CHAPTER 2

PRENATAL LUNG DEVELOPMENT

GAIL H. DEUTSCH, MD, AND HALIT PINAR, MD.

Introduction
Lung development requires integration of multiple regulatory factors that mediate patterns of cell proliferation,
differentiation, migration, and death. These developmental programs, which include transcription factors and signaling molecules, are likely re-enlisted during recovery of
the lung following injury. Understanding these processes
could provide important insight into controlling cell differentiation and regeneration for therapeutic purposes.
This chapter describes the morphological features that
characterize the defined stages of lung development and
also addresses epithelial cell differentiation and vasculogenesis of the airways. In the latter part of the chapter, an
attempt will be made to highlight specific cellular and
molecular events during development that may influence
lung maintenance in adult disease states.

Stages of Lung Development

During gestation, the fetal lung undergoes significant
morphological changes to provide at birth an organ capable of maintaining respiration and gas exchange.
Although lung development is continuous during
embryogenesis, five developmental stages have been
delineated, based on anatomic and histologic characteristics.1 (Table 21). The early embryonic and pseudoglandular stages elaborate the conducting airways; the latter
canalicular, saccular, and alveolar stages are characterized
by reduction of mesenchyme and vascularization to form
a thin air-blood barrier. Birth does not signal the end of

lung development. There is a continuing complex process

of lung growth after birth, permitting changing relationships of airway size, alveolar size, and surface area. At
birth, the newborn infant, with approximately 50 million
alveoli, has the potential to add another 250 million alveoli and increase surface area from approximately 3 to 70
m2.2 There are more than 40 different cell types in the
lung, with different functions.2 How they establish their
final appropriate physical and numerical relationships
with each other is still unknown.
Embryonic Stage (3 to 7 Weeks)
The human fetal lung originates as a ventral diverticulum
that arises from the laryngotracheal groove of the foregut
endoderm.2 The laryngotracheal groove separates
dorsoventrally from the primitive esophagus to form the
tracheal rudiment and, at the same time, gives rise laterally
to the two primary bronchial buds (Figure 21A). The
lung bud grows into adjacent splanchnic mesoderm where
it is induced to branch repeatedly, giving rise to the future
respiratory tree. This primitive lung bud is lined by endodermally derived epithelium, which differentiates into
specialized cells that line both the conducting and respiratory airways.3 Mesenchymal cells condensed around the
primitive airways give rise to blood vessels, smooth muscle, cartilage, and other connective tissues of the lung.4
Pseudoglandular Stage (7 to 16 Weeks)
During the pseudoglandular stage, the stage of branching
morphogenesis, there is rapid proliferation of the primi-

TABLE 21. Stages of Lung Development

Stage

Developmental Age

Major Events

Embryonic

37 wk

Lung budding from the foregut endoderm, with formation of trachea and mainstem bronchi

Pseudoglandular

716 wk

Airway division completed, with formation of 25,000 terminal bronchioles; cartilage, smooth muscle
derived from mesenchyme

Canalicular

1624 wk

Capillarization, with acinar formation; type I and II epithelial cells first differentiate

Saccular

2436 wk

Progressive thinning of epithelial cells; terminal saccular formation; surfactant production

Alveolar (postnatal)

36 wk through infancy
40 wk through infancy

Appearance of true alveoli; alveolar septation and expansion of air spaces

wk = weeks.

2 / Chronic Obstructive Lung Disease

tive airways so that all airway divisions are more or less

completed by 16 weeks.5 This translates into 12 to 17
branches in the upper lobes, 18 to 23 branches in the
middle lobes, and 14 to 23 branches in the lower lobes.2
The branching pattern does not change after this stage
and is similar to that of the adult lung.5 The most peripheral structures, the terminal bronchioles, will further differentiate to form the future respiratory bronchioles and
alveolar ducts. The name pseudoglandular is derived
from the histologic appearance of the lung, which on
cross section consists of hollow tubular-like structures
(glands) surrounded by clusters of mesenchymal cells
(see Figure 21B). The columnar epithelial cells that line
the tubules contain cytoplasmic glycogen; a few become
ciliated as early as 8 weeks while others begin to differentiate into goblet cells.6 During this period, cartilage
begins to form around the larger airways and smooth
muscle forms around airways and major blood vessels.5
Canalicular Stage (16 to 24 Weeks)
The canalicular stage is so named because the potential
air spaces are being canalized and approximated by a
network of capillaries6,7 (see Figure 21C). The pul-

monary acinar units, which eventually contain alveolar

ducts, alveolar sacs, and alveoli, develop during this
period. Acinus is the term applied to the gas exchange
unit associated with a single terminal bronchiole. It follows that primitive lung lobules will have formed by the
beginning of the canalicular stage. Each lobule contains
three to five terminal bronchi and, by the end of 27 weeks,
approximately 25,000 terminal bronchioli.6 A gradual
decrease in mesenchymal tissue results in close apposition of the pulmonary vasculature to the epithelium. By
20 to 22 weeks gestation, type I and type II alveolar cells
can be differentiated from the cuboidal epithelial cells in
the most peripheral parts of the lung.6 Lamellar bodies
associated with surfactant synthesis begin to appear in
the cytoplasm of type II cells. Type I alveolar lining cells,
which differentiate from type II cells, begin their flattening process and attenuate to provide an air-blood interface. The conducting airways have fully developed
smooth muscle, and lymphatic structures now begin to
appear. The developing pulmonary arteries and veins follow the development of the branching airways but lag
behind it somewhat.8,9 By the end of the canalicular
period, the potential air-blood barrier is thin enough to

A.

B.

Prenatal Lung Development / 3

C.

D.

E.

FIGURE 21. Schematic and light-microscopic appearance of human fetal lung at different stages of development.

4 / Chronic Obstructive Lung Disease

support gas exchange.10 The bronchial artery system may

be as critical for lung development as the pulmonary
artery although the role of the bronchial artery in pulmonary differentiation and growth is currently
unknown.6 It has been suggested that the most peripheral
parts of the developing lung are supplied only by the pulmonary arterial vasculature.11
Saccular Stage (28 to 35 Weeks)
The term saccule derives from the saclike appearance of
the most peripheral air spaces, which represent the future
alveolar ducts and alveoli (see Figure 21D). According to
Boyden, each acinus supplied by a terminal bronchiole
has three to four respiratory bronchioles that end in a
transitional duct from which the saccules arise.2 The
major changes that occur during the saccular stage are
further compression of the intervening interstitium, thinning of the epithelium, and the beginning of alveolar septation, with the formation of small mesenchymal ridges.
There is lengthening and widening of saccules distal to
the terminal bronchioles and the addition of the last generations of future alveolar spaces.2 Continual differentation of type I and II alveolar cells occurs during this
period, so that the alveolar epithelial cells become the
most abundant epithelial cells in the lung. The flattened
type I alveolar cells make up the majority of these cells.
Type II cells, ultrastructurally distinguished by their production of lamellar bodies, expand in size and number,
with increased storage of surfactant lipids and less cytoplasmic glycogen.12,13
Alveolar Stage (36 Weeks through Infancy)
Several million alveoli form before birth although this final
stage of lung development primarily occurs during postnatal life.14 The beginning of this stage is not sharply defined;

some alveolar formation probably begins a few weeks earlier. Alveolar formation is closely linked to the deposition of
elastin in the saccular lung.15 Terminal saccules become
invaginated by protrusions from the wall of epithelial cells
and contain a double-walled capillary system16 (see Figure
21E). These protrusions elongate and thin, forming primitive alveoli that at first resemble shallow cups and then
become deeper as development continues.
Postnatal Lung Growth
During the postnatal phase, lung growth is geometric,
and there is no increase in airway number. There is proportionately less growth in the conducting airways in
comparison with alveolar-capillary tissue. Estimates of
the number of alveoli at birth vary widely, but an average
of 50 million is generally accepted.17 These alveoli provide a gas-exchanging surface of approximately 3 to 4
m2.17 Alveoli greatly increase in number after birth, to
reach the adult range of 300 million by 2 years of age18,19
and the surface area of 75 to 100 m2 by adulthood.19
There is substantial remodeling of the parenchyma after
birth, with morphologic changes in the septa.
Alveolarization occurs through the formation of numerous short, blunt tissue crests or ridges, and their protrusion into alveolar sacs increases the internal surface of the
lung. The development of the alveolar crest is closely
linked with elastin deposition and the local proliferation
of interstitial and epithelial cells.15,18

