung lobe at 15.0 days (embryonic weight: 409 mg), showing an abrupt transition from the approximately cuboid epithelium (type I1 cell precursors) lining the prospective pulmonary acinus to the columnar epithelium of the prospective bronchial part of the respiratory tract. The most proximal type I1 cell precursor is indicated (asterisk).The basic structure in the genesis In view of earlier signs of differentiation occasionally found in individual cells (membrane systems in glycogen fields on day 13.6; dense inclusion bodies on day 14.0), local variations apparently occur in the time of onset of differentiation of the type I1 cell in a lung lobe. A possible explanation for this phenomenon is the following. We found indications in the present study (data not shown) that establishment of proximity between the developing pulmonary acinus and the developing capillary system could be a prerequisite for the differentiation of type I1 cell precursors (and likely also other cell types). This suggests that the very process of growth may be a possible determining factor for differentiation. For, according to the concept of spatial coherences (Landsmeer, 1968,1978), growth may lead to a change in spatial relationships, and thus in spatial information given to cells. Therefore, differences in growth rate (feasibly occurring within a lung lobe) may be a basis for the local variations in type I1 cell differentiation observed in the present study. The present transmission and scanning electron microscope studies of lungs of embryos aged 14.2-16.1 days (279-598 mg) confirmed our immunofluorescence findings (Ten Have-Opbroek, 1979) that the type I1 cell precursor is the only type of epithelial cell forming the lining of the prospective pulmonary acini in this interVal. In addition, these studies provided new information by showing that abrupt transitions from the lower cells of the pulmonary acini to the columnar epithelium of the bronchial portion-a phenomenon already observed in our light microscope studies of the embryonic lung (Ten Have-Opbroek, 1979) as well as our electron microscope studies of the adult lung (Ten Have-Opbroek, 1986)of the mouse-are present after prenatal day 14.6. Like Sorokin (1965), we are of the opinion that the of the pulmonary acinus has been called the acinar tubule (Ten HaveOpbroek, 1979). At this stage the pulmonary acinus consists of a short first-orderacinar tubule (1) and two second-orderacinar tubules (2) with a saccular ending (S) (one of which is not visible here; its entrance is indicated by an arrow). x 1,400. appearance of glycogen in an epithelial cell must be considered to be a distinct sign of cell differentiation. In general, this substance may constitute a source of substrates for the production of all lipid bilayers of the cell. Evidence that in type I1 cells glycogen may also specifically provide substrates for surfactant phospholipid biosynthesis has been obtained in biochemical studies performed in the developing rat lung. These studies consisted of in vivo labeling experiments with [U-14Cl-glucosein an organ explant culture system, followed by in vitro assays of labeled products (Farrell and Bourbon, 19861, or experiments concerning the activity (Rijksen et al., 1985) or the inhibition (Bourbon et al., 1987) of enzymes playing a role in glycogenolysis (see also review by Possmayer, 1984). Ultrastructural studies on the fetal rabbit lung have shown that lamellar electron-dense material can be found isolated wthin glycogen areas (Kikkawa et al., 1968).Chi (1985) investigated type I1 cells of developing fetal monkey lungs (Macaca nemestrina, term 168 days) in the gestational period of 135-145 days, and found glycogen Particles in developing and mature multilamellar bodies. This makes it highly probable that the occurrence OfcOmPlex r n m h a n e systems in the glycogen fields of I1 cell precursors found in the present study in the mouse may represent an initial step in the prenatal process of multilamellar body formation. This process may start rather early, because the elaborate membrane systems, which were also observed in the developing human (Campiche et al., 1963)and rat (Hitchcock O'Hare and Sheridan, 1970) lung, have been found in the developing mouse lung as early as day 14.3 (and occasionally even on day 13.6). Our finding that dense bodies (i.e., postulated precursory stages of multilamellar bodies: Kikkawa et al., 1968; Williams and Ma- ULTRASTRUCTURE OF EMBRYONIC TYPE I1 CELL 853 son, 1977) may be localized in or associated with Fukuda, Y., V.J. Ferrans, and R.G. Crystal 1983 The development of apparently glycogen-free areas, as well as others’ find~ u ~ , a l ~ $~ 7’ ~ p ~ ~ ~~ ~~ t ,and U rimmunOa l ing that the scrolled membrane systems or developing Gardner, E., D.J. Gray, and R. ORahilly 1975 Pleura and lungs. In: multilamellar bodies (Campiche et al., 1963; Hitchcock Anatomy, A Regional Study of Human Structure. 