Am J Physiol Lung Cell Mol Physiol 285: L664–L670, 2003.
First published June 6, 2003; 10.1152/ajplung.00306.2002.
Changes in alveolar epithelial cell proportions during
fetal and postnatal development in sheep
S. J. Flecknoe, M. J. Wallace, M. L. Cock, R. Harding, and S. B. Hooper
Department of Physiology, Monash University, Victoria 3800, Australia
Submitted 11 September 2002; accepted in final form 27 May 2003
type I alveolar epithelial cell; type II alveolar epithelial cell;
alveolar stem cell; lung volume; lung development; surfactant protein A; surfactant protein B; surfactant protein C
comprises type I and type II
alveolar epithelial cells (AECs), both of which play
critical roles in the respiratory function of the lung.
Type I AECs are large flattened cells with elongated
cytoplasmic extensions that, collectively, form a large
surface area for gas exchange (24). Type II AECs are
rounded in shape, synthesize and release surfactant,
and are considered to be the main progenitor cell type;
they give rise to new type II cells by division and to
type I cells by transdifferentiation (19, 26). Although it
was previously considered that type I cells are terminally differentiated, recent in vitro (7) and in vivo (9,
10) studies suggest that type I cells are capable of
transdifferentiation into type II cells. Thus it is possible that both AEC types have the potential to transdifferentiate, but the factors that determine the transdifferentiation are not clear. As both cell types are critical
for the respiratory function of the lung after birth, it is
THE ALVEOLAR EPITHELIUM
Address for reprint requests and other correspondence: S. B.
Hooper, Dept. of Physiology, Monash Univ. P. O. Box 13F, Victoria
3800, Australia (E-mail: [email protected]).
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important to understand how the lung attains the
correct proportions of type I and type II AECs during
fetal development and after birth.
Recent studies showing that type I cells have the
potential to transdifferentiate into type II cells indicate
that the degree of strain experienced by AECs is a
critical determinant of their phenotype (7, 9, 10). For
instance, in sheep, a species in which AECs differentiate before birth, sustained increases in fetal lung expansion induce type II cells to transdifferentiate into
type I cells via an intermediate cell type (9). On the
other hand, sustained reductions in lung expansion
promote an increase in type II AEC proportions, most
probably via transdifferentiation of type I into type II
AECs (10). These studies indicate that the basal degree
of lung expansion is an important determinant of the
relative proportions of each AEC phenotype within the
alveolar epithelium.
During fetal development, the future airways of the
lung are filled with liquid, which is secreted across the
pulmonary epithelium into the lung lumen (15). This
liquid leaves the lungs via the trachea, although its
efflux is retarded by adduction of the fetal glottis during apnea (12) and by contraction of the diaphragm
during fetal breathing movements (15). These fetal
activities promote the retention of liquid within the
future airways, which increases in volume from ⬃30
ml/kg at 115 days of gestation to 35–45 ml/kg near
term in fetal sheep (13). Thus in late gestation the fetal
lungs are maintained in a distended state, which is
essential for their growth and development (13, 15).
However, at birth, the removal of liquid from the airways and the entry of air into the lungs reduce resting
lung volumes, because the distending influence of lung
liquid is lost and the creation of surface tension at the
air-liquid interface increases the lung’s recoil properties (13, 15). As a result, end-expiratory lung volumes
decrease from 35–45 ml/kg (3, 13, 17, 21) in fetal sheep
late in gestation to 25–30 ml/kg in air-breathing newborn lambs (8, 16).
In view of the relationship between basal lung expansion and AEC phenotype, we hypothesized that the
proportion of type I and type II AECs would change in
relation to the changes in lung expansion during late
gestation and after birth. Specifically, we hypothesized
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Flecknoe, S. J., M. J. Wallace, M. L. Cock, R. Harding,
and S. B. Hooper. Changes in alveolar epithelial cell proportions during fetal and postnatal development in sheep.
Am J Physiol Lung Cell Mol Physiol 285: L664–L670, 2003.
