Impaired Distal Airway Development in Mice Lacking Elastin
Daniel P. Wendel, Douglas G. Taylor, Kurt H. Albertine, Mark T. Keating, and Dean Y. Li
Program in Human and Molecular Biology & Genetics; Department of Human Genetics; Department of Pediatrics; Howard Hughes
Medical Institute; and Department of Internal Medicine, University of Utah, Salt Lake City, Utah
Elastin is a major component of the mammalian lung, predominantly found in the alveoli. Destruction of alveolar elastic
fibers is implicated in the pathogenic mechanism of emphysema in adults. These data define a role for elastin in the
structure and function of the mature lung, and suggest that
elastin is important for alveogenesis. To investigate the role of
elastin in lung development, we examined mice lacking elastin (Eln⫺/⫺). At birth, the distal air sacs of Eln⫺/⫺ lungs dilate to form abnormally large cavities. This phenotype appears before the synthesis and deposition of alveolar elastin, a
process mediated by myofibroblasts and initiated after postnatal Day 4. Morphometric analyses demonstrate that the
perinatal development of terminal airway branches is arrested
in Eln⫺/⫺ mice. The branching defect is accompanied by
fewer distal air sacs that are dilated with attenuated tissue
septae, a condition reminiscent of emphysema. Elastin expression in the lung parenchyma before alveogenesis is localized
to the mesenchyme surrounding the developing airways, supporting a role for elastin in airway branching. Thus, in addition to its role in the structure and function of the mature
lung, elastin is essential for pulmonary development and is
important for terminal airway branching.
Elastin is an important component of many organ systems
(1, 2). It confers resilience upon structures that undergo
repetitive physiologic stress. In the lung, elastic fibers are
concentrated in a ring around the mouth of each alveolus
(3). These fibers stretch and contract during inhalation
and exhalation. Destruction of elastin is implicated in the
pathogenesis of emphysema (4–6), a condition marked by
thinning and weakening of the alveolar walls that leads to
tissue destruction and abnormal expansion of air sacs.
These data demonstrate that elastin is important for maintaining the structural and functional integrity of the mature lung.
There are five stages of lung development in rodents, on
the basis of gross histologic features. The first four stages,
termed the embryonic, pseudoglandular, canalicular, and
saccular stages, occur during gestation (7–9). At the end of
gestation, saccular-stage lungs have formed alveolar ducts
and air sacs. In mice, the final stage of lung development,
alveogenesis, begins after birth at postpartum Day 4 (P4)
(10–12). Alveogenesis is characterized by the development
of septae that subdivide the terminal air sacs into anatomic
alveoli (8, 11). This process requires that alveolar myofibroblasts form at the tips of the developing septae and
(Received in original form August 20, 1999 and in revised form May 5, 2000)
Address correspondence to: Dean Y. Li, Program in Human Molecular Biology & Genetics, University of Utah, 15 N 2030 E, Salt Lake City, UT
84112-5330. E-mail: [email protected]
Abbreviations: embryonic Day, E; elastin-null, Eln⫺/⫺; wild-type for elastin, Eln⫹/⫹; postpartum Day, P; platelet-derived growth factor, PDGF;
radial alveolar count, RAC; standard deviation, SD; smooth-muscle cell,
SMC.
Am. J. Respir. Cell Mol. Biol. Vol. 23, pp. 320–326, 2000
Internet address: www.atsjournals.org
synthesize elastin. On the basis of the expression of elastin
and the coincident development of septae, Burri and Weibel (12), Emery (13), and Noguchi and colleagues (14) hypothesized that elastin is the driving force of alveogenesis.
Recent studies have correlated disruption of alveolar
elastin expression with abnormal lung development. In
mice deficient for platelet-derived growth factor (PDGF)A, myofibroblasts do not form at the tips of the alveolar
septae and consequently do not express elastin (8). Thus,
PDGF-A⫺/⫺ mice are virtually devoid of elastin expression in the lung parenchyma (10). Surprisingly, lung development in these mice appears normal from gestation to
P4. After P4, the failure of alveogenesis becomes evident
as the distal air sacs become dilated and thin-walled. By
P10 the lungs appear grossly abnormal, and the mice die
by 6 wk of age. These data demonstrate the importance of
alveolar myofibroblasts in depositing elastin and mediating alveogenesis.
