Mesodermal-specific inactivation of Pten causes embryonic and immediate postnatal lethality.
We used Dermo1-Cre
to inactivate Pten
via deletion of exon 5. The expression pattern and efficiency of Dermo1-Cre
in the lung were examined by crossing Dermo1-Cre
mice with ROSA26R-LacZ
reporter mice (Supplemental Figure 1G; supplemental material available online with this article; doi:
). As reported (1
), LacZ activity was widespread throughout the pulmonary mesenchyme, but did not include the endothelial cells in the lung vasculature (Supplemental Figure 1G).
To accomplish mesodermal inactivation of Pten in the lung, heterozygous Ptenfll/+;Dermo-Cre males were crossed with Ptenfl/fl females. The offspring (n = 121) were genotyped using PCR analysis of tail DNA at 3 weeks of age. No Ptenfl/fl;Dermo-Cre (i.e., homozygous deletion) offspring were detected. We therefore determined the percentile of homozygous mutants and WT embryos at different gestational ages (Table ). At E12.5 and E15.5, the mutants accounted for 29% (19 out of 65) and 25% (16 out of 63), respectively, of the total number of embryos, while at E18.5, their number was reduced to 21% (67 out of 311), indicating embryonic lethality between E15.5 and E18.5. During embryonic stages, the mutants showed a wide range of phenotype, from a lack of vascularization in entire embryos at E15.5 (Supplemental Figure 2, A and B; 7 out of 16 mutant embryos: 44%) to a hemorrhagic phenotype at E18.5 (Supplemental Figure 2, C and D; 10 out of 67 mutant embryos: 15%). All other mutant embryos with less severe phenotype died within 2 to 3 hours postnatally, displaying cyanosis, chest retractions, and dyspnea (Supplemental Figure 2E). Measurements of blood oxygenation (Figure I) showed statistically significant differences between Ptenfl/fl (controls) and Ptenfl/fl;Dermo-Cre newborns (97% ± 3.7% vs. 73% ± 8.2%, P < 0.01). A careful study of the embryos at E15.5 showed lack of vascularization in other organs, such as limbs and liver (Supplemental Figure 3, A–J).
Ptenfl/fl;Dermo1-Cre mice suffer embryonic lethality
Absence of Pten in the mesenchyme does not affect lung morphogenesis, but leads to increased mesenchymal cell proliferation.
To validate Pten inactivation in the pulmonary mesoderm, we compared expression patterns and levels of PTEN and phosphorylated protein kinase B (p-AKT) in mutant versus WT lungs by immunohistochemistry (IHC) in E18.5 embryos (Supplemental Figure 1, A–D). When compared with controls, mutant lungs showed an overall decreased PTEN (Supplemental Figure 1, compare B and A). However, recombination was not complete, as some mesodermal-derived cells remained PTEN positive, displaying nuclear immunoreactivity (Supplemental Figure 1B). Consistent with the specificity previously documented for Dermo1-Cre activity, epithelial PTEN immunoreactivity was unperturbed (Supplemental Figure 1B). Using E18.5 lung RNA, quantitative RT-PCR (qRT-PCR) was performed to confirm interruption of Pten gene expression (Supplemental Figure 1E). In Ptenfl/fl;Dermo-Cre lungs, Pten mRNA was decreased (0.58% ± 0.07%, P < 0.01) compared with Ptenfl/fl controls (n = 4 per group). Finally, Western blot analysis (Supplemental Figure 1F) showed that PTEN was reduced in the mutant lungs compared with controls (0.14 ± 0.01 vs. 0.51 ± 0.09, n = 4 per group, P < 0.05).
PTEN is a lipid phosphatase that regulates PIP3 levels, thus negatively modulating the PI3K/AKT pathway. IHC revealed an increase of p-AKT in mutant lungs compared with controls (Supplemental Figure 1, D vs. C), further confirming deregulation of the PI3K pathway. Western blot analysis of E18.5 whole-lung extracts (n = 4 for each group) confirmed increased p-AKT in mutant versus control lungs (0.03 ± 0.004 vs. 0.012 ± 0.00053 P < 0.05; Supplemental Figure 1F).
