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Am J Physiol Lung Cell Mol Physiol
279: L333–L341, 2000.
CFTR modulates lung secretory cell proliferation
and differentiation
JANET E. LARSON,1 JOSEPH B. DELCARPIO,2 MICHELLE M. FARBERMAN,1
SUSAN L. MORROW,1 AND J. CRAIG COHEN3
1
Laboratory of Molecular Genetics, Alton Ochsner Medical Foundation, New Orleans 70121;
and Departments of 2Cell Biology and Anatomy and 3Medicine and Biochemistry,
Louisiana State University School of Medicine, New Orleans, Louisiana 70112
Received 7 July 1999; accepted in final form 28 March 2000
a practical and reliable
technique for in utero transfer of genes via the amniotic fluid. This method results in sustained expression
and high-efficiency adenovirus-mediated transfer to
the fetus. Transfer to the developing epithelium is
accomplished by direct injection of a replication-defective adenovirus into individual amniotic sacs of rodent
fetuses (28). Gene transfer to the fetus in this manner
circumvents the inflammatory response that plagues
adenoviral-mediated gene transfer after birth (28).
Gene transfer is performed at a stage of lung and
intestinal development comparable to that of a 10- to
20-wk-gestation human. At the time of infection, the
rodent airways are lined with multipotential columnar
and cuboidal stem cells; differentiation and further
growth occur after infection and gene transfer. These
undifferentiated epithelial cells are the targets of the
adenovirus (28).
Cystic fibrosis (CF) has been a major target for corrective gene therapy and an ideal candidate for in utero
gene therapy targeting the developing lung. The CF
transmembrane conductance regulator (cftr) gene was
cloned without the prior knowledge of the structure
and function of the protein (26, 27). Structural analysis
suggested that its main function was that of a cAMPregulated chloride channel. Since this discovery, the
protein has been shown to have diverse regulatory
abilities, yet CF disease pathology is still defined in
terms of a lack of a chloride channel. CFTR regulates
other secretory channels (12), mediates vesicular trafficking (3), and affects glycosylation (19, 38). The two
nucleotide binding domains (NBD1 and NBD2) of
CFTR share structural homology with the conserved
sequences of G protein (21). CFTR is degraded by the
ubiquitin-proteasome pathway, which is usually reserved for regulatory proteins that require rapid degradation (35).
CFTR expression is greater in the fetal lung than in
the adult lung. CFTR mRNA and protein show temporal and tissue-specific distribution during development. Protein expression is greatest in the lung during
the first and second trimesters (9, 13, 22, 23, 34).
The significance of the regulatory properties and
expression of the CFTR during development was unclear until our laboratory demonstrated the complete
reversal of the lethal phenotype of the CFTR-deficient
[cftr(⫺/⫺); knockout] mouse after transient in utero
expression of the gene. After gene therapy with a
first-generation adenovirus vector carrying the cftr
gene (Av1CF2), low levels of the cftr gene were detectable
in the fetal gut up to 72 h but not after birth. In utero
gene therapy did not permanently replace the CFTRencoded cAMP-dependent chloride channel, and continuous functioning of CFTR was not required for correction of the intestinal obstruction in cftr(⫺/⫺) mice (20).
The rescued knockout mice exhibited phenotypic
and functional changes in their intestine and lungs
Address for reprint requests and other correspondence: J. E. Larson, Laboratory of Molecular Genetics, Ochsner Medical Foundation,
1516 Jefferson Highway, New Orleans, LA 70121 (janlarson@
hotmail.com).
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
cystic fibrosis transmembrane conductance regulator
THIS LABORATORY HAS DEVELOPED
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L333
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Larson, Janet E., Joseph B. Delcarpio, Michelle M.
Farberman, Susan L. Morrow, and J. Craig Cohen.
CFTR modulates lung secretory cell proliferation and differentiation. Am J Physiol Lung Cell Mol Physiol 279:
L333–L341, 2000.—We have permanently reversed the lethal phenotype in the cystic fibrosis (CF) transmembrane
conductance regulator (CFTR)-deficient (knockout) mouse after in utero gene therapy with an adenovirus containing the
cftr gene. The gene transfer targeted somatic stem cells in
the developing lung and intestine, and these epithelial surfaces demonstrated permanent developmental changes after
treatment. The survival statistics from the progeny of heterozygote-heterozygote matings after in utero cftr gene treatment demonstrated an increased mortality in the homozygous normal pups, indicating that overexpression during
development was detrimental. The lungs of these pups revealed accelerated secretory cell proliferation and differentiation. The extent of proliferation and differentiation in the
secretory cells of the lung parenchyma after in utero transfer
of the cftr gene was evaluated with morphometric and biochemical analyses. These studies provide further support of
the regulatory role of the cftr gene in the development of the
secretory epithelium.