Epithelial Cell Differentiation

Maturation of the epithelium during development starts
in the proximal airways and progresses distally into the
intrapulmonary airways.20 Epithelial cell lineages are
arranged in a distinct proximal-distal spatial pattern in
the airways, which become morphologically apparent

FIGURE 22. Differentiated airway epithelial cells in the proximal trachea and distal alveoli in a human term infant.

Prenatal Lung Development / 5

during the pseudoglandular stage.21 At least 11 different

epithelial cell types have been described in the conducting and respiratory portions of the lung.20 The basal,
secretory, and ciliated cells are the major cell types constituting the pseudostratified portion of the proximal tracheobronchial epithelium while type I and type II
alveolar cells make up the distal respiratory epithelium
(Figure 22). The lineage relationships between the different cell types have not been delineated, and the existence and identity of progenitor cells, which may play a
role in lung injury and repair, are currently under study.
There is some evidence from cell kinetic studies to suggest that basal cells, Clara cells, and type II alveolar cells
are the primary progenitor cells for the pulmonary
epithelium.2227 Following acute lung injury, Clara cells
and type II cells regain the capacity to proliferate and
repopulate the damaged bronchiolar and alveolar epithelium, respectively.2224,28 Lineage studies have not
addressed the relationship of pulmonary neuroendocrine
cells to the putative stem cells (type II cells and Clara
cells) although neuroendocrine cells differentiate morphologically before any other epithelial cell type.29
Bombesin, a peptide secreted by pulmonary neuroendocrine cells, stimulates branching morphogenesis and
lung maturation, in culture and in vivo.30,31
The onset of cellular differentiation is signaled by the
expression of differentiated gene products. Lung-specific
gene products include the surfactant proteins (surfactant
proteins A, B, C, and D [SP-A, SP-B, SP-C, and SP-D])
and Clara cell secretory protein (CCSP).28,3235 The surfactant-associated proteins are abundant phospholipidassociated proteins that are expressed primarily in
alveolar type II epithelial cells32,33,36 although SP-A and
SP-B are also detected in subsets of nonciliated epithelial
(Clara) cells of the conducting airways and tracheobronchial glands.3740 Expression of these genes is extinguished when type II cells undergo terminal
differentiation to type I cells that constitute most of the
gas exchange surface of the alveolus. For the most part,
CCSP is a marker for the proximal Clara cells of the bronchiolar epithelium34,35 (Figure 23). Recent transgenic
studies support a potential role for CCSP in the control of
inflammatory responses; CCSP-deficient mice have an
increased sensitivity to hyperoxia-induced lung injury
characterized by lung edema and induction of proinflammatory cytokine messenger ribonucleic acids
(mRNAs).41 Hepatocyte nuclear factor/forkhead homologue 4 (HFH-4), a winged helix transcription factor, has
been implicated in ciliated cell development (see Figure
23), with mutational abnormalities in the mouse similar
to those seen in Kartageners syndrome.42
Early on in embryonic development, the undifferentiated epithelium coexpresses several lineage markers,
including SP-A, CCSP, and calcitonin gene-related peptide, a marker of neuroendocrine cells.43 It is during the

pseudoglandular stage that pulmonary epithelial cell lineages become restricted to proximal and distal regions of
the airways. Of interest, after bleomycin-induced injury
in the adult lung, there is again coexpression of lineagespecific markers, suggesting that a progenitor-type cell
may be re-enlisted during epithelial repair.44 Tissue
recombination experiments have demonstrated that a
proximal versus a distal epithelial cell phenotype is dictated by soluble factors from the adjacent mesenchyme.45,46 Within a restricted time window of
development, distal lung mesenchyme can reprogram rat
tracheal epithelium to express type II cell differentiation,
and conversely, tracheal mesenchyme can induce distal
lung epithelium to express tracheal cytodifferentiation.45,46 These findings underscore the importance of
epithelial-mesenchymal interactions in coordinating the
precise temporospatial pattern of lung development (see
further discussion below).
Analysis of transcriptional elements in the surfactant
protein genes and CCSP has provided an understanding
of the shared mechanisms of gene regulation and expression in respiratory epithelial cells. A homeodomain protein, Nkx2.1 (also known as thyroid transcription
factor-1 [TTF-1]), and thyroid enhancer binding protein
[T/ebp] plays an essential role in several phases of lung
development, including epithelial cell lineage determination.4750 Nkx2.1 is the earliest marker of the developing
respiratory epithelium, with onset of expression at the
time of lung bud formation from the foregut endoderm.51
Distribution of Nkx2.1 expression in the fetal lung
includes respiratory epithelial cells of the trachea, bronchi,
and developing respiratory tubules although expression is
most robust in distal alveolar cells51,52 (see Figure 23). In
vitro, Nkx2.1 binds and activates SP-A, SP-B, SP-C, and
CCSP promoter elements as well as Nkx2.1 itself.5357
Mice with a null mutation of Nkx2.1 have tracheoesophageal fistulas and fail to form bronchiolar and alveolar structures distal to the lobar bronchi.4850
Pulmonary-specific gene expression including SP-B, SP-C,
and CCSP is extinguished within transgenic lungs, which
do, however, contain ciliated and mucus-secreting cells.50
Thus, Nkx2.1 is felt to function as a master gene that
induces and maintains lung morphogenesis as well as differentiation of certain epithelial cell lineages.

Cellular and Molecular Mechanisms of

Lung Development
During the past few years, major progress has been made in
identifying key determinants of branching morphogenesis
and cellular differentiation within the lung. New insights
have been derived through the characterization of signaling
molecules that regulate gene expression and that are functionally conserved through evolution in invertebrate species
such as Drosophila and Caenorhabditis elegans, which are
functionally conserved through evaluation.58,59 Null muta-

6 / Chronic Obstructive Lung Disease

FIGURE 23. Comparison of N-myc, thryroid transcription factor-1 (TTF-1 or Nkx2.1), hepatocyte nuclear factor/forkhead homologue 4 (HFH-4),
and Clara cell secretory protein (CCSP) messenger ribonucleic acid (mRNA) expression in human fetal xenograft lung, with the appearance of the
alveolar stage of lung development, shown on dark-field photomicrographs of serial sections. Expression of N-myc is not detectable in the
xenograft lung at this time point. The signal for TTF-1 is restricted to distal alveolar spaces whereas CCSP and HFH-4 are localized to the proximal
tracheal and bronchiolar epithelium. (T = trachea; b = bronchi; a = alveoli.)

