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Morphogenetic and functional activity of type II cells in early fetal rhesus monkey lungs.

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THE ANATOMICAL RECORD 234:93-104 (1992)
Morphoge ietic and Functional Activity of Type II Cells in Early Fetal
Rhesus Monkey Lungs
Department of Pulmonology, School of Medicine, University of Leiden, P.O. Box 9602,2300
RC Leiden, The Netherlands (A.A.W.T.H.-0.);Department of Anatomy, School of
Veterinary Medicine, University of California, Davis, California 95616 (C.G.P.)
To evaluate further the role of type I1 alveolar epithelial cells in
primate lung development, lungs of fetal (46 to 155 days gestational age [DGA]),
postnatal, and adult rhesus monkeys were investigated with antibodies against
surfactant protein A (SP-A), Alcian blue (AB) staining, and periodic acid-Schiff
(PAS) staining with/without alpha-amylase pre-treatment.
In adult and postnatal lungs, type I1 cells (cuboid shape; large, roundish nucleus)
displayed a unique cytoplasmic staining for SP-A. In prenatal lungs, a low-columnar to cuboid type of cell with a large, roundish nucleus was first detectable by
62 DGA. It was the only cell type to line the distalmost tubules or buds of the
prospective respiratory tract. It exhibited (initially partial) cytoplasmic staining
for SP-A. AB and PAS stainings showed the presence of acid glycoconjugates and
large apical and/or basal glycogen fields. After 95 DGA, the lining of the distal
respiratory tract additionally displayed flatter cells with immunoreactivity for
SP-A and non-reactive zones. Columnar epithelium (pseudostratified or simple)
never stained for SP-A.
We conclude that morphologically identifiable type I1 cells first appear in fetal
rhesus monkey lungs by 62 DGA (pseudoglandular period). The cells may already
synthesize surfactant and extracellular matrix components. They generate type I
cells, and thus the entire pulmonary acinus lining. These conclusions for the
rhesus monkey fully agree with our earlier conclusions for another primate, the
human, and for rodents. However, as presently shown, primates differ greatly from
rodents with respect to the timing of type I1 cell differentiation (at 29-38% versus
73-75% of gestation or at 22-25% versus 48-49% of prenatal lung development). 0 1992 Wiley-Liss, Inc.
It has been thought for a long time (reviews by Ten I1 cell differentiation and function (i.e., surfactant synHave-Opbroek, 1975, 1979) that type I1 alveolar epi- thesis) start at a much earlier stage of lung developthelial cells first appear prenatally at about the same ment. These studies first detect type I1 cells in embryos
time as type I alveolar epithelial cells, that is, in the with a developmental age (basis: embryonic weight,
period just before birth when primitive alveoli (smooth- Ten Have-Opbroek, 1975) of 14.2 days, i.e., as early as
walled ducts and sacs) are formed (terminal-sac pe- the pseudoglandular period. The early type I1 cells disriod). At that time, cuboid cells lining these ducts and play distinctive features, i.e., a low-columnar to cuboid
sacs start to display multilamellar bodies (MLBI-tracell shape, a large and roundish nucleus, a unique cyditional markers of type I1 cells. MLB are organelles of toplasmic staining for surfactant-associated protein A,
the endocytic pathway that lead to secretion of pulmo- SP-A, and postulated precursory stages of MLB, includnary surfactant, a complex lipid-protein substance that ing a potentially primary structure in prenatal MLB
reduces surface tension at the airlliquid interface and formation (Ten Have-Opbroek et al., 1988, 1990b), i.e.,
thereby, provides mechanical stability to the pulmo- a cytoplasmic inclusion filled with glycogen. Mouse
nary acini (review by Ten Have-Opbroek et al., 1990b). Clara cells display different developmental and funcAlso, based on the appearance of MLB, type I1 cells tional patterns (Ten Have-Opbroek, 1991; Ten Havehave been assumed to start with surfactant synthesis Opbroek and DeVries, 1991). First, they acquire their
in the terminal-sac period (review by Ten Have-Op- adult phenotype and SP-A reactivity only after birth.
Second, their staining pattern for SP-A (apical cap)
broek, 1975).
Our light microscopic (Ten Have-Opbroek, 1975,
1979, 1981, 1991; Van Hemert et al., 1986; Oomen et
al., 1990) and electron microscopic (Ten Have-Opbroek
et al., 1988,1990b; Ten Have-Opbroek, 1991) studies in
Received August 28, 1991; accepted January 15, 1992.
the mouse embryo have been the first to show that type
suggests internalization of SP-A from distal air spaces
during postnatal and adult life. As proposed and discussed (Ten Have-Opbroek et al., 1988,1991),we use
the general term type I1 cell to indicate this cell type
pre- and postnatally.
Our hypothesis based on studies in the mouse-i.e.,
that onset of type I1 cell differentiation and function in
mammals takes place in the pseudoglandular period of
lung development (Ten Have-Opbroek, 1981)-is fully
supported by our studies in another rodent, the rat
(Otto-Verberne and Ten Have-Opbroek, 1987),and in
humans (Otto-Verberne et al., 1988, 1990).Other investigators also report on type I1 cell differentiation in
rodents, based on studies with biochemical cell markers (Jaskoll et al., 1984,1988;Funkhouser et al., 1987;
Edelson et al., 1988;Paine et al., 1988;Slavkin et al.,
1989; Joyce-Brady and Brody, 1990; Williams and
Dobbs, 1990;Woodcock-Mitchell et al., 19901,or ultrastructural, morphometric techniques (Lin and Lechner,
1990;Snyder and Magliato, 1991;Young et al., 1991).
However, as will be discussed, a close comparison with
our results was often not possible. The biochemical results were frequently not related to specific epithelial
cell types in developing lungs. Some studies started at
late gestational ages.
The present study was performed to find out if the
early onset of type I1 cell differentiation and function is
also true for another primate, the rhesus monkey. We
examined fetal, postnatal, and adult rhesus monkey
lungs with light microscopic immunocytochemistry,
using our polyclonal antibodies against human or
mouse surfactant protein, and carbohydrate cytochemistry, using periodic acid Schiff (PAS), alpha-amylase/
PAS, and Alcian blue (AB).