To determine the potential structural causes underlying reduced blood oxygenation in the mutant newborns, histology of E18.5 lungs using H&E staining was examined (Figure , A–D). While no branching or other significant gross structural abnormalities were observed (Figure , B and D vs. A and C), closer examination of mutant lungs (Figure D) revealed a hypercellular mesenchymal compartment compared with the controls (Figure C). This observation was confirmed by detection of mesenchymal-specific increased cell proliferation, as documented by E-cadherin (E-CAD)/PH3 immunofluorescence (Figure , E and F). Due to increased mesodermal cells, the total number of E-CAD–negative cells was higher in mutant lungs compared with controls (Figure G, 363.5 ± 20.7 vs. 223.1 ± 10.2, P < 0.01). The number of E-CAD–negative/PH3-positive cells was nearly 6-fold higher in mutant versus control lungs (17.8% ± 1.1% versus 3.15% ± 0.8% respectively n = 3, P < 0.01) (Figure H).
Mesodermal Pten deficiency affects vasculogenesis.
Differentiated endothelial cells, identified by PECAM, are derived from mesodermally derived progenitor cells, which express Flk1
). These progenitors can be identified in embryonic lungs starting as early as E10.5 (13
). Their formation is tightly associated with mesenchymal and epithelial development during branching morphogenesis (1
). As defective pulmonary vascular formation can cause hypoxemia, we examined PECAM in E15.5 lungs by IHC (Figure , A and B). In Ptenfl/fl;Dermo-Cre
lungs, there was significant reduction in distal capillary network density compared with Ptenfl/fl
controls, the latter exhibiting normal dense capillary plexus adjacent to the developing airway epithelium. In E18.5 control lungs (Figure C), red blood cell–containing capillaries were found in close spatial proximity to the alveolar-air interface. This coupling is critical for normal gas exchange. In contrast, the capillary network in Ptenfl/fl;Dermo-Cre
lungs was distinctly abnormal, with clear uncoupled capillary/airway network, rendering pulmonary units incapable of efficient gas exchange (Figure D). Statistical analysis of the distance between the capillaries and the lumen of airways showed significant increase in the mutant versus control lungs (Figure E, 3.3 μm ± 0.3 μm vs. 0.9 μm ± 0.1 μm, P
< 0.01). More detailed study of the lung ultrastructure showed mutant alveolar spaces were frequently lined by cuboidal cells with “immature” lamellar bodies, while the type 2 cells in control lungs contained surfactant. To better understand the mechanism underlying this phenotype, we assessed the levels of Vegfa
), and Flk1
) at E18.5 by qRT-PCR. Vegfa
was reduced in mutant lungs compared with controls (Figure A, 0.39 ± 0.03 vs. 1, P
< 0.01) as was Flt1
(Figure A, 0.73 ± 0.08 vs. 1, P
< 0.05). There was also a quantifiable increase in Flk1
mRNA in Ptenfl/fl;Dermo-Cre
lungs (Figure A, 11.61 ± 2.6 vs. 1, P
< 0.05). Furthermore, Pecam
mRNA was decreased in the mutant lungs versus controls (Figure A, 0.47 ± 0.1 vs. 1, P
< 0.01), supporting the IHC data shown in Figure , A and B.
Abnormal airway capillary coupling.
Defect in angioblast differentiation in Ptenfl/fl;Dermo-Cre lungs.
Finally, qRT-PCR for Vegfa, using mRNA from E15.5 and E18.5 isolated fibroblasts, revealed no statistically significant difference between control and mutant cells (Figure F, E15.5: 1.067 ± 0.5 vs. 1, P > 0.05; E18.5: 1.16 ± 0.6 vs. 1, P > 0.05). Thus, as the mesenchyme does not appear to be involved, the data suggest the epithelium as the compartment in which the decrease in Vegfa occurs. This conclusion was validated via immunofluorescent (IF) for VEGFA plus E-CAD, which showed markedly decreased VEGFA in E-CAD–positive cells in the mutant lungs (compare Figure , H and G).
