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* From the Division of Pulmonary Biology, Children’s Hospital Medical Center, Cincinnati, OH.
Correspondence to: Jeffrey A. Whitsett, MD, Children’s Hospital Medical Center, Division of Neonatology and Pulmonary Biology, 3333 Burnet Ave, Cincinnati, OH 45229-3039; e-mail: jeff.whitsett{at}chmcc.org
Overview
Transgenic mouse models generated by gene targeting and gene addition have been highly useful for the analysis for gene function in vivo and are providing unexpected insights into pathways mediating lung morphogenesis and repair. Continued progress in the technology for gene addition and targeting in the mouse has made possible the conditional addition, mutation, or deletion of genes in a tissue-specific manner. The reverse tetracycline transactivator (rtTA) system has been of particular utility in generating models in which expression of genes or complementary DNAs can be altered in respiratory epithelial cells of the conducting or peripheral airways. This is accomplished using regulatory elements from the Clara cell secretory protein (CCSP) or surfactant protein (SP)-C promoters. We will summarize progress in generating transgenic activator mice that express rtTA under the control of the CCSP or SP-C promoters. When bred to target transgenic mice, precise temporal-spatial control of gene expression is achieved when the transgenic mouse is exposed to doxycycline administered in the drinking water. Induction can be achieved in utero or in early postnatal mice by treatment of the dam, as doxycycline can cross the placenta or be transferred via maternal milk. When used in concert with gene-targeted animals, the precise temporal-spatial gene expression required for the correction of lung phenotypes can be assessed. The system is also useful in directing conditional expression of Cre-recombinase to inactivate genes in a cell- and tissue-specific manner in vivo.
Specificity of the CCSP Promoter Elements
The promoter element from the CCSP uteroglobin gene has been utilized to express a number of target genes in the conducting airways of transgenic mice, both directly and in a conditional manner. Initial deletion mutants of the CCSP gene demonstrated that lung epithelial cell specificity of target-gene expression was conferred by constructs containing 1.25 to 2.2 kb of the 5' flanking region of the mouse or rat CCSP gene.1 Using the CCSP promoter, complementary DNAs or minigenes were selectively expressed in Clara (nonciliated) respiratory epithelial cells in transgenic mice that were produced by nuclear injection of fertilized oocytes. The CCSP promoter is active in airway cells as early as 14 to 15 days of gestation, increases postnatally, and persists during the postnatal period of development. Expression in tissues other than lung is generally not observed in the mouse, and approximately 50% of airway cells in the conducting airways of the mouse lung express the transgene.
SP-C Promoter Elements
SP-C is expressed only in the lung, being detected in alveolar type-II epithelial cells in the mature lung. The function of both the human and murine SP-C promoter elements was studied in transgenic mice in vivo.2 3 The human SP-C gene is highly active in respiratory epithelial cells as early as days 9 to 10 of gestation4 and increases with advancing gestation, being confined to cells in the lung periphery, and ultimately expressed in bronchiolar and alveolar type-II epithelial cells of the lung. While lung specificity is almost always observed in transgenic mice generated with the SP-C promoter, the level and sites of expression of transgenes vary with the size of the 5' flanking region used,5 and may differ among founders on the basis of copy number and genomic insertion site. In general, transgene expression generated with the mouse SP-C promoter is detected solely in type-II epithelial cells.3 The levels and numbers of cells expressing the transgenes may vary among founder lines, targeting all or only some of the peripheral airway cells and alveolar type-II cells. Expression of the transgene is excluded from type-I epithelial cells.
Utility of the Tetracycline Transactivator System In Vitro and In Vivo
The tetracycline response system developed by Gossen and Bujard6 relies on a fusion protein containing a DNA binding element from Escherichia coli that binds tetracycline and a protein from herpesvirus (VP16) that activates minimal promoter elements when bound to a defined enhancer element consisting of a concatamer of seven (otet) binding sites ([otet]7), which are upstream of an inactive minimal promoter from the cytomegalovirus (CMV). The antibiotic doxycycline binds to the transactivator protein and can either activate (rtTA) or inactivate (tetracycline transactivator) binding of the fusion protein to the target (otet)7 CMV sequence. Binding of rtTA to the (otet)7 element recruits VP16, activating the CMV promoter and downstream gene transcription. When the rtTA is expressed only in Clara cells with the CCSP promoter,7 or in type-II epithelial cells with the SP-C promoter, conditional cell-specific control of target-gene expression is conferred by treatment of the animal with doxycycline. By careful selection of the activator mice, target genes are expressed in a highly defined temporal-spatial and reversible manner under control of doxycycline.