Our present study indicates that morphologically
identifiable and functionally active type I1 cells first
appear in rhesus monkeys by 62 DGA (pseudoglandular period of lung development). These cells play a
primary role in pulmonary acinus development, and
give rise to type I cells. In general, rhesus monkey lung
development follows the pathway proposed for mammals (Ten Have-Opbroek, 1981, 1991).However, primates differ from rodents with respect to the timing of
type I1 cell differentiation (at 29-38% versus 73-75%
of the gestation time or at 22-25% versus 48-49% of
the total time of prenatal lung development). Some of
the observations have been published in an abstract
(Ten Have-Opbroek and Plopper, 1989).
Lung Material, Fixation Procedures
Lung material from fetal, postnatal, and adult
rhesus monkeys (Macaca mulatta, gestation time ca.
165 days) was obtained as described (Tyler et al., 1988;
Plopper et al., 19891,and selected from a collection of
51 fetal (29-165 days gestational age, DGA), eight
postnatal, and five adult monkey lungs.
For immunofluorescence, use was made of 11 fetuses
aged between 46 and 142 DGA; three animals aged 6,
8,and 9 days postnatally (dPN); and three adult animals. Immediately after removal of the lungs from the
thorax, tissue blocks were cut from the peripheral parenchyma and briefly rinsed in PBS (pH 7.2).Some of
these blocks were prepared and used for cryostat sectioning. The fresh-frozen sections (ca. 6 km) were usu-
ally fixed in pro-analysis acetone (Merck, Germany) at
-20°C for 5-10 min (Otto-Verberne and Ten Have-Opbroek, 19871,but sometimes they were only heated at
60°C for 3-5 min to preserve the surfactant lining
layer along the air spaces. Other tissue blocks were
fixed by immersion in Clark's acid (ethanol 100% and
acetic acid, 3:l v/v) or in 1% paraformaldehyde or 1%
paraformaldehyde and 0.1% glutaraldehyde in cacodylate buffer (pH 7.2).After dehydration and embedding in paraffin, the fixed blocks were sectioned at 6
bm thickness.
The same lung material was used for the PAP technique and carbohydrate cytochemistry. In addition, use
was made of paraffin-embedded lung sections derived
from 73 to 155 DGA-old-fetusesand fixed via tracheal
infusion of a mixture of paraformaldehyde and glutaraldehyde in cacodylate buffer (350-400 mOsm, pH 7.2)
(Karnovsky, 1965) at 30 cm fixative pressure as reported (Tyler et al., 1988;Plopper et al., 1989).
Sections stained with hematoxylin and eosin (H&E)
were used to select blocks for (immuno)cytochemistry
as well as for general orientation.
Specific antibodies recognizing type I1 cells on the
basis of a unique, cytoplasmic staining were prepared
in our laboratory by injecting rabbits with the pellet
fraction of bronchoalveolar lavage fluid from adult human lung (Otto-Verberne et al., 1988).The resulting
immune serum was absorbed with serum and crossreacting organs (Otto-Verberne et al., 1988).The SP-A
specificity of the final immune serum, called specific
anti-lavage serum, Human (SALS-Hu)was assessed by
Western blot analysis, in vitro translation assays, and
immunohistochemistry of relevant material (serum,
tissues, organs; fetal/adult) from humans, and also
from some other species including the dog (criteriaSP-A: molecular mass ca. 35 kD; selective, cytoplasmic
staining of type I1 cells; selective blocking of SP-A immunoreactivity by pre-absorption of SALS-Hu with recombinant SP-A, see Otto-Verberne et al., 1988,1990,
1991;Ten Have-Opbroek et al., 1990a, 1991).In addition, we used one of our specific anti-mouse reagents,
SAALS (specific anti-adult lung serum, rabbit antimouse) as a positive control. SAALS specifically reacts
to SP-A and a probably surfactant-related 12 kD protein, and marks type I1 cells in several species strongly
by a unique, cytoplasmic staining, as shown in mice
(Ten Have-Opbroek, 1975,1979,1981;Van Hemert et
al., 19861,rats (Otto-Verberne and Ten Have-Opbroek,
1987; Batenburg et al., 1988), and humans (unpublished observations).
Lung sections were incubated with SALS-Hu or
SAALS for 60 min, and then for 45 min with fluorescein isothiocyanate-conjugated swine anti-rabbit IgG
(150 in PBS, pH 7.2) (Dakopatts, Glostrup, DK; absorbed with human IgG), both incubations being performed at room temperature (RT). Before and after
each incubation the sections were washed in 3-4
changes of PBS (pH 7.2)for 15-20 min. The sections
were mounted under a coverslip in a mixture of 80%
glycerol and 20% PBS (pH 8.0) containing p-phenylene
diamine (1mg/ml, which protects slides from fading),
and examined and photographed in a Leitz Dialux fluorescence microscope with a high pressure HBO 100
mercury lamp and vario-orthomat equipment for photography.
PAP technique
After preincubation with normal goat serum (1:32 in
PBS, pH 7.2) for 15 min a t RT, the lung sections were
exposed to SAALS overnight at 4"C, and then successively for 50 min a t RT to goat anti-rabbit IgG (7S, 1 5 0
in PBS), and rabbit peroxidase-antiperoxidase soluble
complexes (PAP, 1:64 to 1:1024 in PBS; Nordic Immunological Laboratories, Tilburg, The Netherlands). To
enhance the selective type I1 cell staining at early
stages of lung development, the latter two steps were
repeated (PAP technique according to the double
bridge method). Each incubation (30 min) was preceded
and followed by rinsing with 2-4 changes of PBS containing 0.1% Tween 20 for 20 min. When dry, the sections were stained for peroxidase (20 min) according to
Graham and Karnovsky (1966) and counterstained
with methyl green (20 min). After being dipped in alcohol and xylene, the sections were mounted in enthalan.