tions in mouse models have identified several nuclear transcription proteins, which determine respiratory-cell fate
and pattern formation via the activation and repression of
downstream target genes. The recurrent theme emerging
from these studies is that lung development extends in a
coordinated manner from successive epithelial-mesenchymal interactions. Growth factor signaling and induction of
responsive transcription factors mediate this interplay
between developing epithelial and mesenchymal structures.
The following section will detail specific cellular and molecular events during lung morphogenesis, which may be
recalled during tissue injury and regeneration.
Early Lung Differentiation
The lung shares a common embryological origin of other
foregut derivatives including the liver, pancreas, gastrointestinal tract, and thyroid (Figure 24). While many stud-

ies have focused on events critical to organ morphogenesis

and terminal cell differentiation,60 few have revealed how
initial cell type choices are made. In Drosophila, the initial
specification of tissues within the gut endoderm is caused
by signals from overlying mesoderm.61 Studies within the
liver have demonstrated that changes in gene expression
and tissue morphology define discrete phases in organ
development.62 Hepatic gene expression and differentiation begin in the foregut endoderm immediately after the
endodermal epithelium interacts with the cardiac mesoderm, in the 8.5-day-old embryo.63 Recent studies have
demonstrated that fibroblast growth factors (FGFs) emanating from the cardiac mesoderm are sufficient to divert
closely apposed endoderm to express the genetic program
for liver instead of that for pancreas, which is the default
pathway of the ventral foregut endoderm64,65 (see Figure
24). Although the signals that are important in early lung

Prenatal Lung Development / 7

specification are currently unknown, preliminary studies

suggest that dosage-dependent growth factor signaling
from the cardiac mesoderm might play a role (G.H.
Deutsch, unpublished observations) (see Figure 24).
The early pattern of expression of the homeobox gene
Nkx2.1 is consistent with its role in the morphogenesis of
the lung. Targeted disruption of the gene has demonstrated that while Nkx2.1 is not required for the initial
specification of the lung primordia, Nkx2.1 is essential
for further pulmonary development and cell differentiation.49,66 Study of the cis-acting regions in the Nkx2.1
promoter reveals that Nkx2.1 has multiple binding sites
for both ubiquitous and specific transcription factors,
including those of the hepatocyte nuclear factor (HNF)
and GATA zinc finger families,6769 which are required
for the development of the foregut endoderm;7072 HNF3 binding sites have also been identified in the SP-A, SP-B,
and CCSP promoters.53,73,74 The HNF-3 null mutation
results in an early embryonic lethal phenotype, in which
the primitive foregut endoderm fails to close into a tube;
consequently, there is no development of foregut derivatives such as the lung.75

FIGURE 24. Endodermal derivatives (depicted in green) in a 6week human embryo, saggital view. Signals from the cardiac mesoderm are hypothesized to play a role in liver, lung, and thyroid
development. (Redrawn after Larsen, W.J. 1993, Human Embryology.
Churchill-Livingstone, New York)

Pattern Formation of the Lung

In the fully differentiated lung, epithelial cells from the
proximal tracheal, and bronchial tubules greatly differ
from the distal alveolar tubules in morphology and gene
expression. The general principle of pattern formation
states that cells are instructed to assume a specific function according to their position along a concentration
gradient of a signal. Cells located closer to the source of
signal recognize a higher concentration and assume a different phenotype from those cells located at a distance,
which recognize low concentrations of the same signal.
Recognition of their position is followed by the activation
or repression of target genes, which ultimately determine
the regional differences in cell identity along the lung
axis. We have previously discussed how the presence of
proximal versus distal mesenchyme can dictate cell differentiation in adjacent epithelial cells. Early studies have
also shown that the quantity of mesenchyme is influential
in inducing a specific epithelial cell fate.76 A small
amount of recombined mesenchyme is able to direct cell
differentiation toward a bronchiolar phenotype; however,
an increased amount of the same mesenchyme will
induce the epithelial cells to differentiate toward an alveolar phenotype.76 This study implies that variable concentrations of the same signals or morphogens expressed
by the mesenchyme can induce different cell fates in identical epithelial cells.
Homeobox (Hox) genes are transcription factors,
which are known to regulate body axis patterning and
specification of regional identity during embryonic
development,58,77 although the mechanisms by which
they do so are largely unknown. The Hox genes likely regulate downstream factors that influence cell proliferation,
migration, and cell death. The pattern of Hox gene
expression at the beginning of lung development is consistent with its role in elaborating the proximal-distal orientation of the lung and in branching morphogenesis.7880
In the mouse, Hoxb-3 and Hoxb-4 are expressed in the
mesenchyme of the entire developing lung (proximal and
distal) while Hoxb-2 and Hoxb-5 expression are
restricted to the mesenchyme of distal lung buds.78 The
levels of Hox-gene expression decline with advancing
gestational age. Hoxb genes may be involved in patterning
the developing foregut by specifying regional differences
in lung mesenchyme. Hoxb-3 transactivates the rat
Nkx2.1 gene promoter,47 which infers that Hoxb-3 could
be involved in lung-specific gene regulation through
Nkx2.1 as an intermediate. In cultured mouse embryonic
lung, retinoic acid induces Hoxa-5, Hoxb-5 and Hoxb-6
gene expression,81 while Hoxb-5 is negatively regulated
by epidermal growth factor (EGF) and transforming
growth factor- (TGF-).82 Expression of Hox genes in
the adult lung may be relevant to neoplasia as well as to
pathologic disease states such as pulmonary hypertension
and emphysema.79,83 Retinoids have also been shown to

8 / Chronic Obstructive Lung Disease

influence the proximal-distal pattern of lung development. Retinoic acid, in dose-dependent concentrations,
has been demonstrated to favor the growth of proximal
airways and gene expression at the expense of distal
structures.84 It is probable that Hox genes mediate the
retinoic acidinduced alteration in lung patterning.81
Bone morphogenetic proteins (Bmps), members of the
TGF-related family of signaling molecules, are also
implicated in the control of the proximal-distal patterning
of the lung and in branching morphogenesis.85,86 Bone
morphogenetic protein 4 (Bmp-4) ribonucleic acid (RNA)
is restricted to the tips of distal buds and to the adjacent
mesenchyme, locally inhibiting endoderm proliferation
and forcing the outgrowth of lateral branches85 (Figure
25). Inhibition of Bmp signaling in transgenic animals
results in complete proximalization of the respiratory
epithelium, including ciliated cells in the most distal portions of transgenic lungs. It is hypothesized that Bmps provide a concentration gradient to control proximal versus
distal differentiation of the lung endoderm.86 Endodermal
cells located at the periphery of the lung, which are
exposed to high levels of Bmp-4, maintain or assume a distal identity while cells below a certain threshold of the
Bmp-4 signal initiate a proximal differentiation program.

FIGURE 25. Epithelial-mesenchymal interactions in the developing lung during branching morphogenesis. Factors are represented only
at sites where the expression is most abundant. Fibroblast growth factor (FGF) signals in the mesenchyme act as a chemotactic focus for the
epithelium during lung budding. As the bud extends toward the FGF
signals in the mesenchyme, endoderm cells that escape a high bone
morphogenic protein 4 (Bmp-4) signal assume a proximal cellular identity; those that retain a high Bmp-4 signal take on a distal identity. A
high concentration of Bmp-4 signal also serves to locally inhibit endoderm proliferation, thereby inducing the lateral outgrowth of new airway branches. Sonic hedgehog (Shh) at the distal tips functions to
downregulate FGF-10 expression in the mesenchyme, which limits
local budding. Transforming growth factor- (TGF-) signaling also prevents local budding, by decreasing endodermal proliferation and by
stimulating synthesis of matrix components at branch points.(FGFR =
fibroblast growth factor receptor; Hoxb = homeobox b.)