Here, we report that complete disruption of elastin expression results in defective lung development immediately after birth. At birth, the distal air sacs of elastin-null
(Eln⫺/⫺) lungs dilate to form large air-filled cavities. This
is in contrast to PDGF-A⫺/⫺ mice, where ablation of alveolar elastin synthesis is not associated with an aberrant
phenotype before P4. These results indicate that disruption of elastin expression in regions other than the alveoli
account for the pulmonary phenotype of Eln⫺/⫺ mice.
Our data demonstrate that the distal airways fail to develop during the perinatal period in Eln⫺/⫺ mice. Thus, in
addition to the well-established role of elastin in the structure and function of mature alveoli, our results indicate
that elastin expression is important during pulmonary development for distal airway branching.
Materials and Methods
Morphologic Analysis of Murine Lungs
The Eln⫺/⫺ and wild-type for elastin (Eln⫹/⫹) mice used in this
study were C57Bl/6. To collect embryos of known age, pregnancies were staged by plug date. Midday of the vaginal plug was
considered embryonic Day 0.5 (E0.5) in the staging of embryos
(15). The gestation period was 19 d for this strain of mice (16).
Animals were classified as P0 when birth was witnessed, and they
were killed immediately thereafter. The genotype of mice used in
this study was determined by Southern blot or polymerase chain
reaction as described previously (17).
Neonates were killed by asphyxiation with CO2. To avoid
bleeding into the lungs, the animals were bisected laterally just
below the xyphoid process to permit passive drainage of blood.
After the drainage stopped, the mice were decapitated. The inflation state of lungs used in the morphologic and morphometric
studies was standardized by vacuum insufflation. The specimens
were fixed for 6 h in Carnoy’s fixative under 50 mbar vacuum.
This fixative hardens the lungs rapidly and reduces lung collapse
and tissue shrinkage (18). After fixation, the heart, lungs, and
Wendel, Taylor, Albertine, et al.: Eln⫺/⫺ Lung Development
thymus were dissected from the rib cage en bloc, embedded in
paraffin, and sectioned coronally at a thickness of 6 ␮m. Sections
were stained with hematoxylin and eosin (H&E) according to
standard techniques (19).
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age generation numbers of the terminal airways of Eln⫺/⫺ animals at E18.5 and P0.5 were compared with those of their Eln⫹/⫹
siblings. Comparisons between siblings were made to control for
variations in age due to the imprecise nature of using vaginal plug
date to determine date of conception.
Morphometric Analysis
The number of distal air sacs across terminal respiratory units
was estimated by the radial alveolar count (RAC) method (18,
20). A line was drawn from the center of a respiratory bronchiole
to the nearest interlobular septum, to which an intercept line was
drawn perpendicularly. The number of distal air sacs that were
transected by the intercept line were counted. This assessment
was repeated for 10 terminal respiratory units in one random tissue section per mouse.
Volume density of secondary crests was estimated by pointcounting, using a 100-point coherent square lattice (18). Secondary crests were identified as partitions that extended from the
primary septa forming the wall of terminal sacs. We used the adjacent tissue sections to verify that the partitions were not tangential cuts through primary septa. The reference area used to
calculate volume density of secondary crests was defined as the
amount of lung parenchyma as determined by point-counting.
The exclusion of the air sacs from the reference area was important for minimizing the effect of dilated air sacs in Eln⫺/⫺ mice.
A total of 20 nonoverlapping, calibrated fields were analyzed in
one random tissue section per mouse.
