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Journal Vol 47(1)

Use of Transgenic Mice to Study Lung Morphogenesis and Function

James P. Bridges and Timothy E. Weaver

James P. Bridges, Graduate Student of Developmental Biology, and Timothy E. Weaver, Ph.D., Professor of Pediatrics, are in the Division of Pulmonary Biology, Cincinnati Children's Hospital Medical Center and University of Cincinnati College of Medicine, Cincinnati, Ohio.

Abstract

Successful transition to air breathing at birth depends on perinatal maturation of the gas exchange surface, resorption of fluid from the air spaces, and synthesis and secretion of pulmonary surfactant. Genetic mutations that alter lung development and/or cellular differentiation in the prenatal period, lung function in the perinatal period, or lung homeostasis in the postnatal period can lead to neonatal lethality or chronic lung disease. Current knowledge of the molecular pathways that regulate key prenatal, perinatal, and postnatal morphogenetic events has been shaped largely by remarkable advances in transgenic technologies. In this review, selected transgenic mouse models are highlighted to illustrate the power of this technology, which in many cases has provided important insights that otherwise could not have been obtained.

Key Words: development; differentiation; endothelium; epithelium; knockout; mesenchyme; respiratory; vascular

Introduction

Successful initiation and maintenance of air breathing at birth depends on the integration of prenatal, perinatal, and postnatal developmental cues. During the prenatal period, lung morphogenesis leads to formation of conducting airways that terminate in highly vascularized sac-like structures. Perinatal transition to air breathing requires fluid resorption from alveoli to expose a functional gas exchange surface. Septation of the terminal sacs (alveogenesis) leads to rapid postnatal expansion of the gas exchange surface. Sustained ventilation and vascular perfusion are essential for gas exchange in the neonatal and postnatal lung; likewise, maintenance of a sterile gas exchange surface and the ability to rapidly repair the blood-gas barrier after injury are critical for postnatal lung function. Genetic mutations that alter lung development and/or cellular differentiation in the prenatal period, lung function in the perinatal period, or lung homeostasis in the postnatal period can lead to neonatal lethality or chronic lung disease. In this review, we highlight selected transgenic mouse models (summarized in Table 1) that have provided important insight into the molecular pathways that regulate lung morphogenesis in the prenatal period and lung function/homeostasis in the newborn and postnatal animal.

Branching Morphogenesis (Prenatal Development)

Mouse lung morphogenesis commences at embryonic day 9.5 (E9.5), when the ventral foregut endoderm buds into the surrounding splanchnic mesenchyme to form the tracheal-bronchial tubules. Developing bronchi branch into the supporting mesenchyme to form bronchioles and conducting airways that terminate in sac-like structures destined to become alveoli in the postnatal period (for review, see Warburton et al. 2000). Endothelial and smooth muscle cells, which are present in the mesenchyme as soon as the initial lung buds form, promote formation of a vascular network that develops in parallel with the airways. Between E18.0 and 18.5, mature blood vessels of the microvasculature enrobe the terminal sacs, resulting in formation of a blood-gas barrier composed of type I epithelial cells with the underlying basement membrane and endothelial cells.

The fetal respiratory epithelium differentiates into multiple cell types that include ciliated, Clara (nonciliated), goblet, serous cells, neuroepithelial bodies and basal cells in the proximal airways, and type I and type II cells in the terminal sacs/alveoli. Both epithelial cell differentiation and branching morphogenesis are dependent on numerous developmentally regulated transcription factors, including members of the Nkx (e.g., thyroid transcription factor-1 [TTF-1¹]), GATA,² and Fox³ families (for review, see Costa et al. 2001). Inductive autocrine-paracrine signaling between the mesenchyme and adjacent epithelia is modulated in part through fibroblast growth factor (FGF¹), β-catenin/WNT,⁴ bone morphogenetic protein-4 (BMP-4¹), and sonic hedgehog (SHH¹) (Shannon and Hyatt 2004; Warburton et al. 2000). The complex interplay among these secreted factors and their directed transcriptional responses results in the formation of conducting airways and an extensive gas exchange surface.

