Abstract
BackgroundWnt/β-catenin signaling has been suggested to regulate proximal-distal determination of embryonic lung epithelium based upon genetically modified mouse models. The previously identified and characterized small molecule inhibitor IQ1 can pharmacologically decrease the interaction between β-catenin and its transcriptional coactivator p300, thereby enhancing the β-catenin/CBP interaction. Inhibition of the β-catenin/p300 interaction by IQ1 blocks the differentiation of embryonic stem cells and epicardial progenitor cells; however, whether differential coactivator usage by β-catenin plays a role in proximal-distal determination of lung epithelium is unknown.MethodsWe examined the effects of inhibiting the β-catenin/p300 interaction with IQ1 on lung branching morphogenesis in mouse embryos in utero and mouse embryonic lung organ culture ex vivo. The phenotype of IQ1 treated lungs was analyzed by epithelial staining, histology, quantitative PCR and in situ hybridization.ResultsInhibition of the β-catenin/p300 interaction by IQ1 disrupted the distal branching of mouse lung epithelium both in utero and ex vivo. IQ1 proximalized lung epithelium with decreased expression of the genes Bmp4 and Fgf10, hallmarks of distal lung determination, and increased expression of the proximal genes Sox2 and Scgb1a1 (CC10) as shown by quantitative PCR and in situ hybridization. The disruption of branching was reversible ex vivo as branching was reinitiated after removal of IQ1 from the media.ConclusionsThe results demonstrate that the β-catenin/p300 interaction plays a critical role in proximal-distal determination of the epithelium in mouse lung branching morphogenesis and β-catenin/p300 inhibition pharmacologically proximalizes lung epithelium.
Highlights
Wnt/β-catenin signaling has been suggested to regulate proximal-distal determination of embryonic lung epithelium based upon genetically modified mouse models
We found that murine embryonic lung expresses endogenous alkaline phosphatase (ALP) activity in the epithelium
These results suggest that endogenous ALP activity is a specific marker for embryonic mouse lung epithelium and Nitro-blue tetrazolium chloride (NBT)/5-bromo-4-chloro-3’-indolyphosphate p-toluidine salt (BCIP), chromogenic substrate of ALP, can be used to visualize the epithelial branching structure in whole mount mouse lung using bright-field microscopy
Summary
Wnt/β-catenin signaling has been suggested to regulate proximal-distal determination of embryonic lung epithelium based upon genetically modified mouse models. Inhibition of the β-catenin/p300 interaction by IQ1 blocks the differentiation of embryonic stem cells and epicardial progenitor cells; whether differential coactivator usage by β-catenin plays a role in proximal-distal determination of lung epithelium is unknown. We have identified and characterized a number of small molecules that inhibit the protein-protein interaction between β-catenin and either of its two Kat coactivators i.e. Creb binding protein (CBP) or p300. ICG-001 directly inhibits the interaction between β-catenin and CBP via binding to the amino terminus of CBP [8] Using these small molecules, we have previously demonstrated the distinct roles that the two coactivators p300 and CBP in β-catenin dependent transcriptional gene regulation, despite their high degree of identity and even higher homology. The role of β-catenin differential coactivator utilization in lung development and branching morphogenesis is unknown
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