Immunohistochemical controls were performed on
the same lung material with preimmunization serum
(PS) as the primary antibody or omission of one of the
incubation steps. SALS-Hu, SAALS, and PS were used
in serial dilutions with PBS (pH 7.2) to a final concentration of 1:400. For the PAP technique, we used PBS
containing 5% normal monkey serum.
To prevent non-specific background staining in lungs
at early prenatal stages, all sera were additionally absorbed (see Ten Have-Opbroek, 1979) with homogenate
made from arms of fetal Rhesus monkeys aged 61 or 81
Carbohydrate Cytochemistry
To get more information about the presence of carbohydrates or glycoconjugates in type I1 cells a t early
prenatal stages, lung sections (46-81 DGA) were
stained with periodic acid Schiff (PAS) or Alcian blue
(AB). The nuclei were counterstained with hematoxylin and 0.1% nuclear fast red, respectively. To identify
glycogen, some lung sections were exposed to alphaamylase (porcine pancreas; Boerhinger, Mannheim,
Germany) prior t o PAS staining. Exposure to 30 p,g
amylase/ml solvent for 3 hr resulted in a total loss of
PAS staining reactivity.
When applied to lung sections from adult rhesus
monkeys, SALS-Hu (Fig. 1) or SAALS (not shown)
yielded a selective, cytoplasmic staining of cuboid cells
present in the lining of alveolar ducts and sacs, i.e.,
type I1 cells. The large and roundish nuclei of type I1
cells (Fig. 1)were always relatively dark. The remaining lining of the ducts and sacs was dark (Fig. l),indicating that the squamous type I cells present were
not immunoreactive. Cuboid cells with a dark, large,
and roundish nucleus and a cytoplasmic fluorescence
pattern were also observable in the lining of respiratory bronchioles and their outpocketings (not shown;
see Fig. 4). By contrast, the remaining bronchiolar lining-viz., pseudostratified columnar epithelium consisting of ciliated and nonciliated cells with oblong nuclei, and basal cells-was always devoid of staining.
Accordingly, we never found any staining in pseudostratified columnar epithelium lining (sub)terminal
bronchioles or bronchi. The usual absence of a fluorescent extracellular lining layer along alveolar lumens
(Fig. 1) indicated that the surfactant lining had been
removed during tissue processing. Sometimes, surfactant particles that remained behind (Fig. 1) caused
some punctate or diffuse staining over the alveolar
septa. By contrast, when more or less preserved by a
special fixation (heating), extracellular lining layers
along alveolar lumens in adult or postnatal lungs (Fig.
2) stained very strongly, indicating that they contained
SP-A. In postnatal lungs, the prospective (rather
smooth-walled) alveolar ducts and sacs (Fig. 3) were
lined by fluorescent cuboid cells with large and roundish nuclei, dark zones (which likely contain mature
type I cells, see above), and in places also by thinner
fluorescent cells. As reported for other species, the latter cells presumably are intermediate stages in the development of squamous type I cells from cuboid type I1
cells (Ten Have-Opbroek, 1979). Like the prenatal parenchyma (see below), the postnatal parenchyma (Fig.
4) exhibited also small tubules with barely or no visible
lumens and a lining of fluorescent cuboid cells with
large, roundish nuclei. Structures of these kind (lining:
type I1 cells) have been termed acinar tubules or buds
(Ten Have-Opbroek, 1979, 1981), because they are the
basic structures from which all components of the pulmonary acinus derive, as has been shown for the mouse
(Ten Have-Opbroek, 1979, 1981, 19911, the rat (OttoVerberne and Ten Have-Opbroek, 1987), and the human (Otto-Verberne et al., 1988). In the postnatal (Fig.
4) as in the adult lungs, the lining of respiratory bronchioles and their outpocketings contained cuboid cells
with dark, large and roundish nuclei and a cytoplasmic
fluorescence. Likewise, the bronchiolar linings composed of pseudostratified columnar epithelium (Fig. 4)
were never immunoreactive. Alveolar macrophages
present in alveolar outpocketings of respiratory bronchioles or on bronchiolar surfaces (Fig. 4) were usually
brightly fluorescent, which suggests that they internalize SP-A containing particles from nearby or more
distally located extracellular lining layers.
Routine inspection of prenatal lungs revealed that at
46 DGA the branching tubular system of the lung was
lined exclusively by pseudostratified or simple columnar epithelium. Systems of this kind represent the primordial system of the mammalian lung (Ten Have-Opbroek, 1979,1981,1991).Application of SALS-Hu (Fig.
5) or SAALS to sections did not result in any staining
of columnar epithelial (primordial) cells. Occasionally,
some fluorescent particles were seen at luminal surfaces, probably caused by artificial retention of conjugate between epithelial ruffles. Mesenchyme (Fig. 5),
blood vessels, and pleura (not shown) were not immunoreactive. By 62 DGA, the branching tubular system
of the developing lung had a different appearance and
consisted of two different parts: a proximal part with
pseudostratified or simple columnar epithelium, and a
distal part lined by a low-columnar to cuboid cell type
with a large and roundish nucleus. As shown for other
Figs. 1-4. Immunocytochemistry of adult and postnatal rhesus
monkey lungs using SALS-Hu (rabbit anti human SP-A) according to
the indirect immunofluorescence technique.
Fig. 1. Adult lung, alveolar ductslsacs. Cuboid type I1 cells show
specific SP-A reactivity (i.e., a unique cytoplasmic fluorescence pattern) that contrasts strongly with their dark, large and roundish nuclei. Squamous type I cells are not immunoreactive. Surfactant (SPA), which remained behind after tissue processing, causes some
punctate or diffuse fluorescence over the alveolar septa. X 580.
Fig. 2. 9 dPN, future alveolar ductslsacs. The (specifically preserved) extracellular lining layer of these, still smooth-walled, structures displays a bright fluorescence, indicating that it contains SP-A.
x 210.
Fig. 3. 6 dPN, future alveolar ductslsacs. The epithelial lining consists of cuboid cells having a large, roundish nucleus and a cytoplasmic fluorescence (=type 11 cells), fluorescent bands (=intermediate
stages between type I1 and type I cells, tiny arrows), and dark areas
( = mature type I cells). x 220.
