Many of the mice we mention in this review are available form the Jackson Laboratories (http://jaxmice.jax.org
). Most importantly the Jackson Laboratories has also developed useful databases. These include, MGI (Mouse Genome Informatics), which provides access to integrated data on mouse genes and genome features, from sequences and genomic maps to gene expression and disease models (http://www.informatics.jax.org/
). The IMSR
(International Mouse Strain Resource), which is a searchable online database of mouse strains and stocks available worldwide, including inbred, mutant, and genetically engineered mice (http://www.findmice.org/
). And the MPD (Mouse Phenome Database), which is a collaborative collection of baseline phenotypic data on inbred mouse strains. The MPD includes data sets, protocols, projects and publications, and SNPs (http://phenome.jax.org/
1) Epithelial lines
The lung buds from the foregut endoderm around embryonic day (E) 9.5 in mice and continues to develop by branching morphogenesis. By contrast, the trachea and esophagus separate from the foregut just ventral to the lung buds between E10.0 and E11.5 and then increase in length and diameter during development. A number of mouse strains suitable for conditional gene activation in the developing lung have been developed using genes expressed in the foregut endoderm. However, with the exception of Sftpc lines, these are not unique to the developing lung. The choice of line depends partly on the timing of activity required: 1) in the undivided foregut (that is, throughout the entire lung, trachea and esophagus), or 2) more lung-specific. As differentiated cell types appear during lung development they can be targeted with cell type specific mouse lines.
1a) Embryonic epithelial progenitors Sonic hedgehog (Shh)
is expressed in a highly dynamic pattern during embryogenesis. The Shh-Cre
line is a knock-in of a GFP-Cre
fusion protein [73
] and has been shown to activate reporter recombination in the ventral foregut endoderm by E9.5, prior to lung budding and tracheal/esophageal separation [74
]. It has successfully been used to study both tracheal and lung development [11
is expressed in many tissues during embryogenesis including the limb and heart, but is enriched in the lung epithelial precursors at E9.5 [77
]. The Islet1Cre
line is a knock-in strain [78
] and drives Cre expression throughout the pharyngeal endoderm, including the pulmonary epithelial precursors, and in a subset of mesenchymal cells [79
]. Although it is not epithelial specific, it has been successfully used to study Fgf8 function during lung development [80
Transgenic mice driving Cre from a 4.8 kb fragment of the Gata5
) mediate recombination in the developing epicardium and also throughout the developing lung endoderm [81
]. However, onset of recombination and potential activity in the developing trachea has yet to be determined.
The transcription factor Id2
is dynamically expressed in various cell types during embryogenesis and in the adult. The Id2-CreERT2
line is a knock-in of the CreERT2
]. In the developing lung this strain displays tamoxifen-dependent Cre-mediated recombination in the distal epithelial tips and, at a lower level, in a sub-set of mesenchymal cells.
is one of the earliest cardiac-specific markers in vertebrate embryos. The Nkx2-5 Cre
line is a knock-in [84
] and drives recombination in the developing proepicardium and subsequently throughout the myocardium and the first pharyngeal arch [85
]. Recombination occurs in both the foregut endoderm and surrounding mesoderm prior to E9.5 before lung budding or tracheal-esophogeal separation. Nkx2.5-Cre
has successfully been used to inactivate epithelial expression of Sox2
in the developing respiratory tract [34
(also known as Ttf1
) is expressed in the domain of the ventral foregut, which will give rise to both the lung and trachea. Nkx2.1
is also expressed in thyroid progenitors and regions of the developing brain [86
]. An Nkx2.1-Cre
BAC transgenic mouse [87
] functions throughout the lung and tracheal epithelium and has been successfully used in a number of studies [88
Subsequent to budding, the lung epithelium transcribes Sftpc
(SpC, or Surfatant associated protein C
). Since the human SFTPC
promoter is active once endoderm has been committed to becoming lung it is very useful for studying early lung development. The SFTPC
lines are discussed below. As lung development proceeds more restricted progenitor cells, such as bronchiolar progenitors, are hypothesized to exist [76
]. Tools for manipulating gene expression specifically in such progenitor cells would be highly desirable.