L334
CFTR IN DEVELOPMENT
METHODS
Colony Maintenance
The S489X cftr-mutant UNC mouse strain was obtained
from Jackson Laboratories as a fifth-generation backcross.
All animals were housed under standard vivarium conditions
and fed mouse chow (Purina) after being weaned at 28 days
of age. Wood chips were used as bedding, and the mice were
not protected in any manner from exposure to pathogens.
Under these conditions, the S489X cftr-mutant mice develop
intestinal obstruction, and ⬍5% survive into adulthood.
Death occurs from intestinal obstruction in the mice homozygous for the mutant allele during one of two periods postnatally. The first period is within 5 days of birth (⬃50%), and
the second period occurs around weaning (30, 31).
Survival Statistics
Samples of tail DNA were obtained from surviving animals
between 10 and 15 days of age for PCR. Animals were then
tagged and followed until 75 days of age. Experience with the
colony has shown that animals that survive until 75 days of
age [including in utero-treated cftr(⫺/⫺) mice] survive past 1
yr of age. A ␹2 analysis was used to analyze the significance
between observed and expected survival frequencies in the
lacZ gene- and cftr gene-treated populations. A P value of
⬍0.05 was considered significant.
Surgery
The fetuses were treated at 15–16 days gestation with
either Ad5.CMVlacZ or Av1CF2. Initial experiments were
planned around the progeny of heterozygote-heterozygote
matings; therefore, treated groups included knockout
[(⫺/⫺)], heterozygous [(⫺/⫹)], and homozygous normal
[(⫹/⫹)] animals. Subsequent overexpression experiments
utilized homozygous normal-homozygous normal matings.
With amniotic fluid volume as a standard, the vector was
injected in 10% of the known amniotic fluid volume with a
fine-gauge needle. A concentration of 109 plaque-forming
units/ml of each vector was injected into each fetal amniotic
sac, resulting in a final concentration of 108 plaque-forming
units/ml.
Genotyping of Heterozygote-Heterozygote Matings
In addition to the genotyping of live infants, the deceased
fetuses were collected and genotyped when possible. Only
animals in their entirety were used for typing. Wild-type
CFTR and the knockout S489X allele were amplified in
parallel reactions with conditions provided by Jackson Laboratories. Primers CF1248R and CF944 were used for detection of the normal allele. Primers Neo150R and CF944 were
used for detection of the mutant allele. Primer sequences
(provided by Jackson Laboratories) were 5⬘-CTTTGATAGTACCCGGCATAATC-3⬘ for CF1248R, 5⬘-TCGAATTCGCCAATGACAAGAC-3⬘ for Neo150R, and 5⬘-TGAACCTTAGTCCTATGTTGCC-3⬘ for CF944.
Examination of Homozygous Normal Fetuses After In Utero
cftr Treatment
Lung morphology and morphometry light level. At 20–21
days gestation, the lungs and intestines were removed and
weighed. The lethal dose of pentobarbital sodium that the
mothers received inhibited any fetal respiratory effort. Lung
weight-to-total body weight and intestine weight-to-total
body weight ratios were determined. Volume densities of
saccular air space and tissue, airway air space and tissue,
and vessel lumen and tissue wall were estimated by pointcounting morphometry. Light micrographs of the fields were
transferred to the computer by densitometry, and a 121-point
multipurpose test grid was applied. A minimum of 20 fields
were evaluated from each of 12 animals from 4 separate
litters. Differences between groups were evaluated by Student’s t-test. Statistical significance was assigned at P ⬍ 0.05.
