Abstract
BackgroundIn the embryonic mammalian lung, mesenchymal cells act both as a signaling center for epithelial proliferation, differentiation and morphogenesis as well as a source for a multitude of differentiated cell types that support the structure of the developing and mature organ. Whether the embryonic pulmonary mesenchyme is a homogenous precursor pool and how it diversifies into different cell lineages is poorly understood. We have previously shown that the T-box transcription factor gene Tbx2 is expressed in the pulmonary mesenchyme of the developing murine lung and is required therein to maintain branching morphogenesis.MethodsWe determined Tbx2/TBX2 expression in the developing murine lung by in situ hybridization and immunofluorescence analyses. We used a genetic lineage tracing approach with a Cre line under the control of endogenous Tbx2 control elements (Tbx2cre), and the R26mTmG reporter line to trace TBX2-positive cells in the murine lung. We determined the fate of the TBX2 lineage by co-immunofluorescence analysis of the GFP reporter and differentiation markers in normal murine lungs and in lungs lacking or overexpressing TBX2 in the pulmonary mesenchyme.ResultsWe show that TBX2 is strongly expressed in mesenchymal progenitors in the developing murine lung. In differentiated smooth muscle cells and in fibroblasts, expression of TBX2 is still widespread but strongly reduced. In mesothelial and endothelial cells expression is more variable and scattered. All fetal smooth muscle cells, endothelial cells and fibroblasts derive from TBX2+ progenitors, whereas half of the mesothelial cells have a different descent. The fate of TBX2-expressing cells is not changed in Tbx2-deficient and in TBX2-constitutively overexpressing mice but the distribution and abundance of endothelial and smooth muscle cells is changed in the overexpression condition.ConclusionThe fate of pulmonary mesenchymal progenitors is largely independent of TBX2. Nevertheless, a successive and precisely timed downregulation of TBX2 is necessary to allow proper differentiation and functionality of bronchial smooth muscle cells and to limit endothelial differentiation. Our work suggests expression of TBX2 in an early pulmonary mesenchymal progenitor and supports a role of TBX2 in maintaining the precursor state of these cells.
Highlights
In the embryonic mammalian lung, mesenchymal cells act both as a signaling center for epithelial proliferation, differentiation and morphogenesis as well as a source for a multitude of differentiated cell types that support the structure of the developing and mature organ
TBX2 is expressed in a variety of cells excluding the airway epithelium during lung development To define the spatial and temporal expression of Tbx2 mRNA and TBX2 protein during murine lung development in greater detail as previously reported [18], we performed in situ hybridization andimmunofluorescence analysis on lung sections of different developmental stages (Fig. 1)
TBX2 expression was found at low levels in actin, alpha 2, smooth muscle, aorta positive (ACTA2+) and transgelin positive (TAGLN+) bronchial Smooth muscle cell (SMC) and in some scattered endomucin positive (EMCN+) endothelial cells
Summary
In the embryonic mammalian lung, mesenchymal cells act both as a signaling center for epithelial proliferation, differentiation and morphogenesis as well as a source for a multitude of differentiated cell types that support the structure of the developing and mature organ. Mesenchymal cells provide structural support to the respiratory epithelium and the vessels under homeostatic conditions and play an indispensable instructive role at all steps of pulmonary epithelial development in embryogenesis Throughout the extended pseudoglandular stage, which in the mouse ends around E16.5, mesenchymal signals direct the elongation and branching of the lung buds into the bronchial tree [8, 9], and account for their correct proximal-distal patterning and differentiation [10]. The mesenchyme is important for septation of the distal air-sacs, the alveoli, in the canalicular and saccular phases from E16.5 onwards [11, 12]
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