Tropoelastin In Situ Hybridization
Whole lungs were fixed overnight at 4⬚C in 10% buffered formalin, embedded in paraffin, and sectioned. Sections were deparaffinized, hydrated, and treated with Proteinase K. Sections were
incubated in a 0.25% acetic anhydride/0.1 M triethanolamine solution to reduce background staining before being dehydrated
and air-dried. The rat tropoelastin complementary DNA used to
generate the digoxygenin (DIG)-labeled riboprobe was kindly provided by Dr. Richard A. Pierce (Washington University, St. Louis,
MO). RNA probes were labeled and hybridized at 60⬚C according to the manufacturer’s instructions (Genius Kits #3 and #4;
Roche, Indianapolis, IN).
Immunohistochemistry
Whole lungs were fixed overnight at 4⬚C in 10% buffered formalin, embedded in paraffin, and sectioned. Sections were deparaffinized and hydrated. Immunohistochemistry was performed using
␣-smooth-muscle cell (␣-SMC) actin monoclonal mouse immunoglobulin (Ig) G2a (Sigma, St. Louis, MO) and biotinylated goatantimouse IgG2a polyclonal secondary antibody (Amersham, San
Francisco, CA), according to the manufacturer’s instructions.
Airway Branch Quantification
The branching pattern of the airways was quantified by a novel
method using a series of photographs of two-dimensional sections
to discern the three-dimensional structure of the airways. We measured airway branches of the left lungs of littermates. The tissues
were fixed under vacuum, embedded, and sectioned coronally as
described earlier. Each specimen was serially sectioned at a thickness of 6 ␮m. All sections were stained with H&E according to
standard techniques (19), and photographed. The photographs
were a set of images corresponding to sections covering the entire
left lobe of the lung. These photographs were used to manually
trace random paths from the trachea to terminal respiratory
units. For each path, the generation number of the terminal airway was determined. The trachea was assigned generation zero,
and with each succeeding bifurcation, 1 was added to the generation number. As the airways become more distal the generation
number increases, until the terminal air sac is reached. The aver-
Statistical Significance
The morphometric results are shown as means ⫾ standard deviation (SD). Analysis of variance and Student–Newman–Keuls
multiple comparison tests were used to compare results from
Eln⫺/⫺ mice with Eln⫹/⫹ siblings (21). We considered P ⬍ 0.05
as indicating statistical significance.
Results
Morphology of the Eln⫺/⫺ Lung
At E18.5, Eln⫺/⫺ lungs were indistinguishable from Eln⫹/⫹
lungs (Figures 1A and 1B). By this age, the central airways
had formed and begun to differentiate. At birth (P0),
Eln⫺/⫺ lungs had larger distal air sacs and thinner surrounding walls than did their Eln⫹/⫹ siblings (Figures 1C
and 1D). No evidence of hemorrhage, inflammation, apoptosis, cellular debris, or clotting was observed before P0.5
(22). At P0.5 the distal air sacs continued to expand, causing the intervening wall tissue to become progressively
thinner (Figures 1E–1H). By P2.5, areas of atelectasis
were occasionally dispersed among the bullous-like lung
tissue in Eln⫺/⫺ mice (Figures 1I and 1J). No Eln⫺/⫺
mice survived beyond P3.5. No differences were observed
between Eln⫹/⫹ lungs and lungs that were heterozygous
for elastin (D. Wendel and L. Li, unpublished data).
Morphometry of the Eln⫺/⫺ Lung
To quantify the morphologic differences observed between Eln⫹/⫹ and Eln⫺/⫺ lungs, we measured two morphometric parameters: RAC and average volume density
ratio of secondary crests. RAC is a measurement of the
number of terminal air-space units along a line drawn
from the center of a respiratory bronchiole to the nearest
interlobular septum. In Eln⫹/⫹ mice, the RAC increased
from an average of 3.7 to 4.8 (P ⬍ 0.05) between E18.5
and P0.5. By comparison, the RAC remained statistically
unchanged in Eln⫺/⫺ mice, with values of 3.3 at E18.5 to
2.7 at P0.5 (P ⬎ 0.05). Thus, the RAC of Eln⫺/⫺ lungs
failed to increase between E18.5 and P0.5 (Figure 2A).
