Spatial-temporal targeting of lung-specific mesenchyme by a Tbx4enhancer
© Zhang et al.; licensee BioMed Central Ltd. 2013
Received: 6 September 2013
Accepted: 5 November 2013
Published: 13 November 2013
Reciprocal interactions between lung mesenchymal and epithelial cells play essential roles in lung organogenesis and homeostasis. Although the molecular markers and related animal models that target lung epithelial cells are relatively well studied, molecular markers of lung mesenchymal cells and the genetic tools to target and/or manipulate gene expression in a lung mesenchyme-specific manner are not available, which becomes a critical barrier to the study of lung mesenchymal biology and the related pulmonary diseases.
We have identified a mouse Tbx4 gene enhancer that contains conserved DNA sequences across many vertebrate species with lung or lung-like gas exchange organ. We then generate a mouse line to express rtTA/LacZ under the control of the Tbx4 lung enhancer, and therefore a Tet-On inducible transgenic system to target lung mesenchymal cells at different developmental stages. By combining a Tbx4-rtTA driven Tet-On inducible Cre expression mouse line with a Cre reporter mouse line, the spatial-temporal patterns of Tbx4 lung enhancer targeted lung mesenchymal cells were defined. Pulmonary endothelial cells and vascular smooth muscle cells were targeted by the Tbx4-rtTA driver line prior to E11.5 and E15.5, respectively, while other subtypes of lung mesenchymal cells including airway smooth muscle cells, fibroblasts, pericytes could be targeted during the entire developmental stage.
Developmental lung mesenchymal cells can be specifically marked by Tbx4 lung enhancer activity. With our newly created Tbx4 lung enhancer-driven Tet-On inducible system, lung mesenchymal cells can be specifically and differentially targeted in vivo for the first time by controlling the doxycycline induction time window. This novel system provides a unique tool to study lung mesenchymal cell lineages and gene functions in lung mesenchymal development, injury repair, and regeneration in mice.
KeywordsLung mesenchyme Tbx4 lung enhancer Tet-On system
The lung is originally developed from ventral foregut endoderm and surrounding splanchnic mesoderm [1, 2]. Reciprocal interactions between lung mesenchymal and epithelial cells play essential roles in lung organogenesis and homeostasis. In fetal mice, lung epithelial cells are initially specified by Nkx2.1 expression around embryonic day (E) 9.5, followed by lung bud growth, airway branching morphogenesis, and terminal saccular formation . During this developmental process, a wide variety of lung-specific epithelial cells are differentiated from their epithelial progenitor cells. The molecular markers and related animal models to target these epithelial cells are relatively well studied. However, developmental lung mesenchymal progenitor cells and their differentiation are poorly understood. Many unsolved issues of lung mesenchymal biology, such as whether mesenchymal cells in the developing lung are different from those in other organs and whether lung smooth muscle cells in airways and vasculature are derived from the same lung mesenchymal progenitors, remain critical questions in the field of lung research. Furthermore, no animal model is available to specifically target lung mesenchymal cells in order to manipulate gene expression in these cells . Therefore, novel molecular approaches and genetic tools to specifically target lung mesenchyme from the beginning of lung formation are urgently needed.
Tbx4 is a member of the T-box transcription factor family, which play important roles during embryonic development through modulating gene expression . Endogenous Tbx4 gene expression is detected in many mesoderm-derived tissues including lung mesenchyme , but is not specific for lung . However, Menke et al. recently reported that Tbx4 expression in different tissues is controlled by a dispersed group of enhancers at different loci within the Tbx4 genomic structure. One of these is located in the third intron and is conserved among several mammalian species . A 5.5 kb DNA segment from this region is able to drive transgenic reporter expression in the developing lung and trachea at E12.5. However, detailed characteristics of this lung enhancer, including the spatial-temporal pattern of the enhancer activity at different developmental or post-developmental stages, are not known. By taking advantage of this potential lung-specific enhancer of the mouse Tbx4 gene expression, we have generated a new Tbx4 lung enhancer driven-reverse tetracycline transactivator (Tbx4-rtTA) transgenic mouse line. We then developed a lung-specific Tet-On inducible transgenic mouse model by crossing Tbx4-rtTA mice with TetO-Cre mice. Using loxP-mTomato-STOP-loxP-mGFP (mT-mG) reporter mice, we were thus able to identify and define the spatial-temporal pattern of Tbx4-rtTA-targeted lung mesenchymal progenitors and their derived cells during different stages of lung development and adulthood. Thus, our new lung mesenchymal-specific Tet-On inducible genetic system provides a valuable tool for the study of lung mesenchymal cells under both physiological and pathophysiological conditions.