Regulation of Branching Morphogenesis

Elegant recombination experiments45,8790 have demonstrated that mesenchyme is necessary for initial lung budding and branching and that the mesenchyme
surrounding the trachea and mainstem bronchi has a different regional identity than the mesenchyme surrounding the distal lung bud. There is abundant data to suggest
that soluble growth factors are likely mediators of this
interaction.9193 Candidate growth factors whose signaling peptides and cognate receptors are expressed in the
early embryonic mouse lung include FGFs, EGFs, TGFs,
hepatocyte growth factor (HGF), and platelet-derived
growth factor (PDGF).9499 Their influences on lung
development have been demonstrated by gain vs. loss of
function experiments in embryonic mouse lung organ
cultures and in transgenic mice. In general, growth factor
receptors with tyrosine kinase intracellular signaling
domains (EGF, FGF, and PDGF receptors [EGFR, FGFR,
and PDGFR]) stimulate lung morphogenesis100105
whereas those with serine/threonine kinase intracellular
signaling domains (such as the TGF- family) are
inhibitory.106108
Branching of the respiratory tracheae in Drosophila is
considered a conserved genetic paradigm for the morphogenesis of the mammalian lung. Primary branching
in Drosophila is spatially regulated by the branchless gene,
of which FGF-10 is the mammalian homologue.109 The
branchless gene activates the breathless gene in tracheal
cells, to induce their migration and thus primary branching.110,111 Breathless is homologous to the FGFR family.
This FGF homologue-signaling pathway appears to be
evolutionarily conserved in mammals. Fibroblast growth
factor-1 (FOF-1), FGF-7, and FGF-10 are synthesized by
the pulmonary mesenchyme, and they bind to and activate FGFRs on endodermal cells of the developing lung
buds.95,104,112 Fibroblast growth factor-10 may play a
chemotactic role similar to that of branchless in inducing
epithelial branching and cell migration. Transcripts of
FGF-10 are present at discrete sites in the mesenchyme
and have been demonstrated to govern the directional
outgrowth of lung buds93 (see Figure 25). At early
stages, FGFR-2, the receptor for FGF-10, is expressed at
high levels along the entire proximal-distal axis of the respiratory-tract epithelium.113 Disruption of FGF signaling
by the generation of FGF-10deficient mice, or expression of a dominant negative form of its receptor (FGFR2), interferes with branching morphogenesis and
peripheral cell differentiation; transgenic lungs are characterized by only a trachea or a trachea and two primary
bronchi.102,114,115 Thus, it appears that the elaboration of
branched airways involves the directional movement of
epithelial cell precursors toward a localized source of an
FGF ligand, either by cell migration or by outgrowth of
epithelial buds.

Prenatal Lung Development / 9

Despite high-sequence homology and use of the same

receptor, FGF-7 and FGF-10 exert distinct effects on the
developing lung. Rather than being chemotactic, FGF-7
influences airway branching by promoting epithelial cell
proliferation and expansion.91,104,116,117 Misexpression of
FGF-7 in mice during the pseudoglandular stage disrupts
branching morphogenesis and epithelial cell differentiation.118 Transgenic lungs have large cystic spaces that
resemble human congenital cystic adenomatoid malformations. Fibroblast growth factor-7 as a differentiation
factor for the developing lung has been demonstrated in
lung organotypic cultures. In the absence of mesenchyme
or serum, exogenous FGF-7 can induce a type II celllike
phenotype.117,119 Despite evidence that FGF-7 plays a
role in lung development, mice homozygous for a null
mutation in the FGF-7 gene exhibit no apparent pulmonary abnormalities.120 This suggests that other members of the FGF family (perhaps FGF-10) can serve as
functionally redundant molecules.
Many studies have shown that certain signaling and
growth factors are negative regulators of lung epithelial
proliferation, which may play a role in counteracting the
bud-promoting effects of FGFs. Fibroblast growth factor10 is downregulated in lungs of transgenic mice overexpressing sonic hedgehog (Shh),85 which is a secreted
signaling protein that induces gene expression and proliferation of adjacent mesenchymal target cells in the
lung.93,121 Low levels of Shh are expressed throughout the
primitive respiratory epithelium, with high levels seen at
the distal tips85,122 (see Figure 25). Treatment of embryonic lung mesenchymal cells with recombinant Shh
markedly inhibits FGF-10 mRNA expression.123 In the
absence of Shh, FGF-10 expression is no longer restricted
to focal areas but becomes widespread throughout the distal mesenchyme.124 This finding raises the possibility that
one of the roles for Shh signaling in branching is to spatially restrict FGF-10 levels in the mesenchyme surrounding the bud tips.124 Sonic hedgehognull mutant mice
have tracheoesophageal fistulas and bilateral rudimentary
sacs, due to failure of branching and growth after formation of the primary lung buds.121,124 The lung mesenchyme shows enhanced cell death and decreased cell
proliferation.121 Conversely, overexpression of Shh leads
to hypercellularity of the lung, with an absence of normal
alveolar septa because of excessive mesenchyme between
the alveolar spaces.125 Sonic hedgehog expression in the
endoderm may also be essential for the proliferation and
differentiation of the mesoderm, which in turn is required
for proper differentiation of the primitive pulmonary
epithelium.121 Of interest, treatment of cultured lungs
with retinoic acid increases the level of Shh expression
while decreasing the level of FGF-10 expression and the
degree of airway branching.126,127 These data support the
concept that distinct signaling and transcriptional pathways are interrelated during lung development.

The TGF-s represent a family of peptides involved in

cell growth and apoptosis control, connective-tissue formation, and embryonic development.128 Transforming
growth factor-1, TGF-2, and TGF-3 are indirect
mitogens for certain mesenchymal cells and are marked
stimulators of extracellular matrix deposition, including
collagen, fibronectin, and proteoglycans.128 The TGF-s
are also potent inhibitors of epithelial cell proliferation.
The TGF-1 protein is localized at the interface between
epithelial and mesenchymal cells, particularly around the
bronchiolar ducts and airway branch points.96,129 Over
expression of members of the TGF- family, both in
organ culture and in transgenic animals, has a negative
regulatory effect on branching morphogenesis106,108
abrogation of TGF- type II receptor signaling (either
with antisense oligodeoxynucleotides or with blocking
antibodies), stimulates lung morphogenesis twofold to
threefold, and increases expression of the distal NKx2.1
and SPC genes.108
Transforming growth factor-1 has been shown to
downregulate the expression of N-myc and branching
morphogenesis in murine organ lung cultures.106 The
N-myc gene may play a role in lung development by
maintaining cells in an undifferentiated state; its expression is most abundant in undifferentiated progenitor cells
and primitive tumors.130,131 Targeted disruption of the
N-myc gene results in embryonic lethality during the
pseudoglandular stage of development, with a clear
defect in branching morphogenesis; homozygous lungs
consist of only a trachea and mainstem bronchi.132135
Factors Related to Alveolarization
During postnatal life, FGFR-3 and FGFR-4, receptors that
bind several of the FGFs, cooperate to regulate alveolization; a double mutation of these genes results in a postnatal lethal pulmonary phenotype in which there is
failure of alveolar septation, resulting in an emphysematous appearance.136 Lungs of transgenic mice demonstrate an aberration of elastin synthesis. Mutants lacking
the growth factor PDGF-A also exhibit a defect in pulmonary alveogenesis, but this phenotype is apparently
caused by a loss of alveolar smooth-muscle myofibroblasts and parenchymal elastic fibers.137
The defect in alveolization in neonatal EGF receptordeficient mice is also associated with impaired
branching morphogenesis.105 Inactivation of the EGF
receptor affects type-II pneumocyte maturity, with
decreased expression of the distal SPC marker. In contrast, exogenous EGF accelerates alveolar type-II cell differentiation in fetal lungs.138

