IntroductionLung development requires integration of multiple regu-latory factors that mediate patterns of cell proliferation,differentiation, migration, and death. These developmen-tal programs, which include transcription factors and sig-naling molecules, are likely re-enlisted during recovery ofthe lung following injury. Understanding these processescould provide important insight into controlling cell dif-ferentiation and regeneration for therapeutic purposes.

This chapter describes the morphological features thatcharacterize the defined stages of lung development andalso addresses epithelial cell differentiation and vasculo-genesis of the airways. In the latter part of the chapter, anattempt will be made to highlight specific cellular andmolecular events during development that may influencelung maintenance in adult disease states.

Stages of Lung Development During gestation, the fetal lung undergoes significantmorphological changes to provide at birth an organ capa-ble of maintaining respiration and gas exchange.Although lung development is continuous duringembryogenesis, five developmental stages have beendelineated, based on anatomic and histologic characteris-tics.1 (Table 2–1). The early embryonic and pseudoglan-dular stages elaborate the conducting airways; the lattercanalicular, saccular, and alveolar stages are characterizedby reduction of mesenchyme and vascularization to forma thin air-blood barrier. Birth does not signal the end of

lung development. There is a continuing complex processof lung growth after birth, permitting changing relation-ships of airway size, alveolar size, and surface area. Atbirth, the newborn infant, with approximately 50 millionalveoli, has the potential to add another 250 million alve-oli and increase surface area from approximately 3 to 70m2.2 There are more than 40 different cell types in thelung, with different functions.2 How they establish theirfinal appropriate physical and numerical relationshipswith each other is still unknown.

Embryonic Stage (3 to 7 Weeks)The human fetal lung originates as a ventral diverticulumthat arises from the laryngotracheal groove of the foregutendoderm.2 The laryngotracheal groove separatesdorsoventrally from the primitive esophagus to form thetracheal rudiment and, at the same time, gives rise laterallyto the two primary bronchial buds (Figure 2–1A). Thelung bud grows into adjacent splanchnic mesoderm whereit is induced to branch repeatedly, giving rise to the futurerespiratory tree. This primitive lung bud is lined by endo-dermally derived epithelium, which differentiates intospecialized cells that line both the conducting and respira-tory airways.3 Mesenchymal cells condensed around theprimitive airways give rise to blood vessels, smooth mus-cle, cartilage, and other connective tissues of the lung.4

Pseudoglandular Stage (7 to 16 Weeks)During the pseudoglandular stage, the stage of branchingmorphogenesis, there is rapid proliferation of the primi-

CHAPTER 2

PRENATAL LUNG DEVELOPMENTGAIL H. DEUTSCH, MD, AND HALIT PINAR, MD.

TABLE 2–1. Stages of Lung Development

Stage Developmental Age Major Events

Embryonic 3–7 wk Lung budding from the foregut endoderm, with formation of trachea and mainstem bronchi

Pseudoglandular 7–16 wk Airway division completed, with formation of 25,000 terminal bronchioles; cartilage, smooth musclederived from mesenchyme

Canalicular 16–24 wk Capillarization, with acinar formation; type I and II epithelial cells first differentiate

Saccular 24–36 wk Progressive thinning of epithelial cells; terminal saccular formation; surfactant production

Alveolar (postnatal) 36 wk through infancy Appearance of true alveoli; alveolar septation and expansion of air spaces40 wk through infancy

wk = weeks.

tive airways so that all airway divisions are more or lesscompleted by 16 weeks.5 This translates into 12 to 17branches in the upper lobes, 18 to 23 branches in themiddle lobes, and 14 to 23 branches in the lower lobes.2

The branching pattern does not change after this stageand is similar to that of the adult lung.5 The most periph-eral structures, the terminal bronchioles, will further dif-ferentiate to form the future respiratory bronchioles andalveolar ducts. The name pseudoglandular is derivedfrom the histologic appearance of the lung, which oncross section consists of hollow tubular-like structures(glands) surrounded by clusters of mesenchymal cells(see Figure 2–1B). The columnar epithelial cells that linethe tubules contain cytoplasmic glycogen; a few becomeciliated as early as 8 weeks while others begin to differen-tiate into goblet cells.6 During this period, cartilagebegins to form around the larger airways and smoothmuscle forms around airways and major blood vessels.5