The lung enhancer of the mouse Tbx4gene contains genomic DNA sequence elements that are highly conserved across species that have lungs or lung-like gas exchange organs
Generation of a Tbx4-lung enhancer driven Tet-On system that specifically targets lung mesenchyme
Dynamic pattern of Tbx4lung enhancer-driven lung mesenchymal cell targeting during and after development
Differential targeting of smooth muscle cells by the Tbx4lung enhancer in developing lung
In the triple transgenic (Tbx4-rtTA/TetO-Cre/mT-mG) mouse system described above, mGFP expression persists within cells once Cre-mediated floxed-mTomato deletion has occurred. Thus, mGFP-positive cells do not necessarily represent an active status of the Tbx4 lung enhancer-driven rtTA/LacZ expression. Since the TetO-Cre transgene is ubiquitously expressed, the Tet-On induction is dependent upon Tbx4-rtTA/LacZ transgenic expression. Therefore, by comparing the expression pattern of mGFP and LacZ in mouse lungs with different Dox induction time windows, cells that previously expressed Cre in early lung development could be distinguished from the cells that were still actively expressing Cre. Hence, the activity of Tbx4 lung enhancer-mediated targeting to a variety of differentiated lung mesenchymal cells could be analyzed.
Differential targeting of pulmonary endothelial cells by the Tbx4lung enhancer in developing lung
Other lung cell lineages targeted by the Tbx4lung enhancer during lung development
Mesenchymal cells in a variety of tissues are mainly derived from mesoderm during organogenesis. They play important roles in guiding organogenesis, generating the tissue-specific mesenchymal progenitor/stem cells needed for homeostasis in developed organs, and can differentiate into fibroblasts, smooth muscle cells, chondrocytes and other types of mesenchymal cells that support specific tissue functions. However, genetic markers specifically for mesenchymal cells in individual visceral tissue, including lung mesenchyme, have not been well defined. For example, in Dermo1-Cre knockin mice, Dermo1 promoter drives Cre expression in many mesoderm-derived tissues including lung mesenchyme, diaphragm and ventral body walls [17–19]. Similarly, although endogenous Tbx4 expression was detected in the mesenchyme of lung and trachea around E9.25, a few hours after the specification of the lung and trachea primordia by Nxk2.1 and Tbx5 , expression of both Tbx4 and Tbx5 genes is not restricted to lung tissue even though they are important in lung organogenesis . Interestingly, a dispersed group of Tbx4 gene enhancers are found to be responsible for the distinct tissue locations of this gene . Among these, a 5.5 kb fragment of genomic sequences in Tbx4 intron 3 is related to its expression in early embryonic lung. Our DNA sequence analysis suggests that several DNA fragments in this region, particularly an approximately 500 bp fragment in the middle region, are highly conserved in vertebrate species that develop lungs or lung-like gas exchange structures. This suggests that this DNA regulatory element may be important for lung structure formation. However, further experiments to examine individual fragments of the mouse Tbx4 genomic region and compare their gene regulatory activities among different specimens will be needed to understand fully the role of mouse Tbx4 lung enhancer in lung morphogenesis. Furthermore, whether these conserved fragments of DNA sequences in each species are involved in regulating Tbx4 gene expression also needs to be experimentally analyzed.
We have used this lung-specific DNA enhancer of Tbx4 gene to generate a Tet-On inducible transgenic system in mice. In combination with Cre-mediated reporter mice, we have clearly demonstrated that developing lung mesenchymal cells can be specifically marked from the beginning of lung formation, which makes these cells distinguishable from the mesenchymal cells arising from other organs. We have shown for the first time that lung mesenchymal cells can be targeted in an organ-specific manner by using the Tbx4 lung enhancer, rather than the entire endogenous Tbx4 promoter as in the Tbx4-Cre knockin model . This provides a powerful genetic tool with which to study the functions of genes in lung mesenchymal development, and to isolate and trace lung mesenchymal cell lineages during prenatal development through postnatal development to adulthood. More importantly, it provides the potential to generate unique mouse models mimicking lung interstitial diseases without adversely affecting other organs and systems. The molecular mechanisms by which the Tbx4 lung enhancer is activated specifically in lung mesenchymal cells are not yet completely clear, and also will need further investigation. It is possible that specific lung mesenchymal cells may express a unique array of transcription factors that interact and activate the Tbx4 lung enhancer. In contrast, mesenchymal cells in other tissues may lack some of these specific transcriptional activators, resulting in suppression of this particular Tbx4 gene regulatory element, but not endogenous Tbx4 gene expression that can be activated by multiple regulatory elements. Furthermore, our experiments also show that the Tbx4 lung enhancer is not persistently active in all lung mesenchymal cells. Its activity is turned off in some committed and/or differentiated mesenchymal cell lineages at various developmental stages, particularly in pulmonary endothelial cells and vascular smooth muscle cells. Whether active Tbx4 lung enhancer activity is related to lung mesenchymal progenitor cells or is associated with cell differentiation status remains to be determined.