Pulmonary Vasculogenesis
Like lung morphogenesis, pulmonary vascular development is a highly regulated process involving not only the
formation and differentiation of new vessels but also the

10 / Chronic Obstructive Lung Disease

inhibition of further vascular proliferation.8,139 Direct

epithelial-mesenchymal interactions may be essential for
vessel formation,140 with the participation of growth factors and matrix proteins. Pulmonary endothelial cell differentiation is still poorly understood, but studies suggest
that receptors for the vascular endothelial growth factor
(VEGF), located within the mesenchyme, facilitate early
vessel formation within the embryo.140,141 Growth factors
such as FGF-2142 and VEGF143,144 are known stimulants
of endothelial cell migration, proliferation, and transdifferentiation, leading to the appearance of vascular structures. Misexpression of VEGF in transgenic mice under
the control of the SP-C promoter/enhancer induces gross
abnormalities in lung morphogenesis, characterized by
an increase in vascularity and concomitant decrease in
distal acinar tubules and mesenchyme.145 Inhibition of
the VEGF receptor KDR (VEGFR-2) also influences lung
morphology by reducing vascular density and alveolarization in the newborn rat.146
Endothelial monocyte-activating polypeptide II
(EMAPII), localized primarily to the mesenchyme, has
been demonstrated to have a negative effect on neovascularization in the developing lung.147 Its location correlates with the same area of expression as other regulators
of vessel formation, including the VEGF receptors 1 and
2.147 Its continual expression within large vessels in adulthood leads one to theorize that EMAPII might function
postnatally to stabilize or maintain existing vascular
structures.147

References
1. Loosli CG, Potter EL. The prenatal development of the
human lung. Anat Rec 1951;109:3201.
2. Boyden EA. Development and growth of the airways. In:
Hodson WA, editor. Development of the lung. New
York: Marcel Dekker; 1977. p. 335.
3. Ten Have-Opbroek AAW. Lung development in the mouse
embryo. Exp Lung Res 1991;17:11130.
4. Loosli CG, Potter EL. Pre- and postnatal development of
the respiratory portion of the human lung. Am Rev
Respir Dis 1959;80:5.
5. Bucher U, Reid LM. Development of intrasegmental
bronchial tree: the pattern of branching and development of cartilage at various stages of intra-uterine life.
Thorax 1961;16:20718.
6. Boyden EA. Development of human lung. In: Kelley V,
editor. Brennemans practice of pediatrics. Hagerstown
MD: Harper & Row; 1980. p. 117.
7. Boyden EA. The programming of canalization in fetal
lungs of man and monkey. Am J Anat 1976;145:1257.
8. DeMello DE, Sawyer D, Galvin N, Reid LM. Early fetal
development of lung vasculature. Am J Respir Cell Mol
Biol 1997;16:56881.
9. DeMello DE, Reid LM. Embryonic and early fetal development of human lung vasculature and its functional
implications. Pediatr Dev Pathol 2000;3:43949.

10. Burri PH, Moschopulos M. Structural analysis of fetal rat

lung development. Anat Rec 1992;234:399418.
11. Hislop A, Reid LM. Formation of the pulmonary vasculature. In: Hodson WA, editor. Development of the lung.
New York: Marcel Dekker; 1979. p. 3786.
12. Williams M. Development of the alveolar structure of the
fetal rat in late gestation. Fed Proc 1977;36:26539.
13. Juul SE. Lung cell ratios during fetal development in the
primate model of hyaline membrane disease. Am Rev
Respir Dis 1989;139:A289.
14. Meyrick B, Reid LM. Ultrastructure of alveolar lining and
its development. In: Hodson WA, editor. Development
of the lung. New York: Marcel Dekker; 1977. p. 135214.
15. Langston C, Kida K, Reed M, Thurlbeck WM. Human
lung growth in late gestation and in the neonate. Am
Rev Respir Dis 1984;129:60713.
16. Burri PH, Weibel ER. Ultrastructure and morphometry of
the developing lung. In: Hodson WA, editor.
Development of the lung. New York: Marcel Dekker;
1975. p. 2357.
17. Thurlbeck WM. Postnatal growth and development of the
lung. Am Rev Respir Dis 1975;111:80344.
18. Zeltner TB, Caduff JH, Gehr P. The postnatal development and growth of the human lung. I. Morphometry.
Respir Physiol 1987;67:24767.
19. Zeltner TB, Burri PH. The postnatal development and
growth of the lung. II. Morphology. Respir Physiol
1987;67:26982.
20. Ayers MM, Jeffery PK. Proliferation and differentiation in
mammalian airway epithelium. Eur Respir J
1988;1:5880.
21. Ten Have-Opbroek, AAW. The development of the lung in
mammals: an analysis of concepts and findings. Am J
Anat 1981;162:20119.
22. Adamson IY, Bowden DH. The type 2 cell as progenitor of
alveolar epithelial regeneration. A cytodynamic study in
mice after exposure to oxygen. Lab Invest
1974;30:3542.
23. Evans MJ, Cabral LJ, Stephens RJ, Freeman G.
Transformation of alveolar type II cells to type I cells following exposure to NO2. Exp Mol Pathol 1975;22:14550.
24. Evans MH, Cabral-Anderson LJ, Freeman G. Role of the
Clara cell in renewal of the bronchiolar epithelium. Lab
Invest 1978;38:64856.
25. Hook GER, Brody AR, Cameron GS, et al. Repopulation
of denuded tracheas by Clara cells isolated from the
lungs of rabbits. Exp Lung Res 1987;12:31129.
26. Inayama Y, Hook GER, Brody AR, et al. In vitro and in
vivo growth and differentiation of clones of tracheal
basal cells. Am J Pathol 1989;134:53949.
27. Plopper CG, Nishio SJ, Alley JL, et al. The role of the nonciliated bronchiolar epithelial (Clara) cell as the progenitor cell during bronchiolar epithelial differentiation in
the perinatal rabbit lung. Am J Respir Cell Mol Biol
1992;7:60613.
28. Korfhagen TR, Glasser SW, Wert SE, et al. Cis-acting
sequences from a human surfactant protein gene confer
pulmonary-specific gene expression in transgenic mice.
Proc Natl Acad Sci U S A 1990;87:61226.