Gene ablation, through direct disruption of a genetic locus or conditional gene targeting, has permitted identification of factors required for distinct stages of lung morphogenesis. For example, single null mutants of TTF-1 or the FGF-10 receptor (FGF-R2IIIB) produced by the epithelia, or FGF-10, produced by the mesenchyme, result in complete pulmonary agenesis, which demonstrates the importance of these signaling pathways in early branching morphogenesis (De Moerlooze et al. 2000; Kimura et al. 1996; Minoo et al. 1999; Sekine et al. 1999). Similar lung defects have been observed in humans who harbored deletions in chromosome 14q, a region that includes the TITF1 gene (encoding TTF-1), and in newborn infants with mutations in TITF1 (Breedveld et al. 2002; Devriendt et al. 1998; Iwatani et al. 2000). SHH, a potent signaling morphogen produced by the epithelium, is also required for branching morphogenesis. Defects in the SHH signaling pathway have been associated with several human congenital pulmonary malformations (Crisera et al. 1999, 2000; Ioannides et al. 2003). Both direct and respiratory cell-specific targeting of the Shh locus in the mouse resulted in lung hypoplasticity, a loss of peripheral lung tissue, and a developmental delay in type II epithelial cell differentiation (Litingtung et al. 1998; Miller et al. 2004; Pepicelli et al. 1998). The observed defects in mice lacking SHH were not as severe as those observed in Titf-1 and Fgf-10 null embryos, suggesting that SHH acts downstream of these factors. Targeted expression of a transgene encoding diphtheria toxin or a misfolded form of surfactant protein (SP¹)-C in the respiratory epithelium resulted in cytotoxicity associated with severe branching dysmorphogenesis and neonatal demise. This result further underscores the importance of epithelial-mesenchymal cell crosstalk for normal lung development (Bridges et al. 2003; Korfhagen et al. 1990).

Although TTF-1 and FGF-10 are required for early branching morphogenesis, recent data have uncovered the importance of FoxA1 and FoxA2 (previously known has HNF-3α and HNF-3β, respectively) in regulating distal epithelial cell specification and late morphogenetic events. Selective deletion of Foxa2 in pulmonary epithelial cells (Foxa2^Δ/^Δ) of the developing mouse lung has resulted in severe neonatal lung disease with all of the hallmarks of respiratory distress syndrome, including pulmonary surfactant deficiency, severe atelectasis, and hyaline membrane deposition (Wan et al. 2004b). Although lung morphogenesis was reported to be unperturbed in either Foxa1⁻/⁻ (Kaestner et al. 1999; Shih et al. 1999) or Foxa2^Δ/^Δ mice (Wan et al. 2004a), mice deficient in both Foxa1/Foxa2 in the distal lung epithelium exhibited a severe defect in branching morphogenesis (Wan et al. 2005), indicating that these transcription factors have overlapping (compensatory) functions. Transcriptional profiling of Foxa⁻/⁻ mice revealed that levels of SHH and WNT7b, signaling molecules important for morphogenesis, were decreased in the Foxa1/Foxa2 double mutants. The phenotypic similarities in the Shh⁻/⁻ and Foxa1/Foxa2 mutants, coupled with the decrease in SHH mRNA, have suggested that FoxA1 and/or FoxA2 directly or indirectly controls the expression of SHH in the lung epithelium. Collectively, these data indicate that the concerted interplay among the transcription factors TTF-1, FoxA1, and FoxA2 and the secreted factors FGF-10 and SHH play critical roles in the patterning and function of distal lung epithelium. The combined power of transgenic mouse models and microarray technology have greatly facilitated the identification and hierarchical classification of molecules important for normal lung morphogenesis.

