Lineage differentiation of pulmonary alveolar fibroblasts

Rationale:

Alveolar fibroblasts are key cells to the process of alveolar septation as platelet derived growth factor receptor α (PDGFRα) knock-out prevented alveolar myofibroblast differentiation and completely blocked secondary septa formation. Lipofibroblasts have been proposed to be essential for septation, peak in number during septation and regress significantly thereafter. Whether PDGFRα expressing precursors are deriving both lineages of alveolar fibroblasts is not known and transitions in between one or the other fibroblast type are not resolved.

The aims of this work are to analyse the developmental fibroblast lineages, cell-cell transitions and ultimately the role of each cell type and the utility for regenerative septation in adulthood.

Methods:

Laser fluorescent microscopy was performed on 10µm methanol/acetone fixed cryo sections. To follow lineages, cre-reporter mice (mT/mG) expressing tomato fluorescent protein in the membrane of all cells, which switches to GFP upon cre-recombination, were crossed with constitutive PDGFRα-cre or conditional PDGFRα-creER mice. In the conditional mice tamoxifen was injected at postnatal day P2. For identification of active PDGFRα expression, PDGFRα-GFP knock-in mice (Jackson labs) were used. Lung tissue was analysed at various timepoints during fetal and postnatal development. Lipofibroblasts were detected by staining for adipocyte differentiation related protein (ADRP), myofibroblasts by staining for α-smooth muscle actin (αSMA). Tissues were imaged using the Zeiss LSM 710 confocal microscope.

Results:

Immunofluorescence staining revealed a co-expression of PDGFRα-GFP and αSMA in the bronchial compartment as well as in the alveolar space. The expression of ADRP co-localizes with the precursor marker in a subset of cells.

The same pattern of expression could be observed in the constitutive PDGFRα-cre mice. Perinatal lineage tracing experiments with the PDGFRα-creER mouse revealed that postnatal PDGFRα cells derive lipofibroblasts as well as myofibroblasts.

Conclusions:

PDGFRα-GFP mice showed that PDGFRα-expressing precursor cells are differentiating into myo- as well as lipofibroblasts. The constitutive PDGFRα-cre mice revealed restriction of PDGFRα signalling to bronchial smooth muscle cells and alveolar fibroblasts in the lung. Lineage tracing with conditional PDGFRα-creER mice could confirm that postnatal PDGFRα cells derive lipofibroblasts and myofibroblasts.