Key Mechanisms of Early Lung Development

Abstract

Lung morphogenesis requires the integration of multiple regulatory factors, which results in a functional air-blood interface required for gas exchange at birth. The respiratory tract is composed of endodermally derived epithelium surrounded by cells of mesodermal origin. Inductive signaling between these 2 tissue compartments plays a critical role in formation and differentiation of the lung, which is mediated by evolutionarily conserved signaling families used reiteratively during lung formation, including the fibroblast growth factor, hedgehog, retinoic acid, bone morphogenetic protein, and Wnt signaling pathways. Cells coordinate their response to these signaling proteins largely through transcription factors, which determine respiratory cell fate and pattern formation via the activation and repression of downstream target genes. Gain- and loss-of-function studies in null mutant and transgenic mice models have greatly facilitated the identification and hierarchical classification of these molecular programs. In this review, we highlight select molecular events that drive key phases of pulmonary development, including specification of a lung cell fate, primary lung bud formation, tracheoesophageal septation, branching morphogenesis, and proximal-distal epithelial patterning. Understanding the genetic pathways that regulate respiratory tract development is essential to provide insight into the pathogenesis of congenital anomalies and to develop innovative strategies to treat inherited and acquired lung disease.

Keywords

branching morphogenesis epithelium mesenchyme patterning signaling specification

LANDMARKS IN LUNG DEVELOPMENT

During gestation, the fetal lung undergoes significant morphological changes to provide an organ capable of maintaining respiration and gas exchange at birth. Lung development initiates with establishment of a lung cell fate from the ventral foregut endoderm [1]. Subsequently, the laryngotracheal groove arises from the floor of the foregut as a ventral outpouching. The proximal portion of this structure gives rise to the larynx and trachea, whereas the distal portion gives rise to 2 ventrolateral buds that will form the left and right primary bronchi. Division of the foregut by a longitudinal septum separates the trachea and dorsal esophagus. The bronchial buds grow into the adjacent splanchnic mesoderm and undergo reiterative branching to give rise to the future respiratory tree. Although lung development is a continuous process, 5 developmental stages have been delineated based on anatomic and histological characteristics [2] (Fig. 1). Functionally, the embryonic and pseudoglandular stages elaborate the conducting airways of the lung; the later canalicular, saccular, and alveolar stages are characterized by vascularization and reduction of mesenchyme to form a thin air-blood interface. There is a continuing complex process of lung growth postnatally, resulting in rapid expansion of the gas exchange surface area. Concomitant with lung morphogenesis is cell differentiation within the respective tissue compartments. The lung is lined by endodermally derived epithelium, which differentiates into several specialized cell types, including ciliated, basal, Clara, and goblet cells in the proximal airways, and type I and type II cells in the distal alveoli [3]. Mesenchyme originating from splanchnic mesoderm gives rise to the vascular network, smooth muscle, cartilage, and other connective tissue elements that develop in parallel and surround the airways [4].

Figure 1.

Timeline of lung development. Morphological stages and major events in the developing human and mouse lung.

Development of the respiratory tract thus requires a coordinated sequence of processes, which when altered could disrupt key phases of lung development, including specification, growth, and differentiation. Gene ablation in mice (through direct disruption of a genetic locus or conditional gene targeting) and embryonic and lung explant assays have permitted identification of factors required for distinct phases in lung development. The recurrent theme emerging from these studies is that lung development results from successive epithelial-mesenchymal interactions. Growth factor signaling and induction of responsive transcription factors orchestrate this interplay between developing epithelial and mesenchymal structures by influencing cell fate, proliferation, migration, and differentiation. In the subsequent sections, we highlight molecular mechanisms that regulate fundamental aspects of lung development, including specification of a lung cell fate, primary lung bud induction, tracheoesophageal septation, branching morphogenesis, and proximal-distal epithelial differentiation. Cellular and transcriptional programs mediating alveolarization and injury and repair of the lung are not discussed in this review [5]. Molecular regulation of pulmonary vascular development was recently covered in this journal by Galambos and DeMello [6], and reviews of the pulmonary neuroendocrine system and surfactant dysfunction disorders are forthcoming.

SPECIFICATION OF A LUNG CELL FATE

The lung shares a common embryological origin with other foregut endoderm derivatives, including the liver, pancreas, thyroid, and gastrointestinal tract. During early somitogenesis, approximately 8.0 days postcoitum in the mouse, the anterior portion of the embryo invaginates to form the foregut, of which the ventral portion ultimately gives rise to the thyroid, thymus, lung, liver, and ventral pancreas [7] (Fig. 2). Development of the lung can first be identified morphologically by mouse embryonic day 9.5 (E9.5) as 2 lateral buds of proliferating endoderm cells present in the ventral foregut. However, before organ bud formation, between E8.5 and E9.0, presumptive organ domains are delineated by the regional expression of tissue-specific genes along the anterior-posterior axis of the gut endoderm [8,9]. For example, Hex is expressed in the thyroid and liver domains, Nkx2.1 (also named thyroid transcription factor 1 [Titf1] and thyroid enhancer binding protein [T/EBP]) in the thyroid and lung domains, and Pdx1 (pancreatic duodenal homeobox 1) in the pancreas domain [10 –12]. Although many studies have focused on branching morphogenesis and terminal cell differentiation of the lung, the molecular mechanisms underlying the earliest step of lung development, specification, or induction of a lung cell fate from the foregut endoderm are not well understood. Progenitors of the trachea and lung are first visualized in the prospective lung region of the foregut by the expression of Nkx2.1 at E9.0 (Fig. 4A) [13]. Although the timing of when a respiratory cell fate is induced from the foregut endoderm remains incompletely defined, in vitro studies using mouse embryo endoderm explants indicate that diffusible fibroblast growth factor (FGF) signaling emanating from the neighboring heart (cardiac mesoderm) is essential for lung specification [9]. Cardiac mesoderm and FGF influences the anterior-posterior fate of the ventral foregut endoderm in a dose-dependent manner (Fig. 3). Specifically, endoderm cultured in isolation defaults to a pancreatic fate, whereas endoderm exposed to an increasing amount of cardiac mesoderm or FGF results in successive induction of a liver and then a lung or thyroid fate [9,14,15]. These findings correlate with the temporal activation and spatial domain of lineage-specific markers in vivo as well as the intensity of FGF expressed in the heart. Pdx1 is the earliest cell-specific marker to be activated in the foregut endoderm, and its expression is confined to the ventral lip, distal to the developing heart; in contrast, Nkx2.1 comes on in the prospective lung field a half a day later and close to the heart [9,15].

Figure 2.

Schematic view of embryos during lung specification and morphogenesis stages. The ventral portion of the foregut endoderm gives rise to different cell lineages, including the lung. The regional expression of tissue-specific genes along the anterior-posterior (A-P) axis of the gut endoderm delineates organ-specific domains, well before bud formation at the morphogenesis stage of development.

Figure 3.

Proposed model of ventral foregut specification. An increasing threshold of fibroblast growth factor signaling from the heart (red) results in successive induction of a pancreas (blue), liver (pink), and lung/thyroid (green) cell fate from the adjacent foregut endoderm.