First published June 6, 2003; 10.1152/ajplung.00306.2002.—
Basal lung expansion is an important determinant of alveolar epithelial cell (AEC) phenotype in the fetus. Because
basal lung expansion increases toward term and is reduced
after birth, we hypothesized that these changes would be
associated with altered proportions of AECs. AEC proportions were calculated with electron microscopy in fetal and
postnatal sheep. Type I AECs increased from 4.8 ⫾ 1.3% at
91 days to 63.0 ⫾ 3.6% at 111 days of gestation, remained at
this level until term, and decreased to 44.8 ⫾ 1.8% after
birth. Type II AECs increased from 4.3 ⫾ 1.5% at 111 days to
29.6 ⫾ 4.1% at 128 days of gestation, remained at this level
until term, and then increased to 52.9 ⫾ 1.5% after birth.
Surfactant protein (SP)-A, -B and -C mRNA levels increased
with increasing gestational age before birth, but the changes
in SP expression after birth were inconsistent. Thus before
birth type I AECs predominate, whereas after birth type II
AECs predominate, possibly due to the reduction in basal
lung expansion associated with the entry of air into the
lungs.
ONTOGENY OF ALVEOLAR EPITHELIAL CELLS
that the proportion of type I AECs would predominate
in late gestation and increase toward term with increasing lung volume, whereas type II cells would
predominate after birth due to a decrease in the basal
degree of lung expansion. We also hypothesized that
the expression of the surfactant proteins (SP)-A, -B,
and -C would change in parallel with the change in
type II AEC proportions. Consequently, in separate
groups of sheep, we have measured the proportion of
each AEC phenotype and the expression of SP-A, -B,
and -C between 91 days of gestation and term and then
after birth at 2 wk, 8 wk, and 2 yr of age.
METHODS
Analytical Methods
Histological analysis. We chose to identify AECs by morphological criteria using EM as previously described (9, 10), rather
than by light microscopy in conjunction with stains for specific
cell markers. We did this because it is currently unclear which
markers should be used to definitively identify the stem, intermediate, and type I AECs in sheep (see below). Previous studies
have shown that, in sheep (1) and humans (4), the nuclear
diameters of type I and type II AECs are similar, and, therefore,
the chances of counting a nuclear profile of each type are equal
by EM. We have made similar observations in our studies, with
average nuclear diameters of 4–6 ␮m for both type I and type II
AECs (unpublished observations).
After fixation, the right lung was processed for EM (9). The
right lung was separated into the upper, middle, and lower
lobes, and then each lobe was accurately cut into 5-mm slices.
Every second slice from each lobe was further subdivided into
three sections. We then chose six sections at random from
each lobe and cut the tissue into cubes (2 ⫻ 2 ⫻ 2 mm), taking
care to avoid major airways and blood vessels. One tissue
cube from each section was then selected for further processing (i.e., six cubes per lobe for each of the three lobes; 18
tissue cubes per animal). The tissue cubes were washed in 0.1
M cacodylate buffer, incubated in 2% OsO4 (in 0.1 M cacodylate buffer), and embedded in epoxy resin. At least three
epoxy resin/tissue blocks were randomly chosen from each
animal. Ultrathin sections (70–90 nm) were cut with a diaAJP-Lung Cell Mol Physiol • VOL
mond knife, mounted on 200-mesh copper grids, and stained
with aqueous uranyl acetate and Reynolds lead citrate. All
sections were coded, and the observer was blinded to the age
of the animal.
Alveolar epithelial cells were identified under a transmission EM (Joel 100s). For each animal, a minimum of 100
AECs were classified, and the number of nuclear profiles of
each type was counted (9, 10). In a previous study (9), we
performed multiple AEC counts on the same group of fetuses
and found that increasing the number of AECs counted (to
⬎200) did not alter the proportion of each AEC phenotype
obtained following the counting of 100 AECs. We viewed at
least three different sections per animal, ensuring that only
one section per tissue block was analyzed. Identification of
AECs depended on clear visualization of the basement membrane, with all AECs localized to the luminal surface of this
membrane. AECs were categorized as one of four phenotypes:
stem cells, type I AECs, type II AECs, and intermediate
AECs. Alveolar epithelial stem cells (AE stem cells) were
rounded in shape and contained abundant cytoplasmic glycogen; they did not contain lamellar bodies or have any
evidence of a cytoplasmic extension (see Fig. 1 and below).
