Mesenchymal and Epithelial Specific Deletion of Rac1 Prevents Lung Morphogenesis

Abstract

BackgroundEpithelial‐mesenchymal interactions control specification, budding and branching in the developing lung. It is the spatio‐temporal expression of various signaling molecules in the different tissue compartments that drive the patterning of the lung. Our previous work demonstrated that the inhibition of Rac1 in mouse lung explants result in impaired branching morphogenesis and vasculogenesis. This role was primarily due to impaired mesenchymal cell proliferation and differentiation following Rac1 inhibtion. Therefore, we sought to determine the effect of epithelial and mesenchymal Rac1 deletion in vivo on lung development during the pseudoglandular and saccular stages.MethodsTo assess the effect of loss of epithelial or mesenchymal Rac1 on lung development in vivo, Rac1f/f animals were used to conditionally inactivate Rac1 using a Shh‐Cre or Dermo1‐Cre driver respectively. The lungs were then harvested from E13.5, E14.5 and E18.5 mutant (Cre+, Rac1f/f), heterozygous (Cre+,Rac1f/+), and control (Cre−,Rac1+/+) littermates. Upon collection, the lungs were either fixed and processed for embedding and histology, or were used for the extraction of RNA and gene expression analysis. Moreover, human lung explants were cultured in presence or absence of Rac1 inhibitor.ResultsEpithelial deletion of Rac1 resulted in lung hypoplasia starting at E13.5 and up to E18.5. During the early pseudoglandular stage the mutant lungs displayed dilated epithelial tips, and expressed high levels of Fgf10 and Wnt inhibitors. The mesenchymal deletion of Rac1 resulted in lungs that also appeared hypoplastic starting at E14.5 until E18.5. However, these mutant lungs displayed severe hypercellularity starting at E14.5. At E18.5, mesenchymal deletion of Rac1 resulted in severe reduction in saccular spaces. Transcription levels of Wnt inhibitors appeared decreased in the E14.5 mutants. Interestingly, both epithelial and mesenchymal Rac1 deletion resulted in embryonic lethality past E18.5. Using Wnt reporters, we showed that epithelial Rac1 deletion increased Wnt signaling, while mesenchymal Rac1 deletion decreased Wnt signaling. Moreover, we demonstrated that Rac1 is expressed in human fetal lung at different gestational stages. This expression is higher in the mesenchyme as compared to the epithelium. Treating human lung explants with Rac1 inhibitor resulted in impaired explant branching and growth.ConclusionIn summary, both epithelial and mesenchymal Rac1 are required for proper lung development, with opposing effects on Wnt signaling. Moreover, Rac1 seems to play an important role in human lung development. The precise mechanism by which epithelial vs mesenchymal Rac1 act, and the differential signaling molecules it regulates in each compartment require further investigation.Support or Funding InformationThis work is supported by the AHA.