Phosphoinositide 3-kinase alpha-dependent regulation of branching morphogenesis in murine embryonic lung: evidence for a role in determining morphogenic properties of FGF7.

Edward Carter,Stephen G Ward,Malcolm L Watson,John Westwick,Silvia Goldoni,Henry Danahay,Gabriela Miron-Buchacra,David Tosh,Vladimir V Kalinichenko

doi:10.1371/journal.pone.0113555

Edward Carter, Stephen G Ward + Show 7 more

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https://doi.org/10.1371/journal.pone.0113555

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Journal: PloS one	Publication Date: Dec 2, 2014
Citations: 44	License type: CC BY 4.0

Affiliation: University of Bath, Novartis (United Kingdom)

Abstract

Branching morphogenesis is a critical step in the development of many epithelial organs. The phosphoinositide-3-kinase (PI3K) pathway has been identified as a central component of this process but the precise role has not been fully established. Herein we sought to determine the role of PI3K in murine lung branching using a series of pharmacological inhibitors directed at this pathway. The pan-class I PI3K inhibitor ZSTK474 greatly enhanced the branching potential of whole murine lung explants as measured by an increase in the number of terminal branches compared with controls over 48 hours. This enhancement of branching was also observed following inhibition of the downstream signalling components of PI3K, Akt and mTOR. Isoform selective inhibitors of PI3K identified that the alpha isoform of PI3K is a key driver in branching morphogenesis. To determine if the effect of PI3K inhibition on branching was specific to the lung epithelium or secondary to an effect on the mesenchyme we assessed the impact of PI3K inhibition in cultures of mesenchyme-free lung epithelium. Isolated lung epithelium cultured with FGF7 formed large cyst-like structures, whereas co-culture with FGF7 and ZSTK474 induced the formation of defined branches with an intact lumen. Together these data suggest a novel role for PI3K in the branching program of the murine embryonic lung contradictory to that reported in other branching organs. Our observations also point towards PI3K acting as a morphogenic switch for FGF7 signalling.

Highlights

Branching morphogenesis is a key developmental process for the formation of many epithelial organs
To explore the role of PI3K signalling in branching morphogenesis, we used a panel of pharmacological tools with pan-isoform or isoform selectivity as well as inhibitors of various elements of the downstream signalling cascade
Phosphorylated Akt at Ser473 can be used as an indirect method of measuring Akt activation due to this residue being phosphorylated by mTORC2, which functions downstream of Akt [32]

Summary

Introduction

Branching morphogenesis is a key developmental process for the formation of many epithelial organs (lung, pancreas, mammary and salivary glands). During this process, epithelial tubes undergo successive rounds of coordinated branching. The branching program of the lung requires highly organised cross talk between the branching epithelium and the surrounding mesenchymal tissue To facilitate this process, the epithelium and mesenchyme secrete signalling molecules. FGF10 signals through epithelial FGFR2b to induce the budding and elongation of the branching epithelium [4, 6] The importance of this pathway is highlighted by observations from mice deficient in either FGF10 [7] or FGFR2b [8, 9]. Mice lacking FGF7 do not exhibit any lung abnormalities suggesting a redundancy for FGF7 in the branching program [11]

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