Previous studies may have referred to this cell type as either
“progenitor cells” or “immature alveolar type II cells.” Type I
AECs had elongated cytoplasmic extensions, flattened nuclei,
little perinuclear cytoplasm, and few cytoplasmic organelles.
Cytoplasmic extensions are defined as peripheral regions of
cytoplasm that extend along the luminal surface of the epithelial cell basement membrane, with both apical and basolateral membranes running in parallel to each other and
separated by a thin layer of cytoplasm, ⬍0.5 ␮m in thickness
(Fig. 1). Type II AECs were rounded in shape with a rounded
nucleus and had microvilli on their apical surface and abundant cytoplasmic organelles, including lamellar bodies (Fig.
1). The intermediate cells were a heterogeneous group that
displayed characteristics of both type I and type II AECs.
Their classification depended on the presence of a flattened
nucleus and marked cytoplasmic extensions, but they also
contained lamellar bodies and usually had microvilli on their
apical surface (9).
Surfactant Protein Gene Expression
SP-A, SP-B and SP-C mRNA levels in lung tissues from
fetal sheep, lambs, and ewes were quantified by Northern
blot analysis as previously described (18). Total RNA was
extracted from lung tissue, and 20 ␮g were denatured and
electrophoresed in a 1% agarose gel containing 2.2 M formaldehyde. The RNA was then transferred to a nylon membrane (Duralon; Stratagene, La Jolla, CA) by capillary action
and cross-linked to it by ultraviolet light (Hoeffer UVC 500,
Amrad). The membrane was incubated in hybridization
buffer for 3–4 h at 42°C. This was followed by hybridization
with the 32P-labeled SP-A, SP-B, or SP-C cDNA probe (2 ⫻
106 counts 䡠 min⫺1 䡠 ml⫺1) for 24–48 h at 42°C in the same
hybridization buffer. These ovine-specific cDNA probes have
been described previously (18) and were labeled with
[␣-32P]dCTP by the random-priming technique (Oligolabeling kit, Pharmacia).
After hybridization with the labeled probe, the membranes
were washed, sealed in airtight bags, and exposed to a storage phosphor screen for 24–48 h at room temperature. To
standardize the amount of total RNA loaded onto each lane,
the blot was stripped by washing in 0.01⫻ standard salinesodium citrate containing 0.5% SDS at 80°C for 30 min and
was reprobed with a 32P-labeled cDNA probe for 18S rRNA.
We quantified the relative levels of SP-A, SP-B, and SP-C
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Lung tissue was collected from fetal sheep at the gestational ages of 91 (n ⫽ 4), 105 (n ⫽ 4), 111 (n ⫽ 4), 120 (n ⫽ 4),
128 (n ⫽ 5), 132 (n ⫽ 5), 138 (n ⫽ 5), and 142 days (n ⫽ 4),
from lambs at 2 (n ⫽ 4) and 8 wk (n ⫽ 4) after birth and from
adult sheep at 2 yr of age (n ⫽ 5); full term is 145–147 days
of gestation in this breed of sheep, and sexual maturity is
reached within their first year. The ewes, fetuses, and lambs
were all painlessly killed by an intravenous injection of
pentobarbital sodium and weighed, and their lungs were
removed and weighed before the left main bronchus was
ligated and the left lung was removed; portions of the left
lung were frozen in liquid nitrogen for biochemical analysis.
The right lung was fixed at 20 cmH2O via the lung lumen
with 4% paraformaldehyde and 4% glutaraldehyde; tissues
collected from fetuses at 105 days of gestation were not fixed
in glutaraldehyde and could not, therefore, be analyzed by
electron microscopy (EM) for AEC proportions. All ewes,
fetuses, and lambs used in this study were either shamoperated or unoperated controls that were not subjected to
experimental manipulation. All procedures performed on
animals were approved by the Monash University Animal
Welfare Committee according to guidelines established by
the Australian NH & MRC.