Secondary crests are the structures that elongate from
the primary septa of the terminal sacs and partition them
into alveoli. Although this process can be detected at E18.5
and P0.5, complete secondary crest elongation occurs during alveolization. We used the adjacent tissue sections to
verify that the partitions were not tangential cuts through
primary septa. The reference space used in calculating secondary crest volume density was the lung parenchyma, as
determined by point-counting. The average volume density ratio of secondary crests to lung parenchyma in the
Eln⫹/⫹ mice increased from 0.118 to 0.156 (P ⬍ 0.05) between E18.5 and P0.5. The secondary crest volume density
ratio in the lungs of the Eln⫺/⫺ mice decreased from
0.124 to 0.080 (P ⬍ 0.05) over the same developmental period (Figure 2B). The decrease in Eln⫺/⫺ secondary crest
volume density provides independent support for the
RAC measurements. Both RAC and secondary crest vol-
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Figure 2. RAC (A) and secondary crest volume density (B)
graphs. Eln⫹/⫹ (filled bars) are compared with Eln⫺/⫺ (open
bars) lungs. The differences between Eln⫹/⫹ and Eln⫺/⫺ lungs
at E18.5 are not significant, but the differences in both radial alveolar count and secondary crest volume density at P0.5 are significant, with P ⬍ 0.05. Values are means ⫾ SD. *Statistical significance between Eln⫹/⫹ and Eln⫺/⫺ samples.
Tropoelastin Expression in the Murine Lung
Elastin is known to be important in the final stage of lung
development, alveogenesis. However, the phenotype of the
Eln⫺/⫺ lung appeared before this stage. To understand
the role of elastin expression in early lung development,
we performed a series of in situ hybridization experiments.
The results showed that at E16.5, tropoelastin messenger
RNA (mRNA) expression was limited to the mesenchyme
surrounding the airways (Figure 3A). At E17.5, E18.5, and
P0.5, expression was somewhat diminished but continued
to be restricted to the adjacent mesenchyme (Figures 3B–
3E). Our findings are consistent with expression data from
ovine and rat developmental studies (23, 24). The expression pattern of tropoelastin supports a role for elastin in
airway branching.
Figure 1. Morphology of the elastin-deficient lung. H&E stains
of cross-sections from Eln⫹/⫹ (A, C, E, G, and I) and Eln⫺/⫺
(B, D, F, H, and J) lungs. There is no difference between the
lungs of Eln⫹/⫹ and Eln⫺/⫺ mice aged E18.5 (A and B). At
birth (C and D), Eln⫺/⫺ lungs are dilated, thin-walled, and
bullous-like. By P0.5 (E–H), the difference between the Eln⫹/⫹
and Eln⫺/⫺ lungs is striking. Higher magnification of P0.5 sections (G and H) demonstrates that Eln⫺/⫺ distal airways are dilated and the tissue septae are attenuated. The severity of the
phenotype increases by P2.5 (I and J). No Eln⫺/⫺ mice survived
beyond P3.5.
ume density measurements are a ratio of the number of
distal air sacs to reference unit of lung parenchyma. Due
to perinatal growth, the reference space increased in both
Eln⫹/⫹ and Eln⫺/⫺ lungs. Our results indicate that as the
reference space increased between E18.5 and P0.5, there
was no concomitant increase in the number of distal air sacs
for Eln⫺/⫺ lungs.
␣–Smooth-Muscle Actin in the Eln⫺/⫺ Lung
In adult lungs, SMCs exist in the blood vessels, bronchial
walls, and alveolar septae, and are identified by immunostaining for ␣-SMC actin (25). During the perinatal period,
myofibroblasts had not yet fully differentiated and localized to the alveolar septae, and ␣–smooth-muscle actin immunostains only identified the mesenchymal cells surrounding blood vessels and airways (8, 26) (Figure 4). This
immunostain showed no differences between Eln⫹/⫹ and
Eln⫺/⫺ lungs in the expression of ␣-SMC actin by the
mesenchymal cells surrounding the airways.