Prenatal Lung Development / 11

29. Cutz E. Neuroendocrine cells of the lung. An overview of
morphologic characteristics and development. Exp
Lung Res 1982;3:185208.
30. Sunday ME, Hua J, Dai HB, et al. Bombesin increases fetal
lung growth and maturation in utero and in organ culture. Am J Respir Cell Mol Biol 1990;3:199205.
31. King KA, Torday JS, Sunday ME. Bombesin and
[Leu8]phyllolitorin promote fetal mouse lung branching
morphogenesis via a receptor-mediated mechansim.
Proc Natl Acad Sci U S A 1995;92:435761.
32. Weaver TE, Whitsett JA. Function and regulation of
expression of pulmonary surfactant-associated proteins.
Biochem J 1991;273:24964.
33. Wert S, Glasser S, Korfhagen T, Whitsett JA.
Transcriptional elements from the human SP-C gene
direct expression in the primordial respiratory epithelium of transgenic mice. Dev Biol 1993;156:42643.
34. Singh G, Singh J, Katyal SL, et al. Identification, cellular
localization, isolation, and characterization of human
Clara cell-specific 10 KD protein. J Histochem
Cytochem 1988;36:7380.
35. Hackett BP, Shimizu N, Gitlin JD. Clara cell secretory protein gene expression in bronchiolar epithelium. Am J
Physiol 1992;262:L399L404.
36. Kuroki Y, Voelker DR. Pulmonary surfactant proteins. J
Biol Chem 1994;42:259436.
37. Auten RL, Watkins PH, Shapiro DL, Horowitz S.
Surfactant apoprotein A (SP-A) is synthesized in airway
cells. Am J Respir Cell Mol Biol 1990;3:4916.
38. Phelps DS, Floros J. Localization of pulmonary surfactant
proteins using immunohistochemistry and tissue in situ
hybridization. Exp Lung Res 1991;17:98595.
39. Khoor A, Gray ME, Hull WM, et al. Developmental
expression of SP-A and SP-A mRNA in the proximal and
distal respiratory epithelium in the human fetus and
newborn. J Histochem Cytochem 1993;41:13119.
40. Khoor A, Stahlman MT, Gray ME, Whitsett JA. Temporalspatial distribution of SP-B and SP-C proteins and
mRNAs in developing respiratory epithelium of human
lung. J Histochem Cytochem 1994;42:118799.
41. Johnston CJ, Mango GW, Finkelstein JN, Stripp BR.
Altered pulmonary response to hyperoxia in Clara cell
secretory protein deficient mice. Am J Respir Cell Mol
Biol 1997;17:14755.
42. Chen J, Knowles HJ, Hebert JL, Hackett BP. Mutation of
the mouse hepatocyte nuclear factor/forkhead homologue 4 gene results in absence of cilia and random leftright asymmetry. J Clin Invest 1998;102:107782.
43. Wuenschell CW, Sunday ME, Singh G, et al. Embryonic
mouse lung progenitor cells co-express immunohistochemical markers of diverse mature cell lineages. J
Histochem Cytochem 1996;44:11323.
44. Daly HE, Baecher-Allan CM, Paxhia AT, et al. Cell-specific
gene expression reveals changes in epithelial cell populations after bleomycin treatment. Lab Invest
1998;78:393400.
45. Shannon JM. Induction of alveolar type II cell differentiation in fetal tracheal epithelium by grafted distal lung
mesenchyme. Dev Biol 1994;166:60014.

46. Shannon JM, Nielson LD, Gebb SA, Randell SH.

Mesenchyme specifies epithelial differentiation in reciprocal recombinants of embryonic lung and trachea. Dev
Dyn 1998;212:48294.
47. Guazzi S, Lonigro R, Pintonello L, et al. The thyroid transcription factor-1 gene is a candidate target for regulation by Hox proteins. EMBO J 1994;13:333947.
48. Minoo P, Hamdan H, Bu D, et al. TTF-1 regulates lung
epithelial morphogenesis. Dev Biol 1995;172:6948.
49. Kimura S, Yoshinobu H, Pineau T, et al. The T/ebp null
mouse: thyroid-specific enhancer-binding protein is
essential for the organogenesis of the thyroid, lung, ventral forebrain, and pituitary. Genes Dev 1996;10:609.
50. Minoo P, Su G, Drum H, et al. Defects in tracheoesophageal and lung morphogenesis in Nkx2.1(-/-)
mouse embryos. Dev Biol 1999;209:6071.
51. Lazzaro D, Price M, DeFelice M, DiLauro R. The transcription factor TTF-1 is expressed at the onset of thyroid and lung morphogenesis and in restricted regions
of the foetal brain. Development 1991;113:1093104.
52. Ikeda K, Clark JC, Shaw-White JR, et al. Gene structure and
expression of human thyroid transcription factor-1 in respiratory epithelial cells. J Biol Chem 1995;270:810814.
53. Bohinski RJ, DiLauro R, Whitsett JA. The lung-specific
surfactant protein B gene promoter is a target for thyroid transcription factor 1 and hepatocyte nuclear factor
3, indicating common factors for organ-specific gene
expression along the foregut axis. Mol Cell Biol
1994;14:567181.
54. Bruno MD, Bohinski RJ, Huelsman KM, et al. Lung cellspecific expression of the murine surfactant protein A
(SP-A) gene is mediated by interactions between the SPA promoter and thyroid transcription factor-1. J Biol
Chem 1995;270:65316.
55. Yan C, Sever Z, Whitsett JA. Upstream enhancer activity in
the surfactant protein B gene is mediated by thyroid
transcription factor 1. J Biol Chem 1995;270:248527.
56. Kelly SE, Bachurski CJ, Burhans MS, Glasser SW.
Transcription of the lung-specific surfactant protein C
gene is mediated by thyroid transcription factor 1. J Biol
Chem 1996;271:68818.
57. Zhang L, Whitsett JA, Stripp BR. Regulation of Clara cell
secretory protein gene transcription by thyroid transcription factor-1. Biochim Biophys Acta 1997;1350:35967.
58. Lawrence PA, Morata G. Homeobox genes: their function
in Drosophila segmentation and pattern formation. Cell
1994;78:1819.
59. Salser SJ, Kenyon C. Patterning C. elegans: homeotic cluster genes, cell fates and cell migrations. Trends Genet
1994;10:15964.
60. Hogan BLM. Morphogenesis. Cell 1999;96:22533.
61. Immergluck K, Lawrence PA, Bienz M. Induction across
germ layers in Drosophila mediated by a genetic cascade.
Cell 1990;62:2618.
62. Zaret KS. Early liver differentiation: genetic potentiation
and multilevel growth control. Curr Opin Genet Dev
1998;8:52631.
63. Gualdi R, Bossard P, Zheng M, et al. Hepatic specification
of the gut endoderm in vitro: cell signaling and transcriptional control. Genes Dev 1996;10:167082.