One possibility for the changes in Flk1
levels is that mesodermal-specific Pten
inactivation affects endothelial cell differentiation even though Dermo1-Cre
as shown in this and other reports is not active in this cell lineage (1
). Therefore, we examined the differentiation of endothelial cells by generating triple-transgenic mice consisting of the Ptenfl/fl;Dermo-Cre
embryos in a Flk1LacZ
reporter background (14
). In these mice, LacZ
is under the control of the endogenous Flk1
is an early marker of angioblasts, and its expression is maintained in mature endothelial cells together with Flt1
). Significant increase in Flk1
-driven LacZ activity was found throughout the entire mutant lungs compared with controls (Figure , C vs. B). Ablation of mesodermal Pten
therefore does not interfere with specification and amplification of angioblasts. However, analysis of PECAM, a marker for mature endothelial cells in Ptenfl/fl;Dermo-Cre
lungs, revealed impaired differentiation of angioblasts into mature endothelial cells and blood vessels when compared with controls. We therefore analyzed sections from E18.5 Ptenfl/fl;Dermo-Cre;Flk1LacZ
lungs and examined them for PECAM. The mutant lungs showed increased FLK1-positive/PECAM-negative cells (Figure , E vs. D), confirming the conclusion that angioblasts are indeed increased in the mutant lungs. Finally, we examined the tridimensional structure of lung vasculature by injecting FITC leptin in the left ventricle of mutant and control hearts, observing again a significant defect in vasculogenesis in mutant versus controls (Supplemental Video 1). In sum, the results strongly support the conclusion that lack of PTEN in the mesodermal lineages inhibits differentiation of angioblasts into mature endothelial cells.
Dermo1-Cre–mediated Pten inactivation affects ontogeny of mesodermal cell lineages.
To determine the impact of mesodermal Pten inactivation on the emergence and differentiation of lung mesenchymal cell lineages, expression of a number of cell markers was examined by IHC and qRT-PCR. Immunofluorescence showed decreased α-SMA, a smooth muscle differentiation marker in E18.5 mutant lungs (Figure , B versus A). Adipose differentiation-related protein (ADRP), a lipid droplet–associated protein expressed early during adipose differentiation and a marker of lipofibroblasts in the lung, was decreased in Ptenfl/fl;Dermo-Cre lungs compared with Ptenfl/fl (Figure , D vs. C). Finally, in E18.5 lungs, the capillary network was misaligned with corresponding respiratory airways (i.e., airway/capillary uncoupling or dysplasia) in the mutants, as shown by IHC for PECAM (Figure , F vs. E). Interestingly, double IHC for PDGFrα (marker for fibroblasts) and E-CAD (marker for epithelial cells) and Sirius red staining for collagen, a product of fibroblasts, showed increased PDGFrα-positive/E-CAD–negative cells (Figure , H vs. G, statistical analysis: Figure L, 2% ± 0.2% vs. 11% ± 0.02%, P < 0.01). Collagen deposition was also increased in the Ptenfl/fl;Dermo-Cre lungs compared with controls (Figure , J vs. I). These IHC results were validated by qRT-PCR for the different cell lineage markers, which showed a trend toward reduced expression of α-Sma (Figure K, 0.52 ± 0.3 vs. 1, P > 0.05) and Adrp (Figure K, 0.03 ± 0.00001 vs. 1, P < 0.01) in primary cultures of E18.5 mutant fibroblasts compared with controls. Pecam was also reduced in the mutant versus control lungs (Figure K, 0.47 ± 0.1 vs. 1, P < 0.01). Collagen 1 by Sircol assay was increased in the mutant lungs versus controls (Figure M, 83.13 μg/ml ± 7.2 vs. 39.23 μg/ml ± 6.3, P < 0.01).
Arrested mesenchymal cell differentiation in Ptenfl/fl;Dermo-Cre lungs.
Using flow cytometry to confirm these observations, we gated the mesenchymal progenitor cells by Hoechst staining in E17.5 mutant and control lungs (n
= 14 for each). In the embryonic lung, 2 subtypes of side populations are thought to exist (15
). One is identified as CD45–
E-SP cells, believed to differentiate into endothelial cells. The other is identified by markers CD45–
E-SP, with a gene profile consistent with a smooth muscle precursor (15
). At E17.5, we observed more than a 5-fold increase in the number of CD45–
E-SP and CD45–
E-SP cell populations in Ptenfl/fl;Dermo-Cre
lungs versus controls (respectively 1.1% versus 0.2% and 2.9% versus 0.5%) (Figure N). Therefore, mesenchymal PTEN controls the size of the 2 putative lung mesenchymal progenitor cell populations.