1b) Alveolar type II cells
The 3.7 Kb fragment of the human SFTPC
promoter is one of the most widely used promoters to generate constitutive and conditional transgenic mouse strains that target the respiratory epithelium [3
]. The advantage of the SFTPC
promoter is that it is very lung-specific and off-target effects on other organs are extremely rare. In the adult lung SFTPC
promoter activity is restricted to alveolar type II cells and subsets of cuboidal bronchiolar cells. The most widely distributed strains are the SFTPC-rtTA
] and SFTPC-Cre
]. The SFTPC-rtTA
lines are particularly useful since dox application from E6.5 to E10.5 only targets the progenitor pool of the distal lung epithelium, the parathyroid and the thymus. However, targeting of neuroendocrine cells with these lines was not observed [58
]. During organogenesis expression levels of morphogenic genes dynamically change spatially and temporally. With the rtTA line it was possible to activate and inactivate signaling pathways that regulate morphogenic genes in specific compartments at defined times. These studies led to a better understanding of temporal windows for FGF signaling and allowed the process of lung organogenesis to be dissected in greater detail [59
]. Most prenatal studies were done using SFTPC-rtTA
line 1, which expresses rtTA at high levels, but also shows dox independent gene activation especially around birth and postnatally. Dox independent expression can result in embryonic lethal phenotypes, as it was the case for overexpression of FGF7 and VEGF [54
]. To overcome these limitations, a second founder line (SFTPC-rtTA line2)
was characterized [90
], which expresses lower levels of rtTA and demonstrates less off-dox effects. While line 2 has been demonstrated to work after E14.5 and in adult mice, activation in the developing embryonic endoderm has not been tested.
Both lines have been widely distributed in the scientific community.
transgenic strain contains the human SFTPC
promoter fragment, which drives a rabbit β globin intron, followed by Cre recombinase. SFTPC-Cre
directs recombination throughout the lung epithelium starting at E10.5 [91
]. This strain has also been widely used and has contributed significantly to our understanding of lung development [94
]. There are reports of toxic effects on some genetic backgrounds [95
]. More recently, it has been demonstrated that the SFTPC-Cre
line directs recombination in the male germline, which may have confounded previous studies resulting in the apparent toxicity (B.R. Stripp, personal communication). As long as this transgene is transmitted through the female germline it is very useful to study embryonic lung development.
is a knock-in of both CreERT2
and rtTA cassettes just after the stop codon of the endogenous Sftpc
]. This very flexible strain drives both CreERT2
and rtTA expression in mature adult type II cells. It has already proved to be useful for lineage-tracing experiments and will undoubtedly also be widely used for manipulating gene expression.
Other SFTPC-lines are summarized in . There are alternative promoters (e.g. ABCA3, C/EBPα), which could be potentially used to target alveolar type II cells. However, these promoters will not be lung specific.
Embryonic epithelial progenitors
1c) Alveolar type I cells
Aquaporin 5 (Aqp5)
, is expressed predominantly in salivary and lacrimal glands, cornea, trachea, and distal lung [98
]. In rat and human lungs, Aqp5
is specifically expressed in alveolar type I (AT1) cells and not in alveolar type II (AT2) cells. In mice Aqp5
expression has also been found in AT2 cells [100
]. A Cre-IRES-DsRed cassette has been inserted into exon 1 of the endogenous Aqp5
locus generating the Aqp5-Cre-IRES-DsRed
, or ACID
]. Analysis with the ROSA-mT/mG reporter [102
], demonstrated that recombination had occurred in a very high fraction of AT1 cells in the distal lung and not in AT2 cells. However, AT2 recombination in other genetic backgrounds cannot yet be ruled out. This is the first transgenic mouse engineered to express Cre in AT1 cells and it should be very useful for studies of AT1 turnover and function.