12 / Chronic Obstructive Lung Disease

64. Jung J, Zheng M, Goldfarb M, Zaret KS. Initiation of
mammalian liver development from endoderm by
fibroblast growth factors. Science 1999;284:19982003.
65. Deutsch GH, Jung J, Zheng M, et al. A bipotential precursor population for pancreas and liver within the embryonic endoderm. Development 2001. [In press].
66. Kimura S, Ward JM, Minoo P. Thyroid-specific enhancerbinding protein/thyroid transcription factor 1 is not
required for the initial specification of the thyroid and
lung primordia. Biochimie 1999;81:3217.
67. Lai E, Clark KL, Burley SK, Darnell JE Jr. Hepatocyte
nuclear factor 3/forkhead or winged helix proteins: a
family of transcription factors of diverse biologic function. Proc Natl Acad Sci U S A 1993;90:104213.
68. Ikeda K, Shaw-White JR, Wert SE, Whitsett JA. Hepatocyte
nuclear factor 3 activates transcription of thyroid transcription factor 1 in respiratory epithelial cells. Mol Cell
Biol 1996;16:362636.
69. Shaw-White JR, Bruno MD, Whitsett JA. Gata-6 activates
transcription of thyroid transcription factor-1. J Biol
Chem 1999;274:265864.
70. Monaghan AP, Kaestner KH, Grau E, Schutz G.
Postimplantation expression patterns indicate a role for
the mouse forkhead/HNF-3 alpha, beta and gamma
genes in determination of the definitive endoderm,
chordamesoderm and neuroectoderm. Development
1993;119:56378.
71. Ang S-L, Wierda A, Wong D, et al. The formation and
maintenance of the definitive endoderm lineage in the
mouse: involvement of HNF-3/forkhead proteins.
Development 1993;119:130115.
72. Kuo CT, Morrisey EE, Anandappa R, et al. GATA4 transcription factor is required for ventral morphogenesis
and heart tube formation. Genes Dev 1997;11:
104860.
73. Alcorn JL, Gao E, Chen Q, et al. Genomic elements
involved in transcriptional regulation of the surfactant
protein-A gene. Mol Endocrinol 1993;7:107285.
74. Bingle CD, Hackett BP, Moxley M, et al. Role of HNF-3
alpha and HNF-3 beta in Clara cell secretory protein
gene expression in bronchiolar epithelium. Biochem J
1995;308:197202
75. Ang S-L, Rossant J. HNF-3B is essential for node and
notochord formation in mouse development. Cell
1994;78:56174.
76. Masters JRW. Epithelial-mesenchymal interaction during
lung development: the effect of mesenchymal mass. Dev
Biol 1976;51:98108.
77. Krumlauf, R. Hox genes in vertebrate development. Cell
1994;78:191201.
78. Bogue CW, Lou LJ, Vasavada H, et al. Expression of Hoxb
genes in the developing mouse foregut and lung. Am J
Respir Cell Mol Biol 1996;15:16371.
79. Kappen C. Hox genes in the lung. Am J Respir Cell Mol
Biol 1996;15:15662.
80. Volpe MV, Martin A, Vosatka RJ, et al. Hoxb-5 expression
in the developing mouse lung suggests a role in branching morphogenesis and epithelial cell fate. Histochem
Cell Biol 1997;108:495504.

81. Bogue CW, Gross I, Vasavada H, et al. Identification of

Hox genes in newborn lung and effects of gestational
age and retinoic acid on their expression. Am J Physiol
1994;266:L44854.
82. Chinoy MR, Volpe MV, Cilley RE, et al. Growth factors
and dexamethasone regulate Hoxb5 protein in cultured
murine fetal lungs. Am J Physiol 1998;274:L61020.
83. Golpon HA, Geraci MW, Moor MD, et al. HOX genes in
human lung-altered expression in primary pulmonary
hypertension and emphysema. [In press.] Am J
Pathology 2001;158:95566.
84. Cardoso WV, Williams MC, Mitsialis SA, et al. Retinoic
acid induces changes in the pattern of airway branching
and alters epithelial cell differentiation in the developing
lung in vitro. Am J Respir Cell Mol Biol 1995;12:46476.
85. Bellusci, S, Henderson R, Winnier G, et al. Evidence from
normal expression and targeted misexpression that bone
morphogenetic protein-4 (Bmp-4) plays a role in mouse
embryonic lung morphogenesis. Development
1996;122:1693702.
86. Weaver M, Yingling YM, Dunn NR, et al. Bmp signaling regulates proximal-distal differentiation of endoderm in
mouse lung development. Development 1999;126:400515.
87. Alescio T, Cassini A. Induction in vitro of tracheal buds by
pulmonary mesenchyme grafted on tracheal epithelium.
J Exp Zool 1962;150:8394.
88. Spooner BS, Wessells NK. Mammalian lung development:
interactions in primordium formation and bronchial
morphogenesis. J Exp Zool 1970;175:44554.
89. Wessells NK. Mammalian lung development: interactions
in formation and morphogenesis of tracheal buds. J Exp
Zool 1970;175:45566.
90. Hilfer SR, Rayner RM, Brown JW. Mesenchymal control
of branching pattern in the fetal mouse lung. Tissue Cell
1985;17:52338.
91. Nogawa H, Ito T. Branching morphogenesis of embryonic
mouse lung epithelium in mesenchyme-free culture.
Development 1995;121:101522.
92. Deterding RR, Shannon JM. Proliferation and differentiation of fetal rat pulmonary epithelium in the absence of
mesenchyme. J Clin Invest 1995;95:296372.
93. Bellusci S, Grindley J, Emoto H, et al. Fibroblast growth factor 10 (FGF10) and branching morphogenesis in the
embryonic mouse lung. Development 1997;124:486778.
94. Snead ML, Luo W, Oliver P, et al. Localization of epidermal growth factor precursor in tooth and lung during
embryonic development. Dev Biol 1989;134:4209.
95. Fu YM, Spirito P, Yu ZX, et al. Acidic fibroblast growth factor in the developing rat embryo. J Cell Biol
1991;114:126173.
96. Pelton RW, Saxena B, Jones M, et al. Immunohistochemical
localization of TGF beta 1, TGF beta 2, and TGF beta 3 in
the mouse embryo: expression patterns suggest multiple
roles during embryonic development. J Cell Biol
1991;115:1091105.
97. Han RN, Liu J, Tanswell AK, Post M. Expression of basic
fibroblast growth factor and receptor: immunolocalization studies in developing rat fetal lung. Pediatr Res
1992;31:43540.

Prenatal Lung Development / 13

98. Han RN, Mawdsley C, Souza P, et al. Platelet-derived
growth factors and growth-related genes in rat lung. III.
Immunolocalization during fetal development. Pediatr
Res 1992;31:3239.
99. Ohmichi H, Koshimizu U, Matasumoto K, Nakamura T.
Hepatocyte growth factor (HGF) acts as a mesenchymederived factor during fetal lung development.
Development 1998;125:131524.
100. Warburton D, Seth R, Shum PG, et al. Epigenetic role of
epidermal growth factor expression and signalling in
embryonic mouse lung branching morpogenesis. Dev
Biol 1992;149:12333.
101. Seth R, Shum L, Wu F, et al. Role of epidermal growth factor expression in early mouse embryo lung branching
morphogenesis in culture: antisense oligodeoxynucleotide strategy. Dev Biol 1993;158:5559.
102. Peters K, Werner S, Liao X, et al. Targeted expression of a
dominant negative FGF receptor blocks branching morphogenesis and epithelial differentiation of the mouse
lung. EMBO J 1994;13:3296301.
103. Souza P, Kuliszewski M, Wang J, et al. PDGF-AA and its receptor influence early lung branching via an epithelial-mesenchymal interaction. Development 1995;121:255967.
104. Post M, Souza P, Liu J, et al. Keratinocyte growth factor
and its receptor are involved in regulating early lung
branching. Development 1996;122:310715.
105. Miettinen P, Warburton D, Bu D, et al. Impaired lung
branching morphogenesis in the absence of functional
EGF receptor. Dev Biol 1997;186:22436.
106. Serra R, Pelton RW, Moses HL. TGFB1 inhibits branching
morphogenesis and N-myc expression in lung bud
organ cultures. Development 1994;120:215361.
107. Kaartinen V, Voncken JW, Shuler CA, et al. Abnormal lung
development and cleft palate in mice lacking TGF-3
indicates defects of epithelial-mesenchymal interaction.
Nat Genet 1995;11:41521.
108. Zhao J, Bu D, Lee M, et al. Abrogation of transforming
growth factor-beta type II receptor stimulates embryonic mouse lung branching morphogenesis in culture.
Dev Biol 1996;180:24257.
109. Sutherland D, Samkovlis C, Krasnow MA. Branchless
encodes a Drosophila FGF homolog that controls tracheal cell migration and patterning of branching. Cell
1996;87:1091101.
110. Glazer L, Shilo BZ. The Drosophila FGF-R homolog is
expressed in the embryonic tracheal system and appears
to be required for directed tracheal extension. Genes
Dev 1991;5:697705.
111. Lee T, Hacohen N, Krasnow M, Montell DJ. Regulated
Breathless receptor tyrosine kinase activity required to
pattern cell migration and branching in the Drosophila
tracheal system. Genes Dev 1996;10:291221.
112. Yamasaki M, Miyake A, Tagashira S, Itoh N. Structure and
expression of the rat mRNA encoding a novel member
of the fibroblast growth factor family. J Biol Chem
1996;271:1591821.
113. Peters KG, Chen WG, Williams LT. Two FGF receptor
genes are differentially expressed in epithelial and mesenchymal tissues during limb formation and organogenesis in the mouse. Development 1992;114:23343.