Fig. 4. 6 dPN, respiratory bronchiole (RB) opening into a future
alveolar duct (AD). The lining contains some cuboid cells with a cytoplasmic fluorescence pattern (tiny arrows), i.e., SP-A containing
type I1 cells. The pseudostratified columnar epithelium of RB never
shows any immunoreactivity. Alveolar macrophages (M) present on
the surface of RB or in alveolar outpocketings are usually strongly
positive, probably due to uptake of SP-A from the air spaces. The lung
parenchyma contains many acinar tubules or buds lined by type I1
cells only (arrowhead) (see Figs. 6-8, 12). x 410.
Figs. 5-8. Immunocytochemistry of early prenatal rhesus monkey
lungs using SALS-Hu (rabbit anti human SP-A) according to the indirect immunofluorescence technique.
Fig. 5.46 DGA. The lining of the original branching tubular system,
the primordial system of the lung (P),is pseudostratified or simple
columnar, and is not immunoreactive. x 390.
Fig. 6. 62 DGA. The distalmost branches of the prospective respiratory tract are lined by low-columnar or cuboid cells, with a large
and roundish nucleus, that exhibit a prominent fluorescence in apical
(andlor basal) cytoplasmic areas (ie., SP-A containing type I1 cells).
These branches are the basic structures in pulmonary acinus development, and are termed acinar tubules (A). There is always a n abrupt
transition between the alveolar epithelium of the acinar tubule
(=fluorescent, cuboid) and the bronchial epithelium of the terminal
bronchiole (TB)(dark, columnar). x 330.
Fig. 7.81 DGA, terminal bronchiole (TB) opening into three acinar
buds. The cuboid or low-columnar type I1 cells constituting these buds
frequently display a bright fluorescence throughout the cytoplasm.
x 380.
Fig. 8. 81 DGA, lung parenchyma containing a dark terminal bronchiole (TB) with a fluorescent acinar bud, a pulmonary artery branch
(PA), and some fluorescent acinar tubules (A). The light-emission
intensity may be different in places but all cuboid (type 11) cells lining
the acinar tubules show a cytoplasmic immunoreactivity for SP-A.
x 380.
mammals, these parts represent the prospective bronchial system and pulmonary acinus, respectively. (Ten
Have-Opbroek 1979, 1981, 1991). Specific staining for
SP-A (Fig. 6) was also first detectable by 62 DGA, and
was restricted to the low-columnar or cuboid epithelium lining the distalmost tubules or buds (termed: acinar tubules or buds, see above). The fluorescent staining was initially mainly observable along the apical
(Fig. 6) and/or basal cell borders but was soon found
throughout the cytoplasm (Figs. 7, 8). All of the lowcolumnar or cuboid cells displayed SP-A specific fluorescence, beit with a widely varying intensity. This
latter phenomenon hampered optimal recording of the
overall staining pattern in photomicrographs. There
was always a sharp demarcation (Figs. 6, 7) between
the distal and proximal parts of the branching tubular
system in the developing lungs (criteria: approximately cuboid epithelium with large and roundish nuclei; presence of SP-A reactivity versus pseudostratified or simple columnar epithelium with oblong nuclei;
absence of SP-A reactivity). By 81 DGA, the distal part
of the prospective respiratory tract consisted of one
(Fig. 7) to five (not shown) generations of fluorescent
tubules or buds (acinar tubuledbuds). After application of SALS-Hu, fluorescence was most striking in the
distalmost buds or tubules; SAALS yielded a more
equal staining over all generations, which may be
caused by its reactivity to two type I1 cell proteins,
namely, SP-A and a 12 kD protein (Van Hemert e t al.,
1986). As in earlier stages, SALS-Hu or SAALS never
provided any staining of connective tissue or blood vessels (Fig. 8).
In older rhesus monkey fetuses (84-95 DGA), lowcolumnar or cuboid fluorescent cells still were the only
type of epithelial cells to line the distal part of the
prospective respiratory tract. After 95 DGA, we observed locally a more squamous (instead of cuboid) fluorescence pattern and after 128 DGA sometimes also a
total absence of staining, a pattern as found in neonatal lungs (see Fig. 3). As mentioned above, these images presumably reflect the presence of differentiating
or mature type I cells. Immunohistochemical controls
using PS instead of SALS-Hu or SAALS, and other
controls were always negative.
lamina of larger or smaller tubules of the original
branching tubular system of the lung (primordial system). The pseudostratified or simple columnar (primordial) epithelium was almost or fully devoid of staining.
By 62 DGA (Fig. 9), a striking positive reaction was
observable in the low-columnar or cuboid cells lining
the distal branches of the prospective respiratory tract
(acinar tubules). The corresponding basal laminas
were also frequently positive. Columnar epithelial cells
with oblong nuclei lining more proximal branches (terminal bronchioles) showed some positive reactivity
(Fig. 9). In even more proximally located branches, i.e.,
(pre)terminal bronchioles, on the contrary, the staining
was usually restricted to the basal lamina (not shown).
After treatment with PAS, primordial epithelial
cells at 46 DGA exhibited positive (pink or red) granules in their cytoplasm (not shown). Low-columnar or
cuboid cells present by and after 62 DGA (Fig. 10) displayed a highly prominent (red) staining in their apical
and/or basal cytoplasmic areas. The pattern or intensity of staining was not influenced by cellular
height or location. Terminal bronchioles (Fig. 10) usually showed a less pronounced staining of their lining
epithelium. The regular finding of PAS positive material in lumens (not shown) suggested that lack of PAS
reactivity in epithelial cells (Fig. 10) was caused by
artificial loss of intracellular substance.
The PAS reactivity in all epithelial cells could completely be blocked by pre-treating sections with alphaamylase.