Vascular Morphogenesis

Embryonic blood vessel development occurs by the following two mechanisms: (1) vasculogenesis, the process by which new vessels form de novo from angioblasts, and (2) angiogenesis, the process by which new vessels sprout from pre-existing ones. Numerous growth factors, transcription factors, and signaling pathways affect vascular development, including vascular endothelial growth factor (VEGF¹)/VEGF-receptor (VEGFR¹), angiopoietin/Tie-2, and Notch/Jagged, as well as transforming growth factor (TGF¹)-β, hypoxia inducible factor, and the homeobox protein HEX1 (for review, see Pauling and Vu 2004). However, determining the precise roles these factors play in the developing lung vasculature has proven difficult due, in part, to early embryonic lethality in gene-targeted mice.

The vascular system of the lung can be divided into the bronchial and pulmonary systems. The bronchial system provides nutrients to the lung while the pulmonary system ultimately regulates gas exchange. The pulmonary system is further subdivided into the proximal vasculature, consisting of large, medium, and small pulmonary arteries, and the peripheral vasculature, consisting of microvessels and capillaries that enrobe the alveoli at the site of gas exchange (for review, see Stenmark and Abman 2005). Initial studies using canalization and barium infusion techniques provided valuable anatomical information on late stage vessel development but offered only limited insight into early vascular development and the underlying developmental processes (Burri 1984; Effmann 1982). Investigators in an ontological study of pulmonary vasculature in the mouse (deMello et al. 1997) used transmission electron microscopy to analyze early (E9-12) developmental timepoints and casting of the vasculature combined with scanning electron microscopy for later (E13-17) timepoints. Their results suggested that the proximal vessels arise through angiogenesis, as evidenced by sprouting of vessels from the primitive aorta and left atrial chamber, whereas the distal vessels arise primarily through vasculogenesis, as evidenced by the appearance of vascular lakes surrounding the lung buds. The authors concluded that the two processes occur concurrently and that fusion of the proximal and peripheral vessels on E13-E14 resulted in a complete vascular circuit.

Studies in transgenic mice provided additional insight into the process described above. VEGFR-2, also known as FLK1 in the mouse, is expressed in mesodermal cells before endothelial differentiation and is considered to be the earliest marker of endothelial cells (Kabrun et al. 1997; Shalaby et al. 1995; Yamaguchi et al. 1993). Analyses of mice expressing LacZ knocked into the Flk1 locus have indicated that endothelial cells forming the pulmonary arteries are connected to the proximal aortic sac and the peripheral vascular network surrounding the distal lung buds (Schachtner et al. 2000). Importantly, connections between the proximal and peripheral circulations of the lung were detected at E10.5, 1 day after initial lung bud formation. This transgenic study suggested that the proximal circulation can also form by vasculogenesis, similar to the aorta (DeRuiter et al. 1993; Schachtner et al. 2000), and that connections between the proximal and peripheral circulations occur much earlier in pulmonary development than was previously thought.

Pulmonary vascular development and branching morphogenesis of the epithelium occur simultaneously. Signaling between the epithelium and adjacent mesenchyme is critical for pulmonary vascular cell proliferation and survival (Gebb and Shannon 2000). SHH is synthesized and secreted by pulmonary epithelial cells and signals through its primary receptor Patched-1 (PTC), which is located in the mesenchyme. Targeted ablation of the locus encoding Shh in mice has been shown to result in decreased VEGF-A, platelet endothelial cell adhesion molecule (PECAM¹), an endothelial cell marker, and mRNA transcripts of several smooth muscle-associated genes that include α-smooth muscle actin, serum response factor cofactor protein (SRFCP), and myosin heavy chain 11 (MYH11) (Miller et al. 2004). Reduced pulmonary vascular development in SHH-deficient mice was associated with the loss of peripheral lung tissue, underscoring the interdependence of epithelial-mesenchymal crosstalk in survival, growth, and differentiation of the pulmonary vasculature.

