Characterization of The Role of Primary Cilia During Human Pulmonary Development

Introduction: Lung development is a highly intricate process that encompasses a series of regulated signaling pathways, including the Hedgehog pathway (HH), ultimately leading to the establishment of mature and functional pulmonary structures. Throughout development, components of the HH pathway such as SHH, PTCH1 and HHIP are found to be spatially and temporally regulated. Primary cilia play a vital role in this process, as the HH pathway relies entirely on them. They act as a sensory organelle that transduce signals essential for epithelial and mesenchymal interactions. This study aims to characterize the role of primary cilia during lung development in humans. Methods: Immunofluorescence (IF) stainings were performed on formalin-fixed paraffin-embedded (FFPE) human fetal lung tissues between 10 and 23 weeks of gestation (n=31). Co-staining of ARL13B (cilia) and ACTA2 (smooth muscle cell marker) was performed to quantify the percentage of ACTA2+ cells harboring primary cilia (ACTA2+ ARL13B+). Additionally, to characterize the role of primary cilia in lung organogenesis, human fetal lung explants from 10 to 15 weeks of gestation were treated with 0.5 mM or 1 mM of chloral hydrate (CH) for 48 hours to inhibit primary ciliogenesis. RT-qPCR was used to assess the expression of different genes ( ACTA2, SHH, SOX2, SOX9, PTCH1 and HHIP ) following CH treatment. Results: Our results showed a significant decrease in ACTA2+ ARL13B + cells as pulmonary development progressed, from 15% at 10 weeks to 3% at 20 weeks or older (r 2 = 0.7130; p < 0.001). Next, we assessed the effect of primary cilia inhibition in these ACTA2+ cells harboring primary cilia. Our data showed that ACTA2+ ARL13B+ cell percentage in fetal lung explants is reduced following treatment with CH, confirming the inhibition of primary ciliogenesis. Furthermore, we observed decreased expression levels of Hedgehog pathway genes ( SHH, PTCH1 and HHIP), as well as ACTA2 and SOX9, while SOX2 remained unaffected in the explants after treatment. Conclusion: These findings suggest that primary cilia are more prevalent during early stages of lung development and diminish as the lung matures, reflecting a transition from a proliferative state to a more differentiated, specialized state. During development, primary cilia appear to play a critical role in regulating key components of the HH pathway. this work is funded by Region Grand-Est. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.