Figure 4.

Key phases of early mouse lung development. (A) Lung and tracheal progenitors (arrow) are identified at embryonic day 9.0 (E9.0) by the expression of Nkx2.1 (purple), which also denotes the thyroid (arrowhead). (B) At E9.5, the right and left lung buds arise from the ventral-lateral aspect of the foregut, invading into the adjacent mesoderm. (C) Concurrent with primary lung bud formation, the tracheal primordium forms from the ventral aspect of the foregut, separating from the dorsal foregut/primitive esophagus by a tracheoesophageal septum. (D) The 3.7-kb human Sftpc promoter is expressed at high levels in the developing and mature lung and has been used to drive ectopic expression of many genes of interest (here the reporter gene beta-galactosidase blue staining) into the early respiratory epithelium [112] (Sftpc-Cre mice provided by Dr. Anne-Karina Perl and Dr. Jeffrey Whitsett, Cincinnati Children's Hospital Medical Center). (E) From E10.5 to E16.5, the epithelium undergoes branching morphogenesis, a reiterative process involving bud outgrowth, elongation, and dichotomous subdivision of terminal units. (F) As this occurs, a proximodistal gradient is established in the developing lung with bronchial and alveolar differentiation, distinguished by CC10 (red) and Sftpc (green) expression, respectively (Sftpc antibody provided by Dr. Susan Wert, Cincinnati Children's Hospital Medical Center).

All foregut derivatives are specified within the domain of Foxa1 and Foxa2 gene expression, transcription factors that play a significant role in foregut differentiation and morphogenesis [16,17]. A null mutation in Foxa2 results in failure of the foregut endoderm to close into a tube; consequently, no organs, including the lung bud, can form [18]. In vitro, Foxa2 activates transcription of the homeodomain gene Nkx2.1 [19] and is thought to cooperate with Nkx2.1 to define cell lineage within the pulmonary epithelium [11,13,20]. Early on, Nkx2.1 is expressed in all pulmonary epithelial cells but becomes progressively restricted to proximal Clara cells and distal type II cells with development [21,22]. Targeted disruption of this gene has demonstrated that while Nkx2.1 plays an essential role in several phases of lung development, including tracheoesophageal septation, branching morphogenesis, and terminal cell differentiation, it is not required for specification [11,13,23]. Mice deficient in Nkx2.1 form lungs, although they are arrested distal to the lobar bronchi and contain only ciliated and mucus-secreting cells, characteristic of proximal lung [11,13,24]. In humans, heterozygous loss of function mutations in the NKX2.1 gene, localized to chromosome 14q, has been described in infants and children with recurrent respiratory distress and thyroid and central nervous system dysfunction [25 –27].

To date, a compound null Gli2^−/−/Gli3^−/− mutation is the only genetic model in which the tracheal and lung anlage fail to arise from the foregut endoderm [28]; the thymus, liver, and pancreas form are but hypoplastic. The Gli family of zinc finger transcription factors has been implicated in transduction of sonic hedgehog (SHH) signal in the endoderm, and Gli1, Gli2, and Gli3 are expressed in the splanchnic and early lung mesenchyme [29,30]. Of interest, this phenotype is much more severe than that seen in Shh- null mutants, who are born with a lung, albeit abnormal [31] (see below). Mice with single mutations in Gli2 or Gli3 have tracheal stenosis and lung lobation and branching defects, similar to those seen in Pallister-Hall syndrome, caused by frame-shift mutations in the Gli3 gene [28,30,32].

PRIMARY LUNG BUD FORMATION

In mice, primary lung buds are evident at E9.5 (Fig. 4B). Akin to specification, lung budding and branching morphogenesis arise from dynamic and reciprocal interactions between the early epithelium and mesenchyme. Soluble factors have long been implicated in branching morphogenesis, since it was discovered that transposition of early-stage distal lung mesenchyme onto the tracheal epithelium can induce supernumerary branching as well as type II cell differentiation with lamellar bodies and surfactant protein C (Sftpc) expression [33]. Studies in Drosophila have determined that primary branching of the lung is spatially regulated by an FGF ligand, the branchless gene [34]. The branchless gene activates an FGF receptor (breathless) in the endoderm to induce tracheal development [35,36]. This FGF signaling pathway appears to be evolutionarily conserved in mammals because FGF10 and its receptor FGFR2b (expressed throughout the respiratory epithelium) are critical for lung bud formation. Fgf10, present at discrete sites in the mesenchyme around the distal lung buds, locally induces and guides bud growth by promoting endodermal proliferation and chemoattraction [37 –39]. Deletion of either Fgf10 or Fgfr2b interferes with primary lung budding and peripheral cell differentiation; transgenic lungs are characterized by only a trachea or trachea and 2 bronchi [40 –43]. The similar phenotypes of Fgf10- and Fgfr2b-deficient mice suggest that FGF10 from the mesenchyme binds to FGFR2b in the epithelium to activate proliferation and migration of the epithelial cells, leading to directional outgrowth of the lung bud (Fig. 5A).

Figure 5.

Models of the interactions among key molecules in primary bud formation and branching of the lung. Mesenchyme is depicted in light gray and endodermal epithelium in dark gray. (A) Fgf10 expression (dotted pattern) in the distal mesenchyme is regulated by Tbx genes and retinoic acid. FGF10 diffuses and binds to FGFR2b in the epithelium to induce a bud through proliferation and directional outgrowth (white arrow). (B) As the lung bud elongates, FGF10-FGFR2b signaling in the distal epithelium (black gradient) induces Bmp4 and Spry2. Epithelial WNT signaling also promotes Fgfr2b and Bmp4 expression, which stimulates epithelial proliferation and bud outgrowth. In contrast, SPRY2 controls the size of the lung bud by repressing FGF signaling and proliferation. In the proximal mesenchyme, transforming growth factor-beta1 represses Fgf10 expression and promotes synthesis of extracellular matrix (horizontal striped pattern), including collagen and fibronectin, which might serve to prevent ectopic budding. (C) Elongation of the lung bud arrests because sonic hedgehog (SHH) in the distal epithelium negatively regulates Fgf10. This is counterbalanced by Hhip1, which maintains Fgf10 expression by sequestering the SHH protein. Low SHH levels in the proximal region of the bud allows for higher FGF10-FGFR2b signaling (2 black gradients at the lateral sides) and induction of lateral buds. Lateral bud branching might be facilitated by canonical WNT signaling, which induces fibronectin and alpha-smooth muscle actin in the mesenchyme near the tip of lung bud. Dkk1 inhibits WNT signaling around the distal bud. (D) Resultant secondary lung branches with transforming growth factor-beta1 and extracellular matrix localized to the mesenchyme at the branch point, as well as around the proximal region. A new cycle of bud induction and outgrowth continues from B. As well as their role in branching, WNTs and bone morphogenetic proteins in the distal epithelium influence proximal-distal differentiation of the lung.

