Spatial-temporal targeting of lung-specific mesenchyme by a Tbx4enhancer

Abstract

BackgroundReciprocal 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.ResultsWe 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.ConclusionsDevelopmental 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.

Transcription factor TBX4 regulates myofibroblast accumulation and lung fibrosis

“…Tbx4lineage cells include lipofibroblasts and a few endothelial cells (Figure 1, G and H). These results are consistent with recent data indicating that Tbx4-Cre labels undifferentiated mesenchyme, airway smooth muscle, vascular smooth muscle, and mesothelium (22,26,27). The locations of tdTomatolabeled (tdTlabeled) cells were identical in the lungs of both strains.…”

Lung Pericytes and Resident Fibroblasts

“…Thus, embryonic Tbx4 enhancer activity and adult Col1 transcription appear to match in spatially separated mesenchymal populations with the features of pericytes and resident fibroblasts. 8,10,21 The pattern of PDGFR-a expression also matches this connection. A large population of PDGFR-a þ , a-SMA þ myofibroblasts position themselves adjacent to PDGF-A þ epithelial cells and play a crucial role in generating the septae between alveoli before embryonic day 18.5.…”

“…8,35 Among the populations traced by Tbx4, by either labeling throughout embryogenesis or only after embryonic day 15.5, were NG2 þ a-SMA À pericytes and NG2 À a-SMA þ perialveolar adipophilin-stained lipofibroblasts. Thus, embryonic Tbx4 enhancer activity and adult Col1 transcription appear to match in spatially separated mesenchymal populations with the features of pericytes and resident fibroblasts.…”

PBX transcription factors drive pulmonary vascular adaptation to birth

“…Cre allele or Tbx4-rtTA TetO-Cre transgenes, as previously described (28,39 For microarray, lungs from 3 Pbx1/2-CKO or 3 control mice were pooled at each stage. RNA (1 μg) was analyzed from each pooled sample at the University of Wisconsin -Madison Gene Expression Center using a Mouse Gene 1.0 Array (Affymetrix).…”

“…At P7, when PH was detected by the PAAT/PAET ratio, we detected no statistically significant reduction in the number of vessels outlined by vWF staining (Figure 3 (Figure 3I). To determine whether this decrease in capillary volume is sufficient to cause PH, we used doxycycline to induce Tbx4-rtTA TetO-Cre-mediated deletion of Pbx1/2 at E17 (referred to hereafter as Pbx-iCKO mice), which resulted in loss of PBX1 expression in the distal mesenchyme but not in the proximal VSM (Supplemental Figure 4, A-F) (39). As was observed in Pbx1/2-CKO mice, the Pbx-iCKO mutants had lung simplification and decreased capillary density relative to controls ( Figure 3, J-Q).…”