Secreted growth factors in the WNT family are also key regulators of cell-cell interactions during embryogenesis and organogenesis. WNT proteins bind membrane-spanning receptors of the Frizzled family, which signal through multiple pathways that include the ß-catenin/LEF-TCF pathway and culminate in the activation of target genes responsible for cell proliferation, survival, and differentiation. Expression of WNT7b is restricted to epithelial cells in the developing lung. Disruption of the locus encoding Wnt7b in mice has been shown to result in respiratory failure and neonatal lethality (Shu et al. 2002). Morphological and histochemical analyses revealed lung hypoplasticity associated with a decrease in mesenchymal cell proliferation/survival and defects in the smooth muscle component of large pulmonary vessels that were detectable as early as E12.5. The vascular defects led to dilation and hemorrhage of the large vessels upon expansion of the lungs at birth. Interestingly, the vascular network surrounding the distal airways and smooth muscle lining the proximal airways were unperturbed.

While WNT7b is required for the integrity of large pulmonary vessels, the transcription factor FoxF1 has been shown to be important for development of the pulmonary microvasculature. FoxF1 haploinsufficiency has been shown to result in perinatal lethality from pulmonary hemorrhage in approximately 50% of Foxf1^+/− mice (Kalinichenko et al. 2001, 2002; Mahlapuu et al. 2001); however, 40% of the Foxf1^+/− mice compensated for the allelic loss by upregulating FoxF1 expression, which resulted in normal lung development and survival. Hemorrhage in newborn low-expressing Foxf1^+/− mice was associated with fusion of the lung lobes and developmental defects in the peripheral lung saccules and microvasculature. Expression of factors important for morphogenesis and vessel formation was decreased in mutant lungs, including PECAM, BMP-4, and a delayed expression of FGF-10. Further analysis using transcriptional profiling has recently revealed a marked decrease in the Notch-2 receptor and HES-2, a downstream target of Notch-2 signaling (Kalinichenko et al. 2004). Although effects on the lung vasculature were not reported, mice with a hypomorphic allele of Notch-2 exhibited neonatal lethality associated with defects in kidney development and hemorrhaging in the embryonic heart and eyes, consistent with a role for Notch-2 in vascular development and/or vessel integrity (McCright et al. 2001). These mouse models highlight factors that are important in distinct stages of lung vascular development, including the formation, maintenance, and integrity of the vascular network.

Transition to Air Breathing (Perinatal Development)

Successful transition to air breathing at birth depends on perinatal maturation of the blood-gas barrier, resorption of fluid from the air spaces, and synthesis and secretion of pulmonary surfactant. Formation of the blood-gas barrier begins between E18 and E18.5 and continues into the alveolarization phase of the developing perinatal lung. The creation of a functional blood-gas barrier is dependent on thinning of the saccular septae and juxtaposition of the capillaries of the developing vasculature with terminally differentiated type I cells of the alveoli. Abnormalities in pulmonary vasculature, including formation of the blood-gas barrier, have been reported to contribute to the pathogenesis of neonatal lung diseases, including alveolar capillary dysplasia and bronchopulmonary dysplasia (deMello 2004; Parker and Abman 2003).