Ultrastructural Morphometry and Morphology
Tissue preparation. Isolated lungs were dissected and immersion fixed with gentle agitation in 100 ml of 0.1 M sodium
cacodylate-buffered 2.5% glutaraldehyde at ambient temperature. Tissue slices were fixed overnight in fresh fixative,
then rinsed three times for 30 min each in chilled 0.1 M
cacodylate buffer. The lungs were then cut into 1-mm cubes
and postfixed in aqueous 1.0% osmium tetroxide, en bloc
stained with 0.5% aqueous uranyl acetate, dehydrated in
acetone, and infiltrated and embedded in Polybed 812 (Polysciences). Thin sections were obtained on a Reichert-Jung
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(6). Intestines from the in utero cftr gene-treated
cftr(⫺/⫺) mice did not develop the intestinal pathology of crypt dilation or goblet cell hypertrophy that
proves fatal for the untreated knockout mice. The
intestinal crypt cells in the untreated knockout mice
were deficient in both intracellular uptake of Calcium Green-AM and purinergic receptors. Both of
these deficiencies were partially corrected in the
rescued knockout mice, and the restored cells were
clonally distributed along the villi (6).
The phenotypic changes in the lungs were predominantly in the airways. Clara cells, which are the predominant secretory cell in the terminal airways,
showed marked differences in ultrastructure in animals treated in utero with the cftr gene (6). Clara cells
in knockout mice treated in utero with the cftr gene
showed significant increases in vesicle number and
dilated smooth endoplasmic reticulum. Untreated
knockout mice had an increase in secreted glycoconjugates containing ␣-(2,6)-sialic acid and fucose (both
implicated in the pathogenesis of CF) compared with
that in control animals. Lectin histochemistry located
these glycoconjugates to the airway epithelial cell surface. The in utero treated knockout mice had an increase in this material as well, but it was contained in
intracellular vesicles. These data suggested that the
cftr gene restored regulated secretion in both the lung
and intestine and was required for normal differentiation of secretory cell populations.
Here, we examine the survival of our treated and
untreated adult populations from heterozygote-heterozygote matings. We describe the lethal effects of excessive CFTR expression during normal development of
the lung in additional experiments with homozygous
normal [cftr(⫹/⫹)] populations. We present data that
shows that CFTR expression during lung development
increases epithelial proliferation and enhances secretory cell differentiation. These findings are consistent
with the CFTR protein possessing a secretory cell missing (scm) function that is required for normal development of secretory cells in the lungs, intestines, and
other organs (6).
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CFTR IN DEVELOPMENT
DNA Content
DNA was isolated from tissue with the commercially available method of Trisolv (Biotecx Laboratories, Houston, TX).
The tissues were homogenized in the reagent, and chloroform
was added, which yielded a top aqueous phase, an interphase, and a bottom organic phase. DNA was precipitated
from the interphase and organic phase and dissolved in 8 mM
NaOH after being washed. The DNA concentration was measured by spectrophotometry (5).
Statistical Comparison of Biochemical
and Morphometric Analysis
t-Tests were applied for comparison of observations between homozygous normal animals treated in utero with the
lacZ gene and homozygous normal animals treated in utero
with the cftr gene. A P value ⬍ 0.05 was considered significant. Data are expressed as means ⫾ SD.
RESULTS
Effects of In Utero cftr Gene Therapy
on Surviving Population Statistics
of Heterozygote-Heterozygote Matings
Uniform correction of a lethal autosomal recessive
defect such as CF would result in a normal adult
population distribution, with an expected Mendelian
ratio of 1:2:1. Adult animals (75 days) from heterozygote-heterozygote matings treated in utero at 15–16
days gestation with the cftr gene were genotyped to
evaluate the effect of transgene expression on genotype-specific survival. These data are presented in Table 1.
The population ratios of adult survivors (⬎75 days)
after in utero treatment with the cftr gene varied
significantly from expected (Table 1). Only 8% of the
surviving adult population was cftr(⫹/⫹) (expected
percentage of 25%; P ⬍ 0.001). The intrauterine treatment of fetuses with cftr resulted in twice as many
surviving knockout mice as treated homozygous normal genotypes. A ␹2 analysis of the two surviving
populations found them to be significantly different
(P ⬍ 0.001). After rescue of the 11 surviving knockout
mice shown above, this laboratory has produced ⬎30
additional knockout adults, including some that are
offspring from knockout-heterozygote breeding. Thus
transient expression of the cftr gene during development reversed the lethal phenotype in the cftr(⫺/⫺)
mice but was lethal to the cftr(⫹/⫹) mice.
Mice dying during the perinatal period were genotyped when possible; many appeared to be cyanotic.