Airway Branch Quantification
The morphologic analysis of Eln⫺/⫺ lungs and the expression of elastin mRNA surrounding the airways before
birth suggests that elastin is required for the generation of
airways. Previous studies of airway branch morphology
have used pulmonary casting. Those studies focused on
the airway branching patterns of adult humans, guinea
pigs, and rats (27–29). We found the techniques used in
those studies were inappropriate for our studies of embryonic and neonatal lungs. In casting experiments, silicone
compounds are injected through the trachea and allowed
Wendel, Taylor, Albertine, et al.: Eln⫺/⫺ Lung Development
Figure 3. Tropoelastin expression in the Eln⫹/⫹ lung. In situ
hybridization to sections of Eln⫹/⫹ lungs using DIG-labeled antisense tropoelastin probe. Blue color indicates tropoelastin expression. Tropoelastin expression at E16.5 in the lung (A) is restricted to the pulmonary vasculature and to the mesenchyme
immediately surrounding the airways. In older lungs (B, C, D,
and E) there is little tropoelastin expression in the lung parenchyma, and it is localized to the airways. At E18.5 (C and D), only
limited expression was detected. No signal was detected in Eln⫺/⫺
lungs (data not shown) or in in situ hybridizations using sense
controls (F).
to polymerize. The surrounding lung tissue is cleared, leaving behind a solid cast of the pulmonary airway architecture. Due to the small size and fragility of E18.5 and P0.5
lungs and trachea, casting methods failed.
To compare the branching patterns of Eln⫹/⫹ and
Eln⫺/⫺ lungs, we used two-dimensional sections to reconstruct the three-dimensional structure of the airway network of the left lung (Figure 5). This method allowed us to
determine the number of branch points from the trachea
to randomly selected terminal air sacs of Eln⫹/⫹ and
Eln⫺/⫺ lungs. To control for small variations in age, only
littermates were compared. We determined the average
and SD of the generation number of terminal airways. Between E18.5 and P0.5, the average generation of the terminal airway in the Eln⫹/⫹ lung increased from 16 ⫾ 5.1 to
20 ⫾ 1.7 (P ⬍ 0.05) Over the same time period, the
branching pattern of Eln⫺/⫺ lungs remained statistically
unchanged, from 16 ⫾ 2.4 to 14 ⫾ 0.6 (Figure 6). Thus,
while the number of airway generations increased between E18.5 and P0.5 in Eln⫹/⫹ mice, the formation of
distal airways was arrested in Eln⫺/⫺ lungs.
Discussion
Elastin Is Essential for Lung Development
Here, we show that elastin is required for lung development.
Previously, Burri and colleagues hypothesized that elastin
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Figure 4. Immunostains of P0.5 Eln⫹/⫹ and Eln⫺/⫺ lungs for
␣-SMC actin. Staining in Eln⫹/⫹ and Eln⫺/⫺ lungs (A and B) is
detected in the SMCs surrounding blood vessels (V) and to a
lesser extent around airways (A). Septal myofibroblasts within
the alveoli are not detected in either Eln⫹/⫹ or Eln⫺/⫺ lungs
during the perinatal period. No difference is observed in mesenchymal ␣-SMC actin expression of Eln⫹/⫹ and Eln⫺/⫺ lungs.
is a critical morphogenetic force in the process of alveogenesis (30). This hypothesis was based on the observation
that elastin is predominantly expressed in the lung parenchyma at the tips of the developing alveolar septae. Recently, studies of PDGF-A⫺/⫺ mice have been used to
support this hypothesis (8, 10). In these mice myofibroblasts fail to form, thereby disrupting alveolar elastin expression and resulting in defective lung morphology after
P4. However, none of this previous work directly investigated the role of elastin during lung development. By studying Eln⫺/⫺ mice, we demonstrated that elastin is essential
for lung development.