VEGF-A, commonly referred to as VEGF, is an important regulator of cardiovascular and pulmonary vascular development. Precisely controlled levels of VEGF expression are critical for normal vascular morphogenesis because deletion of a single VEGF allele in mice has been reported to result in failure of blood vessel development and early embryonic lethality (Carmeliet et al. 1996; Ferrara et al. 1996). In the saccular stage of lung development, VEGF expression is restricted to epithelial cells, and it promotes vascular morphogenesis through the binding of two receptors localized to the endothelium, VEGFR1 and VEGFR2 (Gebb and Shannon 2000; Ng et al. 2001). VEGF exists as three alternatively spliced isoforms in the mouse, VEGF-120, 164, and 188, which differ in their binding affinity to heparin sulfate proteoglycans in the extracellular matrix. The importance of the VEGF-164 and/or 188 isoforms in regulating the formation of the pulmonary blood-gas barrier was demonstrated in a mouse model engineered to express only the highly diffusible VEGF-120 isoform (Galambos et al. 2002). These mice exhibited an alveolar capillary dysplasia-like phenotype that included decreased numbers of capillaries per airway unit and failure of the basement membranes, between capillary endothelial cells and type I cells, to fuse. This study indicated an essential role for heparin-sulfate binding isoforms of VEGF in signaling to the endothelial cells for the formation of the gas exchange surface of the lung. Targeted deletion of endothelial nitric oxide synthase, an enzyme involved in the formation of the potent vasodilator nitric oxide, also resulted in lethal neonatal respiratory distress associated with a marked thickening of the saccular septae and misalignment of capillaries with the developing epithelium (Han et al. 2004). These mouse models highlight two important factors in the formation of the pulmonary blood-gas barrier and provide insight into the pathogenesis of neonatal lung diseases associated with vascular dysgenesis.

In the late stages of lung development, the air spaces are filled with fluid that must be removed rapidly, before the onset of respiration at birth. The epithelium of the lung expresses amiloride-sensitive epithelial sodium channels (ENaCs¹) that mediate sodium transport and fluid adsorption. ENaC is a heterotrimeric protein that consists of three homologous subunits, alpha, beta, and gamma. Mice that harbor a targeted mutation of the alpha subunit of ENaC have been shown to develop respiratory distress and to die within 40 hr after birth (Hummler et al. 1996). Functional and histological analyses showed abolished sodium transport in the lung epithelium of EnaC-deficient mice and failure to clear airway fluid at birth. This mouse model establishes a critical role for ENaC in adaptation to air breathing at birth.

Elastic fibers in the terminal sac/alveolar wall combined with surface tension resulting from a thin liquid layer on the epithelial surface generate a high collapsing force at end expiration. Spreading of a phospholipid-rich film (pulmonary surfactant) on the epithelial cell surface dramatically reduces surface tension and prevents alveolar collapse during expiration. Surfactant deficiency results in widespread alveolar collapse (atelectasis) that compromises gas exchange and frequently requires respiratory support. Pulmonary surfactant is synthesized and secreted into the air spaces by alveolar type II epithelial cells. Dipalmitoylphosphatidylcholine (DPPC¹), the most abundant phospholipid component of surfactant, is primarily responsible for the surface tension-reducing properties of the lipid film. The results of numerous in vitro studies have indicated that rapid formation and maintenance of a stable DPPC-rich surface film requires at least one of the two hydrophobic surfactant proteins, SP-B or SP-C. The importance of SP-B for surfactant function was confirmed by disrupting the Sftpb locus in mice (Clark et al. 1995). Sftpb^−/− mice died of respiratory distress syndrome shortly after birth. Similarly, human infants with SFTPB mutations resulting in SP-B deficiency died of respiratory failure even with aggressive postnatal care (Hamvas et al. 1994; Nogee et al. 1993, 1994, 2000). In contrast, disruption of genetic loci encoding the surfactant proteins SP-A (Ikegami et al. 1997, 1998; Korfhagen et al. 1998a), SP-D (Botas et al. 1998; Korfhagen et al. 1998b), or SP-C (Glasser et al. 2001, 2003) did not perturb surfactant function in newborn mice, underscoring the importance of SP-B for adaptation to air breathing at birth. Importantly, deficiency of FoxA2 in respiratory epithelial cells of the peripheral lung was shown to result in deficiency of both surfactant proteins and lipids, leading to severe respiratory distress syndrome (Wan et al. 2004b).