The predominant genotype of mice that died within
72 h after birth and that had received in utero cftr
gene treatment was cftr(⫹/⫹) (71%). This high perinatal mortality explained the decreased proportion
of treated homozygous normal mice surviving to
adulthood (Table 1). Only 9% of the deceased genotyped mice were homozygous knockouts [cftr(⫺/⫺)],
and the remaining 21% were heterozygotes. Although perinatal mortality did occur in the control
(Ad5.CMVlacZ-infected) mice, the genotype distribution of the deceased pups suggested a more random
effect. The genotype of perinatal deceased lacZ genetreated mice was 23% homozygous normal, 73% heterozygous, and 4% knockout.
Comparison of survivors of litters that received adenovirus containing the lacZ gene in utero and litters
Table 1. Adult survivors of intrauterine treatment
No. of Survivors
Treated mice
Control mice
Untreated mice
⫺/⫺
⫺/⫹
⫹/⫹
11 (15)
0
0
57 (77)
7 (43)
57 (52)
6 (8)
9 (57)
52 (48)
Nos. in parentheses, percent survivors. Treated mice received
cystic fibrosis transmembrane conductance regulator (cftr) gene.
Control mice received lacZ gene. ⫺/⫺, CFTR deficient (knockout);
⫺/⫹, heterozygous; ⫹/⫹, homozygous normal. Only 8% of the surviving adult population after cftr gene treatment was homozygous
normal, with an expected frequency of 25% if the population were to
follow normal population genetics (as defined by the classic Mendelian ratio of 1:2:1). Both untreated and control-treated populations
had an increased percentage of homozygous normal mice than would
be expected in a lethal autosomal recessive disorder. The Mendelian
ratio expected in this population is 2:1. Surgery had no affect on
survival. After rescue of the 11 surviving knockout mice, this laboratory has produced additional knockout adults that are the result of
knockout-heterozygote breeding.
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Ultracut E ultramicrotome equipped with a diamond knife
(Diatome), collected on 300-mesh copper grids, and poststained with lead citrate. They were examined with a JEOL
1210 transmission electron microscope (EM) at 60 kV.
Nuclear counts. Ten tissue blocks from each individual
animal were thin sectioned as described in Tissue preparation. A ribbon of sectioned material was located under low
magnification in the EM. The entire contents of an area
outlined by a set of grid bars was recorded on Kodak 4489
electron-microscopic film by taking overlapping images at an
initial magnification setting of ⫻2,000. Negatives were photographically enlarged to achieve a final magnification of
⫻6,000 and constructed into montages for visual analysis.
Montages were analyzed for the presence of alveolar type I
and type II cells, undifferentiated epithelial cells, endothelial
cells, and interstitial cells.
Type II cell differentiation. The size of the morphometric
test lattice was determined previously on the basis of the size
of the organelle of interest (mitochondria and secretory granules) and the frequency of its appearance in sectioned tissue
(36, 37). A lattice consisting of intersections 1.0 cm apart was
used in determining the volume percent of mitochondria and
cytoplasmic granules. Lightly stippled cytoplasm was identified as glycogen. Volume densities for mitochondria, lamellar
bodies, and percent glycogen (Vmt,l,g) are expressed as percent volumes and were calculated by dividing the number of
points [grid intersections (Pi)] falling on a structure of interest by the total number of points (Pt) within that cell
[(Vmt,l,g ⫽ Pi/Pt) ⫻ 100]. A ribbon of sectioned material was
located under low magnification in the EM. As described in
Nuclear counts, the entire contents of an area outlined by a
set of grid bars were recorded on Kodak 4489 electronmicroscopic film by taking overlapping images at an initial
magnification of ⫻2,000. Negatives were photographically
enlarged to achieve a final magnification of ⫻19,500 and
constructed into montages. Thirty cells were counted from
each treatment group. Differences between volume densities
in groups were evaluated by Student’s t-test. Significance
was assigned at P ⬍ 0.05. All morphometric analyses were
done blinded by three independent investigators.
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CFTR IN DEVELOPMENT
Somatic Growth Parameters of In Utero cftr
Gene-Treated cftr(⫹/⫹) Mice
Previous work by this laboratory (28) with the reporter gene lacZ demonstrated that injection of adenovirus into the amniotic fluid targets the fetal intestine
and the lung epithelium specifically. It would be expected that these would be the primary organs to
reflect toxicity. In our studies of heterozygote-heterozygote matings, many of the cftr(⫹/⫹) animals treated in
utero with the cftr gene had appeared cyanotic. This
suggested that the cause of death was cardiorespiratory.