Another feature of our Tbx4-rtTA-mediated Tet-On Cre inducible system is the combination of the Cre-mediated reporter and LacZ reporter systems. Cre-mediated mGFP expression is the marker for cells that had and/or have induced Cre expression, while LacZ expression indicates active Tbx4 lung enhancer activity at the time of examination. Therefore, using this system, we have been able to determine the dynamic changes and fates of lung mesenchymal cells with Tbx4 lung enhancer activity. In early lung buds, the majority of these mesenchymal progenitors can be marked using this Tbx4 lung enhancer, and these cells are able to differentiate to endothelial cells and smooth muscle cells of both airway and vasculature. However, the multi-potent differentiation potential of these mesenchymal progenitor cells is reduced during the course of lung development, as the majority of endothelial cells and vascular smooth muscle cells are neither labeled by Cre-mediated mGFP expression if Dox induction is given after E11.5 and E15.5, respectively, nor detected for LacZ expression. However, lung airway smooth muscle cells and subtypes of fibroblasts (lipofibroblast and myofibroblast) are positive for induced mGFP expression even if Dox is given at late gestation. Therefore, differential targeting of mouse lung mesenchymal progenitors and related cell lineages can be achieved by controlling the time windows of Dox induction.
Interestingly, a proportion of mesenchymal cells in postnatal developing and even adult lung are still active for the Tbx4-lung enhancer. These cells are located adjacent to alveolar epithelial cells and alveolar endothelial cells of adult lung alveolar structure, and are positive for NG2 or SMA staining [see Additional file 2], suggesting that these may be pericytes and myofibroblasts. Therefore, our Tbx4-lung enhancer driven targeting driver line has the potential to be used in the study of adult lung diseases, such as asthma, emphysema and interstitial pulmonary fibrosis. It could be used to create new disease models, to determine the response of targeted cell lineages to specific injuries and/or to test intervention approaches to prevent or attenuate pathological processes. Moreover, understanding developmental lung mesenchymal specificity is also important when considering the future design of cell-based therapies. For example, the role of bone marrow-derived mesenchymal stem cells in lung injury repair and regeneration is controversial, although their effects on modulating immune and inflammatory responses are well recognized . This raises the question whether mesenchymal stem cells derived from lung may function better in repairing lung structures than those originating from other organs such as bone marrow . Our newly generated Tet-On system will make it possible to compare mesenchymal stem cells of lung with those of non-lung origins in mice by marking developing lung mesenchymal cells with prenatal Dox induction. Comparison of these two groups of cells and characterization of their molecular signatures under both physiological and pathological conditions will greatly advance our knowledge about lung biology and pulmonary diseases.
Although mesenchymal cells in developing lung display complicated heterogeneous phenotypes, they are probably derived from splanchnic mesoderm surrounding lung progenitors in foregut endoderm. We have found an evolutionally conserved Tbx4 lung enhancer that is active specifically in lung mesenchymal progenitors and subsets of their derived cells. Therefore, with our newly created Tbx4 lung enhancer-driven Tet-On inducible system, lung mesenchymal cells can be specifically and differentially targeted for the first time by controlling the doxycycline induction time window. This novel system provides a unique tool to study lung mesenchymal cell lineages and gene functions in lung mesenchymal development, injury repair, and regeneration in mice.