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ONTOGENY OF ALVEOLAR EPITHELIAL CELLS
mRNA and 18S rRNA by measuring the total integrated
density of each band using a phosphorimager and ImageQuant software (Molecular Dynamics, Sunnyvale, CA).
Data Analysis
Data are expressed as the means ⫾ SE, and the level of
significance used was P ⬍ 0.05. The changes in the proportions of each AEC type were determined separately by oneway ANOVA. In all Northern blots, the total integrated
density of each SP-A, SP-B (the density of the two SP-B
transcripts were summed), and SP-C transcript was divided
by the total integrated density of the 18S rRNA band for that
sample (lane) to account for minor differences in total RNA
loading between lanes. As a result, the band densities are
presented as a ratio of the 18S rRNA band density and,
therefore, have no units. To compare values from different
Northern blots, we expressed values from each age group as
a percentage of the mean value obtained from the same
128-day fetuses that were run on all blots. For each surfactant protein, differences between age groups were determined by one-way ANOVA following log10 transformation to
normalize the data.
at 142 days’ gestation, in 2- and 8-wk-old lambs, or in
2-yr old sheep (Fig. 2).
Type I AECs. The proportion of type I AECs increased from 4.8 ⫾ 1.3% at 91 days of gestation to
63.0 ⫾ 3.6% at 111 days of gestation. Similar proportions of type I AECs were maintained throughout the
rest of gestation (120 days, 66.9 ⫾ 3.2%; 128 days,
64.8 ⫾ 0.5%; 132 days, 71.6 ⫾ 2.6%; 138 days, 63.2 ⫾
2.3%; 142 days, 68.9 ⫾ 3.6%). In contrast, at 2 wk after
birth, the proportion of type I AECs had decreased to
44.8 ⫾ 1.8% and remained at this level at 8 wk and 2 yr
of age (45.5 ⫾ 2.9%) (Fig. 3).
RESULTS
AEC Phenotypes
Undifferentiated AE stem cells. At 91 days of gestation, most epithelial cells (93.8 ⫾ 2.0%) were undifferentiated AE stem cells, but the proportion of this cell
type was reduced to 30.3 ⫾ 3.2% by 111 days of gestation (Fig. 2). The proportion of undifferentiated AE
stems cells continued to decrease with increasing fetal
age, reaching 2.5 ⫾ 0.4% at 120 days, 1.0 ⫾ 0.4% at 128
days, 0.2 ⫾ 0.2% at 132 days, and 0.4 ⫾ 0.2% at 138
days; too few stem cells could be counted at these ages
for statistical comparison. No stem cells were observed
AJP-Lung Cell Mol Physiol • VOL
Fig. 2. Changes in the proportions of undifferentiated alveolar epithelial stem cells (F) and intermediate AECs (E) before (from 91 to
142 days of gestation) and after birth (from 2 wk to 2 yr of age). For
each cell type, values that do not share a common letter are significantly different from one another.
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Fig. 1. Electron micrographs of alveolar epithelial cells (AECs) at different
stages of gestation. Cells that are at an
early stage of differentiation from a
stem cell into either a type I (A, C) or
type II (B) AEC are depicted in A–C. D:
a fully differentiated type I cell. E: a
differentiated type II cell with numerous lamellar bodies. Arrows indicate
the position of tight junctions between
adjacent cells, arrowheads indicate cytoplasmic extensions, and the bar represents 2 ␮m; n, nucleus; lb, lamellar
bodies; g, glycogen; c, capillary.