To evaluate possible toxic effects of in utero cftr gene
therapy on the lungs and intestines, animals homozygous for the normal cftr allele were bred, and the
fetuses were treated at 15–16 days gestation with
either Ad5.CMVlacZ, serving as control, or Av1CF2.
Only cftr(⫹/⫹) mice were used because these animals
exhibited the greatest sensitivity to CFTR in utero
toxicity. Treated mice were evaluated at 21 days gestation immediately before birth because of the high
mortality in the perinatal period. Both the lung and
intestine were evaluated.
Somatic and organ growth in the in utero cftr genetreated cftr(⫹/⫹) mice varied markedly from the in
utero lacZ gene-treated cftr(⫹/⫹) mice. The body
weights of the in utero cftr gene-treated cftr(⫹/⫹) mice
were significantly decreased compared with those in
the control animals (Table 2). Absolute lung weights
were also significantly decreased. Despite the lung
weight decrease, the lung weight-to-body weight ratios
in the in utero cftr gene-treated cftr(⫹/⫹) mice were
significantly increased. The intestine weight and intestine weight-to-body weight ratio were slightly increased in the in utero cftr gene-treated cftr(⫹/⫹) mice,
but the differences did not approach significance.
These data suggested that lung growth was either
spared or accelerated after in utero cftr gene therapy in
the cftr(⫹/⫹) mice, and their somatic growth suffered.
DNA Content of Lungs and Intestines After In Utero
cftr Gene Treatment
DNA content was measured in the lung and intestine
as an indicator of cell number because these two characteristics have been shown to be directly related to
each other (40, 41). There was a significant increase in
the amount of DNA per milligram of lung weight of the
in utero cftr gene-treated cftr(⫹/⫹) mice (Table 3).
These findings agreed with the increase in lung
weight-to-body weight ratio in the same group and
suggested that the increase in the lung weight-to-body
weight ratio was due to increased cellularity in these
lungs. Thus the treated mice did not have pulmonary
hypoplasia as defined by a decrease in organ size due to
a decreased cell number (7). In fact, the increase in
lung weight-to-body weight ratio and increased DNA
were consistent with a hyperplastic lung.
The DNA content in the intestines between the two
groups showed no significant difference, and this corresponded to the unchanged intestine weight between
the two groups. The intestine DNA content-to-body
weight reached significance because somatic growth
was so substantially decreased in the in utero cftr
gene-treated cftr(⫹/⫹) animals.
Table 2. Somatic and organ growth after in utero cftr gene treatment
Homozygous
Normal Litters
Control mice
Treated mice
P value
n
11
13
Body
Weight, g
Lung Weight,
mg
Lung-to-Body
Weight Ratio
Intestine
Weight, mg
Intestine-to-Body
Weight Ratio
1.28 ⫾ 0.1
0.99 ⫾ 0.06
⬍0.001
37.0 ⫾ 5.6
31.7 ⫾ 4.7
0.045
0.027 ⫾ 0.003
0.034 ⫾ 0.004
0.031
36.6 ⫾ 13.0
42.3 ⫾ 13.5
0.32
0.029 ⫾ 0.010
0.035 ⫾ 0.009
0.20
Values are means ⫾ SD; n, no. of mice.
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that were born without surgical intervention showed
that the surgical procedure itself had no impact on the
homozygous normal perinatal mortality. A surviving
adult population carrying a lethal autosomal recessive
disorder consists of a normal population distribution of
two-thirds heterozygous and one-third homozygous
normal. Both untreated (no surgery) and control lacZ
gene-treated populations had a higher percentage of
homozygous normal animals than would be expected in
a lethal autosomal recessive disorder (P ⬍ 0.05; Table
1). The homozygote-to-heterozygote ratio approached
1:1 in our colony and demonstrated a selection advantage for homozygous normal survival in this strain.
Surgery did not change this proportion. Therefore, the
large perinatal loss seen in the cftr(⫹/⫹) population
treated in utero with the cftr gene was specifically due
to the overexpression of the gene during development.