Expression of Fibroblast Growth Factors in Respiratory Epithelial Cells In Vivo
The fibroblast growth factor (FGF) family of polypeptides comprise a diverse group of growth factors that bind to and activate transmembrane receptor tyrosine kinases involved in growth and differentiation of various tissues.8 9 A number of FGF family members are expressed with distinct temporal and spatial characteristics in the lung, and include FGF-1, FGF-2, FGF-7, FGF-9, FGF-10, and FGF-18.10 11 12 13 14 15 16 Likewise, FGF receptors (FGF-Rs), particularly the FGF-R2IIIb isoform, are expressed in the respiratory epithelium of the developing lung.10 17 18 Targeted deletion of FGF-10 caused pulmonary and limb agenesis in vivo,19 20 findings similar to those seen in the disruption of FGF-R2 signaling in vivo,10 21 22 demonstrating a strict requirement for FGF-10 and FGF-R2 in lung formation in vivo. FGFs have a profound effect on lung epithelial cell proliferation, fluid transport, and cell differentiation in the postnatal lung. Exogenous FGF-7 enhances epithelial cell proliferation in the adult lung,23 and protects the lung from various injuries.24 25 26 27 However, expression of FGF-7 in the developing lung of transgenic mice was lethal at approximately day 15 of gestation.28 Thus, study of the chronic effects of FGF on the postnatal lung has not been discerned.
Conditional Expression of FGF-7 in the Respiratory Epithelium
In order to determine the effects of increased FGF expression on the developing and postnatal lung, FGF-7 and FGF-10 were placed under conditional control of the rtTA regulatory system, in which the growth factors were expressed in respiratory epithelial cells under control of doxycycline (Fig 1) . SP-C rtTA and CCSP rtTA times (otet)7 CMV FGF-7 double transgenic mice were utilized to assess prenatal and postnatal effects of FGF-7 on lung morphogenesis, differentiation, and remodeling.29 Doxycycline treatment of the dam enhanced expression of FGF-7 in the fetal lung in utero, causing cystadenomatoid malformations of the lung. When controlled by the CCSP activator, however, the FGF-7 messenger RNA (mRNA) was detected only in the presence of doxycycline in the prenatal or postnatal lung. In the postnatal double transgenic mice, FGF-7 mRNA was detected in type-II epithelial cells and in peripheral conducting airways, causing marked hyperplasia of the respiratory epithelium. Expression of thyroid transcription factor-1, aquaporin-5, and the type-II cell markers, proSP–C and SP-B, was markedly induced by expression of FGF-7. In spite of marked alveolar proliferation and remodeling, withdrawal of doxycycline blocked expression of the FGF transgene and reversed the abnormalities in the lung. This model should be highly useful in discerning the role of FGF-7 in the regulation of pulmonary genes and defining mechanisms involved in alveolar remodeling.
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SP-C rtTA and CCSP rtTA times (otet)7 CMV FGF-10 double transgenic mice were generated in the same activator lines used to express FGF-7.30 As seen in the FGF-7 studies, expression of FGF-10 with CCSP activator mice required doxycycline, while doxycycline-independent expression was observed in mice in the postnatal lung bearing the SP-C rtTA transgene. In the fetal lung, FGF-10 mRNA was markedly increased by doxycycline when administered to the dam. Postnatally, expression of FGF-10 caused multifocal pulmonary tumors. The tumors were highly differentiated, adenomatous lesions, consisting of cells with characteristics of type-II epithelial cells. The tumor cells contained lamellar bodies and expressed SPs in high abundance. Mice in which CCSP rtTA controlled expression of FGF-10 also developed adenomatous polyps in the conducting airways after doxycycline treatment. Both tumor cells and adenomatous changes were readily reversed by removal of doxycycline. Similar to findings in the FGF-7 expressing mice, thyroid transcription factor-1, SP-B, SP-C, and BrdU incorporation were increased in the adenomas induced by FGF-10. In fetal mice, expression of FGF-10 caused adenomatous changes and disruption of branching morphogenesis. Tumors were generally more organized and less cystic in the FGF-10–expressing mice, compared to the more generalized cellular hyperplasia seen following expression of FGF-7. Thus, conditional expression of a single polypeptide, FGF-10 was sufficient to produce highly differentiated adenomatous lung tumors in vivo. Removal of FGF-10 signaling caused remarkable and rapid remodeling of the tumors. The mechanisms by which FGF induces cellular hyperplasia and tumor formation should provide insight to pathways mediating lung remodeling in acute and chronic lung diseases.
Summary
The doxycycline-regulatable rtTA system has proven useful for the generation of transgenic mice in which polypeptides are expressed in the conducting and peripheral airways of lungs. Future advances with these transgenic systems include the ability to control gene insertion or deletion at genomic loci in transgenic mice, making possible the regulated addition or deletion of target genes in lung cells and their daughter cells. Since the disruption of a number of genes is lethal prior to lung formation, generation of transgenic mice with conditionally controlled Cre-recombinase in lung epithelium should also prove useful in elucidating the roles of critical regulatory pathways in the lung.
Footnotes
Abbreviations: CCSP = Clara cell secretory protein; CMV = cytomegalovirus; FGF = fibroblast growth factor; FGF-R = fibroblast growth factor receptor; mRNA = messenger RNA; (otet)7 = concatamer of seven (otet) binding sites; rtTA = reverse tetracycline transactivator; SP = surfactant protein
References
This article has been cited by other articles:
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  M. Nanjundan and F. Possmayer Pulmonary phosphatidic acid phosphatase and lipid phosphate phosphohydrolase Am J Physiol Lung Cell Mol Physiol, January 1, 2003; 284(1): L1 - L23. [Abstract] [Full Text] [PDF]
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