Early mesodermal Pten inactivation leads to increased FGF10 signaling.
AKT-mediated phosphorylation of β-catenin (β-CAT) on Ser552 drives nuclear localization of β-CAT and activation of WNT target genes. To determine whether mesodermal Pten
inactivation increases phosphorylation of β-CAT on Ser552 in the mesenchyme, we performed IHC for E-CAD and β-CATSer552
(Figure , G and H), the form of β-CAT specifically phosphorylated by the PTEN/AKT pathway (16
). We found significantly increased E-CAD–negative/ β-CATSer552
–positive cells in mutant lungs compared with controls (Figure J, 1.3% ± 0.1 vs. 0.3% ± 0.1, P
< 0.01). Co-IF for β-CAT and mesenchymal markers including PECAM or PDGFRα showed that these cells are negative for these markers (data not shown). We did not observe β-CATSer552
–positive cells in the epithelium. In our model, absence of mesenchymal Pten
affected lipofibroblast, VSMC, and parabronchial SMC (PSMC) differentiation likely due to increased levels of Ser552
β-CAT, a recognized positive regulator of stem cell homeostasis downstream of the AKT pathway.
Increase in mesenchymal progenitor cells is due to increased β-CAT and FGF10 signaling.
The pulmonary mesenchyme is the source of many signaling factors, the most important of which is FGF10, which interacts with receptor tyrosine kinases (RTKs). Fgf10
expression by the mesenchyme is positively regulated by WNT and FGF9 signaling and is negatively regulated by sonic hedgehog (SHH) (2
). Deletion of Fgf10
leads to lung agenesis, suggesting a key role in mediating mesenchymal-epithelial interactions that are necessary for lung morphogenesis (17
). FGF10 is the major FGFR2b ligand during the pseudoglandular stage. FGF10 is secreted exclusively by the mesenchyme and acts on the epithelium through FGFR2b.
Because of the increase in β-CATSer552
expression was analyzed by in situ hybridization (ISH). Fgf10
mRNA was increased in mutant lungs compared with controls (Figure , B vs. A). qRT-PCR analysis for Fgf10
confirmed the ISH results (Figure I, 2.09 ± 0.48 vs. 1, P
< 0.05). A downstream pathway activated by FGF10 in the epithelium is β-CAT signaling (3
). To examine whether the observed increased FGF10 and increased mesenchymal β-CATSer552
stimulates β-CAT signaling, we generated triple transgenic mice composed of Ptenfl/fl;Dermo-Cre
mice and Batgal
, the latter a WNT signaling reporter (19
), and analyzed the embryos at E15.5 (Figure , C–F). β-CAT signaling was increased in lung epithelium and mesenchyme of the Ptenfl/fl;Dermo-Cre;Batgal
lungs compared with controls (Figure , D and F vs. C and E).
Mesenchymal Pten inactivation leads to expansion of the distal epithelial progenitor cell domain characterized by ID2 and SPC expression.
Since activated β-CAT is recognized as controlling stem cell homeostasis in lung epithelial cells, we performed IF for epithelial markers in E18.5 control and mutant lungs (Figure , C–H). Double IF with antibodies to E-CAD and FGFR2 showed increased FGFR2 in E18.5 Ptenfl/fl;Dermo-Cre lungs compared with controls (Figure , B vs. A). qRT-PCR confirmed this observation by showing a 4-fold increase of Fgfr2b mRNA in mutant lungs (Figure I, 4.1 ± 0.3 vs. 1, P < 0.01). qRT-PCR showed increased expression for Fgf9 and Fgf7, 2 additional growth factors made by the lung mesothelium/epithelium (Fgf9) and by the mesenchyme (Fgf7), (Figure I, Fgf9: 2.6 ± 0.5 vs. 1, P < 0.05; Fgf7: 5.7 vs. 1 ± 1.6, P < 0.05). To validate these results, we also examined the expression of FGF10/FGFR2b downstream and positive regulator targets by qRT-PCR. As expected, compared with the controls, Wnt2b, Spry2, Etv4, Etv5, and Bmp4 were increased, while Shh, Ptch1, and Gli1 decreased in the mutant lungs (Figure I, Wnt2b: 3.03 ± 1.2 vs. 1, NS; Spry2: 2.46 ± 0.58 vs. 1, P < 0.05; Etv4: 1.26 ± 0.15 vs. 1, NS; Etv5: 1.56 ± 0.24 vs. 1, P < 0.05; Bmp4: 1.26 ± 0.06 vs. 1, P < 0.01; Figure I, Shh: 0.6 ± 0.3 vs. 1, P < 0.05; Ptch1: 0.47 ± 0.1, P < 0.05; Gli1: 0.5% ± 0.06 vs. 1, P < 0.01).