Podopladin, or T1 alpha, a gene with unclear function, is expressed in mouse AT1 cells and lymphatics [103
]. By contrast, the rat podopladin gene is specific for AT1 cells. A modified rat BAC containing internal ribosome entry site (IRES)-green fluorescent protein (GFP) in the podoplanin 3′UTR has been generated (RTIbac) [104
]. RTIbac-transgenic mice expressed rat podoplanin in AT1 cells and in the brain, and expression in AT2 cells, airways, and vascular endothelium was not detected. Modifications of this BAC to express the rtTA or Cre recombinase could make this construct useful for targeting ATI cells.
1d) Bronchiolar Clara Cells
Secretoglobin1a1 (Scgb1a1, also known as CCSP, CC10 and CCA) is expressed in all bronchiolar Clara cells, and at lower levels in most tracheal Clara-like cells. In the rat Scgb1a1 is also expressed in AT2 cells. A rat Scgb1a1 promoter fragment has been used for making various transgenic lines.
A 2.3Kb fragment of the rat Scgb1a1
promoter is sufficient to direct expression in mouse Clara cells [1
]. This promoter was subsequently used to generate two independent Scgb1a1-rtTA
mouse lines. The first line [56
] has been widely distributed within the research community and used successfully in multiple studies. Lineage-tracing showed that this strain has efficient activity in many bronchiolar Clara cells and also a sub-set of AT2 cells [59
]. The Scgb1a1-rtTA
line 2 targets most Clara cells, but retains little AT2 cell activity [43
is also active in the uterus. However, using luciferase reporter mice no luciferase activity was detected in whole uterus homogenates [56
The rat Scgb1a1
promoter has also been used to generate a Scgb1a1-rtTA2S-M2
which uses the newer more-sensitive version of rtTA [68
]. This strain is reported to have no basal activity and increased doxycycline sensitivity. However, it also has some activity in AT2 cells.
transgenic strain was generated using the rat promoter fragment inserted upstream of the coding sequence for Cre [105
]. Lineage tracing shows that this strain directs recombination in the bronchiolar cells, but not in any alveolar cells [107
], demonstrating that the insertion site of the transgene has a strong effect on expression pattern.
“knock-in” mouse strain was generated by inserting an IRES-CreER™ cassette into the 3′ UTR of the endogenous mouse Scgb1a1
]. This line was used for detailed lineage-tracing studies, which revealed that it provides specific, tamoxifen dose-dependent Cre activity in up to 90% of bronchiolar Clara cells, and up to 7% of AT2 cells. However, it also displays some tamoxifen-independent activity.
A knock-in Tgfb3-Cre
] has recently been used to manipulate Notch signaling in the postnatal lung airways [109
]. Reporter analysis suggested that this strain targets the majority (~90%) of Clara cells.
There is evidence to suggest that not all Clara cells are functionally equivalent [110
]. Transgenic strains, which target specific sub-sets of Clara cells would be highly desirable for the lung research community.
1e) Ciliated cells
Foxj1 is a transcription factor which is expressed in all multiciliated cells including those of the lung, oviducts, ependyma and testes [112
], and various cells with motile cilia [113
]. A 1Kb fragment of the human FOXJ1
promoter was shown to be sufficient to direct reporter gene expression specifically in all of these cell types in adult mice [116
]. This promoter was subsequently used to generate FOXJ1-Cre
], and FOXJ1-CreERT2
transgenic mice [28
]. Both lines drive efficient recombination in ciliated cells of the respiratory tract and have been useful for gene knock-out and lineage-tracing studies. In particular, the FOXJ1-CreERT2
mice were used to determine the average half-life of ciliated cells in the mouse airways [118
]. The FOXJ1-CreERT2
strain also unexpectedly directs recombination very efficiently in pericytes (J.R. Rock, B.L.M. Hogan, personal communication). This may reflect a low level of endogenous pericyte Foxj1 expression, or be due to the insertion site of the transgene. Similarly, a recent paper has shown that the same human FOXJ1
promoter can drive expression in human ciliated cells, but also some basal cells, growing at an air-liquid interface [119
]. Basal cell expression was not observed in the transgenic animals. However, any future transgenic strains generated with this promoter should be screened for basal cell and pericyte activity.