Canalicular Stage (16 to 24 Weeks)The canalicular stage is so named because the potentialair spaces are being “canalized” and approximated by anetwork of capillaries6,7 (see Figure 2–1C). The pul-

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monary acinar units, which eventually contain alveolarducts, alveolar sacs, and alveoli, develop during thisperiod. “Acinus” is the term applied to the gas exchangeunit associated with a single terminal bronchiole. It fol-lows that primitive lung lobules will have formed by thebeginning of the canalicular stage. Each lobule containsthree to five terminal bronchi and, by the end of 27 weeks,approximately 25,000 terminal bronchioli.6 A gradualdecrease in mesenchymal tissue results in close apposi-tion of the pulmonary vasculature to the epithelium. By20 to 22 weeks’ gestation, type I and type II alveolar cellscan be differentiated from the cuboidal epithelial cells inthe most peripheral parts of the lung.6 Lamellar bodiesassociated with surfactant synthesis begin to appear inthe cytoplasm of type II cells. Type I alveolar lining cells,which differentiate from type II cells, begin their flatten-ing process and attenuate to provide an air-blood inter-face. The conducting airways have fully developedsmooth muscle, and lymphatic structures now begin toappear. The developing pulmonary arteries and veins fol-low the development of the branching airways but lagbehind it somewhat.8,9 By the end of the canalicularperiod, the potential air-blood barrier is thin enough to

A.

B.

Prenatal Lung Development / 3

FIGURE 2–1. Schematic and light-microscopic appearance of human fetal lung at different stages of development.

C.

D.

E.

support gas exchange.10 The bronchial artery system maybe as critical for lung development as the pulmonaryartery although the role of the bronchial artery in pul-monary differentiation and growth is currentlyunknown.6 It has been suggested that the most peripheralparts of the developing lung are supplied only by the pul-monary arterial vasculature.11

Saccular Stage (28 to 35 Weeks)The term “saccule” derives from the saclike appearance ofthe most peripheral air spaces, which represent the futurealveolar ducts and alveoli (see Figure 2–1D). According toBoyden, each acinus supplied by a terminal bronchiolehas three to four respiratory bronchioles that end in atransitional duct from which the saccules arise.2 Themajor changes that occur during the saccular stage arefurther compression of the intervening interstitium, thin-ning of the epithelium, and the beginning of alveolar sep-tation, with the formation of small mesenchymal ridges.There is lengthening and widening of saccules distal tothe terminal bronchioles and the addition of the last gen-erations of future alveolar spaces.2 Continual differenta-tion of type I and II alveolar cells occurs during thisperiod, so that the alveolar epithelial cells become themost abundant epithelial cells in the lung. The flattenedtype I alveolar cells make up the majority of these cells.Type II cells, ultrastructurally distinguished by their pro-duction of lamellar bodies, expand in size and number,with increased storage of surfactant lipids and less cyto-plasmic glycogen.12,13

Alveolar Stage (36 Weeks through Infancy)Several million alveoli form before birth although this finalstage of lung development primarily occurs during postna-tal life.14 The beginning of this stage is not sharply defined;

4 / Chronic Obstructive Lung Disease

some alveolar formation probably begins a few weeks ear-lier. Alveolar formation is closely linked to the deposition ofelastin in the saccular lung.15 Terminal saccules becomeinvaginated by protrusions from the wall of epithelial cellsand contain a double-walled capillary system16 (see Figure2–1E). These protrusions elongate and thin, forming prim-itive alveoli that at first resemble shallow cups and thenbecome deeper as development continues.