Several studies have implicated retinoic acid signaling in regulating Fgf10 expression and bud formation [44,45]. Deficiency of vitamin A or compound null mutations of the retinoic acid receptor genes results in severe abnormalities of the lung, including tracheoesophageal fistula, pulmonary hypoplasia, and agenesis of the left lung [46,47]. In the early lung, retinoic acid synthesis and use are most prominent in the foregut, where the primary lung buds are emerging [48]. Disruption of retinoic acid signaling in E8.5 foregut explants interferes with Fgf10 expression and primary bud formation [44].

T-box (Tbx) transcription factors have also been shown to be positive regulators of Fgf10 expression (Fig. 5A). In chick embryos, Tbx4 and Fgf10 are coexpressed in the foregut mesoderm adjacent to endodermal Nkx2.1 expression in the early lung field. Misexpression of Tbx4 induces ectopic Fgf10 expression and ectopic buds, which express Nkx2.1 [49]. In mouse embryonic lung cultures, repression of Tbx4 and Tbx5 expression through antisense oligonucleotides suppresses Fgf10 expression in the mesenchyme and eliminates formation of new lung branches [50]. Thus, expression of factors in the early foregut mesoderm, including retinoic acid, Fgf10, and Tbx transcription factors are critical for induction of the primary lung bud.

TRACHEOESOPHAGEAL SEPTATION

Concurrent with formation of the primary lung bud is septation of the trachea from the esophagus. The early expression of Nkx2.1 at E9.0 demarcates the dorsoventral boundary of the foregut endoderm, distinguishing the lung primordium from the esophagus [13] (Fig. 4C). This dorsoventral separation is absent or incomplete in mouse embryos with null mutations in Nkx2.1 and Shh, resulting in a tracheoesophageal fistula, as well as lung hypoplasia [13,31,51]. In contrast to Nkx2.1^−/− mice, which have a defect in distal lung differentiation, normal proximodistal differentiation of epithelial cells is preserved in Shh^−/− mice with expression of both Sftpc and Clara cell protein 10 (CC10) genes. Haploinsufficiency of Foxf1, a Shh target, causes esophageal atresia and tracheoesophageal fistula, which is also present in Gli2^−/−/Gli3^+/− mice, compound retinoic acid receptor mutants, and Tbx4 deficiency [28,31,49,52,53]. Human malformations of the foregut, including laryngeal clefts, tracheoesophageal fistula, and esophageal and tracheal atresia, occur in isolation or part of more complex syndromes, including VACTERL, Smith-Lemli-Opitz, and Pallister-Hall, the latter a defect in the SHH signaling pathway [54 –56]. Although many signaling molecules regulate both tracheal and lung morphogenesis, Fgf10- and Fgfr2-null mutants have normal septation of the foregut, indicating that formation of the proximal and distal respiratory tree may be genetically distinguishable. Lineage analysis in the lung using Cre recombinase to activate a floxed reporter gene under control of the doxycyline-dependent 3.7-kb human Sftpc gene promoter (Fig. 4D) also suggests that the progenitor cells of the trachea and bronchi differ in origin from those that will form the distal airways and alveoli [57].

LEFT-RIGHT ASYMMETRY

In mice and humans, the lung has a left-right asymmetric pattern. Asymmetry of the lung lobes is dependent on early determinants of left-right axis specification that is largely regulated by Tgf-beta–related genes, such as activin receptor II, Lefty1, Lefty II, Nodal, and Pitx2. Of interest, the majority of the left-right determinant genes are expressed in the node and/or lateral plate mesoderm from E8.0 to E8.5, well before the primary lung bud forms at E9.5. In mice, disruption of activin receptor IIB, Foxj1, Pitx2, Pkd2, or Nodal results in right pulmonary isomerism (bilateral 4-lobed lungs) [58 –62], whereas inversin- and Lefty1-deficient mice show left pulmonary isomerism (bilateral single lobe) [63,64]. As well as a lung phenotype, there is global asymmetry with cardiac defects and visceral situ inversus.

BRANCHING MORPHOGENESIS

Branching morphogenesis results in formation of the conducting airways down to the terminal bronchioles, a process involving bud outgrowth, elongation, and subdivision of terminal units [65] (Fig. 4E). The size and shape of the lung bud are regulated by both positive and negative signaling between the growing epithelial bud and the mesenchyme. As well as primary lung bud induction (see above), FGF10-FGFR2b signaling controls secondary and subsequent bud formation. FGF7, which shares high-sequence homology to FGF10 and utilizes the same receptor, also influences lung branching by promoting epithelial cell proliferation and expansion [66 –69]. Misexpression of FGF7 using the Sftpc or rat Clara cell secretory protein promoter in mice during the pseudoglandular stage alters lung branching with large cystic spaces in the distal lung [70].

Several signaling pathways have been shown to be negative regulators of lung epithelial proliferation, which appear to play a role in counteracting the bud-promoting effect of FGFs. During elongation of the bud, FGF10-FGFR2b signaling induces Sprouty2 (Spry2), as well as bone morphogenetic protein 4 (BMP4) in the distal tip of the epithelium (Fig. 5B) [39,53,71,72]. SPRY family members are inducible negative regulators of FGF signaling, as well as other tyrosine kinase signaling pathways; mSPRY2 inhibits mitogen-activated protein kinase activity in mouse lung epithelial cells in response to FGF10 stimulation [73]. Reduction of Spry2 results in increased branching, cell proliferation, and cell differentiation in cultured embryonic mouse lungs [74]. Conversely, overexpression of Spry2 in the distal epithelium under control of the Sftpc promoter impairs branching with decreased epithelial cell proliferation [72]. Thus, SPRY2 inhibition of FGF10-FGFR2b signaling appears to control the size of the lung bud by limiting its outgrowth.

Bud formation is also controlled by SHH signaling. Shh is highly expressed in the epithelium of the distal lung buds, from where it diffuses to activate signaling in the adjacent mesenchyme through its receptor patched (Ptch1) and the Gli transcription factors [75] (Fig. 5C). Data from lung explants and transgenic mice suggest that the role of SHH signaling is to spatially restrict FGF10 levels in the mesenchyme surrounding the bud tips, thus limiting further bud outgrowth [31,37,76]. In Shh-null mutant mice, Fgf10 is no longer restricted to focal areas but distributes broadly throughout the distal mesenchyme; transgenic lungs are cystic with a defect in branching, and the mesenchyme shows enhanced cell death and decreased cell proliferation [31,51]. In contrast, Fgf10 expression is repressed in lung mesenchyme of transgenic mice that overexpress Shh throughout the epithelium [75]. Overexpression of Shh also leads to hypercellularity of the lung, with excessive mesenchyme between the alveolar spaces [75]. Thus, SHH not only regulates FGF10 expression but also serves as a trophic factor for the lung mesenchyme. SHH induction of the hedgehog interacting protein Hhip1 in the distal mesenchyme may contribute to the regulation of FGF10 expression [77]. Hhip1 attenuates SHH signaling by sequestration of the Hedgehog protein, thus releasing SHH-mediated repression of FGF10 [77] (Fig. 5C). In Hhip1-null animals, branching is severely inhibited because of increased Hedgehog signaling and repression of Fgf10 [78].