Lung Maturation (Postnatal Development)

Formation of the gas exchange surface begins just before birth and continues in the postnatal period in both humans and mice. The growth of secondary septae into peripheral lung saccules results in the formation of alveoli, dramatically expanding the functional gas exchange surface. In the fully mature human lung, approximately 400 million alveoli provide a gas exchange surface of 100 to 150 m². The alveolar epithelium is composed predominantly of large cells with a very thin cytoplasmic profile (type I epithelial cells) that cover 90% of the alveolar surface. Type I epithelial cells, together with the underlying basal lamina and capillary endothelium, comprise the air-blood barrier. Adequate gas exchange in the maturing lung depends on (1) expansion of alveolar air spaces during the postnatal period of rapid somatic growth, (2) prevention of alveolar collapse at end-expiration, (3) maintenance of a sterile gas exchange surface, and (4) the ability to rapidly repair the air-blood barrier after epithelial cell injury.

Septation of the terminal sacs (alveogenesis) is the primary mechanism by which the gas exchange surface is expanded in the postnatal lung. Alveogenesis, which begins on postnatal day 4 in the mouse, depends on the deposition of elastin within the walls of the terminal sacs. This key morphogenetic event is under the control of PDGF-A, a growth factor that induces the proliferation and migration of smooth muscle cells in the postnatal lung (Lindahl et al. 1997). In Pdgfa^−/− mice, alveolar smooth muscle cells fail to relocate to terminal sacs, which results in a lack of elastin deposition, a failure to form secondary septae and, ultimately, respiratory failure (Bostrom et al. 1996). The elastin null mouse exhibits an even more severe phenotype in which the terminal sacs are abnormally dilated at birth before formation of secondary septae (Wendel et al. 2000). Scaffolding proteins involved in the assembly of elastic fibers also play a critical role in postnatal lung morphogenesis. A deficiency of fibulin-5, an elastin-binding protein that organizes and links elastic fibers to cell surface integrins, has been shown to result in general elastinopathy-associated pulmonary abnormalities (apparent on the first day of life) and progressive air space enlargement (Yanagisawa et al. 2002). These two transgenic models indicate that elastin and elastin-binding proteins play critical roles in postnatal lung maturation by regulating postnatal branching of the distal airways and subsequent formation of secondary septae.

Several transgenic mouse models have provided insight into signaling pathways that regulate alveogenesis. Elevated levels of adenosine in adenosine deaminase null mice have been shown to result in inflammation and alveolar enlargement that led to respiratory failure at 3 wk of age (Blackburn et al. 2000). Aberrant adenosine signaling was associated with altered expression of genes regulating apoptosis, proliferation, and vascular development (Banerjee et al. 2004). Forced expression of transforming growth factor (TGF¹)-α in the distal respiratory epithelium of transgenic mice also resulted in air space enlargement, as well as fibrosis, that was detectable as early as 1 wk of age (Hardie et al. 1997; Korfhagen et al. 1994). This phenotype was similar in many respects to that observed in lungs of human infants with bronchopulmonary dysplasia (BPD¹), suggesting that TGF-α may play a role in the pathogenesis of this disease. Some aspects of the BPD phenotype were also detected in mice in which the genes encoding FGFR-3 and FGFR-4 were disrupted (Weinstein et al. 1998). Interestingly, lung morphogenesis was normal in mice in which only one FGFR was disrupted, indicating that FGFR-3 and FGFR-4 act cooperatively to direct alveogenesis in the postnatal lung. Collectively, these transgenic mouse models demonstrate that perturbation of the complex molecular pathways that regulate alveogenesis contributes to the development of chronic and/or fatal lung disease.

