Phosphoinositide 3-Kinase Alpha-Dependent Regulation of Branching Morphogenesis in Murine Embryonic Lung: Evidence for a Role in Determining Morphogenic Properties of FGF7

<div><p>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.</p></div

Inhibition of PI3K does not affect the differentiation of the lung epithelium nor does it affect their proliferation.

<p><b>A.</b> Immunofluorescence images of the lung markers SOX9 (top panels), Nkx2.1 (middle panels) and pro-SPC (bottom panels) in isolated lung epithelium cultured for 96 hours with 100 ng/ml FGF7 with or without 10 µM ZSTK474. 0.1% DMSO was used a vehicle control along with FGF7. <b>B.</b> Immunofluorescence images of Ki67 (top panels) and phospho-histone H3 (PH3, lower panels) in isolated lung epithelium cultured over 96 hours with 100 ng/ml FGF7 alone or in combination with 10 µM ZSTK474. <b>C.</b> Percentage of Ki67 positive cells in isolated lung epithelium cultured with either FGF7 alone or in combination with ZSTK474. Data pooled from 20 images per condition. Images representative of at least 12 isolates per condition. Scale bar  = 20 µm. NS  =  Not Significant.</p

Inhibition of mTORC2 but not mTORC1 promotes epithelial branching.

<p><b>A.</b> Light microscope images of E12.5 murine lung explants cultured in either 0.1% DMSO (Control), 1 µM rapamycin or 0.1 µM AZD8055. Images representative of at least 12 explants per condition are shown. <b>B.</b> Percentage increase in epithelial branching over 24 and 48 hours relative to the number of branches at initial isolation. Bars show mean ± s.e.m from n = 12. <b>C.</b> Western blot analysis for levels of phosphorylated S6 ribosomal protein (p-S6), phosphorylated Akt (p-Akt), total Akt and ERK in explants cultured for 48 hours. Total Akt and phosphorylated Akt bands are visualised from different gels containing an equal amount of the same protein lysate. Protein bands representative of 3 separate experiments. Scale bar  = 0.5 mm. *** P<0.001, NS  =  Not Significant, compared with control.</p

PI3K inhibition promotes branching morphogenesis through manipulation of FGF7 signalling.

<p><b>A.</b> Sites of intervention for PI3K signalling and apparent effects on branching morphogenesis. Inhibitory compounds are presented in red text with apparent biological effect in green. Pharmacological inhibition of either PI3K alpha, Akt or dual inhibition of mTORC1/2 potentiates epithelial branching. Inhibition of either PI3K beta or mTORC1 has no observable effects on branching. <b>B.</b> FGF10 and FGF7 signalling during lung branching morphogenesis. Lung epithelium is displayed in blue surrounded by orange mesenchyme. Red represents FGF10 producing cells. Classically, FGF10 promotes branching whereas FGF7 induces the enlargement of the epithelium (left panel). In a setting where PI3K is inhibited (right panel) FGF7 signalling through FGFR2b is switched to an FGF10 signalling event. This has the outcome of inducing the formation of branches that would not otherwise develop.</p

The alpha isoform of PI3K regulates lung branching morphogenesis.

<p><b>A.</b> Light microscope images of E12.5 murine lung explants cultured with 0.1% DMSO (Control), 10 µM BYL719 or 10 µM GSK2636771. Images representative of at least 12 explants per condition are shown. <b>B.</b> Percentage increase in epithelial branching over 24 and 48 hours relative to the number of branches at initial isolation. Bars show mean ± s.e.m from n = 12. <b>C.</b> Western blot analysis for levels of phosphorylated Akt (p-Akt), total Akt and ERK in explants following 48 hours of culture. Total Akt and phosphorylated Akt bands are visualised from different gels containing an equal amount of the same protein lysate. Protein bands representative of 3 separate experiments. Scale bar  = 0.5 mm. *** P<0.001, NS  =  Not Significant, compared with control.</p

Inhibition of Akt promotes epithelial branching.

<p><b>A.</b> Light microscope images of E12.5 murine lung explants cultured with 0.1% DMSO (Control) or 0.1, 1 or 10 µM Akt inhibitor VIII over 48 hours. Images representative of at least 12 explants for each condition are shown. <b>B.</b> Percentage increase in epithelial branching over 24 and 48 hours relative to the number of branches at initial isolation. Bars show mean ± s.e.m from n = 12 <b>C.</b> Western blot analysis for levels of phosphorylated Akt (p-Akt), total Akt and ERK in explants following 48 hours of culture. Protein bands representative of 3 separate experiments. Scale bar  = 0.5 mm. * P<0.05, *** P<0.001, compared with control.</p

Inhibition of PI3K promotes epithelial branching in murine lung explants.

<p><b>A.</b> Light microscope images of E12.5 murine lung explants cultured with either 0.1% DMSO (Control) or 0.1, 1 or 10 µM ZSTK474 over 48 hours. Images representative of at least 12 explants per condition are shown. Scale bar  = 0.5 mm <b>B.</b> Percentage increase in epithelial branching over 24 and 48 hours relative to the number of branches at initial isolation. Bars show mean ± s.e.m from n = 20 <b>C.</b> Western blot analysis for levels of phosphorylated Akt (p-Akt), total Akt and ERK in explants following 48 hours of culture. Total Akt and phosphorylated Akt bands are visualised from different gels containing an equal amount of the same protein lysate. Protein bands representative of 3 separate experiments. <b>D.</b> Confocal image of E-Cadherin expression in lung explants cultured with or without 10 µM ZSTK474 for 48 hours. Scale bar  = 200 µm. *** P<0.001 * P<0.05 * NS  =  Not Significant, compared with control.</p

FGF7-treated lung epithelium develop branches with defined lumen and organised epithelium following PI3K inhibition.

<p><b>A.</b> Light microscope images of isolated E12.5 murine lung epithelium cultured with either 100 ng/ml FGF7 alone (left panels) or in combination with 10 µM ZSTK474 (right panels) over 96 hours. 0.1% DMSO was used as a vehicle control along with FGF7. Images representative of at least 20 isolates are shown. Scale bar  = 200 µm. <b>B.</b> Z-stack confocal images of top and mid sections from isolated lung epithelium cultured with 100 ng/ml FGF7 alone or in combination with 10 µM ZSTK474 showing expression of the epithelial marker E-Cadherin and actin cytoskeleton. Images representative of three experiments. Scale bar  = 20 µm.</p