Elastin Is Required for Distal Airway Branching
Our characterization of Eln⫺/⫺ lungs demonstrates that
elastin is required for the formation of distal airways during lung development. At E18.5 (term ⫽ E19), the average
number of airway generations in both Eln⫹/⫹ and Eln⫺/⫺
lungs was 16. Approximately 24 h later, at P0.5, the generation number in Eln⫹/⫹ lungs increased to 20 while the
generation number in Eln⫺/⫺ lungs remained the same
(Figure 6). Thus, while distal airways continue to branch
postnatally during normal lung development, in Eln⫺/⫺
mice this process is arrested. The arrested distal airway
branching explains why the onset of morphologic defects
in Eln⫺/⫺ lung development occurs at birth, before the
development of morphologic defects in PDGF-A⫺/⫺ mice
after P4. In PDGF-A⫺/⫺ mice there is an absence of the
myofibroblasts that are critical to the formation of septae
and the synthesis of elastin during alveogenesis. This cellular defect results in PDGF-A⫺/⫺ lungs that are virtually
devoid of parenchymal elastin (10). Despite these severe
molecular and cellular defects, PDGF-A⫺/⫺ lung morphology appeared normal before P4. In contrast, lung development in Eln⫺/⫺ mice is disrupted at birth. Our re-
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Figure 5. Comparison of terminal air sac generation counting in Eln⫹/⫹ and Eln⫺/⫺
lungs. Five serial sections from
the left lobe of Eln⫹/⫹ (A,
C, E, G, and I) and Eln⫺/⫺
(K, M, O, Q, and S) lungs at
P0.5 are shown. Schematic
drawings illustrate how generation numbers were counted
in Eln⫹/⫹ (B, D, F, H, and
J) and Eln⫺/⫺ (L, N, P, R,
and T) lungs. The main air
passages in the sections are
represented in the schematics. The generation number is
determined by counting from
the trachea (generation zero)
and adding 1 at each bifurcation. As shown here, murine
lungs tend toward nondichotomous branching.
sults indicate that before its well-established role in alveolar
structure and function, elastin plays an essential role in
terminal airway branching.
Prenatal Elastin Expression in the Lung Parenchyma
Localizes to Airways
Expression data support the conclusion that elastin is required for terminal airway branching. Previously, it was
suggested that there are three phases of elastin mRNA expression during murine development of the lung parenchyma (8). The first phase of elastin expression, evident
during the pseudoglandular stage of lung development, is
localized to the mesenchyme surrounding the airways and
occurs at E16.5. The second phase occurs immediately before birth, when heavy elastin expression is noted throughout the lung parenchyma. The third and final stage of expression involves synthesis of elastin during alveogenesis
by septal myofibroblasts after P4. In contrast to the previous study, we did not observe here a second phase of ubiquitous elastin expression immediately before birth (Figure
3). We did observe that before birth, parenchymal elastin
expression is confined to the mesenchyme surrounding the
developing airways. Thus, the spatial expression of elastin
before alveogenesis supports a role for elastin in airway
branching.
Arrested Branching Reduces RAC and Secondary Crest
Volume Density
The arrest of airway branching morphogenesis in Eln⫺/⫺
lungs is associated with a reduction in the RAC and secondary crest volume density. These indices measure the
number of distal air sacs in relation to a reference unit of
lung tissue. For RAC, the reference unit is the distance between a respiratory bronchiole and the nearest interlobular septum. For secondary crest volume density, the refer-
Figure 6. Average terminal airway generations of sibling pairs.
The lungs of Eln⫹/⫹ (filled bars)
are compared with those of their
Eln⫺/⫺ (open bars) siblings. At
E18.5, there is no significant
difference between the number
of branches in Eln⫹/⫹ and
Eln⫺/⫺ lungs. By P0.5, the difference in the number of airway
generations between Eln⫹/⫹
and Eln⫺/⫺ lungs is apparent
and significant, P ⬍ 0.05. Values are means ⫾ SD. *Statistical significance between Eln⫹/⫹
and Eln⫺/⫺ samples.