Postnatal Lung GrowthDuring the postnatal phase, lung growth is geometric,and there is no increase in airway number. There is pro-portionately less growth in the conducting airways incomparison with alveolar-capillary tissue. Estimates ofthe number of alveoli at birth vary widely, but an averageof 50 million is generally accepted.17 These alveoli pro-vide a gas-exchanging surface of approximately 3 to 4m2.17 Alveoli greatly increase in number after birth, toreach 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 afterbirth, with morphologic changes in the septa.Alveolarization occurs through the formation of numer-ous short, blunt tissue crests or ridges, and their protru-sion into alveolar sacs increases the internal surface of thelung. The development of the alveolar crest is closelylinked with elastin deposition and the local proliferationof interstitial and epithelial cells.15,18

Epithelial Cell DifferentiationMaturation of the epithelium during development startsin the proximal airways and progresses distally into theintrapulmonary airways.20 Epithelial cell lineages arearranged in a distinct proximal-distal spatial pattern inthe airways, which become morphologically apparent

FIGURE 2–2. Differentiated airway epithelial cells in the proximal trachea and distal alveoli in a human term infant.

during the pseudoglandular stage.21 At least 11 differentepithelial cell types have been described in the conduct-ing and respiratory portions of the lung.20 The basal,secretory, and ciliated cells are the major cell types con-stituting the pseudostratified portion of the proximal tra-cheobronchial epithelium while type I and type IIalveolar cells make up the distal respiratory epithelium(Figure 2–2). The lineage relationships between the dif-ferent cell types have not been delineated, and the exis-tence and identity of progenitor cells, which may play arole in lung injury and repair, are currently under study.There is some evidence from cell kinetic studies to sug-gest that basal cells, Clara cells, and type II alveolar cellsare the primary progenitor cells for the pulmonaryepithelium.22–27 Following acute lung injury, Clara cellsand type II cells regain the capacity to proliferate andrepopulate the damaged bronchiolar and alveolar epithe-lium, respectively.22–24,28 Lineage studies have notaddressed the relationship of pulmonary neuroendocrinecells to the putative stem cells (type II cells and Claracells) although neuroendocrine cells differentiate mor-phologically before any other epithelial cell type.29

Bombesin, a peptide secreted by pulmonary neuroen-docrine cells, stimulates branching morphogenesis andlung maturation, in culture and in vivo.30,31

The onset of cellular differentiation is signaled by theexpression of differentiated gene products. Lung-specificgene products include the surfactant proteins (surfactantproteins A, B, C, and D [SP-A, SP-B, SP-C, and SP-D])and Clara cell secretory protein (CCSP).28,32–35 The sur-factant-associated proteins are abundant phospholipid-associated proteins that are expressed primarily inalveolar type II epithelial cells32,33,36 although SP-A andSP-B are also detected in subsets of nonciliated epithelial(Clara) cells of the conducting airways and tracheo-bronchial glands.37–40 Expression of these genes is extin-guished when type II cells undergo terminaldifferentiation to type I cells that constitute most of thegas exchange surface of the alveolus. For the most part,CCSP is a marker for the proximal Clara cells of the bron-chiolar epithelium34,35 (Figure 2–3). Recent transgenicstudies support a potential role for CCSP in the control ofinflammatory responses; CCSP-deficient mice have anincreased sensitivity to hyperoxia-induced lung injurycharacterized by lung edema and induction of pro-inflammatory cytokine messenger ribonucleic acids(mRNAs).41 Hepatocyte nuclear factor/forkhead homo-logue 4 (HFH-4), a winged helix transcription factor, hasbeen implicated in ciliated cell development (see Figure2–3), with mutational abnormalities in the mouse similarto those seen in Kartagener’s syndrome.42

Early on in embryonic development, the undifferenti-ated epithelium coexpresses several lineage markers,including SP-A, CCSP, and calcitonin gene-related pep-tide, a marker of neuroendocrine cells.43 It is during the

Prenatal Lung Development / 5

pseudoglandular stage that pulmonary epithelial cell lin-eages become restricted to proximal and distal regions ofthe airways. Of interest, after bleomycin-induced injuryin the adult lung, there is again coexpression of lineage-specific markers, suggesting that a progenitor-type cellmay be re-enlisted during epithelial repair.44 Tissuerecombination experiments have demonstrated that aproximal versus a distal epithelial cell phenotype is dic-tated by soluble factors from the adjacent mes-enchyme.45,46 Within a restricted time window ofdevelopment, distal lung mesenchyme can reprogram rattracheal epithelium to express type II cell differentiation,and conversely, tracheal mesenchyme can induce distallung epithelium to express tracheal cytodifferentia-tion.45,46 These findings underscore the importance ofepithelial-mesenchymal interactions in coordinating theprecise temporospatial pattern of lung development (seefurther discussion below).