Analysis of epithelial differentiation suggests increased FGF signaling.
As we observed increased FGF10 activity in the epithelium of the mutant lungs, we investigated whether this change affected epithelial differentiation along the pulmonary proximal and distal domains (20
). IHC for SPC, T1α, and CC10 markers for alveolar type 2 (distal domain), alveolar type 1 (distal domain) and Clara cells (proximal domain), respectively, showed an increase of alveolar type 2 (SPC-positive) cells, while the Clara cells (CC10-positive) and type 1 cells (T1α-positive) were unchanged in the Ptenfl/fl;Dermo-Cre
lungs compared with Ptenfl/fl
controls (Figure , C–H). Western blot analysis demonstrated a statistically significant increase in SPC expression (marker for alveolar type 2 cells) in the mutant compared with controls (Figure J, SPC: 0.2 ± 0.003 vs. 0.09 ± 0.003, in arbitrary units, P
< 0.05). In addition, comparison of CC10 and T1α expression showed no statistical difference between the 2 groups (data not shown). Finally, we performed IHC for TTF1 (NKX2.1), the first transcriptional factor present in the lung during embryogenesis (21
), and for ID2, a marker of distal lung progenitor cells (22
). The increase in TTF1-positive cells (Figure , L vs. K) and ID2/E-CAD–double-positive cells (Figure , N vs. M) in the mutant lungs compared with the control confirmed the expansion of the distal domain. Western blot results further confirmed the latter results, showing an increase of TTF1 (0.13 ± 0.006 vs. 0.23 ± 0.006, P
< 0.01) in the mutants compared with controls (Figure J). Overall, these data suggest that the increase in FGF10 signaling in the epithelium, due to Pten
mesenchymal inactivation, is the likely cause of distal epithelial domain expansion.
Mesenchymal Pten inactivation phenocopies ACD.
As the phenotype of the lungs with mesenchymal Pten inactivation resembled what has been described in newborn infants as ACD, we examined the hypothesis that the PTEN/PI3K/AKT pathway is activated in the lungs of ACD patients. For this purpose, we performed PTEN and p-AKT IHC on 5 different and independent lung samples from newborn human infants who died with ACD diagnosis and compared these to lungs of newborns who died of causes unrelated to ACD. ACD patients showed a decrease of PTEN staining and a corresponding increase in p-AKT–positive cells compared with lungs from control patients. While PTEN staining was readily detectable (Figure , B vs. A), the number of p-AKT–positive cells was limited in the control lungs (Figure , D vs. C). Statistical analysis showed an increase in the percentage of p-AKT–positive cells in the ACD lungs compared with controls (Figure F, 1.4 ± 0.4 vs. 0.07 ± 0.05, P < 0.05).
Human newborn ACD lungs display decreased PTEN and increased p-AKT.
ACD has been associated with mutations in Forkhead box protein F1 (FOXF1
) in humans and heterozygous Foxf1
loss of function in mice (23
). Accordingly, Foxf1
mRNA were measured by qRT-PCR. We found decreased expression of both genes in mutant compared with control lungs (Figure E, Foxf1
: 0.59 ± 0.015 vs. 1, P
< 0.01; Foxc2
: 0.57 ± 0.13 vs. 1, P
< 0.05). These data support the involvement of the PTEN/PI3K/AKT pathway in the pathogenesis of ACD and justify further analysis of this pathway in our newly described mouse model of a lethal human congenital lung disease.