Wendel, Taylor, Albertine, et al.: Eln⫺/⫺ Lung Development
ence unit is the area of lung parenchyma as determined by
point-counting. These reference units continue to increase
during development as the lung parenchyma volume expands. Thus, a failure to increase the number of air sacs,
caused by arrested branch development, will result in a reduction of the RAC and secondary crest volume density.
As predicted, RAC and secondary crest volume density
values were reduced in Eln⫺/⫺ mice at P0.5 when airway
branching morphogenesis was arrested.
Mechanism of Impaired Airway Branching
in Eln⫺/⫺ Lungs
One explanation for the diminished airway branching in
Eln⫺/⫺ mice is destruction of weakened lung tissue between E18.5 and P0.5. This destruction could be either a result of mechanical manipulation or the product of inspiratory stress. First, it is unlikely that experimental manipulation
of the lungs in this study caused tissue destruction. Our
method minimized physical handling by keeping the lungs
within the chest cavity during the application of vacuum
and fixative. Only after being fixed were the lungs dissected and manipulated. Using these methods, we found
no broken tissue fragments or other evidence of destruction in E18.5, P0, and P0.5 specimens. Second, it is unlikely
that the Eln⫺/⫺ pulmonary phenotype is caused by inspiratory stress. Our fixation method exposed both prenatal and postnatal lungs to supraphysiologic stress. The
lungs in our study were fixed under 50 mbar of vacuum
pressure, compared with the standard 23 mbar used for
adult mice (6). If elastin is required to maintain the structural integrity of terminal airways during inspiration, then
prenatal E18.5 Eln⫺/⫺ lungs, fixed at more than twice the
pressure used for adult lungs, should show evidence of tissue destruction. However, at E18.5 the lungs of Eln⫺/⫺
mice were indistinguishable from the lungs of their Eln⫹/⫹
siblings. In addition, mice killed immediately upon birth
(P0) took few if any breaths. Despite this limited exposure
to respiratory stress, Eln⫺/⫺ lungs were clearly morphologically different from Eln⫹/⫹ lungs at P0 (Figures 1C
and 1D). Thus, our studies indicate that neither mechanical manipulation nor physiologic stress are primary factors
in the arrest of terminal airway branching observed in P0.5
Eln⫺/⫺ lungs.
A method for excluding the role of tissue destruction in
the development of the Eln⫺/⫺ pulmonary phenotype
would be to culture lung explants in vitro. Such techniques
isolate organ systems from physiologic stress. Previously,
we used explanted aortic organ culture to exclude hemodynamic stress as a significant pathogenic factor in defective
arterial morphogenesis in Eln⫺/⫺ mice (17). However,
explant cultures of whole lungs at E18.5 or later are complicated by their size and structural complexity, and result
in problems with tissue viability and developmental arrest.
Indeed, use of murine lung explant cultures to study branching is documented for midgestational developmental (31, 32).
Conclusions
Our characterization of the Eln⫺/⫺ lung demonstrates that
elastin is required early in pulmonary development for distal airway branching. Because elastin regulates cellular pro-
325
liferation, migration, and arterial morphogenesis, we postulate that elastin is a key morphogenetic factor required
for terminal airway branching (17, 33, 34). Thus, in addition
to its role as an important structural element in the formation of alveoli, we anticipate that elastin signals mesenchymal cells to form the distal airways. With further advancements in explant culture technologies, the Eln⫺/⫺ mouse
will be an invaluable tool for elucidating the molecular
and cellular role of elastin in branching morphogenesis.
Acknowledgments: The authors thank Zhengming Wang for assistance with the
in situ hybridization protocol and Drs. Robert Mecham, John Hoidal, Shannon
J. Odelberg, and Lisa D. Urness for guidance, discussion, and critical comments
on the manuscript. This research was supported in part by National Institutes of
Health grants K08 HL03490-03 and T35 HL07744-06 (D.Y.L.), and HL62875
(K.H.A.); a University of Utah Seed Grant (D.Y.L.); and American Heart Association grant-in-aid (K.H.A. and D.Y.L.). D.Y.L. is a Charles E. Culpeper
Medical Scholar and was supported in part by the Rockefeller Brothers Fund.
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