1f) Basal cells
Published gene expression data have so far not identified a gene that is expressed exclusively in airway basal cells [27
]. A split-Cre, or viral, approach may be necessary for airway-specific basal cell genetic manipulation.
) and Keratin 14
) promoters have been used to target basal cells in the airway epithelium. All mouse and human airway epithelial basal cells express Krt5 [120
]. A human 6kb KRT5
promoter fragment was cloned by the Fuchs lab and successfully used to target epidermal basal cells [122
]. Using the same promoter fragment KRT5-CreER2T
transgenic mice were generated and used for cell lineage tracing in the airways. These studies demonstrated that airway basal cells are stem cells [27
]. This strain has subsequently been used for studying the control of basal cell function [124
] and should prove to be generally useful for manipulating gene expression in tracheal basal cells. However, the KRT5-CreER2T
transgenic strain is limited as it directs recombination in only about 15% of basal cells in the adult trachea. Moreover, the high levels of transgene activity in the skin and oral epithelium makes this strain extremely difficult to use for studies of oncogenes, as the mice will develop skin and oral tumors before the trachea is affected.
is expressed in roughly 30% of mouse tracheal basal cells [120
]. Transgenic mice containing the human KRT14
promoter linked to CreERT
were generated for use in the skin [126
]. In the trachea these KRT14-CreERT
transgenic mice allow tmx-induced recombination in an extremely small population of basal cells at steady-state [125
]. Following naphthalene injury, most surviving basal cells upregulate Krt14
and also express the transgene [127
]. This mouse has not yet been used to manipulate gene expression in airway basal cells. However, a K14-rtTA mouse [128
] has been used, in combination with the tetO-Cre strain [58
], to direct gene expression in the trachea [44
]. These data show the importance of careful control experiments, as in the K14-rtTA strain tracheal Clara cells were sensitive to dox exposure.
1g) Neuroendocrine cells
Pulmonary neuroendocrine (NE) cell differentiation depends on genes which are conserved in the nervous system of many organisms, for example Ascl1
, and Gfi1
]. A rat NE cell specific promoter has been identified [133
] and recently used in an adenovirus to direct gene expression specifically to NE cells [134
]. While no transgenic mouse strains, that specifically target NE cells have been generated, such strains could be very useful for studying NE cell function, or their putative role as an airway epithelial stem cell niche [135
2) Mesenchymal lines
There is still much disagreement over the numbers of different lung mesenchymal cell types and their best markers [136
]. The challenge of the available mesenchymal mouse strains is that they are not lung-specific and that many of their expression patterns are highly variable depending on the integration of the transgene. Pod1
(also known as Tcf21
) is highly expressed in the mesenchyme of the developing lung, kidney, heart and intestine and may be a useful promoter for more restricted mesenchymal gene manipulation [137
genes may also provide useful mesenchymal promoters. Tbx2-5
are expressed in the developing lung mesenchyme [138
] and are often used as reporters of embryonic mesenchymal fate [139
]. However, the expression patterns of Pod1
and the Tbx
genes in the adult lung mesenchyme have yet to be determined.
(also known as Twist2-Cre
) is a knock-in of Cre [141
] that displays robust recombination in mesenchymal and mesothelial lineages in the lung [142
]. It has been widely used for manipulating gene expression in the developing lung mesenchyme [143
is a knock-in of Cre, replacing the Mesp1
coding region, and drives expression throughout the anterior mesoderm from early gastrulation [145
]. It has been successfully used to manipulate gene expression in the developing lung mesoderm [80
Fibroblast specific protein 1 (FSP1), also known as S100A4, has been reported as a fibroblast specific gene, but is also induced in epithelial cells during injury and tumor progression [146
]. The FSP1 promoter has been used to generate various FSP1-Cre
mice with variable success [148
]. The use of these mice to study mesenchymal cells in the lung remains controversial.