DNA vector construction
rtTA2s-M2 DNA fragment was amplified from pTet-On Advanced vector (Clontech, Mountain View, CA, USA) using primers 5′-CGGCCCCGAATTCACCATGTCTAGA-3′ and 5′-ACGCGTCGACACTTAGTTACCCGGGGAGCATG-3′, and subcloned to EcoRI/SalI digested pBluescript KS II to generate pBS-rtTA. A 5.5 kb DNA fragment of mouse Tbx4 lung enhancer and a 0.9 kb DNA fragment of HSP68 minimal promoter were obtained by NotI and SmaI/SfoI digestion of pDBM3 , respectively, and subcloned into pBS-rtTA to generate pBS-Tbx4-rtTA. Finally, IRES-T-LacZ DNA fragment was obtained by digesting pNTR-lacZ-PGKNeolox plasmid provided by Dr. Vesa Kaartinen at University of Michigan, and inserted to pBS-Tbx4-rtTA to produce an intact transgenic vector pBS-Tbx4-rtTA-LacZ. The prokaryotic part of the vector was then removed by NotI digestion and the 12 kb transgenic DNA fragment was isolated for C57BL/6 pronuclear injection.
Mouse strains, breeding, and genotyping
Tbx4-rtTA mouse line was generated at the University of California at Los Angeles (UCLA) Transgenic Core facility. The founders were identified by genomic DNA PCR using primers 5′-GGA AGG CGA GTC ATG GCA AGA-3′ and 5′-AGG TCA AAG TCG TCA AGG GCA T-3′. The transgenic mice were further verified by DNA Southern blot for genomic DNA digested with XhoI and SalI. DIG-labeled nonradioactive detecting system (Roche, Indianapolis, IN, USA) was used.
The TetO-Cre mouse line was originally provided by Dr. Jeffrey Whitsett at Cincinnati Children’s Hospital [23, 24]. mT-mG double fluorescent Cre reporter mice were obtained from Jackson Laboratory (Bar Harbor, ME, USA) . All mice were bred in C57BL/6 background. Fetal lung mesenchymal-specific Cre expression was induced by Dox administration from different gestational stages by feeding the pregnant mice with both Dox food (625 mg/kg; TestDiet, Richmond, IN, USA) and Dox water (0.5 mg/ml; Sigma, St. Louis, MO, USA). In postnatal pups, Cre induction was initiated by a single intraperitoneal injection of Dox (100 mg/kg body weight) followed by Dox oral administration, the same as described above for the entire induction period. All reported mouse studies were approved by the Institutional Animal Care and Use Committee at the Saban Research Institute of Children’s Hospital Los Angeles.
Fluorescence protein and immunofluorescence detection
Mouse embryos or lung tissues were dissected. For mT and mG detection, mouse embryos or tissues were directly imaged under a Leica MZFLIII fluorescence dissecting microscope. The embryos or tissues were then fixed with 4% buffered paraformaldehyde and embedded either in optimal cutting temperature (OCT) compound for frozen section or in paraffin for regular histological section. Tissue frozen sections were washed in PBS, and mounted with VECTASHIELD medium with DAPI (Vector Laboratories, Burlingame, CA, USA). Immunofluorescence staining was performed following methods published previously . The related antibodies were: rabbit anti-GFP (Santa Cruz Biotechnology, Santa Cruz, CA, USA), goat anti-GFP (Abcam, Cambridge, MA, USA), mouse anti-cytokeratin and mouse anti-SMA (Sigma), rabbit anti-PECAM-1 (LSBio, Seattle, WA, USA), rabbit anti-lacZ (MP Biomedicals, Solon, OH, USA), mouse anti-ADRP (BioGenex, Fremont, CA, USA), rabbit anti-NG2 chondroitin sulfate proteoglycan (Millipore, Billerica, MA, USA), hamster anti-T1α (DSHB at the University of Iowa) and mouse anti-CGRP (Sigma). Fluorescent signals were detected using a Zeiss LSM710 confocal microscope at the Imaging Core Facility of the Saban Research Institute of Children’s Hospital Los Angeles. All experiments were repeated at least five times and data represent consistent results.
Lung specimens were briefly fixed with 4% paraformaldehyde in PBS containing 2 mM MgCl2 and embedded in OCT. Lung frozen sections were incubated with X-gal reaction buffer following the procedures described in a previous publication .
Calcitonin gene-related peptide
Green fluorescent protein
Optimal cutting temperature
Polymerase chain reaction
Platelet endothelial cell adhesion molecule
Reverse tetracycline transactivator
α-smooth muscle actin.
We thank Dr. Esteban Fernandez at the Cell Imaging Core of CHLA for helping with confocal imaging and Dr. Vesa Kaartinen for providing the plasmid containing IRES-T-lacZ fragment. This research was supported by NIH/NHLBI grant R21-HL109932 and R01-HL068597 (WS), California Institute of Regenerative Medicine Training Grant (WX and YL), and The Saban Research Institute of Children’s Hospital Los Angeles Pilot Project Grant (WS).
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