The transforming growth factor-beta (TGFβ1) subfamily is also implicated in branching morphogenesis, specifically in regulating epithelial cell proliferation and extracellular matrix deposition [79]. Tgfβ1 is expressed in the mesenchyme and preferentially accumulates around the bronchiolar ducts and airway branch points where collagen, fibronectin, and proteoglycans are also present [80]. Overexpression of Tgfβ1 in organ culture or misexpression of Tgfβ1 in the distal epithelium of transgenic mice has a negative regulatory effect on branching morphogenesis, whereas abrogation of TGFß type II receptor signaling stimulates lung branching with increased epithelial cell proliferation [81,82]. Because TGFß1 has been shown to repress Fgf10 expression in the lung mesenchyme [76], it may be part of the mechanism that prevents budding in the proximal parts of the lung by inhibiting FGF10 and promoting synthesis of extracellular matrix.

Bone morphogenetic proteins are signaling molecules that play multiple roles in lung development. BMP4 is present in the early mesenchyme but is not present in the epithelium until E11–12, where it is localized to the distal lung buds [83 –85]. Although there has been some debate on the role of epithelial-derived BMP4 in branching morphogenesis, recent data in which BMP receptor type Ia or BMP4 was disrupted in the epithelium using the Sftpc-Cre transgene indicate that autocrine BMP signaling promotes proliferation and morphogenesis of the distal epithelium [39,84,86] (Fig. 5B).

Functional roles for WNT signaling in lung branching have also been reported. Detection of nuclear localized beta-catenin and Tcf/Lef transcription factors, components of the WNT pathway, can monitor activation of canonical WNT signaling. These factors are increased in the distal lung epithelium at the sites of active branching, as well as in the mesenchyme adjacent to proximal airways where the smooth muscle will form [87,88]. Disruption of canonical WNT signaling in the lung by targeted deletion of beta-catenin or expression of the diffusible WNT inhibitor Dickkopf 1 (Dkk1) prevents distal lung proliferation and differentiation [89,90]. This defect has been linked to failure of Fgfr2b induction in the distal lung epithelium where WNT signaling is inhibited [90] (Fig. 5B). Furthermore, Dkk1-treated lung explants show a reduction in fibronectin and alpha-smooth muscle actin expression in lung mesenchymal cells, as well as impaired vascular development [88] (Fig. 5C). Fibronectin deposition is essential for cleft formation during lung bud branching [91]. In contrast to the above findings, lungs of homozygous Wnt5a-mutant embryos exhibit overbranching of the distal airways; inhibition of lung maturation; and increased expression of Fgf10, Bmp4, and Shh [92]. WNT5a is a noncanonical WNT, normally expressed in the lung mesenchyme and distal epithelium at E12 [92,93]. Further studies are needed to clarify whether there are distinct functions of canonical and noncanonical WNTs.

PROXIMAL-DISTAL PATTERNING AND CELL DIFFERENTIATION

Epithelial cell lineages are arranged in a distinct proximal-distal spatial pattern in the airways, which become morphologically apparent during the pseudoglandular stage [1]. Ciliated, basal, secretory, and neuroendocrine cells are the major cell types constituting the proximal epithelium, whereas type I and type II alveolar cells make up the distal epithelium. 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 still under investigation [94]. There is evidence from cell injury models to suggest that basal cells, Clara cells, and type II alveolar cells can function as progenitors, because they have the capacity to proliferate and repopulate the damaged epithelium after acute lung injury [95 –100]. Of interest, early on in embryonic development, the undifferentiated epithelium coexpresses several lineage markers, including Sftpa, CC10, and calcitonin gene-related peptide, the latter a marker of neuroendocrine cells [101]. Cells positive for Clara- and type II–specific markers have also been identified in the adult lung in the bronchioalveolar duct region and become more numerous after bleomycin- or naphthalene-induced injury [102,103]. In vitro, these coexpressing cells self-renew and give rise to cells that express markers for Clara, type I, or type II cells, providing the best evidence that multipotential cells in the lung exist and become reenlisted during epithelial repair.

Although ciliated cells have been considered to be terminally differentiated, recent data suggest that these cells can also proliferate and repopulate the epithelium in response to naphthalene injury or partial pneumonectomy [104,105]. However, a genetic lineage–labeling approach using Foxj1-GFP mice provides conflicting data by demonstrating that ciliated cells change their morphology in response to lung injury but do not proliferate, self-renew, or transdifferentiate during repair of the epithelium [94]. The winged helix transcription factor Foxj1 is necessary for ciliated cell development in the lung as well as left-right axis patterning, with mutational abnormalities in the mouse similar to those seen in Kartagener syndrome [106].

Tissue recombination experiments have demonstrated that a proximal versus distal epithelial phenotype is dictated by soluble factors from the adjacent mesenchyme; within a restricted time window, distal lung mesenchyme can reprogram the tracheal epithelium to take on a type II cell phenotype, and, conversely, tracheal mesenchyme can induce proximal differentiation (ciliated and mucous cells) in distal lung epithelium [107,108]. Early studies have also shown that the quantity of mesenchyme is influential in inducing a specific epithelial cell fate [109]. 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. Thus, an increasing concentration of the same signals or morphogens expressed by the mesenchyme can induce different cell fates in identical epithelial cells.

BMP4 and canonical WNTs both serve as morphogens, which regulate proximal-distal cell fate in the lung along a gradient of signal (Fig. 5D). Inhibition of BMP signaling through transgenic expression of BMP antagonists (noggin, gremlin) in the epithelium promotes proximalization of the lung with ciliated and Clara cells in the peripheral compartment [83,110]. A similar phenotype results from the targeted deletion of beta-catenin or overexpression of the WNT inhibitor Dkk1 in the distal lung epithelium [90,111]. Remarkably, constitutively activating WNT signaling in embryonic lung endoderm using the Sftpc-Cre transgene results in a lineage switch with the presence of intestinal cells, including Paneth and goblet cells, in the pulmonary epithelium [87]. Hence, insight into metaplasia comes from knowledge of pathways regulating normal lineage commitment and differentiation in the developing lung.

CONCLUSION

Lung morphogenesis, like other developmental processes, depends on complex interactions that are tightly controlled both temporally and spatially. Extensive investigation over the past decade has uncovered select molecular processes that regulate different components of lung development. Table 1 recapitulates the principal factors involved and the genetic evidence that supports their role in lung morphogenesis and/or cell differentiation. Although the clinical relevance of these processes in human developmental anomalies and perinatal lung diseases remains to be fully elucidated, a better understanding of the molecular mechanisms of lung development will provide important insight into understanding congenital disorders of the respiratory tract, as well as controlling cell differentiation and regeneration for therapeutic purposes.

Table 1.