Adipocyte lipid-binding protein 2 (aP2, also known as fatty acid binding protein 4, Fabp4) is expressed in alveolar type II cells and interstitial lipofibroblasts. The mouse aP2
promoter was used to generate aP2-Cre
]. While this aP2-Cre
line does not target AT2 cells it does target a subset of alveolar fibroblasts. Induction of recombination with the aP2-CreERT2
in adult mice was not observed (A.K. Perl, unpublished data).
) promoter was used to generate SM22-rtTA
mice. This line provides a “Tet-On” tool that allows the inducible expression of genes in smooth muscle cells [150
]. Expression is mainly in the vascular smooth muscle and the SM22
promoter was used to generate several transgenic and knock-in CreERT2
-expressing lines with varying expression patterns: with the highest levels detected in the aorta, intestine and uterus. However, none of these lines is particularly efficient, even in the vascular smooth muscle [151
(Smooth Muscle alpha Actin,
) is expressed in all smooth muscle cells in the adult, and also transiently in myocardiocytes and skeletal muscle during embryonic development [152
]. In lung parenchyma SMA is expressed during alveolarization, realveolarization, and during the development of lung fibrosis after bleomycin or hyperoxic injury [154
]. Mice with a murine αSMA-Cre
transgene express Cre in the airway smooth muscle and lung vasculature [158
]. This line is not inducible which limits postnatal studies. More recently an αSMA-CreERT2
BAC transgenic line has been generated and shown to exhibit tamoxifen-dependent Cre activity in all adult smooth muscle, including the lung airways and vasculature [159
]. These mice have off target Cre activity only in a small number of cardiomyocytes and should be very useful for future lung studies.
(Smooth Muscle Myosin Heavy Chain,
is expressed in all smooth muscle cells. Two independent mouse strains expressing Cre from a fragment of the mouse SMMHC
promoter have been generated. The expression of the transgene is somewhat variable [160
]. This promoter leads to spurious CRE activity in some tissues due to expression in male and female germline [47
]. By contrast, a SMMHC-CreERT2
BAC transgenic strain shows inducible Cre activity in all smooth muscle, including the airway smooth muscle and lung vasculature [22
]. This strain should be useful for manipulation of gene expression in perivascular and peribronchiolar smooth muscle in the postnatal lung.
To direct Cre expression to the mesothelium of internal organs including the liver, gut and lung a Wt1-Cre
YAC (yeast artificial chromosome) transgenic strain was generated [162
], This has been shown to be active throughout the lung mesothelium from early developmental stages [163
]. However, it may be expressed at low levels in mesenchymal lineage of the embryonic lung and needs to be used with caution (B.L.M. Hogan personal communication). An inducible version would be useful for adult studies.