Analysis of transcriptional elements in the surfactantprotein genes and CCSP has provided an understandingof the shared mechanisms of gene regulation and expres-sion in respiratory epithelial cells. A homeodomain pro-tein, Nkx2.1 (also known as thyroid transcriptionfactor-1 [TTF-1]), and thyroid enhancer binding protein[T/ebp] plays an essential role in several phases of lungdevelopment, including epithelial cell lineage determina-tion.47–50 Nkx2.1 is the earliest marker of the developingrespiratory epithelium, with onset of expression at thetime of lung bud formation from the foregut endoderm.51

Distribution of Nkx2.1 expression in the fetal lungincludes respiratory epithelial cells of the trachea, bronchi,and developing respiratory tubules although expression ismost robust in distal alveolar cells51,52 (see Figure 2–3). Invitro, Nkx2.1 binds and activates SP-A, SP-B, SP-C, andCCSP promoter elements as well as Nkx2.1 itself.53–57

Mice with a null mutation of Nkx2.1 have tracheoe-sophageal fistulas and fail to form bronchiolar and alveo-lar structures distal to the lobar bronchi.48–50

Pulmonary-specific gene expression including SP-B, SP-C,and CCSP is extinguished within transgenic lungs, whichdo, however, contain ciliated and mucus-secreting cells.50

Thus, Nkx2.1 is felt to function as a “master gene” thatinduces and maintains lung morphogenesis as well as dif-ferentiation of certain epithelial cell lineages.

Cellular and Molecular Mechanisms ofLung Development

During the past few years, major progress has been made inidentifying key determinants of branching morphogenesisand cellular differentiation within the lung. New insightshave been derived through the characterization of signalingmolecules that regulate gene expression and that are func-tionally conserved through evolution in invertebrate speciessuch as Drosophila and Caenorhabditis elegans, which arefunctionally conserved through evaluation.58,59 Null muta-

tions in mouse models have identified several nuclear tran-scription proteins, which determine respiratory-cell fateand pattern formation via the activation and repression ofdownstream target genes. The recurrent theme emergingfrom these studies is that lung development extends in acoordinated manner from successive epithelial-mesenchy-mal interactions. Growth factor signaling and induction ofresponsive transcription factors mediate this interplaybetween developing epithelial and mesenchymal structures.The following section will detail specific cellular and mole-cular events during lung morphogenesis, which may berecalled during tissue injury and regeneration.

Early Lung DifferentiationThe lung shares a common embryological origin of otherforegut derivatives including the liver, pancreas, gastroin-testinal tract, and thyroid (Figure 2–4). While many stud-

6 / Chronic Obstructive Lung Disease

ies have focused on events critical to organ morphogenesisand terminal cell differentiation,60 few have revealed howinitial cell type choices are made. In Drosophila, the initialspecification of tissues within the gut endoderm is causedby signals from overlying mesoderm.61 Studies within theliver have demonstrated that changes in gene expressionand tissue morphology define discrete phases in organdevelopment.62 Hepatic gene expression and differentia-tion begin in the foregut endoderm immediately after theendodermal epithelium interacts with the cardiac meso-derm, in the 8.5-day-old embryo.63 Recent studies havedemonstrated that fibroblast growth factors (FGFs) ema-nating from the cardiac mesoderm are sufficient to divertclosely apposed endoderm to express the genetic programfor liver instead of that for pancreas, which is the defaultpathway of the ventral foregut endoderm64,65 (see Figure2–4). Although the signals that are important in early lung

FIGURE 2–3. 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 thealveolar stage of lung development, shown on dark-field photomicrographs of serial sections. Expression of N-myc is not detectable in thexenograft 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 proximaltracheal and bronchiolar epithelium. (T = trachea; b = bronchi; a = alveoli.)

specification are currently unknown, preliminary studiessuggest that dosage-dependent growth factor signalingfrom the cardiac mesoderm might play a role (G.H.Deutsch, unpublished observations) (see Figure 2–4).