ONTOGENY OF ALVEOLAR EPITHELIAL CELLS
SP-B. Relative to the mean 128-day value, SP-B
mRNA levels were low at 91 (37.5 ⫾ 4.1%), 105 (45.8 ⫾
6.6%), and 111 (40.6 ⫾ 7.0%) days of gestation and
increased to 100.0 ⫾ 2.6% at 128 days. SP-B mRNA
levels continued to increase after 128 days to reach
168.7 ⫾ 22.7 and 264.2 ⫾ 40.6% of the 128-day value at
138 and 142 days of gestation, respectively. At 2 wk
after birth, SP-B mRNA levels had decreased to
170.0 ⫾ 11.6% of the 128-day value and remained at
similar values at 8 wk after birth (160.5 ⫾ 8.4%) and at
2 yr of age (135.0 ⫾ 5.6%), which was not significantly
different from values at 128 days of gestation (Fig. 4B).
SP-C. Relative to the 128-day values, SP-C mRNA
levels were low at 91 (22.8 ⫾ 3.0%), 105 (26.1 ⫾ 4.5%),
and 111 days (34.0 ⫾ 10.9%) of gestation and increased
to 100.0 ⫾ 4.3% at 128 days of gestation. SP-C mRNA
levels continued to increase after 128 days of gestation
to reach 217.1 ⫾ 55.1 and 223.7 ⫾ 40.0% of the 128-day
value at 138 and 142 days of gestation, respectively. At
Type II AECs. At 91 days of gestation, only 1.2 ⫾
0.9% of all AECs was of the type II cell phenotype. A
similar proportion of type II AECs was present at 111
days (4.3 ⫾ 1.5%), but by 120 days, the proportion of
type II AECs had increased to 25.1 ⫾ 3.9% (Fig. 3). The
proportion of type II AECs remained at similar levels
throughout the rest of gestation (128 days, 28.5 ⫾
2.2%; 132 days, 22.4 ⫾ 3.1%; 138 days, 33.4 ⫾ 1.7; 142
days, 30.0 ⫾ 3.7%). In contrast, the proportion of type
II AECs increased from 30.0 ⫾ 3.7% at 142 days of
gestation to 52.9 ⫾ 1.5% at 2 wk following birth and
remained at this level at 8 wk and at 2 yr of age (53.4 ⫾
2.7%) (Fig. 3).
Intermediate AECs. At 91 days of gestation, only
0.1 ⫾ 0.1% of AECs was of an intermediate phenotype.
Although this level did not change significantly for the
remainder of gestation (111 days, 2.4 ⫾ 1.1%; 120 days,
5.5 ⫾ 0.7%; 128 days, 5.7 ⫾ 1.3%; 132 days, 5.8 ⫾ 2.1%;
138 days, 3.0 ⫾ 1.4%; 142 days, 1.1 ⫾ 0.4%), it tended
to increase between 120 and 132 days of gestation
before declining again by 138 days (Fig. 2). After birth,
the proportions of intermediate AECs remained at low
levels (2 wk, 2.2 ⫾ 0.9%; 8 wk, 1.2 ⫾ 0.5%), but these
cells were still observed at 2 yr of age (⬃1%; Fig. 2).
Surfactant Protein mRNA Levels
SP-A. Expressed as a percentage of the mean value
for 128-day fetuses, SP-A mRNA levels were low at 91
(15.8 ⫾ 1.6%), 105 (16.5 ⫾ 1.1%), and 111 days (17.8 ⫾
1.3%) of gestation, before increasing to 100.0 ⫾ 10.7%
at 128 days of gestation. SP-A mRNA levels continued
to increase after 128 days of gestation to reach 208.4 ⫾
67.5 and 400.2 ⫾ 80.9% of the 128-day value at 138 and
142 days of gestation, respectively. By 8 wk after birth,
SP-A mRNA levels had increased to 728.0 ⫾ 56.2% of
the 128-day value and remained at similar values
(646.0 ⫾ 65.5%) at 2 yr of age (Fig. 4A).
AJP-Lung Cell Mol Physiol • VOL
Fig. 4. Changes in surfactant protein (SP)-A (A), SP-B (B), and SP-C
(C) mRNA levels in lung tissue before (from 91 to 142 days of
gestation) and after birth (from 2 wk to 2 yr of age). Values are
expressed as a percentage of 128-day (d) mRNA expression. In each
panel, values that do not share a common letter are significantly
different from one another.