Pulmonary phenotypes in mutant mice

Gene symbol	Gene name	Domain of expression	Mutant lung phenotype	Reference
Specification and primary lung bud formation
Gli2/3	GLI-Kruppel family member GLI2/3	M	Absent lung anlage	28
Fgf10	Fibroblast growth factor 10	M	Lung agenesis	42
Fgfr2b	Fibroblast growth factor receptor 2b	E	Lung agenesis	43
RARα/β2^*	Retinoic acid receptor-alpha/beta	E and M	Left lung agenesis and right lung hypoplasia, tracheoesophageal fistula	47
Tracheoesophageal septation

Shh^*	Sonic hedgehog	E	Tracheoesophageal fistula, impaired branching	31
Foxf1^*	Forkhead box F1	M	Tracheoesophageal fistula, impaired branching	53
Titf1^* (Nkx2.1)	Thyroid transcription factor 1	E	Tracheoesophageal fistula, impaired branching, absent Clara and type II cells	11
Left-right asymmetry

Acvr2b	Activin receptor IIB	NR	Right pulmonary isomerism	58
Foxj1^*	Forkhead box J1	E	Right pulmonary isomerism, defective ciliogenesis	59
Pitx2	Paired-like homeodomain transcription factor 2	M	Right pulmonary isomerism	60
Pkd2	Polycystic kidney disease gene 2 product	NR	Right pulmonary isomerism	61
Nodal	Nodal	NR	Right pulmonary isomerism	62
Invs	Inversin	NR	Left pulmonary isomerism	63
Lefty1	Left-right determination factor 1	NR	Left pulmonary isomerism	64
Branching morphogenesis

Adam17	A disintegrin and metallopeptidase domain 17	E	Impaired branching	113
Egfr	Epidermal growth factor receptor	E and M	Impaired branching	114
Fgf9	Fibroblast growth factor 9	E and P	Impaired branching, reduced mesenchyme	115
Foxa1/a2	Forkhead box A1/A2	E	Impaired branching, absent ciliated, Clara, type II cells	116
Gata6	GATA-binding protein 6	E	Impaired branching	117
Hhip^*	Hedgehog-interacting protein	M	Impaired branching, left pulmonary isomerism	78
Itgα3	Integrin α3	E	Impaired branching	118
Nfib	Nuclear factor I/B	E and M	Impaired branching	119
Nmyc	Neuroblastoma myc-related oncogene	E	Impaired branching	120
Sox11	SRY-box-containing gene 11	E	Impaired branching	121
Tcf21	Transcription factor 21	M	Impaired branching	122
Timp3	Tissue inhibitor of metalloproteinase 3	E	Impaired branching	123
Proximal-distal patterning

Catnnβ1^*	Beta-catenin	E	Defect in distal lung development, impaired branching	111
Noggin	Noggin	M	Defect in distal lung development	85
Wnt5a	Wingless-related MMTV integration site 5A	E and M	Overexpansion of the distal airways	124
Epithelial differentiation

Cutl1	Cut-like 1	E	Defect in type I and type II cell differentiation	125
Ep300	E1A binding protein p300	M	Defect in Clara cell and type II cell differentiation	126
Lamα5	Laminin-alpha5	E and P	Defect in type I and type II cell differentiation	127
Ascl1(Mash1)	Achaete-scute complex homolog-like 1	E	Absent neuroendocrine cells	128
Pdpn (T1α)	Podoplanin	E	Defect in type I cell differentiation	129
Pthlh	Parathyroid hormone-like peptide	E	Defect in type II cell differentiation	130
Tgfβ3	Transforming growth factor-beta3	E and P	Defect in type II cell differentiation	131

Table adapted from Cardoso and Lu [132].

E indicates lung epithelium; M, lung mesenchyme; P, pleura; U, unspecified expression in lung epithelium and/or mesenchyme; NR, not reported.

Denotes more than 1 function of the gene in lung development.

References

Ten Have-Opbroek

. The development of the lung in mammals: an analysis of concepts and findings. Am J Anat 1981;162:201–219.

Potter

Loosli

. Prenatal development of the human lung. Am J Dis Child 1951;82:226–228.

Ten Have-Opbroek

. Lung development in the mouse embryo. Exp Lung Res 1991;17:111–130.

Loosli

Potter

. Pre- and postnatal development of the respiratory portion of the human lung with special reference to the elastic fibers. Am Rev Respir Dis 1959;80:5–23.

Bourbon

Boucherat

Chailley-Heu

Delacourt

. Control mechanisms of lung alveolar development and their disorders in bronchopulmonary dysplasia. Pediatr Res 2005;57:38R–46R.

Galambos

Demello

. Molecular mechanisms of pulmonary vascular development. Pediatr Dev Pathol 2007;10:1–17.

Lawson

Meneses

Pedersen

. Cell fate and cell lineage in the endoderm of the presomite mouse embryo, studied with an intracellular tracer. Dev Biol 1986;115:325–339.

Wells

Melton

. Early mouse endoderm is patterned by soluble factors from adjacent germ layers. Development 2000;127:1563–1572.

Serls

Doherty

Parvatiyar

Wells

Deutsch

. Different thresholds of fibroblast growth factors pattern the ventral foregut into liver and lung. Development 2005;132:35–47.

10.

Barbera

Martinez JP

Clements

Thomas

. The homeobox gene Hex is required in definitive endodermal tissues for normal forebrain, liver and thyroid formation. Development 2000;127:2433–2445.

11.

Kimura

Hara

Pineau

. 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;10:60–69.

12.

Ahlgren

Jonsson

Edlund

. The morphogenesis of the pancreatic mesenchyme is uncoupled from that of the pancreatic epithelium in IPF1/PDX1-deficient mice. Development 1996;122:1409–1416.

13.

Minoo

Drum

Bringas

Kimura

. Defects in tracheoesophageal and lung morphogenesis in Nkx2.1(-/-) mouse embryos. Dev Biol 1999;209:60–71.

14.

Jung

Zheng

Goldfarb

Zaret

. Initiation of mammalian liver development from endoderm by fibroblast growth factors. Science 1999;284:1998–2003.

15.

Deutsch

Jung

Zheng

Lora

Zaret

. A bipotential precursor population for pancreas and liver within the embryonic endoderm. Development 2001;128:871–881.

16.

Sasaki

Hogan

. Differential expression of multiple fork head related genes during gastrulation and axial pattern formation in the mouse embryo. Development 1993;118:47–59.

17.

Monaghan

Kaestner

Grau

Schutz

. 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;119:567–578.

18.

Ang

Rossant

. HNF-3 beta is essential for node and notochord formation in mouse development. Cell 1994;78:561–574.

19.

Ikeda

Shaw-White

Wert

Whitsett

. Hepatocyte nuclear factor 3 activates transcription of thyroid transcription factor 1 in respiratory epithelial cells. Mol Cell Biol 1996;16:3626–3636.

20.

Guazzi

Price

De Felice

Damante

Mattei

Di Lauro

. Thyroid nuclear factor 1 (TTF-1) contains a homeodomain and displaysa novel DNA binding specificity. Embo J 1990;9:3631–3639.

21.

Lazzaro

Price

de Felice

Di Lauro

. 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;113:1093–1104.

22.

Ikeda

Clark

Shaw-White

Stahlman

Boutell

Whitsett

. Gene structure and expression of human thyroid transcription factor-1 in respiratory epithelial cells. J Biol Chem 1995;270:8108–8114.

23.