The proportion of rescued homozygous knockout animals was also lower than expected for the number of
surviving heterozygotes (Table 1). The low percentage
of cftr(⫺/⫺) animals in both the surviving mice and the
mice that succumbed during the perinatal period indicated that some loss occurred prenatally and intrauterine rescue was not 100% efficient with this protocol.
Although we could not label individual fetuses in utero,
a number of fetuses were identified by inspection during injections as growth retarded. These litters subsequently produced confirmed knockout [(⫺/⫺)] mice.
The in utero cftr gene-treated knockout mice were
smaller at weaning and remained so throughout adulthood. These observations as well as the numerous
resorbed fetuses observed during surgery suggested an
earlier requirement for the cftr gene in the fetus that
was not corrected by replacement of the gene at 15–16
days gestational age.
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CFTR IN DEVELOPMENT
Table 3. DNA content of lungs and intestines after in utero cftr gene treatment
Homozygous Normal
Litters
Control mice
Treated mice
P value
n
␮g DNA/mg lung
␮g lung DNA/g
body wt
␮g DNA/mg
intestine
␮g intestine DNA/g
body wt
11
13
12.78 ⫾ 3.47
20.44 ⫾ 2.89
0.008
291.45 ⫾ 44.2
374.74 ⫾ 44.7
0.003
5.39 ⫾ 2.07
6.01 ⫾ 2.6
0.30
145.7 ⫾ 23.7
213.2 ⫾ 45.4
0.002
Values are means ⫾ SD; n, no. of mice.
Volume Proportion of Lung Structure
Evaluation of Parenchymal Cell Type
A nuclear count was obtained as an index of the
number of specific cell types in the proliferating airexchanging parenchyma (2). This ultrastructural morphometric analysis was done because at this stage of
gestation, many cell types share cellular markers,
making histochemical and immunohistochemical techniques of little value. The parenchyma was analyzed
Evaluation of Epithelial Cell Maturity
To substantiate the role of the cftr gene in epithelial
cell differentiation, the type II cells were examined by
ultrastructure. Because the type II cells are the primary secretory cells in the parenchymal epithelium
and produce lung surfactant, their differentiation has
been examined in detail. With differentiation, the volume proportion of lamellar bodies (containing surfactant) and mitochondria increases and the volume proportion of glycogen decreases (2, 32, 43). The results of
the ultrastructural morphometric analysis are described in Table 6. The volume proportion of lamellar
bodies increased from 5% in the control mice to 9.5% in
the in utero cftr-treated animals (P ⫽ 0.008). Glycogen,
thought to be a substrate for future surfactant production and an indicator of an immature cell, represented
12% of the volume in the control cells and none of the
Table 4. Fetal lung percent volume densities
Homozygous Normal
Litters
Control mice
Treated mice
P value
n
6
6
Values are means ⫾ SD; n, no. of mice.
%Saccular
Wall
%Saccular
Air
%Airway
Wall
%Airway
Lumen
%Vessel
59.6 ⫾ 1.5
69.2 ⫾ 1.7
0.001
21.9 ⫾ 2.1
14.2 ⫾ 1.9
0.025
8.4 ⫾ 0.9
8.7 ⫾ 0.7
0.63
4.6 ⫾ 1.0
2.7 ⫾ 0.05
0.048
5.5 ⫾ 0.5
5.2 ⫾ 0.04
0.42
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Morphometric analysis was used for statistical evaluation of lung structure. When growth alterations such
as hyperplasia occur, morphometry can delineate the
specific changes in different tissue compartments of
the lung. The percentage of saccular air in the animals
treated with cftr was significantly decreased, and the
percentage of saccular wall was significantly increased
(Table 4). Normally, as the lung develops, the volume
density of air increases and the mesenchyme and epithelia become more attenuated. The epithelial proliferation that occurred as a result of the cftr gene treatment did not allow this attenuation to happen.
