Cell Fate Decisions in the Developing Lung

Abstract

The epithelium of the adult lung can be broadly segmented into two different categories: a bronchiolar region consisting of conducting airways which are responsible for conducting air towards the alveoli, and an alveolar region where gas exchange takes place (Herriges and Morrisey, 2014). During embryogenesis, the airways are first laid down through an iterative process of branching morphogenesis, which terminates at the alveoli. Throughout gestation the lungs are filled with fluid, but at the moment of birth this fluid is expelled and lungs must be competent to function for gas exchange and enable breathing (Szoták-Ajtay et al., 2020). Research has suggested that a multipotent progenitor population of epithelial cells resides in the distal tip of the developing lung (Nikolić and Rawlins, 2017) and is capable of differentiating into both bronchiolar and alveolar cells. Clarifying which tip cells differentiate to form the different lung compartments, and what factors promote lineage-restriction and competence loss are questions that remain to be addressed. Exposure to glucocorticoids has previously been demonstrated to accelerate lung maturation (Laresgoiti et al., 2016). However the changes in tip cell fate decisions and lung morphology induced by glucocorticoids are not well understood. To address these questions I have taken a clonal lineage-tracing approach to label lung tip epithelial progenitors. A range of tamoxifen doses were administered to two different mouse models, representing either a biased (tip cells only) or unbiased (random) labelling approach, to ensure an appropriate number of initial cells were being labelled. Different conditions and protocols were tested to image entire lung lobes. A protocol where wholemount immunostaining is combined with CUBIC-1A clearing, and imaged on a confocal microscope has been most successful. Using these optimised conditions I have imaged entire E18.5 embryonic mouse lung lobes following lineage-labelling. I have developed an image analysis pipeline to assess cell fate outcomes in the context of the overall branching structure in both the biased and unbiased lineage tracing models. Embryonic mouse lungs exposed to glucocorticoids in drinking water from embryonic day 12 - 15 showed larger, more robust alveolar regions with increased expression of mature alveolar markers. Those exposed by intraperitoneal injection at embryonic day 13 showed swelling of the distal regions, increased lumen space, and decreased branching. To explore the mechanism of glucocorticoid activity in human lungs, organoids were derived from late-stage (17 post-conception weeks or older) embryos and treated with a variety of glucocorticoids or other maturation reagents. These showed varying levels of expression of alveolar type II markers by both immunohistochemistry and quantitative reverse transcription polymerase chain reaction. These glucocorticoid exposures were repeated in mouse lung explants, which demonstrated that the maturation effect of glucocorticoids is achieved through multiple signalling mechanisms, affecting a variety of cell types throughout the lung. My results will facilitate future studies of whole organ clonal analysis and of branching structure morphology. They also provide deeper insight into the morphology changes that occur during dexamethasone treatment, and provide potential avenues to explore the signalling mechanism by which glucocorticoids activate lung maturation.