Kimura

Ward

Minoo

. Thyroid-specific enhancer-binding protein/thyroid transcription factor 1 is not required for the initial specification of the thyroid and lung primordia. Biochimie 1999;81:321–327.

24.

Minoo

Hamdan

Warburton

Stepanik

deLemos

. TTF-1 regulates lung epithelial morphogenesis. Dev Biol 1995;172:694–698.

25.

Devriendt

Vanhole

Matthijs

de Zegher

. Deletion of thyroid transcription factor-1 gene in an infant with neonatal thyroid dysfunction and respiratory failure. N Engl J Med 1998;338:1317–1318.

26.

Iwatani

Mabe

Devriendt

Kodama

Miike

. Deletion of NKX2.1 gene encoding thyroid transcription factor-1 in two siblings with hypothyroidism and respiratory failure. J Pediatr 2000;137:272–276.

27.

Krude

Schutz

Biebermann

. Choreoathetosis, hypothyroidism, and pulmonary alterations due to human NKX2–1 haploinsufficiency. J Clin Invest 2002;109:475–480.

28.

Motoyama

Liu

Ding

Post

Hui

. Essential function of Gli2 and Gli3 in the formation of lung, trachea and oesophagus. Nat Genet 1998;20:54–57.

29.

Hui

Slusarski

Platt

Holmgren

Joyner

. Expression of three mouse homologs of the Drosophila segment polarity gene cubitus interruptus, Gli, Gli-2, and Gli-3, in ectoderm- and mesoderm-derived tissues suggests multiple roles during post-implantation development. Dev Biol 1994;162:402–413.

30.

Grindley

Bellusci

Perkins

Hogan

. Evidence for the involvement of the Gli gene family in embryonic mouse lung development. Dev Biol 1997;188:337–348.

31.

Pepicelli

Lewis

McMahon

. Sonic hedgehog regulates branching morphogenesis in the mammalian lung. Curr Biol 1998;8:1083–1086.

32.

Shin

Kogerman

Lindstrom

Toftgard

Biesecker

. GLI3 mutations in human disorders mimic Drosophila cubitus interruptus protein functions and localization. Proc Natl Acad Sci USA 1999;96:2880–2884.

33.

Shannon

Hyatt

. Epithelial-mesenchymal interactions in the developing lung. Annu Rev Physiol 2004;66:625–645.

34.

Sutherland

Samakovlis

Krasnow

. Branchless encodes a Drosophila FGF homolog that controls tracheal cell migration and the pattern of branching. Cell 1996;87:1091–1101.

35.

Glazer

Shilo

. The Drosophila FGF-R homolog is expressed in the embryonic tracheal system and appears to be required for directed tracheal cell extension. Genes Dev 1991;5:697–705.

36.

Lee

Hacohen

Krasnow

Montell

. Regulated Breathless receptor tyrosine kinase activity required to pattern cell migration and branching in the Drosophila tracheal system. Genes Dev 1996;10:2912–2921.

37.

Bellusci

Grindley

Emoto

Itoh

Hogan

. Fibroblast growth factor 10 (FGF10) and branching morphogenesis in the embryonic mouse lung. Development 1997;124:4867–4878.

38.

Park

Miranda

Lebeche

Hashimoto

Cardoso

. FGF-10 is a chemotactic factor for distal epithelial buds during lung development. Dev Biol 1998;201:125–134.

39.

Weaver

Dunn

Hogan

. Bmp4 and Fgf10 play opposing roles during lung bud morphogenesis. Development 2000;127:2695–2704.

40.

Peters

Werner

Liao

Wert

Whitsett

Williams

. Targeted expression of a dominant negative FGF receptor blocks branching morphogenesis and epithelial differentiation of the mouse lung. Embo J 1994;13:3296–3301.

41.

Min

Danilenko

Scully

. Fgf-10 is required for both limb and lung development and exhibits striking functional similarity to Drosophila branchless. Genes Dev 1998;12:3156–3161.

42.

Sekine

Ohuchi

Fujiwara

. Fgf10 is essential for limb and lung formation. Nat Genet 1999;21:138–141.

43.

De Moerlooze

Spencer-Dene

Revest

Hajihosseini

Rosewell

Dickson

. An important role for the IIIb isoform of fibroblast growth factor receptor 2 (FGFR2) in mesenchymal-epithelial signalling during mouse organogenesis. Development 2000;127:483–492.

44.

Desai

Malpel

Flentke

Smith

Cardoso

. Retinoic acid selectively regulates Fgf10 expression and maintains cell identity in the prospective lung field of the developing foregut. Dev Biol 2004;273:402–415.

45.

Desai

Chen

. Distinct roles for retinoic acid receptors alpha and beta in early lung morphogenesis. Dev Biol 2006;291:12–24.

46.

Wilson

Roth

Warkany

. An analysis of the syndrome of malformations induced by maternal vitamin A deficiency: effects of restoration of vitamin A at various times during gestation. Am J Anat 1953;92:189–217.

47.

Mendelsohn

Lohnes

Decimo

. Function of the retinoic acid receptors (RARs) during development (II): multiple abnormalities at various stages of organogenesis in RAR double mutants. Development 1994;120:2749–2771.

48.

Malpel

Mendelsohn

Cardoso

. Regulation of retinoic acid signaling during lung morphogenesis. Development 2000;127:3057–3067.

49.

Sakiyama

Yamagishi

Kuroiwa

. Tbx4-Fgf10 system controls lung bud formation during chicken embryonic development. Development 2003;130:1225–1234.

50.

Cebra-Thomas

Bromer

Gardner

Lam

Sheipe

Gilbert

. T-box gene products are required for mesenchymal induction of epithelial branching in the embryonic mouse lung. Dev Dyn 2003;226:82–90.

51.

Litingtung

Lei

Westphal

Chiang

. Sonic hedgehog is essential to foregut development. Nat Genet 1998;20:58–61.

52.

Mendelsohn

Larkin

Mark

. RAR beta isoforms: distinct transcriptional control by retinoic acid and specific spatial patterns of promoter activity during mouse embryonic development. Mech Dev 1994;45:227–241.

53.

Mahlapuu

Enerback

Carlsson

. Haploinsufficiency of the forkhead gene Foxf1, a target for sonic hedgehog signaling, causes lung and foregut malformations. Development 2001;128:2397–2406.

54.

Ming

Roessler

Muenke

. Human developmental disorders and the Sonic hedgehog pathway. Mol Med Today 1998;4:343–349.

55.

Kim

Hui

. The VACTERL association: lessons from the Sonic hedgehog pathway. Clin Genet 2001;59:306–315.

56.

Roux

Wolf

Mulliez

. Role of cholesterol in embryonic development. Am J Clin Nutr 2000;71:1270S–1279S.

57.

Perl

Wert

Nagy

Lobe

Whitsett

. Early restriction of peripheral and proximal cell lineages during formation of the lung. Proc Natl Acad Sci USA 2002;99:10482–10487.

58.

. The signaling pathway mediated by the type IIB activin receptor controls axial patterning and lateral asymmetry in the mouse. Genes Dev 1997;11:1812–1826.

59.