Type I1 Cell Differentiation and Function
Our studies in adult and newborn rhesus monkeys
show that type I1 cells present in the lining of peripheral air spaces contain surfactant protein A (SP-A, 35
kD). Type I cells, which occupy the remaining lining of
these air spaces, do not comprise this substance. Pseudostratified columnar epithelium with ciliated and
nonciliated cells lining terminal and respiratory bronchioles is also devoid of SP-A. The fact that distal nonciliated columnar epithelial cells lack SP-A is not surprising because they have been shown to be mucous
cells in the rhesus monkey (Tyler and Plopper, 1985;
Plopper et al., 1989; Plopper, 1990) as well as in huPAP technique
mans (Ten Have-Opbroek e t al., 1991). The correspondApplication of SAALS to prenatal lung sections ac- ing columnar secretory cells in rodents, also termed
cording to the PAP technique yielded similar pictures Clara cells, have a different phenotype and do express
a s observed in our immunofluorescence studies. There SP-A, being mainly postnatally and in their apical dowas a prominent cytoplasmic staining of low-columnar main (Oomen et al., 1990; Ten Have-Opbroek, 1991;
or cuboid cells lining the distal generations of the pro- Ten Have-Opbroek and DeVries, 19911, which suggests
spective respiratory tract (acinar tubules/buds). Be- that they internalize this substance from the air spaces
tween 62 and 106 DGA, the distalmost cells in these after birth. The present finding of SP-A containing
linings were usually low-columnar, and the more prox- type I1 cells in respiratory bronchioles is not uneximal cells cuboid (see Fig. 10); after 106 DGA, the di- pected in view of the usual presence of type I cells (destalmost cells frequently were also cuboid. These tem- rived from type I1 cells) in their lining (present study;
poral differences in height-also
observed in our Tyler and Plopper, 19851, and has also been reported
immunof luorescence studies-had little or no inf lu- for humans (Ten Have-Opbroek et al., 1991), dogs (Ten
ence on the SAALS-immunoreactivity. Application of Have-Opbroek et al., 1990a1, and mice (Ten Have-OpPS to prenatal lung sections and other controls did not broek, 1986). Type I1 cells in the rhesus monkey prove
to exhibit the same selective ( =cytoplasmic) staining
result in any staining of epithelium or other tissues.
pattern for SP-A as type I1 cells in other species such as
Carbohydrate cytochemistry
the mouse (Ten Have-Opbroek, 1975, 1981, 1991;
After application of AB to lung sections at 46 DGA, Oomen et al., 19901, the rat (Otto-Verberne and Ten
a positive (light blue) staining was found a t the basal Have-Opbroek, 19871, the human (Otto-Verberne et
Figs. 9-1 0. Carbohydrate cytochemistry of early prenatal rhesus
monkey lungs. (Fig. 9), AB staining; (Fig. lo), PAS staining without
pretreatment with alpha-amylase.
Fig. 9. 62 DGA, terminal bronchiole (TB, tangentially sectioned)
opening into acinar tubules (A), one of which ends into a saccule (S).
The low-columnar or cuboid type I1 cells of the acinar tubules (and the
basal laminas) contain acid glycoconjugates (light blue staining,
arrow), which suggests synthesis of extracellular matrix components.
x 300.
al., 1988, 1990, 1991; Ten Have-Opbroek et al., 19911,
and the dog (Ten Have-Opbroek et al., 1990a).
As appears from our present studies in rhesus monkey fetuses, type I1 cells are first detectable in developing lungs by 62 DGA, i.e., in the pseudoglandular
period of lung development. The cells are identifiable
by means of “adult” distinctive features, namely, an
approximately cuboid cell shape, a large and roundish
nucleus, and a selective, cytoplasmic staining for SP-A.
The initial restriction of SP-A pools to apical or basal
cell borders can possibly be explained by the fact that
rough endoplasmic reticulum seems to concentrate in
those (relatively glycogen-free) peripheral cytoplasmic
areas at early developmental stages as has been shown
for the mouse (Ten Have-Opbroek et al., 1988) and humans (Otto-Verberne et al., 1988). SP-A expression of
type I1 cells in developing rhesus monkey lungs appears not to be influenced by initial differences in cellular height (low-columnar or cuboid), an observation
also made in mice (Ten Have-Opbroek, 1979, 1991;
Oomen et al., 19901, rats (Otto-Verberne and Ten
Have-Opbroek, 1987), and humans (Otto-Verberne et
al., 1988, 1990). However, as shown by the varying
fluorescence intensity, individual type I1 cells may dif-
Fig. 10. 73 DGA, terminal bronchiole (TB,tangentially sectioned)
opening into acinar tubules (A). Glycogen (red, =dark staining) predominates in the epithelium of the prospective pulmonary acinus,
where it may serve as a substrate for the synthesis of surfactant and
other lipids (Ten Have-Opbroek et al. 1988, 1990).As shown by some
acinar tubules (asterisks), type I1 cells easily lose their glycogen during tissue processing. PA, pulmonary artery branch. x 260.
fer with respect to the level of SP-A expression. In
other words, rhesus monkey type I1 cells are probably
able to express one of their specific functions, synthesis
of SP-A, from the time of their appearance (62 DGA)
onward. Similar conclusions have been drawn from immunohistochemical studies in some other species, i.e.,
the mouse (14.2 DGA), the rat (16 DGA), and the human (70-84 DGA) (reviews by Ten Have-Opbroek et
al., 1988,1990b). Further evidence for an early onset of
surfactant protein synthesis in humans has been provided by the finding of mRNA coding for SP-A in human fetuses aged 17-18 weeks (Otto-Verberne et al.,
Our general conclusion for normal mammalian
(adult, fetal) type I1 cells-i.e., that phenotype (lowcolumnar or cuboid cell shape; large and roundish nucleus) and function (SP-A synthesis) come to expression
at about the same time-has proved to be true for type
I1 cells of alveolar or papillary tumors in mice (Rehm et
al., 1988) and dogs (Ten Have-Opbroek et al., 1990a) as
well. This general statement is fully compatible with
our observations in the rhesus monkey that phenotype
and SP-A expressions may get lost concurrently when
type I1 cells transform into type I cells. Similar findings
acinar tubule
derivative structures
acinar tubule
Figs. 11-1 2. Rhesus monkey lung development follows the pathway
proposed for mammals (Ten Have-Opbroek, 1981).