The early pattern of expression of the homeobox geneNkx2.1 is consistent with its role in the morphogenesis ofthe lung. Targeted disruption of the gene has demon-strated that while Nkx2.1 is not required for the initialspecification of the lung primordia, Nkx2.1 is essentialfor further pulmonary development and cell differentia-tion.49,66 Study of the cis-acting regions in the Nkx2.1promoter reveals that Nkx2.1 has multiple binding sitesfor both ubiquitous and specific transcription factors,including those of the hepatocyte nuclear factor (HNF)and GATA zinc finger families,67–69 which are requiredfor the development of the foregut endoderm;70–72 HNF-3– binding sites have also been identified in the SP-A, SP-B,and CCSP promoters.53,73,74 The HNF-3β null mutationresults in an early embryonic lethal phenotype, in whichthe primitive foregut endoderm fails to close into a tube;consequently, there is no development of foregut deriva-tives such as the lung.75

Prenatal Lung Development / 7

Pattern Formation of the LungIn the fully differentiated lung, epithelial cells from theproximal tracheal, and bronchial tubules greatly differfrom the distal alveolar tubules in morphology and geneexpression. The general principle of pattern formationstates that cells are instructed to assume a specific func-tion according to their position along a concentrationgradient of a signal. Cells located closer to the source ofsignal recognize a higher concentration and assume a dif-ferent phenotype from those cells located at a distance,which recognize low concentrations of the same signal.Recognition of their position is followed by the activationor repression of target genes, which ultimately determinethe regional differences in cell identity along the lungaxis. We have previously discussed how the presence ofproximal versus distal mesenchyme can dictate cell differ-entiation in adjacent epithelial cells. Early studies havealso shown that the quantity of mesenchyme is influentialin inducing a specific epithelial cell fate.76 A smallamount of recombined mesenchyme is able to direct celldifferentiation toward a bronchiolar phenotype; however,an increased amount of the same mesenchyme willinduce the epithelial cells to differentiate toward an alve-olar phenotype.76 This study implies that variable con-centrations of the same signals or morphogens expressedby the mesenchyme can induce different cell fates in iden-tical epithelial cells.

Homeobox (Hox) genes are transcription factors,which are known to regulate body axis patterning andspecification of regional identity during embryonicdevelopment,58,77 although the mechanisms by whichthey do so are largely unknown. The Hox genes likely reg-ulate downstream factors that influence cell proliferation,migration, and cell death. The pattern of Hox geneexpression at the beginning of lung development is con-sistent with its role in elaborating the proximal-distal ori-entation of the lung and in branching morphogenesis.78–80

In the mouse, Hoxb-3 and Hoxb-4 are expressed in themesenchyme of the entire developing lung (proximal anddistal) while Hoxb-2 and Hoxb-5 expression arerestricted to the mesenchyme of distal lung buds.78 Thelevels of Hox-gene expression decline with advancinggestational age. Hoxb genes may be involved in patterningthe developing foregut by specifying regional differencesin lung mesenchyme. Hoxb-3 transactivates the ratNkx2.1 gene promoter,47 which infers that Hoxb-3 couldbe involved in lung-specific gene regulation throughNkx2.1 as an intermediate. In cultured mouse embryoniclung, retinoic acid induces Hoxa-5, Hoxb-5 and Hoxb-6gene expression,81 while Hoxb-5 is negatively regulatedby epidermal growth factor (EGF) and transforminggrowth factor-β (TGF-β).82 Expression of Hox genes inthe adult lung may be relevant to neoplasia as well as topathologic disease states such as pulmonary hypertensionand emphysema.79,83 Retinoids have also been shown to