Tamakoshi

Itakura

Chandra

. Roles of the Foxj1 and Inv genes in the left-right determination of internal organs in mice. Biochem Biophys Res Commun 2006;339:932–938.

60.

Lin

Kioussi

O'Connell

. Pitx2 regulates lung asymmetry, cardiac positioning and pituitary and tooth morphogenesis. Nature 1999;401:279–282.

61.

Pennekamp

Karcher

Fischer

. The ion channel polycystin-2 is required for left-right axis determination in mice. Curr Biol 2002;12:938–943.

62.

Lowe

Yamada

Kuehn

. Genetic dissection of nodal function in patterning the mouse embryo. Development 2001;128:1831–1843.

63.

Morishima

Yasui

Nakazawa

Ando

Ishibashi

Takao

. Situs variation and cardiovascular anomalies in the transgenic mouse insertional mutation, inv. Teratology 1998;57:302–309.

64.

Meno

Shimono

Saijoh

. Lefty-1 is required for left-right determination as a regulator of lefty-2 and nodal. Cell 1998;94:287–297.

65.

Hogan

. Morphogenesis. Cell 1999;96:225–233.

66.

Nogawa

Ito

. Branching morphogenesis of embryonic mouse lung epithelium in mesenchyme-free culture. Development 1995;121:1015–1022.

67.

Post

Souza

Liu

. Keratinocyte growth factor and its receptor are involved in regulating early lung branching. Development 1996;122:3107–3115.

68.

Shiratori

Oshika

Ung

. Keratinocyte growth factor and embryonic rat lung morphogenesis. Am J Respir Cell Mol Biol 1996;15:328–338.

69.

Cardoso

Itoh

Nogawa

Mason

Brody

. FGF-1 and FGF-7 induce distinct patterns of growth and differentiation in embryonic lung epithelium. Dev Dyn 1997;208:398–405.

70.

Simonet

DeRose

Bucay

. Pulmonary malformation in transgenic mice expressing human keratinocyte growth factor in the lung. Proc Natl Acad Sci USA 1995;92:12461–12465.

71.

Hyatt

Shangguan

Shannon

. FGF-10 induces SP-C and Bmp4 and regulates proximal-distal patterning in embryonic tracheal epithelium. Am J Physiol Lung Cell Mol Physiol 2004;287:L1116–L1126.

72.

Mailleux

Tefft

Ndiaye

. Evidence that SPROUTY2 functions as an inhibitor of mouse embryonic lung growth and morphogenesis. Mech Dev 2001;102:81–94.

73.

Tefft

Lee

Smith

Crowe

Bellusci

Warburton

. mSprouty2 inhibits FGF10-activated MAP kinase by differentially binding to upstream target proteins. Am J Physiol Lung Cell Mol Physiol 2002;283:L700–L706.

74.

Tefft

Lee

Smith

. Conserved function of mSpry-2, a murine homolog of Drosophila sprouty, which negatively modulates respiratory organogenesis. Curr Biol 1999;9:219–222.

75.

Bellusci

Furuta

Rush

Henderson

Winnier

Hogan

. Involvement of Sonic hedgehog (Shh) in mouse embryonic lung growth and morphogenesis. Development 1997;124:53–63.

76.

Lebeche

Malpel

Cardoso

. Fibroblast growth factor interactions in the developing lung. Mech Dev 1999;86:125–136.

77.

Chuang

McMahon

. Vertebrate hedgehog signalling modulated by induction of a hedgehog-binding protein. Nature 1999;397:617–621.

78.

Chuang

Kawcak

McMahon

. Feedback control of mammalian hedgehog signaling by the hedgehog-binding protein, Hip1, modulates Fgf signaling during branching morphogenesis of the lung. Genes Dev 2003;17:342–347.

79.

Moses

Yang

Pietenpol

. TGF-beta stimulation and inhibition of cell proliferation: new mechanistic insights. Cell 1990;63:245–247.

80.

Heine

Munoz

Flanders

Roberts

Sporn

. Colocalization of TGF-beta 1 and collagen I and III, fibronectin and glycosaminoglycans during lung branching morphogenesis. Development 1990;109:29–36.

81.

Serra

Pelton

Moses

. TGF beta 1 inhibits branching morphogenesis and N-myc expression in lung bud organ cultures. Development 1994;120:2153–2161.

82.

Zhou

Dey

Wert

Whitsett

. Arrested lung morphogenesis in transgenic mice bearing an SP-C-TGF-beta 1 chimeric gene. Dev Biol 1996;175:227–238.

83.

Weaver

Yingling

Dunn

Bellusci

Hogan

. Bmp signaling regulates proximal-distal differentiation of endoderm in mouse lung development. Development 1999;126:4005–4015.

84.

Bellusci

Henderson

Winnier

Oikawa

Hogan

. Evidence from normal expression and targeted misexpression that bone morphogenetic protein (Bmp-4) plays a role in mouse embryonic lung morphogenesis. Development 1996;122:1693–1702.

85.

Weaver

Batts

Hogan

. Tissue interactions pattern the mesenchyme of the embryonic mouse lung. Dev Biol 2003;258:169–184.

86.

Eblaghie

Reedy

Oliver

Mishina

Hogan

. Evidence that autocrine signaling through Bmpr1a regulates the proliferation, survival and morphogenetic behavior of distal lung epithelial cells. Dev Biol 2006;291:67–82.

87.

Okubo

Hogan

. Hyperactive Wnt signaling changes the developmental potential of embryonic lung endoderm. J Biol 2004;3:11.

88.

De Langhe

Sala

Del Moral

. Dickkopf-1 (DKK1) reveals that fibronectin is a major target of Wnt signaling in branching morphogenesis of the mouse embryonic lung. Dev Biol 2005;277:316–331.

89.

Mucenski

Nation

Thitoff

. Beta-catenin regulates differentiation of respiratory epithelial cells in vivo. Am J Physiol Lung Cell Mol Physiol 2005;289:L971–L979.

90.

Shu

Guttentag

Wang

. Wnt/beta-catenin signaling acts upstream of N-myc, BMP4, and FGF signaling to regulate proximal-distal patterning in the lung. Dev Biol 2005;283:226–239.

91.

Sakai

Larsen

Yamada

. Fibronectin requirement in branching morphogenesis. Nature 2003;423:876–881.

92.

Xiao

. Wnt5a regulates Shh and Fgf10 signaling during lung development. Dev Biol 2005;287:86–97.

93.

Dean

Miller

Smith

Dufort

Lang

Niswander

. Canonical Wnt signaling negatively regulates branching morphogenesis of the lung and lacrimal gland. Dev Biol 2005;286:270–286.

94.

Rawlins

Hogan

. Epithelial stem cells of the lung: privileged few or opportunities for many? Development 2006;133:2455–2465.

95.

Adamson

Bowden

. The type 2 cell as progenitor of alveolar epithelial regeneration: a cytodynamic study in mice after exposure to oxygen. Lab Invest 1974;30:35–42.

96.

Evans

Cabral

Stephens

Freeman

. Transformation of alveolar type 2 cells to type 1 cells following exposure to NO2. Exp Mol Pathol 1975;22:142–150.