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Fig. 3. Changes in the proportions of type I (F) and type II (E)
alveolar epithelial cells before (from 91 to 142 days of gestation) and
after birth (from 2 wk to 2 yr of age). For each cell type, values that
do not share a common letter are significantly different from one
another.
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ONTOGENY OF ALVEOLAR EPITHELIAL CELLS
8 wk after birth, SP-C mRNA levels had increased to
332.0 ⫾ 19.4% of the 128-day value (Fig. 4C) but then
decreased to 223.0 ⫾ 29.1% at 2 yr of age.
DISCUSSION
AJP-Lung Cell Mol Physiol • VOL
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In view of the relationship between fetal lung expansion and AEC differentiation (9, 10), we hypothesized
that the proportion of type I and type II AECs would
change in parallel with the changes in basal lung
expansion that occur during late gestation and at birth.
We found that during the early canalicular stage of
fetal lung development [91 days of gestation (1)], most
(⬃94%) AECs were undifferentiated AE stem cells with
very few type I (⬃4%) or type II (⬃1%) cells. By 111
days of gestation, a substantial proportion of the AECs
(⬃63.0%) had differentiated into the type I cell phenotype, although very few type II AECs were present at
this stage (⬃4%). However, by 120 days of gestation,
which coincides with the beginning of the alveolar
stage of lung development (1), the proportion of type II
AECs had increased to ⬃25%. Despite the increase in
lung liquid volume from 120 days of gestation until
near term (15), the proportions of type I and type II
cells did not change during this period. However,
within 2 wk of birth, the proportion of type I AECs
decreased from ⬃63% to ⬃44%, whereas the proportion
of type II cells almost doubled from ⬃30% to ⬃53%;
these proportions persisted into adult life. Our findings
demonstrate that type I and type II cell differentiation
occurs rapidly (10–20 days) at defined, yet different,
stages of gestation in fetal sheep. Type I AECs differentiate at an earlier stage of development (91–111
days of gestation) than type II AECs, which rapidly
appear over a 9-day period (111–120 days). The
changes in the proportions of type I and type II AECs
that occur soon after birth are consistent with the
changes in end-expiratory lung expansion that occur at
birth (13, 15), and these proportions persist into adult
life.
Previous in vitro studies have shown that sustained
cell stretch may be an important determinant of AEC
phenotype (7, 11, 25). More recently we have provided
evidence to indicate that, in vivo, sustained increases
in fetal lung expansion induce type II AECs to transdifferentiate into type I AECs via an intermediate cell
type (9). In contrast, sustained reductions in fetal lung
expansion increase the proportion of type II AECs,
most probably via the transdifferentiation of type I into
type II cells (10). The findings of these studies indicate
that sustained increases in lung expansion promote
differentiation into the type I cell phenotype, whereas
reduced lung expansion promotes differentiation into
the type II cell phenotype. As the basal degree of lung
expansion in the fetus is greater than in the newborn
(13, 15), it is not surprising that type I AECs predominate before birth, accounting for ⬃65% of all AECs;
this finding is consistent with previous studies in fetal
sheep (1, 6, 9). Furthermore, the large increase in the
proportion of type I AECs between 91 and 111 days of
gestation coincides with the exponential-like increase
in potential air space volume of the lung at this stage of
development in sheep (1). The stimulus for this increase in type I AEC proportions is unknown, but as
the increase occurs in advance of the increase in type II
cell numbers it is likely to result from direct differentiation of undifferentiated stem cells into type I cells,
as previously suggested (1) (Fig. 1). Indeed, a number
of the type I cells identified at this stage of gestation
had significant amounts of perinuclear cytoplasm and
contained some glycogen but were identified as type I
cells due to the presence of cytoplasmic extensions (see
Fig. 1). It is possible that this increase in type I AEC
numbers is causally related to the increase in potential
air space volume that occurs at this stage of gestation
(1). This, in turn, may be determined by the ability of
the fetal glottis and fetal breathing movements to
restrict lung liquid efflux and, therefore, maintain an
internal distending pressure on the lung.