FIGURE 2–4. Endodermal derivatives (depicted in green) in a 6-week human embryo, saggital view. Signals from the cardiac meso-derm are hypothesized to play a role in liver, lung, and thyroiddevelopment. (Redrawn after Larsen, W.J. 1993, Human Embryology.Churchill-Livingstone, New York)

influence the proximal-distal pattern of lung develop-ment. Retinoic acid, in dose-dependent concentrations,has been demonstrated to favor the growth of proximalairways and gene expression at the expense of distalstructures.84 It is probable that Hox genes mediate theretinoic acid–induced alteration in lung patterning.81

Bone morphogenetic proteins (Bmps), members of theTGF-β–related family of signaling molecules, are alsoimplicated in the control of the proximal-distal patterningof the lung and in branching morphogenesis.85,86 Bonemorphogenetic protein 4 (Bmp-4) ribonucleic acid (RNA)is restricted to the tips of distal buds and to the adjacentmesenchyme, locally inhibiting endoderm proliferationand forcing the outgrowth of lateral branches85 (Figure2–5). Inhibition of Bmp signaling in transgenic animalsresults in complete proximalization of the respiratoryepithelium, including ciliated cells in the most distal por-tions of transgenic lungs. It is hypothesized that Bmps pro-vide a concentration gradient to control proximal versusdistal differentiation of the lung endoderm.86 Endodermalcells located at the periphery of the lung, which areexposed to high levels of Bmp-4, maintain or assume a dis-tal identity while cells below a certain threshold of theBmp-4 signal initiate a proximal differentiation program.

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Regulation of Branching MorphogenesisElegant recombination experiments45,87–90 have demon-strated that mesenchyme is necessary for initial lung bud-ding and branching and that the mesenchymesurrounding the trachea and mainstem bronchi has a dif-ferent regional identity than the mesenchyme surround-ing the distal lung bud. There is abundant data to suggestthat soluble growth factors are likely mediators of thisinteraction.91–93 Candidate growth factors whose signal-ing peptides and cognate receptors are expressed in theearly embryonic mouse lung include FGFs, EGFs, TGFs,hepatocyte growth factor (HGF), and platelet-derivedgrowth factor (PDGF).94–99 Their influences on lungdevelopment have been demonstrated by gain vs. loss offunction experiments in embryonic mouse lung organcultures and in transgenic mice. In general, growth factorreceptors with tyrosine kinase intracellular signalingdomains (EGF, FGF, and PDGF receptors [EGFR, FGFR,and PDGFR]) stimulate lung morphogenesis100–105

whereas those with serine/threonine kinase intracellularsignaling domains (such as the TGF-β family) areinhibitory.106–108

Branching of the respiratory tracheae in Drosophila isconsidered a conserved genetic paradigm for the mor-phogenesis of the mammalian lung. Primary branchingin Drosophila is spatially regulated by the branchless gene,of which FGF-10 is the mammalian homologue.109 Thebranchless gene activates the breathless gene in trachealcells, to induce their migration and thus primary branch-ing.110,111 Breathless is homologous to the FGFR family.This FGF homologue-signaling pathway appears to beevolutionarily conserved in mammals. Fibroblast growthfactor-1 (FOF-1), FGF-7, and FGF-10 are synthesized bythe pulmonary mesenchyme, and they bind to and acti-vate FGFRs on endodermal cells of the developing lungbuds.95,104,112 Fibroblast growth factor-10 may play achemotactic role similar to that of branchless in inducingepithelial branching and cell migration. Transcripts ofFGF-10 are present at discrete sites in the mesenchymeand have been demonstrated to govern the directionaloutgrowth of lung buds93 (see Figure 2–5). At earlystages, FGFR-2, the receptor for FGF-10, is expressed athigh levels along the entire proximal-distal axis of the res-piratory-tract epithelium.113 Disruption of FGF signalingby the generation of FGF-10–deficient mice, or expres-sion of a dominant negative form of its receptor (FGFR-2), interferes with branching morphogenesis andperipheral cell differentiation; transgenic lungs are char-acterized by only a trachea or a trachea and two primarybronchi.102,114,115 Thus, it appears that the elaboration ofbranched airways involves the directional movement ofepithelial cell precursors toward a localized source of anFGF ligand, either by cell migration or by outgrowth ofepithelial buds.