97.

Evans

Cabral-Anderson

Freeman

. Role of the Clara cell in renewal of the bronchiolar epithelium. Lab Invest 1978;38:648–653.

98.

Plopper

Nishio

Alley

Kass

Hyde

. 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;7:606–613.

99.

Hong

Reynolds

Watkins

Fuchs

Stripp

. Basal cells are a multipotent progenitor capable of renewing the bronchial epithelium. Am J Pathol 2004;164:577–588.

100.

Giangreco

Reynolds

Stripp

. Terminal bronchioles harbor a unique airway stem cell population that localizes to the bronchoalveolar duct junction. Am J Pathol 2002;161:173–182.

101.

Wuenschell

Sunday

Singh

Minoo

Slavkin

Warburton

. Embryonic mouse lung epithelial progenitor cells co-express immunohistochemical markers of diverse mature cell lineages. J Histochem Cytochem 1996;44:113–123.

102.

Daly

Baecher-Allan

Paxhia

Ryan

Barth

Finkelstein

. Cell-specific gene expression reveals changes in epithelial cell populations after bleomycin treatment. Lab Invest 1998;78:393–400.

103.

Kim

Jackson

Woolfenden

. Identification of bronchioalveolar stem cells in normal lung and lung cancer. Cell 2005;121:823–835.

104.

Park

Wells

Zorn

Wert

Laubach

Fernandez

Whitsett

. Transdifferentiation of ciliated cells during repair of the respiratory epithelium. Am J Respir Cell Mol Biol 2006;34:151–157.

105.

Park

Wells

Zorn

Wert

Whitsett

. Sox17 influences the differentiation of respiratory epithelial cells. Dev Biol 2006;294:192–202.

106.

Chen

Knowles

Hebert

Hackett

. Mutation of the mouse hepatocyte nuclear factor/forkhead homologue 4 gene results in an absence of cilia and random left-right asymmetry. J Clin Invest 1998;102:1077–1082.

107.

Shannon

. Induction of alveolar type II cell differentiation in fetal tracheal epithelium by grafted distal lung mesenchyme. Dev Biol 1994;166:600–614.

108.

Shannon

Nielsen

Gebb

Randell

. Mesenchyme specifies epithelial differentiation in reciprocal recombinants of embryonic lung and trachea. Dev Dyn 1998;212:482–494.

109.

Masters

. Epithelial-mesenchymal interaction during lung development: the effect of mesenchymal mass. Dev Biol 1976;51:98–108.

110.

Yang

Zhang

Shu

Blair

Morrisey

. The bone morphogenic protein antagonist gremlin regulates proximal-distal patterning of the lung. Dev Dyn 2001;222:667–680.

111.

Mucenski

Wert

Nation

. Beta-catenin is required for specification of proximal/distal cell fate during lung morphogenesis. J Biol Chem 2003;278:40231–40238.

112.

Wert

Glasser

Korfhagen

Whitsett

. Transcriptional elements from the human SP-C gene direct expression in the primordial respiratory epithelium of transgenic mice. Dev Biol 1993;156:426–443.

113.

Zhao

Chen

Peschon

. Pulmonary hypoplasia in mice lacking tumor necrosis factor-alpha converting enzyme indicates an indispensable role for cell surface protein shedding during embryonic lung branching morphogenesis. Dev Biol 2001;232:204–218.

114.

Miettinen

Warburton

. Impaired lung branching morphogenesis in the absence of functional EGF receptor. Dev Biol 1997;186:224–236.

115.

Colvin

White

Pratt

Ornitz

. Lung hypoplasia and neonatal death in Fgf9-null mice identify this gene as an essential regulator of lung mesenchyme. Development 2001;128:2095–2106.

116.

Wan

Dingle

. Compensatory roles of Foxa1 and Foxa2 during lung morphogenesis. J Biol Chem 2005;280:13809–13816.

117.

Yang

Zhang

Whitsett

Morrisey

. GATA6 regulates differentiation of distal lung epithelium. Development 2002;129:2233–2246.

118.

Kreidberg

Donovan

Goldstein

. Alpha 3 beta 1 integrin has a crucial role in kidney and lung organogenesis. Development 1996;122:3537–3547.

119.

Grunder

Ebel

Mallo

. Nuclear factor I-B (Nfib) deficient mice have severe lung hypoplasia. Mech Dev 2002;112:69–77.

120.

Moens

Auerbach

Conlon

Joyner

Rossant

. A targeted mutation reveals a role for N-myc in branching morphogenesis in the embryonic mouse lung. Genes Dev 1992;6:691–704.

121.

Sock

Rettig

Enderich

Bosl

Tamm

Wegner

. Gene targeting reveals a widespread role for the high-mobility-group transcription factor Sox11 in tissue remodeling. Mol Cell Biol 2004;24:6635–6644.

122.

Quaggin

Schwartz

Cui

. The basic-helix-loop-helix protein pod1 is critically important for kidney and lung organogenesis. Development 1999;126:5771–5783.

123.

Gill

Pape

Khokha

Watson

Leco

. A null mutation for tissue inhibitor of metalloproteinases-3 (Timp-3) impairs murine bronchiole branching morphogenesis. Dev Biol 2003;261:313–323.

124.

Zhang

Choi

Litingtung

Chiang

. Sonic hedgehog signaling regulates Gli3 processing, mesenchymal proliferation, and differentiation during mouse lung organogenesis. Dev Biol 2004;270:214–231.

125.

Ellis

Gambardella

Horcher

. The transcriptional repressor CDP (Cutl1) is essential for epithelial cell differentiation of the lung and the hair follicle. Genes Dev 2001;15:2307–2319.

126.

Shikama

Lutz

Kretzschmar

. Essential function of p300 acetyltransferase activity in heart, lung and small intestine formation. Embo J 2003;22:5175–5185.

127.

Nguyen

Kelley

Schlueter

Meyer

Senior

Miner

. Epithelial laminin alpha5 is necessary for distal epithelial cell maturation, VEGF production, and alveolization in the developing murine lung. Dev Biol 2005;282:111–125.

128.

Ito

Udaka

Yazawa

. Basic helix-loop-helix transcription factors regulate the neuroendocrine differentiation of fetal mouse pulmonary epithelium. Development 2000;127:3913–3921.

129.

Ramirez

Millien

Hinds

Cao

Seldin

Williams

. T1alpha, a lung type I cell differentiation gene, is required for normal lung cell proliferation and alveolus formation at birth. Dev Biol 2003;256:61–72.

130.

Rubin

Kovacs

De Paepe

Tsai

Torday

Kronenberg

. Arrested pulmonary alveolar cytodifferentiation and defective surfactant synthesis in mice missing the gene for parathyroid hormone-related protein. Dev Dyn 2004;230:278–289.

131.

Kaartinen

Voncken

Shuler

. Abnormal lung development and cleft palate in mice lacking TGF-beta 3 indicates defects of epithelial-mesenchymal interaction. Nat Genet 1995;11:415–421.

132.

Cardoso

. Regulation of early lung morphogenesis: questions, facts and controversies. Development 2006;133:1611–1624.