4) Endothelial lines
Multiple independent transgenic strains express Cre recombinase under the control of the mouse Tek
promoter (Endothelial-Specific Receptor Tyrosine Kinase
, also known as Tie2
), Some of these have been very widely used [165
]. In these strains, reporter gene activity was detected in most endothelial cells and blood islands of the extra embryonic mesoderm by E7.5, in the dorsal aorta by E8.5 and in all blood vessels and some blood cells examined at E11.5, indicating that Cre was active in early vascular progenitors, endothelial cells and some hematopoietic cells. This promoter leads to spurious Cre activity in some tissues due to expression in male and female germline [47
5) Reporters of signaling pathway activity
Wnt signaling pathways play divergent roles during development, homeostasis and repair and play a major role in stem cell proliferation and differentiation. Three transgenic reporter lines for Wnt pathway activity have been generated 1), TOPGAL
, which reports epithelial Wnt signaling [169
], 2) BATGAL
with sporadic epithelial and mesenchymal activity [170
] and 3) Axin2-lacZ
, which is useful to study proximal lung and mesenchymal Wnt signaling [171
]. A recent study compared these lines during development and after naphthalene injury [172
]. A new reporter line, TCF/Lef:H2B-GFP
, has not yet been tested in the lung [173
Signals through the Notch receptors are used throughout embryonic development and in the adult to control cellular fate choices. CP-EGFP
(also known as TNR) transgenic mice have a transgenic Notch reporter with an enhanced green fluorescent protein (EGFP) placed under the control of 4 tandem copies of a CBF1 (also known as Rbpj) responsive element (4 CBF1 binding site consensus sequences and the basal SV40 promoter) [174
]. This strain has been shown to faithfully report Notch activity in the adult trachea [176
are knock-ins of Cre, replacing the Notch1
coding region (Notch1 Intramembrane Proteolysis) and allow lineage studies of descendents of cells after Notch 1 activation [164
]. However, this Cre line identifies each cell lineage, which has previously experienced Notch activity and does not report on current signaling events. Comparison of various Notch reporter lines will shed better light on cell fate decisions and lineage relationships and lead to a better understanding of stem cell biology and interactions of epithelium, mesenchyme, mesothelium and endothelium during development and repair.
6) Virus-mediated transient lung transgenics
The lung is exposed to the external environment and multiple groups have taken advantage of this by administering viruses to manipulate gene expression in the adult mouse lung epithelium. The most widespread system is intranasal administration of an adenovirus expressing Cre from a ubiquitous CMV promoter (AdenoCre) to activate the expression of oncogenes and model lung cancer [178
]. A similar adenovirus-based approach has been taken to transiently overexpress specific genes throughout the lung epithelium (for example, [179
]). More recently, adenoviruses using Scgb1a1
, rat SftpC
, or rat CGRP promoter fragments to direct Cre expression to specific epithelial cell types have been developed [134
]. Lentiviral vectors containing specific promoters for manipulating gene expression in restricted adult lung epithelial cell types have also been reported [180
]. In addition, Adeno-Associated Virus (AAV) transduction of mouse lung epithelial progenitors has also been demonstrated [182
]. The use of viral systems is likely to become more widespread over the next few years, particularly for epithelial studies.
Transgenic mice have been instrumental in developing our current understanding of lung embryonic development, adult homeostasis and repair. However, it is important to remember that all transgenic approaches have limitations, which can only be overcome by integrating findings from different lines and performing all the appropriate controls. New developments in mouse conditional genetics have the potential to further enhance our understanding of lung development and disease. Moreover, optimizing mouse strains of the existing doxycycline and Cre systems will increase flexibility and improve experimental design. For example, using the newer more dox-sensitive rtTA gene activation (rtTA2S
-M2), or extremely low doses of tamoxifen in CreER based transgenic mice, will allow recombination in single cells and enable clonal cell type-specific gene manipulation. Due to the lack of lung-specific mesenchymal and endothelial gene expression, more lines need to be characterized for their usefulness in targeting specific subsets of mesenchymal and endothelial cells. On the other hand, complex targeting systems, such as the split-Cre will be helpful to target subsets of epithelial progenitor cells or specific mesenchymal cell lineages. Recently the applicability of the Flipase system to the lung has been demonstrated by combining Cre and FLP to independently control recombination of p53 and kRas in lung tumor progression [51
]. Development of tools based on flippase and viruses will further expand the combinatorial use of the existing mouse lines and help to develop newer lines, possibly overcoming the problems of off-target activation, lack of cell type specificity and lack of adult regulation. In addition, the generation of new publicly-available floxed alleles by the International Mouse Knock out Consortium (http://www.knockoutmouse.org/
) and the use of transgenic mice expressing conditional RNA interference constructs, should facilitate mouse conditional genetic analysis.