FIGURE 2–5. Epithelial-mesenchymal interactions in the develop-ing lung during branching morphogenesis. Factors are represented onlyat sites where the expression is most abundant. Fibroblast growth fac-tor (FGF) signals in the mesenchyme act as a chemotactic focus for theepithelium during lung budding. As the bud extends toward the FGFsignals in the mesenchyme, endoderm cells that escape a high bonemorphogenic protein 4 (Bmp-4) signal assume a proximal cellular iden-tity; those that retain a high Bmp-4 signal take on a distal identity. Ahigh concentration of Bmp-4 signal also serves to locally inhibit endo-derm proliferation, thereby inducing the lateral outgrowth of new air-way branches. Sonic hedgehog (Shh) at the distal tips functions todownregulate FGF-10 expression in the mesenchyme, which limitslocal budding. Transforming growth factor-β (TGF-β) signaling also pre-vents local budding, by decreasing endodermal proliferation and bystimulating synthesis of matrix components at branch points.(FGFR =fibroblast growth factor receptor; Hoxb = homeobox b.)

Despite high-sequence homology and use of the samereceptor, FGF-7 and FGF-10 exert distinct effects on thedeveloping lung. Rather than being chemotactic, FGF-7influences airway branching by promoting epithelial cellproliferation and expansion.91,104,116,117 Misexpression ofFGF-7 in mice during the pseudoglandular stage disruptsbranching morphogenesis and epithelial cell differentia-tion.118 Transgenic lungs have large cystic spaces thatresemble human congenital cystic adenomatoid malfor-mations. Fibroblast growth factor-7 as a differentiationfactor for the developing lung has been demonstrated inlung organotypic cultures. In the absence of mesenchymeor serum, exogenous FGF-7 can induce a type II cell–likephenotype.117,119 Despite evidence that FGF-7 plays arole in lung development, mice homozygous for a nullmutation in the FGF-7 gene exhibit no apparent pul-monary abnormalities.120 This suggests that other mem-bers of the FGF family (perhaps FGF-10) can serve asfunctionally redundant molecules.

Many studies have shown that certain signaling andgrowth factors are negative regulators of lung epithelialproliferation, which may play a role in counteracting thebud-promoting effects of FGFs. Fibroblast growth factor-10 is downregulated in lungs of transgenic mice overex-pressing sonic hedgehog (Shh),85 which is a secretedsignaling protein that induces gene expression and prolif-eration of adjacent mesenchymal target cells in thelung.93,121 Low levels of Shh are expressed throughout theprimitive respiratory epithelium, with high levels seen atthe distal tips85,122 (see Figure 2–5). Treatment of embry-onic lung mesenchymal cells with recombinant Shhmarkedly inhibits FGF-10 mRNA expression.123 In theabsence of Shh, FGF-10 expression is no longer restrictedto focal areas but becomes widespread throughout the dis-tal mesenchyme.124 This finding raises the possibility thatone of the roles for Shh signaling in branching is to spa-tially restrict FGF-10 levels in the mesenchyme surround-ing the bud tips.124 Sonic hedgehog–null mutant micehave tracheoesophageal fistulas and bilateral rudimentarysacs, due to failure of branching and growth after forma-tion of the primary lung buds.121,124 The lung mes-enchyme shows enhanced cell death and decreased cellproliferation.121 Conversely, overexpression of Shh leadsto hypercellularity of the lung, with an absence of normalalveolar septa because of excessive mesenchyme betweenthe alveolar spaces.125 Sonic hedgehog expression in theendoderm may also be essential for the proliferation anddifferentiation of the mesoderm, which in turn is requiredfor proper differentiation of the primitive pulmonaryepithelium.121 Of interest, treatment of cultured lungswith retinoic acid increases the level of Shh expressionwhile decreasing the level of FGF-10 expression and thedegree of airway branching.126,127 These data support theconcept that distinct signaling and transcriptional path-ways are interrelated during lung development.