In contrast to type I AECs, few morphologically distinct type II AECs were present at 111 days (⬃4%),
after which the proportion of type II cells rapidly increased to ⬃25% by 120 days of gestation. The marked
increases in mRNA levels for SP-A (fivefold), SP-B
(2.5-fold), and SP-C (threefold) over this gestational
age period (111–128 days of gestation) are consistent
with the increase in the proportion of type II AECs at
this time. However, expression of the surfactant proteins, particularly SP-B and SP-C, was also evident
well before a significant number of morphologically
distinct type II AECs (containing lamellar bodies)
could be identified. This raises the interesting question
as to whether expression of the surfactant proteins can
be reliably used to distinguish between AE stem cells
and type II AECs early in gestation. The mechanisms
for the rapid increase in type II cell proportions between 111 and 120 days of gestation are unknown, but
it is unlikely that corticosteroids are involved, as
plasma cortisol concentrations do not begin to increase
until ⬃135 days of gestation in fetal sheep (2). Whatever the mechanisms, our data indicate that the period
between 111 and 120 days of gestation is critical for
type II cell differentiation in fetal sheep in vivo.
Our finding that the proportions of type I and type II
AECs did not change significantly between 120 days of
gestation and near term (142 days) was unexpected
considering the large increase in lung luminal volume
that occurs over this time (13). In fetal sheep, lung
liquid volumes increase from ⬃30 ml/kg (⬃70 mls) at
115 days of gestation to 35–45 ml/kg (⬃170 ml) near
term (13). If the mechanical load experienced by AECs
is the primary determinant of their phenotype, as previously proposed (9, 10), these data indicate that despite the increase in lung luminal volume, the mechanical load experienced by individual AECs does not
change between 120 days of gestation and term. This
could result from lung growth as well as from the
considerable amount of structural remodeling that occurs in the lung over this period of development, resulting in marked changes in tissue mechanics.
Our study is the first to document the changes in the
proportions of type I and type II AECs before and after
ONTOGENY OF ALVEOLAR EPITHELIAL CELLS
AJP-Lung Cell Mol Physiol • VOL
pansion of the lungs substantially increases at this
time. Previous studies have shown that phasic stretch
of type II AECs in culture increases the expression of
the surfactant proteins (20, 22, 23), although a direct
effect on type II cell differentiation was not examined.
Before 128 days of gestation, expression of SP-A, -B,
and -C was low, which is consistent with the low
number of type II AECs at this stage of gestation, but
markedly increased with the appearance of morphologically distinct type II AECs at ⬃120 days of gestation.
However, despite the fact that the proportion of type II
AECs did not increase further between 120 days and
term, SP-A, -B, and -C mRNA levels increased over this
period; SP-A increased by 400%, SP-B by 250%, and
SP-C by 220% between 128 and 142 days of gestation.
This increase in surfactant protein expression, without
a corresponding increase in type II AEC proportions,
indicates that surfactant protein expression per type II
cell may increase over this period. The mechanisms
involved are unknown but could be related to the
increase in endogenous fetal cortisol levels at this time.
However, the large increase in the proportion of type II
AECs after birth (from ⬃30–53%) had an inconsistent
effect on surfactant protein expression. This increase
in type II AEC proportions (77% increase) at 2 wk of
age was not accompanied by a similar increase in SP-A,
-B, or -C mRNA levels, although SP-A expression was
increased at 8 wk and 2 yr of age. After birth, we found
that expression of SP-A, -B, and -C showed a different
pattern of expression. Compared with values measured
in late gestation (142 days), after birth SP-A mRNA
levels increased, and SP-B mRNA levels decreased,
whereas SP-C mRNA levels were elevated at 8 wk but
not at 2 wk or 2 yr of age. These data indicate that
expression of the SP-A, -B, and -C genes is differentially regulated after birth, although the potential
mechanisms are unclear.