Fig. 11. The two lung buds arising from the primitive foregut (in the
rhesus monkey, by 27 DGA) develop into the primordial systems of
the right and left lungs. The primordial tubules are lined by pseudostratified or simple columnar, primordial epithelium. The development of the primordial system (in the rhesus monkey, between 27 and
62 DGA) takes place in the first part of the pseudoglandular period,
which lasts in the rhesus monkey from ca. 27 to 95 DGA.
have been reported first for the mouse (Ten Have-Opbroek, 1979), and later also for the rat (Otto-Verberne
and Ten Have-Opbroek, 1987) and the human (OttoVerberne et al., 1988).
The present investigations on carbohydrate cytochemistry in rhesus monkey fetuses suggest a n early
onset of some other type I1 cell functions as well. First,
by and after 62 DGA, type I1 cells display a n abundance of glycogen, which among other things has been
shown specifically to provide substrates for surfactant
lipid biosynthesis and to play a distinct role in prenatal
multilamellar body formation (see introduction, and
Ten Have-Opbroek et al., 1988, 1990b). The present
finding of a n abundance of glycogen in type I1 cells at
that time is in agreement with ultrastructural observations in peripheral lung areas in the rhesus monkey
great alveolar (type II) cell or precursor
cii3.- small alveolar (type 1 ) cell or precursor
Fig. 12. Differentiation of the primordial system into the prospective bronchial and respiratory systems starts in the second part of
the pseudoglandular period, that is, in the rhesus monkey by 62
DGA. The basic structure in the development of the respiratory unit,
the pulmonary acinus, is the acinar tubule or sprout (lining: type I1
cells). The pulmonary acinus grows by budding of type I1 cells. In the
canalicular and following periods, type I1 cells lining acinar tubules
may develop into type I cells (that is, in the rhesus monkey, by 95
DGA). By this, acinar tubules transform into derivative structures
(alveolar ducts, sacs, pouches). The abrupt demarcation between alveolar (cuboid, and later also squamous) epithelium and bronchial
(columnar) epithelium is always evident.
(Tyler et al., 1988, 1989), and also with those in the
mouse (Ten Have-Opbroek et al., 1988,1990b) and the
human (Otto-Verberne et al., 1988) at comparable
stages. Second, by and after 62 DGA, type I1 cells contain acid glycoconjugates, which suggests that the cells
may synthesize basal lamina components.
Primary Role of Type I1 Cells in Pulmonary
Acinus Development
The present study indicates that the prospective respiratory system, and thus its units, the pulmonary
acini are first detectable in developing rhesus monkey
lungs by 62 DGA. The respiratory system, and the
more proximally located prospective bronchial system,
develop at that time from the undifferentiated primordial system present (see interpretive diagrams for
TABLE 1. Aspects of lung development in primates and rodents in relation to gestation time'
First appearance
Lung primordium
Type I1 cells
'Expressed in days of gestational age (DGA), and as a percentage of total gestation time. For details on other developmental data, see present
study; Ten Have-Opbroek (1981, 1991); Otto-Verberne and Ten Have-Opbroek (1987); Otto-Verberne et al. (1988).
'Based on photographs of embryos (Heuser and Streeter, 1941).
mammalian lung development, Fig. 11,12). This major
developmental step takes place in the middle of the
pseudoglandular period of lung development, which
lasts in the rhesus monkey from approximately 27
DGA (time of appearance of the lung primordium) to 95
DGA (see below). Our present study further shows
that, also in the rhesus monkey, type I1 cells play a key
role in pulmonary acinus formation (see Fig. 12). First,
between 62 and 95 DGA (i.e., in the last part of the
pseudoglandular period), type I1 cells are the only cells
to line and generate the prospective pulmonary acinus
and its components, the acinar tubules and buds. Second, after 95 DGA (ie., in the canalicular, terminalsac, and alveolar periods), type I1 cells also give rise to
a flatter cell type in the lining of the prospective pulmonary acinus, i.e., precursors of type I cells. This observation is in line with our earlier ultrastructural observations in rhesus monkey lungs, i.e., low-cuboid
cells line shallow depressions in prospective respiratory bronchioles (Tyler et al., 1988). Third, also in later
prenatal and postnatal stages of lung development,
proliferation and budding of type I1 cells seem to be
responsible for the final maturation of the pulmonary
acinus including alveolarization. A comparable developmental pattern-proliferation and budding of epithelial cells from the tracheal lining into the underlying mesenchyme-has
been reported for rhesus
tracheal submucosal glands (Plopper et al., 1986b).
Our present conclusion concerning a primary role of
type I1 cells in pulmonary acinus formation in the
rhesus monkey fully agrees with our findings in some
other species, i.e., the mouse (Ten Have-Opbroek, 1979,
1981,1991; Oomen et al., 19901,the rat (Otto-Verberne
and Ten Have-Opbroek, 1987), and the human (OttoVerberne et al., 1988,1990). In summary, we conclude
that the histologic pattern of lung development in the
rhesus monkey corresponds with the patterns found for
rodents and humans (reviews by Ten Have-Opbroek,
1981, 1991; Ten Have-Opbroek et al., 1988).
Timing of Lung Development in Primates and Rodents
The timing of lung development in primates and rodents shown in Tables 1and 2 is based on data from the
literature (Heuser and Streeter, 1941) and our own
data (see Table 1). Comparison of the time course of
lung development in rhesus monkeys and humans on
the one hand and mouse and rats on the other hand
reveals (Table 1) that the timing of type I1 cell differentiation is different for primates and rodents, i.e., at
29-38% and 73-75% of the total gestation time, respectively. However, as shown in Table 1, the lung primordium appears in these two mammalian classes also
at different times (at 9-16% versus 48-50% of gestation). Taking this into account, the onset of type I1 cell
differentiation in primates and rodents seems to take
place after a more or less similar developmental interval (20-25% of the total gestation time). As reported
(Ten Have-Opbroek, 1981), this major interval covers
the establishment of the primordial system. The second
major developmental interval (Table l),which extends
from the onset of type I1 cell differentiation to term,
covers the prenatal development of the respiratory and
bronchial systems (Ten Have-Opbroek, 1981). As appears from Table 1, this specific period does show distinct species differences. In primates it lasts about
twice as long as in rodents (62-71% versus 25-27% of
the total gestation time).