114. Min H, Danilenko DM, Scully SA, et al. FGF-10 is

required for both limb and lung development and
exhibits striking functional similarity to Drosophila
branchless. Genes Dev 1998;12:315661.
115. Sekine K, Ohuchi H, Fujiwara M, et al. FGF10 is essential
for limb and lung formation. Nat Genet 1999;21:13841.
116. Shiratori M, Oshika E, Ung LP, et al. Keratinocyte growth
factor and lung morphogenesis. Am J Respir Cell Mol
Biol 1996;15:32838.
117. Cardoso WV, Itoh A, Nogawa H, et al. FGF-1 and FGF-7
induce distinct patterns of growth and differentiation in
embryonic lung epithelium. Dev Dyn 1997;208:398405.
118. Simonet WS, DeRose ML, Bucay N, et al. Pulmonary malformation in transgenic mice expressing human keratinocyte growth factor in the lung. Proc Natl Acad Sci
U S A 1995;92:124615.
119. Shannon JM, Gebb SA, Nielson LD. Induction of alveolar
type II cell differentiation in embryonic tracheal epithelium in mesenchyme-free culture. Development
1999;126:167588.
120. Guo L, Degenstein L, Fuchs E. Keratinocyte growth factor
is required for hair development but not for wound
healing. Genes Dev 1996;10:16575.
121. Litingtung Y, Lei L, Westphal H, Chiang C. Sonic hedgehog is
essential to foregut development. Nat Gene 1998;20:5861.
122. Bitgood MJ, McMahon AP. Hedgehog and BMP genes are
coexpressed at many diverse sites of cell-cell interaction
in the mouse embryo. Dev Biol 1995;172:12638.
123. Lebeche D, Malpel S, Cardoso WV. Fibroblast growth factor interactions in the developing lung. Mech Dev
1999;86:12536.
124. Pepicelli CV, Lewis P, McMahon AP. Sonic hedgehog regulates branching morphogenesis in the mammalian
lung. Curr Biol 1998;8:10836.
125. Bellusci S, Furuta Y, Rush MG, et al. Involvement of sonic
hedgehog (Shh) in mouse embyronic lung growth and
morphogenesis. Development 1997;124:5363.
126. Cardoso WV, Mitsialis SA, Brody JS, Williams MC. Retinoic
acid alters the expression of pattern-related genes in the
developing rat lung. Dev Dyn 1996;207:4759.
127. Rush MG, Perkins DR. Induction of sonic hedgehog and
patched by retinoic acid in the developing rat lung in
vitro. Pediatr Res 1997;41:51A.
128. Moses HL, Yang EY, Pietenpol JA. TGF- stimulation and
inhibition of cell proliferation. New mechanistic
insights. Cell 1990;63:2457.
129. Heine UI, Munoz EF, Flanders KC, et al. Colocalization of
TGF-1 and collagen I and III, fibronectin, and glycosaminoglycans during lung branching morphogenesis. Development 1990;109:2936.
130. Jakobovits A, Schwab M, Bishop JM, Martin GR.
Expression of N-myc in teratocarcinoma stem cells and
mouse embryos. Nature 1985;318:18891.
131. Zimmerman KA, Yancopoulos GD, Collum RG, et al.
Differential expression of myc family genes during
murine development. Nature 1986;319:7803.
132. Stanton BR, Perkins AS, Tessarollo L, et al. Loss of N-myc
function results in embryonic lethality and failure of the
epithelial component of the embryo to develop. Genes
Dev 1992;6:223547.

14 / Chronic Obstructive Lung Disease

133. Charron J, Malynn BA, Fisher P, et al. Embryonic lethality
in mice homozygous for a targeted disruption of the Nmyc gene. Genes Dev 1992;6:224857.
134. Moens CB, Auerbach AB, Conlon RA, et al. A targeted
mutation reveals a role for N-myc in branching morphogenesis in the embryonic mouse lung. Genes Dev
1992;6:691704.
135. Sawai S, Shimono A, Watamatus Y, et al. Defects of embryonic organogenesis resulting from the targeted disruption of the N-myc gene in the mouse. Development
1993;117:144555.
136. Weinstein M, Xu X, Ohyama K, Deng C-X. FGFR-3 and
FGFR-4 function cooperatively to direct alveogenesis in
the murine lung. Development 1998;125:361523.
137. Bostrom H, Willetts K, Pekny M, et al. PDGF-A signaling
is a critical event in lung alveolar myofibroblast development and alveogenesis. Cell 1996;85:86373.
138. Plopper CG, St George JA, Read LC, et al. Acceleration of
type II cell differentiation in fetal rhesus monkey lung
by administration of EGF. Am J Physiol 1992;262:
L31321.
139. Scavo LM, Ertsey R, Chapin CJ, et al. Apoptosis in the
development of rat and human fetal lungs. Am J Respir
Cell Mol Biol 1998;18:2131.

140. Gebb SA, Shannon JM. Tissue interactions mediate early

events in pulmonary vasculogenesis. Dev Dyn
2000;217:15969.
141. Yamaguchi TP, Dumont DJ, Conlon RA, et al. flk-1, an fltrelated receptor tyrosine kinase is an early marker for
endothelial cell precursors. Development 1993;118:48998.
142. Shing Y, Folkman J, Sullivan R, et al. Heparin affinity:
purification of a tumor-derived capillary endothelial cell
growth factor. Science 1984;223:12969.
143. Plate KH, Breier G, Weich H, Risau W. Vascular endothelial growth factor is a potential tumour angiogenesis factor in human gliomas in vivo. Nature 1992;359:8458.
144. Kim J, Li B, Winer J, et al. Inhibition of vascular endothelial cell growth factor-induced angiogenesis suppresses
tumour growth in vivo. Nature 1993;362:8414.
145. Zeng X, Wert SE, Federici R, et al. VEGF enhances pulmonary vasculogenesis and disrupts lung morphogenesis in vivo. Dev Dyn 1998;211:21527.
146. Jakkula M, Le Cras TD, Gebb S, et al. Inhibition of angiogenesis decreases alveolarization in the developing rat
lung. Am J Phyiol Lung Cell Mol Physiol 2000;279:L6007.
147. Schwarz M, Lee M, Zhang F, et al. EMAP II: a modulator
of neovascularization in the developing lung. Am J
Physiol Lung Cell Mol Physiol 1999;276:L36575.