Our study demonstrates, for the first time, that the
relative proportions of type I and type II AECs reverse
after birth. Before birth the type I cell phenotype predominates, whereas after birth the type II cell phenotype predominates, and these proportions persist into
adult life. We suggest that the changes in the proportion of AEC phenotypes at birth result from an increase
in lung recoil and a decrease in the degree of lung
expansion at this time, possibly due to type I to type II
cell transdifferentiation. In view of the effect of birth
on AEC phenotypes, it will be important to determine
the effect of preterm birth on the proportion of AECs,
particularly in the very preterm infant. These infants
can be born with few, if any, fully differentiated AECs,
and, therefore, the majority of AECs must differentiate
after birth in an environment completely different from
that which they are exposed during in utero development.
We are indebted to Alison Thiel for expert technical assistance,
particularly in the extraction of RNA samples and the generation of
Northern blots, as well as Samantha Louey for assistance in obtaining lung tissue from 8-wk- and 2-yr-old sheep.
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birth, and our data are consistent with previous studies reporting the relative proportions of type I and type
II AECs in fetal sheep before birth (1, 6) as well as in
adult humans (4) and rats (5). We have demonstrated
that the proportion of type II AECs increases whereas
the proportion of type I AECs decreases after birth, and
we propose that these changes result from an associated reduction in the basal degree of lung expansion.
Although discrepancies exist in the literature as to the
precise timing for the decrease in lung liquid volume
around the time of birth, most recent studies agree
that fetal lung liquid volumes are maintained at 35–45
ml/kg up until 1–3 days before labor in sheep (3, 13, 15,
17, 21). However, the oldest gestational age examined
in this study was 142 days, which is ⬃5 days before
labor in the breed of sheep we used and 2–4 days before
the period when fetal lung liquid volumes arguably
decrease. Our previous studies indicate that, unless
there is an external complicating factor (e.g., oligohydramnios), fetal lung liquid volumes do not decrease
before labor onset (17). The presence or absence of
oligohydramnios, which can cause reductions in lung
liquid volumes (13), was not examined at the time of
lung liquid volume measurement in some recent studies focusing on this question (3, 21).
The mechanism for the change in AEC proportions
after birth is unknown but could have resulted only
from type II cell proliferation, type I cell apoptosis, or
from type I-to-type II AEC transdifferentiation. Although it was originally considered that type I AECs
were terminally differentiated, recent in vitro and in
vivo studies have provided evidence to suggest that
type I cells are not terminally differentiated but can
transdifferentiate into type II cells, particularly in response to reduced levels of lung expansion (10). Consequently, we suggest that a sustained reduction in
basal lung expansion, associated with the onset of
gaseous ventilation at birth, induces some type I AECs
to transdifferentiate, possibly via an intermediate cell
type, into type II AECs. A dependent relationship between reduced lung expansion and differentiation into
the type II cell phenotype after birth may have functional advantages. In particular, it may increase the
lung’s potential to produce surfactant at a time when
the recoil of the lung increases and the mechanism that
opposes its collapse changes from the internal distension
by liquid to external bracing by a semirigid structure
(chest wall) that is relatively compliant at birth (8).
It is also possible that, with the onset of air breathing after birth, the larger tidal volumes and the resultant increase in phasic stretch of the lungs are responsible for the increase in type II cell proportions at this
time. In the fetus, individual breathing movements are
essentially isovolumetric, with a tidal volume of ⬍1%
of lung volume (13). This is because the viscosity of
lung liquid is much greater than air and the fetal chest
wall is very compliant; as a result, parts of the chest
wall are drawn in when the diaphragm contracts (14).
However, after birth, tidal volumes increase to ⬃20%
of end-expiratory lung volumes (functional residual
capacity) (8), indicating that the breath-by-breath ex-
L669
L670
ONTOGENY OF ALVEOLAR EPITHELIAL CELLS
DISCLOSURES
This work was funded by the National Health and Medical Research Council of Australia.
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AJP-Lung Cell Mol Physiol • VOL
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