More accurate information on the issue of species
differences can be obtained (Table 2) by relating the
duration of the two major specific developmental periods to the total time covering prenatal lung development. In this light, establishment of the bronchial and
respiratory systems proves to last in primates about 1.5
times longer than in rodents (75-78% versus 5142%).
For the development of the primordial system it is of
course just the reverse (22-25% versus 48-49%). Possible mechanisms for the shorter duration of this period
in primates might be the following. First, a higher
growth rate of primordial epithelium might enable primates to accomplish a primordial system comparable
in size to that in rodents within a shorter time. Second,
this formation is terminated at an earlier time-point by
a faster development of the peripheral capillary system
in primates. Proximity to primordial epithelial cells
possibly promotes their differentiation to type I1 cells
(Ten Have-Opbroek et al., 1988). Finally, and maybe
most likely, the development of the bronchial and respiratory systems does not need a primordial system
with a mammalian-specific critical mass. Anyhow, the
longer duration of the second specific developmental
period in primates is not surprising in view of the complexity of the final bronchial and respiratory systems
(length and number of generations of branches) in
higher mammals.
In the evaluation presented here, we have left the
period of postnatal lung development out of consideration for clarity reasons. This period deals only with
final structuring and growth of the already existing
and functioning bronchial and respiratory systems.
Moreover, it is an extremely long period, because it
virtually extends t o the end of the puberal or growth
Although the bronchial system starts to develop at
the same time as the respiratory system in the rhesus
TABLE 2. Duration of specific periods in lung development in primates and rodents’
Prenatal lung development2
Primordial system development3
Bronchial and respiratory
systems development4
‘Expressed in DGA, and as a percentage of the total time of prenatal lung development.
2Calculated from the time of appearance of the lung primordium until term (see Table 1).
31nterval between the detection of the lung primordium and the appearance of type I1 cells (see Table 1).
41nterval between the appearance of type I1 cells and term (see Table 1).
monkey, i.e., by 62 DGA (present study), the proper
process of bronchial epithelial differentiation in the
respiratory tract as expressed by the appearance of ciliated cells starts earlier, that is, in the trachea by 46
DGA (Plopper et al., 1986a). After that time, bronchial
epithelial differentiation proceeds in a distal direction
throughout the airway tree (Tyler et al., 1989; Plopper,
Our conclusion that type I1 cell differentiation and
function in the rhesus monkey (Macaca mulatta) start
by 62 DGA, that is, in the pseudoglandular period of
lung development fully agrees with others’ observations in the macaque. In the same species (Macaca muZatta) (Kerr et al., 1975), the cells lining the peripheral
tubules of the developing lung become cuboid in height
from the 50th to 75th day of gestation, when the “glandular” pattern is still characteristic. At the same time,
their cellular organelles increase in number, short microvilli and terminal bars become obvious a t their apical surfaces, and capillaries with thickened endothelial
cells come to lie in close proximity. In the pig-tail monkey, Macaca nemestrzna (Boyden, 19761, which has a
similar gestation time (about 168 days), peripheral rosettes of epithelial buds destined to develop into the
respiratory units of the lung (Boyden, 1976) are first
noticeable about the 57th gestational day; their epithelium is made of closely packed cuboid cells. Our conclusion for the rhesus monkey that type I1 cells start to
give rise to type I cells by 95 DGA, is also supported by
these studies in the macaque. As reported, flatter cells
occur in the originally cuboid epithelial lining of peripheral tubules by 100 (Kerr et al., 1975) or at 106
(Boyden, 1976) days gestational age and thereafter.
The underlying mechanism, capillary invasion of overlying cuboid cells, starts by the gestational age of 75
days in the rhesus monkey (Kerr et al., 1975), and 80
days in the pig-tail monkey (Boyden, 1976).However, a
possible alveolar nature of the cuboid cells is never
discussed (Kerr et al., 1975; Boyden, 1976).On the contrary, it has generally been thought for a long time
that cuboid epithelium in developing and adult mammalian lungs represents (prospective)bronchial epithelium (reviews by Ten Have-Opbroek, 1981; Ten HaveOpbroek et al., 1991).
However, by contrast, it is not feasible to relate our
time-points of type I1 or type I cell differentiation in the
rhesus monkey (and other mammalian species) to timepoints of other investigators in rodents for the following reasons. One group of investigators reports not on
the first appearance of type I1 cells but on the expression of a differentiated type I1 cell function at the end
of gestation such as a peak in alkaline phosphatase
activity (Edelson et al., 1988), adult changes in the
pattern of cytokeratin synthesis (Paine et al., 19881,or
correlations between membrane and secretory differential markers like cell-surface glycoconjugates and
surfactant phospholipids (Joyce-Brady and Brody,
1990), or provides quantitative ultrastructural data on
type I1 cell differentiation a t late gestational ages (Lin
and Lechner, 1990; Snyder and Magliato, 1991; Young
et al., 1991).
A second group of investigators, on the other hand,
does report on the first appearance of type I1 or type I
cells based on antigenic or biochemical marker studies
in developing mouse or rat lungs. However, the relation between markers and specific epithelial cell types
in developing lungs is not or not sufficiently explained.