4th ed. Saunders, OHare and Sheridan, 1970) may be so localized, is not Philadelphia, London, Toronto, chap. 29, PP. 294-298. incompatible with our conclusions concerningmulti- h d b l o e d , J.F. 1976 The embryonic and Postnatal growth of rat and mouse. IV.Prenatal growth of organs and tissues: Age determination, lamellar body formation. For these glycogen-free areas and general growth ~d Anat,, 95r8-33, could be due to uptake of substance (i.e., glycogen or Gray’s Anatomy 1973 The respiratory system. In: Gray’s Anatomy. R. its degradation products such as lipids) by the strucWarwick and P.L. W1niams, eds. 35th ed. Longman, Edinburgh, Chap. 8, p. 1200. tures in question Or loss Of this substance during the Hilfer, S.R. 1983 Development of terminal buds in the fetal mouse lung. preservation or dehydration procedures in our and 0thIn: scanning Eledron M~~OSCOPY. Om Johari, ed. SEM Inc., AM.F. ers’ studies. Further investigation on the Drocess of OHare, Chicago. Val. 3. DD. 1387-1401. multilamellar body formationis in progress. Our earlier Hitchcock OHareY k, and M.N. Sheridan 1970 Electron microscopic observations on the morphogenesis of the albino rat lung with special conclusion (Otto-Verberne and Ten Have-Opbroek, reference to pulmonary epithelial cells. Am. J . Anat., 127:181-206. 1987a) that production of surfactant-associated protein Kikkawa, Y., E.K. Motoyama, and L. Gluck 1968 Study of the lungs of may be a basic property of the type I1 cell, which comes fetal and newborn rabbits. Am. J. Pathol., 52:177-209. to expression as soon as the cell type appears, is in line Landsmeer, J.M.F. 1968 Les coherences spatiales et l’equilibre spatial dans la region carpienne. Acta Anat., 70 (Suppl. 54-1):1-84. with our hypothesis concerning an early onset of mul- Landsmeer, J.M.F. 1978 Spatial aspects of extremity development. In: tilamellar body formation. XIXth MorphologicalCongress Symposium, Charles University, PraIn an earlier report (Ten Have-Opbroek, 19791, we gue. E. Klika, ed. Univerzita Karlova, Prague, pp. 149-159. have proposed the term “precursor of the type I1 alveo- ORahilly, R. 1986 The pleurae and lungs. In: E. Gardner, D.J. Gray, and R. O’Rahilly, eds. Anatomy. A Regional Study of Human Struclar epithelial cell” for prenatal and postnatal cells havture. 5th ed. Saunders, Philadelphia, London, Toronto, Chap. 29, ing an approximately cuboid shape and a large, more p. 299. or less round nucleus, and containing the type I1 cell- Otto-Verberne, C.J.M., and A.A.W. Ten Have-Opbroek 1987a Development of the pulmonary acinus in fetal rat lung: A study based on an specific antigen, because these cells lack the multilaantiserum recognizing surfactant-associated proteins. Anat. Emmellar inclusion bodies characteristic of mature type I1 bryol., 175:365-373. cells. However, our present finding that morphologi- Otto-Verberne, C.J.M., and A.A.W. Ten Have-Opbroek 1987b Immunological detection of the type-I1 alveolar epithelial cell or its precally identifiable precursory stages of multilamellar cursor before week 20 of human gestation. Anat. Rec., 218:102A. bodies are very probably present in embryonic type I1 Pease, D.C. 1964 Histological techniques for electron microscopy. 2nd cells around the time of their appearance in the lung ed. Academic, New York, London, pp. 234-236. implies that this fundamental difference between de- Possmayer, F. 1984 Biochemistry of pulmonary surfactant during fetal development and in the perinatal period. In: B. Robertson, L.M.G. veloping and mature type I1 cells presumably does not van Golde, and J.J. Batenburg, eds. Pulmonary Surfactant. Elsevier, exist. We now believe that the term “precursor of the Amsterdam, pp. 295-355. type I1 alveolar epithelial cell” is artificial. We therefore Rehm, S., J.M. Ward, A.A.W. Ten Have-Oubroek. L.M. Anderson. G. propose to choose the general term “type I1 alveolar Singh, S.L. Katyal, and J.M. Rice 1988 Mouse papillary lung t&ors transplacentally induced by N-nitrosoethylurea: Evidence for alveoepithelial cell” or a synonym (great alveolar cell, type lar type I1 cell origin by comparative light microscopic, ultrastrucI1 pneumonocyte) to indicate this cell type in all of its t u r d and immunohistochemical studies. Cancer Res., 48~148-160. morphological manifestations. Reynolds, E.S. 1963 The use of lead citrate at high pH as an electron $zEzz ACKNOWLEDGMENTS The authors are indebted to Dr. L.A. 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