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Ultrastructural features of type II alveolar epithelial cells in early embryonic mouse lung.

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THE ANATOMICAL RECORD 221:846-853 (1988)
Ultrastructural Features of Type II Alveolar
Epithelial Cells in Early Embryonic Mouse Lung
Department of Anatomy and Embryology, Faculty of Medicine, University of Leiden,
2300 RC Leiden, The Netherlands
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
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.
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.
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 Polaron@apparatus 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
15 kV.
Primordial Epithelium
The pseudostratified (or sometimes simple) columnar
epitheiium that lined the distal part of the primordial s
Basal lamina
Dense body
Golgi apparatus
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
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
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
Cell arrangement
Cell shape’
Nucleus shape
Golgi apparatus
Glycogen pattern
Membrane systems
associated with
glycogen fields
Dense inclusion
Type I1 cell precursor
(Low) col.
Low col./cub.
Cub./low col.
Cub./(low col.)
‘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
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
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.
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.
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-
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
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
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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,
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The authors are indebted to Dr. L.A. Ginsel (Laboratory of Electron Microscopy, University of Leiden) for
his critical reading of the manuscript, and wish to thank
Mr. J.H. Lens, Mr. C.J. van der Sijp, and Mr. H.G.
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ultrastructure, features, epithelium, alveolar, embryonic, lung, mouse, typed, early, cells
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