The volume proportion of the remaining lung structures (airways and vessels) was unchanged between
the two groups (Table 4). At the time of in utero cftr
therapy, the large conducting airways were already
formed and differentiation of the proximal portions of
the airways had begun. The most rapid proliferation
was occurring in the developing respiratory part of the
lung, i.e., the distal tubules that would develop into
saccules and eventually alveoli. The morphometric
analysis established that the cftr gene had its most
discernible effect on the cells that were rapidly proliferating at the time of infection and not on those that
were already undergoing differentiation. The overexpression of the cftr gene in utero resulted in increased
cellularity in the parenchyma (air-exchanging area of
lung) but in decreased complexity. Although not hypoplastic by the criteria of cell number, lung development
was defective and met the definition of structural hypoplasia (7).
for alveolar type I, type II, undifferentiated, endothelial, and interstitial cells. These results are summarized in Table 5. In the control (LacZ gene-treated)
lungs, 22% of the cells had large pools of lightly stippled material identified as glycogen in their cytoplasm,
had no other differentiating features, and were defined
as undifferentiated. In the treated animals, there were
no glycogen-containing cells in the parenchyma (P ⬍
0.001). Although the proportion of type II cells remained the same within the two groups (21.2 and
20.1%, respectively), there was an increase in type I
cells from 10.1–20.1% in the in utero cftr-treated animals (P ⫽ 0.004). Together, the type I and type II cells
comprise the epithelial surface of the lung parenchyma. Type I cells are thought to be the terminally
differentiated cells of the epithelium, and type II cells
are their precursors (1). This suggested that the cftr
gene played a role in differentiation as well as in
proliferation of the lung epithelium. There was also an
increase in interstitial cells in the treated animals,
again indicating a role in proliferation. There was no
difference in the proportion of vascular endothelial
cells in the groups.
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CFTR IN DEVELOPMENT
Table 5. Peripheral cell type variation
Homozygous Normal
Litters
Control mice
Treated mice
P value
%Interstitial
%Alveolar
Type I
%Alveolar
Type II
%Endothelial
%Undifferentiated
19.0 ⫾ 2.2
32.5 ⫾ 3.1
0.002
10.1 ⫾ 1.6
19.7 ⫾ 2.4
0.004
21.2 ⫾ 3.2
20.1 ⫾ 2.7
0.80
27.2 ⫾ 2.9
27.6 ⫾ 3.4
0.93
22.3 ⫾ 3.2
0
⬍0.001
Values are means ⫾ SD.
DISCUSSION
Previous studies (reviewed in Ref. 18) of the pathogenesis of CF have alluded to the lack of correlation
between disease pathology and the pattern of CFTR
expression in the respiratory tissues. It has been observed that CF pathology starts in the small airways
where there is little CFTR expression. CFTR is
present, however, in the distal airway epithelium during lung development, and expression follows temporal
and tissue-specific patterns. Lung development begins
proximally with the formation of the large conducting
airways and proliferation of the epithelium, which
progresses distally. Once the proliferation has diminished, differentiation occurs in this same cephalocaudal pattern (15, 33). In developing airways, CFTR
distribution follows this same cephalocaudal pattern of
maturation and differentiation (9). The cellular distribution of CFTR protein and mRNA changes with the
degree of differentiation of the epithelial cell. In more
immature lungs, CFTR is not polarized to one domain
of the cell but is diffusely expressed. As the lung
matures and the cells become differentiated, the localization of CFTR mRNA and protein shifts to a more
apical distribution and bears a similarity to that seen
in the adult lung (9, 23). This suggests that the role of
CFTR changes with the differentiated state of the cell.
At the time of infection, these lungs consisted of
blind airways lined with multipotential columnar and
cuboidal stem cells (33). The airway cells were the
primary targets of the adenovirus because the respiratory portion of the lung didn’t yet exist (28). Yet the
volume proportion of the lungs indicated that most of
the cellular proliferation from in utero treatment with
the cftr gene occurred in the respiratory portion of the
lung. These data indicate that the cftr gene works in
trans, altering the cellular environment. This is also
evidence that CFTR affects proliferation of predominantly undifferentiated cell types. The airways showed
mild cellular proliferation as evidenced by a decrease
in airway lumen. However, the volume proportion of
airway was unchanged between the two groups because airway structural development had occurred before infection. The upper airway cells were beginning
to differentiate at the time of infection and were largely
unaffected by CFTR. In contrast, the largest effects of
the gene were in the respiratory epithelium, which was
undifferentiated and proliferating at the time of infection.
These data explain the findings of Whitsett et al.