Prenatal Lung Development / 9

The TGF-βs represent a family of peptides involved incell growth and apoptosis control, connective-tissue for-mation, and embryonic development.128 Transforminggrowth factor-β1, TGF-β2, and TGF-β3 are indirectmitogens for certain mesenchymal cells and are markedstimulators of extracellular matrix deposition, includingcollagen, fibronectin, and proteoglycans.128 The TGF-βsare also potent inhibitors of epithelial cell proliferation.The TGF-β1 protein is localized at the interface betweenepithelial and mesenchymal cells, particularly around thebronchiolar ducts and airway branch points.96,129 Overexpression of members of the TGF-β family, both inorgan culture and in transgenic animals, has a negativeregulatory effect on branching morphogenesis106,108

abrogation of TGF-β type II receptor signaling (eitherwith antisense oligodeoxynucleotides or with blockingantibodies), stimulates lung morphogenesis twofold tothreefold, and increases expression of the distal NKx2.1and SPC genes.108

Transforming growth factor-β1 has been shown todownregulate the expression of N-myc and branchingmorphogenesis in murine organ lung cultures.106 The N-myc gene may play a role in lung development bymaintaining cells in an undifferentiated state; its expres-sion is most abundant in undifferentiated progenitor cellsand primitive tumors.130,131 Targeted disruption of theN-myc gene results in embryonic lethality during thepseudoglandular stage of development, with a cleardefect in branching morphogenesis; homozygous lungsconsist of only a trachea and mainstem bronchi.132–135

Factors Related to AlveolarizationDuring postnatal life, FGFR-3 and FGFR-4, receptors thatbind several of the FGFs, cooperate to regulate alveoliza-tion; a double mutation of these genes results in a post-natal lethal pulmonary phenotype in which there isfailure of alveolar septation, resulting in an emphysema-tous appearance.136 Lungs of transgenic mice demon-strate an aberration of elastin synthesis. Mutants lackingthe growth factor PDGF-A also exhibit a defect in pul-monary alveogenesis, but this phenotype is apparentlycaused by a loss of alveolar smooth-muscle myofibro-blasts and parenchymal elastic fibers.137

The defect in alveolization in neonatal EGF recep-tor–deficient mice is also associated with impairedbranching morphogenesis.105 Inactivation of the EGFreceptor affects type-II pneumocyte maturity, withdecreased expression of the distal SPC marker. In con-trast, exogenous EGF accelerates alveolar type-II cell dif-ferentiation in fetal lungs.138

Pulmonary VasculogenesisLike lung morphogenesis, pulmonary vascular develop-ment is a highly regulated process involving not only theformation and differentiation of new vessels but also the

inhibition of further vascular proliferation.8,139 Directepithelial-mesenchymal interactions may be essential forvessel formation,140 with the participation of growth fac-tors and matrix proteins. Pulmonary endothelial cell dif-ferentiation is still poorly understood, but studies suggestthat receptors for the vascular endothelial growth factor(VEGF), located within the mesenchyme, facilitate earlyvessel formation within the embryo.140,141 Growth factorssuch as FGF-2142 and VEGF143,144 are known stimulantsof endothelial cell migration, proliferation, and transdif-ferentiation, leading to the appearance of vascular struc-tures. Misexpression of VEGF in transgenic mice underthe control of the SP-C promoter/enhancer induces grossabnormalities in lung morphogenesis, characterized byan increase in vascularity and concomitant decrease indistal acinar tubules and mesenchyme.145 Inhibition ofthe VEGF receptor KDR (VEGFR-2) also influences lungmorphology by reducing vascular density and alveolar-ization in the newborn rat.146

Endothelial monocyte-activating polypeptide II(EMAPII), localized primarily to the mesenchyme, hasbeen demonstrated to have a negative effect on neovascu-larization in the developing lung.147 Its location corre-lates with the same area of expression as other regulatorsof vessel formation, including the VEGF receptors 1 and2.147 Its continual expression within large vessels in adult-hood leads one to theorize that EMAPII might functionpostnatally to stabilize or maintain existing vascularstructures.147

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