Continued synthesis and secretion of pulmonary surfactant by alveolar type II epithelial cells is essential for postnatal lung function. The results of studies in compound transgenic Sftpb^−/− mice, in which expression of SP-B was regulated by administration of doxycycline, indicated that SP-B was absolutely required for postnatal lung function and that a 75% decrease in SP-B concentration in the air spaces resulted in respiratory failure (Melton et al. 2003). Surprisingly, SP-C deficiency had no effect on gas exchange in Sftpb^−/− mice, indicating that SP-B was sufficient for surfactant function (Glasser et al. 2001). However, Sftpc^−/− mice progressively developed interstitial lung disease (ILD¹) similar to that detected in humans carrying a mutation in one allele of the SFTPC gene (Glasser et al. 2003; Nogee 2001, 2004; Thomas et al. 2002). The severity of ILD in Sftpc^−/− mice was dramatically altered by the genetic background, consistent with the influence of one or more modifier genes (Glasser et al. 2003). Transgenic expression of an SFTPC mutation associated with ILD in humans resulted in disruption of lung morphogenesis (Bridges et al. 2003). The mutant SP-C proprotein induced cytotoxicity in the presence of two wild type Sftpc alleles, demonstrating that the misfolded protein produced a dominant negative effect in both transgenic mice and human patients.

In addition to SP-B and SP-C, pulmonary surfactant contains two hydrophilic proteins, SP-A and SP-D. The results of in vitro studies predicted that SP-A would be an important regulator of surfactant homeostasis. However, alveolar surfactant composition, pool size, and function were shown to be essentially normal in Sftpa1^−/− mice (Korfhagen et al. 1998a). Unexpectedly, SP-D, which does not pellet with the surface active, large aggregate fraction of surfactant, was found to play an important role in surfactant homeostasis. Alveolar surfactant pool size was significantly increased in Sftpd^−/− mice (Botas et al. 1998; Korfhagen et al. 1998b). Using compound transgenic mice, SP-D was reported to influence the ultrastructure of surfactant and to modulate the uptake and catabolism of surfactant by type II epithelial cells (Ikegami et al. 2005). SP-D deficiency was also linked to the development of emphysema in gene-targeted mice (Hawgood et al. 2002; Wert et al. 2000). Overall, transgenic mouse models have provided important insights into the function of surfactant proteins that otherwise could not have been deduced.

Recent studies in gene-targeted mice have also identified an important role for SP-A and SP-D in innate host defense of the air spaces. Over the course of a day, millions of micro-organisms are inhaled, providing the potential for colonization of the air spaces, epithelial cell injury, and, ultimately, disruption of gas exchange. Surfactant provides a physical barrier to inhaled pathogens because it contains several antimicrobial proteins, including SP-A and SP-D, which directly kill pathogens and/or facilitate the opsonization and clearance of pathogens by alveolar macrophages. Disruption of genes encoding Sftpa1 or Sftpd in mice has been shown to result in increased susceptibility to airway infection by a wide variety of micro-organisms (LeVine et al. 1997, 1998, 1999a, 2000, 2001, 2002; Li et al. 2002; Linke et al. 2001). Similarly, disruption of the locus encoding lysozyme, an antimicrobial protein associated with tubular myelin in rodents, was shown to result in increased lung bacterial burden, systemic dissemination of pathogen, and mortality (Ganz et al. 2003; Markart et al. 2004). In contrast, transgenic mice expressing elevated levels of lysozyme in the air spaces were reported earlier to exhibit decreased bacterial burden and mortality after intratracheal infection with Gram-negative or Gram-positive bacteria (Akinbi et al. 2000). Thus, transgenic mouse models have contributed to our understanding of the mechanisms by which the host rapidly clears inhaled micro-organisms, thereby avoiding recruitment/activation of immune cells and constitutive inflammation.