(39), who used the cftr gene under the direction of the
surfactant protein (SP) C and 10-kDa Clara cell (CC10)
promoters in transgenic mice and evaluated the effects
of expression in utero. These investigators found that
somatic growth and lung development of the CFTR
transgenic mice were identical to that of their nontransgenic littermates. The use of the SP-C and CC10
promotors ensured CFTR expression in more differentiated cell types because each of these proteins become
specific markers of their respective cell types (type II
and Clara cell) as they differentiate (25, 29, 42). Although expression of chimeric genes with the SP-C
promoter was observed as early as 11 days gestation in
the mouse (10, 39), rodent fetal lungs at 15 days gestation do not demonstrate any detectable messages for
SP-C (14). A dramatic increase in SP-C expression
occurs as gestation progresses (14, 39). Likewise, a
progressive increase in CC10 protein is apparent only
Table 6. Comparative differentiation in type II cells
Homozygous Normal
Litters
%Lamellar
Bodies
%Glycogen
%Mitochondria
Maturational changes
Control mice
Treated mice
P value
Increases
5.06 ⫾ 0.94
9.52 ⫾ 1.29
0.008
Decreases
12.31 ⫾ 2.11
0
⬍0.001
Increases
4.34 ⫾ 0.52
4.96 ⫾ 0.48
0.37
Values are means ⫾ SD.
Downloaded from http://ajplung.physiology.org/ by 10.220.33.2 on June 18, 2017
volume in the in utero cftr gene-treated cells (P ⬍
0.001). Figure 1, A and B, are type II cells from control
animals (lacZ gene treated) showing large pools of
glycogen and a paucity of lamellar bodies. Figure 1, C
and D, are type II cells from in utero cftr gene-treated
animals demonstrating the lack of glycogen and an
increase in lamellar body volume proportion. These
data confirmed that the cftr gene affects differentiation
of the secretory epithelium in addition to proliferation.
The effects of overexpression of CFTR in utero on
epithelial cell proliferation were readily apparent on
histological examination. Figure 2A demonstrates the
normal saccular formation of a homozygous wild-type
mouse at 21 days gestation after in utero treatment
with the lacZ gene. The lung of a homozygous wild-type
mouse treated in utero with the cftr gene (Fig. 2B)
exhibited the hypercellularity documented by the biochemical and morphometric analyses above. The proliferating cells filled the saccular spaces and impeded
adequate air exchange. This unchecked proliferation of
the epithelium inhibited normal lung development and
caused structural hypoplasia. This lung pathology was
the most likely cause of the high perinatal mortality in
the in utero cftr gene-treated cftr(⫹/⫹) mice.
CFTR IN DEVELOPMENT
L339
from day 18 gestation in the rodent. The most marked
increase occurs from birth to 1 wk of age (4, 29).
Whitsett et al. (39) acknowledged that CFTR expression in their transgenic mice was diffuse and not confined to the membrane surface. They stated that the
lack of toxicity observed may have resulted from failure of the respiratory cells to insert or activate excess
CFTR protein. Our studies suggest that their findings
of unchanged growth, differentiation, or function were
most likely due to the timing of the their hybrid cftr
Fig. 2. Proliferation of lung parenchyma in control
(lacZ gene-treated) and cftr gene-treated fetuses. A:
normal saccular formation of a homozygous wild-type
mouse at 21 days gestation after in utero treatment
with the lacZ gene. B: lung of a homozygous wild-type
mouse treated in utero with the cftr gene exhibited
hypercellularity. The proliferating cells filled the saccular spaces and impeded adequate air exchange. Original magnification, ⫻20.
Downloaded from http://ajplung.physiology.org/ by 10.220.33.2 on June 18, 2017
Fig. 1. Differentiation in type II cells in control (lacZ
gene-treated) and cystic fibrosis transmembrane conductance regulator (cftr) gene-treated fetuses as shown
by transmission electron microscopy. a and b: immature type II cells from control fetuses are easily identified by the presence of large glycogen lakes (g) and
reduced number of lamellar bodies (l). These contrast
markedly to more mature appearing type II cells from
cftr gene-treated fetuses (c and d), which contain no
glycogen but have more lamellar bodies. *, Alveolar
lumen. Original magnification: ⫻6,000 in a and b;
⫻8,300 in c and d.
L340
CFTR IN DEVELOPMENT
We are indebted to Carol L. Thouron and Michele F. St. Onge for
excellent technical assistance with preparation of tissues, to Cathy
C. Vial for excellent assistance in preparation of tissues for electron
microscopy, and to Michael L. Johnson for assistance with ultrastructural morphometry.
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