Studies on mouse lung development (Jaskoll et al.,
1984, 1988; Slavkin et al., 1989) conclude that type I1
cells first appear at 16.5 DGA in vivo, that is, a t the
beginning of the canalicular period. This conclusion is
based on the detection of SP-A expression in a number
of cuboid cells at that time. However, morphologically
identifiable (and SP-A reactive) type I1 cells are
present in developing mouse lungs as early as embryonic day 14.2, and are the only cells to line the future
respiratory unit a t earlier stages (Ten Have-Opbroek,
1975, 1979, 1991; Ten Have-Opbroek et al., 1988,
1990b). The relatively late detection and the small
number of reactive type I1 cells in developing and adult
lungs (Jaskoll et al., 1984, 1988; Slavkin et al., 1989)
suggest that only a subpopulation of mouse type I1 cells
has been stained by the antibody used (anti human
SP-A; source: human alveolar proteinosis lavage material). Studies on rat lung development using antigenic or biochemical cell markers, provide some different time-points for the first appearance of type I1 or
type I cells. Surprisingly, only one study (Funkhouser
et al., 1987) relates labeling to cell morphology. This
study using a monoclonal antibody to the luminal surface of rat type I1 cells (JBR-1, Funkhouser et al., 1987)
detects labeling on or in cuboid cells as early as 14
DGA, that is two days earlier in the pseudoglandular
period than we reported (Otto-Verberne and Ten HaveOpbroek, 1987). The labeled structure (Funkhouser et
al., 1987) was found at a level caudal to the tracheal
bifurcation and ascribed to staining of the epithelial
lining of the two embryonic main stem bronchi. The
authors conclude that progenitors of type I1 cells at
that level already express the JBR-1 reactive type I1
cell antigen by day 14. As mentioned but not shown
(Funkhouser et al., 19871, the antigen continued to be
expressed by cuboid cells lining the proximal embryonic respiratory ducts between 14 and 18 DGA, and
finally appeared on selected cells in the smallest ducts
by 19 DGA. However, there is some evidence that the
JBR-1 antibody initially reacts to endothelium of embryonic pulmonary arteries, and later also to a subpopulation of type I1 cells. First, unlike the embryonic
main bronchi (lining: pseudostratified columnar epithelium with oblong nuclei, Ten Have-Opbroek, 1981,
19911, embryonic arteries have a prominent cuboid endothelial lining. Second, as shown by the ultrastructural studies in adult rat lungs (Funkhouser e t al.,
19871, some endothelial surfaces do react to JBR-1.
Third, double labeling studies which we performed
with JBR-1 and anti SP-A a t 19 DGA (unpublished
observations) detect a distinct JBR-1 reactivity (cuboid
fluorescent cells) in pulmonary artery branches accompanying bronchioles and sometimes also in smaller
vessels, whereas the cuboid epithelium of prospective
respiratory structures (present from 16 DGA onward
and consisting of type I1 cells, Otto-Verberne and Ten
Have-Opbroek, 1987) always labels with anti SP-A but
only infrequently with JBR-1. This infrequent labeling
of type I1 cells by JBR-1 at 19 DGA-also observable in
photomicrographs of adult rat lungs (see Funkhouser
et al., 1987)-indicates that JBR-1 reacts to a subpopulation of type I1 cells by the end of gestation. In other
words, the monoclonal antibody JBR-1 does not provide
a suitable marker for studies on type I1 cell differentiation. The antigenic determinants recognized do not
represent a consistent feature of type I1 cells, and are
not type I1 cell specific. Other studies in rats also conclude t h a t type I1 cells start to differentiate in the
pseudoglandular period but at a later time (on 16 DGA;
Williams and Dobbs, 1990; Woodcock-Mitchell e t al.,
1990). Type I cells are said to be first detectable in the
same period, by 15 DGA (Williams and Dobbs, 1990).
The reagents used for specific cell detection are the
lectin Muclura pomiferu, MPA (Williams and Dobbs,
1990) or antibodies to keratin No. 18 (WoodcockMitchell et al., 1990) for type I1 cells, and antibodies to
a n unknown antigenic determinant for type I cells
(Williams and Dobbs, 1990). As mentioned (Williams
and Dobbs, 1990), MPA binding is detectable indeed on
16 DGA and thereafter but, as shown in the illustrations, it has a widespread distribution. The authors do
not correlate this labeling to the specific lung cell types
present. Criteria that can be used for this purpose-i.e.,
the shape and size of individual epithelial cells and
their nuclei, and the arrangement as a simple or pseudostratified epithelium (Ten Have-Opbroek et al.,
1988, 1990b)-are not optimally visualized in the photomicrographs (see Williams and Dobbs, 19901, due to a
high background staining in the fluorescence pictures
and the use of phase contrast images for routine orientation. Despite this fact, however, i t is evident that by
and after 16 DGA MPA binding occurs in cells scattered in the lining of larger tubules with pseudostratified or simple columnar epithelium (i.e., bronchial epithelium) and smaller peripheral tubules with
approximately cuboid epithelium (i.e., alveolar epithelium consisting of type I1 cells), see Otto-Verberne and
Ten Have-Opbroek, 1987. Because the labeling pattern
of the peripheral tubules is discontinuous, and even
smaller structures like acinar buds ( = newly formed
type I1 cells) seem to be devoid of staining (see illustrations in Williams and Dobbs, 19901, MPA binding
apparently involves a subpopulation of type I1 cells,
i.e., probably the more mature cells that occur more
proximally and have a more or less cuboid shape. As
appears from the pictures, antibodies to keratin No. 18
(Woodcock-Mitchell et al., 1990) also label both bronchial epithelial and type I1 cells in developing lungs.
The monoclonal antibody for type I cells has a widespread reactivity as well (see photomicrographs in
Williams and Dobbs, 1990). On and after 16 DGA, this
antibody seems to bind to bronchial epithelial cells and
a subpopulation of type I1 cells; by 15 DGA, i t seems to
react occasionally to the columnar epithelium of the
original branching tubular system of the lung, the primordial system (cf. Otto-Verberne and Ten Have-Opbroek, 1987). In the adult rat lung (Dobbs et al., 19881,
there is also some reactivity with type I1 cells.
In summary, our conclusions for the rhesus monkey
(and some other mammalian species) concerning the
first appearance of type I1 or type I cells cannot be
compared to recent conclusions for rodents, because the
markers used for cell identification in the rodent studies lack the required ( = unique and persistent) cell
specificity. However, our conclusions fully agree with
others’ findings in the macaque.
The authors thank Mr. Erwin C. P. de Vries and Mrs.
Alison J . Weir for technical assistance, Mrs. Joyce W.
Wetselaar for making the explanatory drawings, Mr.
Jan H. Lens for photographic assistance, and Mrs. E. A.
van der Kwast and Mrs. G.N. Augustina for secretarial
This work was supported in part by Public Health
Service grants HD 24959 and HL 28988, and the Netherlands Asthma Foundation.
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