Summary of Ongoing Research and Future Perspectives

Creative use of promoters has led to the development of strategies for conditional gene expression. In the tetracycline (tet¹)-inducible system, mice carrying the transgene of interest under of control of the tet-operator (tetO¹) are bred with transgenic mice carrying the reverse tetracycline response transactivator (rtTA¹), under control of a cell/tissue-specific promoter (Gossen et al. 1995; Kistner et al. 1996). Transgene expression is induced only in bitransgenic mice after treatment with doxycycline, which enables rtTA to bind to tetO elements and activate transcription. The combination of a cell-specific promoter (driving rtTA expression) with the timing of doxycycline administration or withdrawal permits the transgene to be switched "on" or "off" in subsets of cells (e.g., alveolar type II epithelial cells or bronchiolar Clara cells) (Perl et al. 2002b). A variation of this approach is conditional gene deletion, which permits temporal and/or cell-specific silencing of the targeted gene. This strategy involves breeding of three independent mouse lines, including a line in which the gene of interest has been "floxed" (i.e., loxP sites are inserted into the 5′ and 3′ regions surrounding a gene target), a transgenic line carrying the cre-recombinase gene under control of tetO, and a separate transgenic line carrying rtTA under control of a cell/tissue-specific promoter. Administration of doxycycline to triple transgenic mice results in expression of cre-recombinase, which excises DNA between the loxP sites in specific cells (determined by the promoter driving rtTA). Development of this important tool facilitates the study of genetic loci that when disrupted, produce embryonic lethality before lung organogenesis (Wan et al. 2004b). Conditional gene deletion/replacement has also been used in cell lineage tracing experiments to identify lung progenitor cells (Perl et al. 2002a).

Refinement of experimental approaches such as those briefly mentioned above will provide new opportunities to generate novel, informative mouse models. For example, targeted transgenesis, in which the transgene is introduced into the hypoxanthine-guanine phosphoribosyl transferase (Hprt) locus by homologous recombination, avoids the variable gene expression (and potentially variable phenotype) associated with random integration of the transgene (Farhadi et al. 2003). Targeted insertion of RNAi, coupled with conditional and cell-specific expression, could provide a powerful new approach to reversible gene silencing in vivo. Finally, the advent and refinement of technologies such as laser capture microdissection, microarray, and proteomic instrumentation will greatly enhance our ability to identify the molecular pathways underlying altered phenotype in transgenic mouse models.

Conclusion

This review is by no means a comprehensive listing of all of the transgenic mouse models generated to study lung morphogenesis and homeostasis in the prenatal and perinatal periods. For example, the many transgenic models generated to study lung injury and the repair processes critical for maintenance of a functional blood-gas barrier are not discussed. Rather, selected transgenic models are highlighted to illustrate the power of transgenic technology, which in many cases has provided important insights that otherwise could not have been obtained. Key to the development of this technology has been the identification of lung- and cell-specific promoters, which have enabled investigators to target transgene expression to proximal or distal respiratory epithelium, endothelium, or smooth muscle cells. The identification and characterization of new promoters will significantly expand the ability to manipulate the temporal and spatial expression of selected genes in transgenic mice.

Acknowledgments

The authors gratefully acknowledge the support of the National Heart, Lung and Blood Institute (HL56285, HL61646, and HL56387), helpful suggestions from Drs. Ann Akeson and Tim Le Cras, and the excellent secretarial assistance of Ann Maher.

¹Abbreviations used in this article: BMP-4, bone morphogenetic protein-4; BPD, bronchopulmonary dysplasia; DPPC, dipalmitoylphosphatidylcholine; ENaC, epithelial sodium channel; FGF, fibroblast growth factor; ILD, interstitial lung disease; PECAM, platelet endothelial cell adhesion molecule; rtTA, reverse tetracycline response transactivator; SHH, sonic hedgehog; SP, surfactant protein; tet, tetracycline; tetO, tet-operator; TGF, transforming growth factor; TTF-1, thyroid transcription factor-1; VEGF, vascular endothelial growth factor; VEGFR, VEGF-receptor.

²GATA is a family of zinc transcription factors that bind GATA consensus sequence elements.

³Fox, the forkhead box family of transcription factors, was previously known as the HNF-3 family (hepatocyte nuclear factor-3)

⁴Wnt, the int-1 (integration-1) gene, was originally identified as a locus activated by mouse mammary tumor virus insertion and subsequently renamed to Wnt-1, a member of the Wnt family.

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