Formation and differentiation of multiple mesenchymal lineages during lung development is regulated by beta-catenin signaling

Abstract

Background: The role of ss-catenin signaling in mesodermal lineage formation and differentiation has been elusive.

Methodology: To define the role of ss-catenin signaling in these processes, we used a Dermo1(Twist2)(Cre/+) line to target a floxed beta-catenin allele, throughout the embryonic mesenchyme. Strikingly, the Dermo1(Cre/+); beta-catenin(f/-) conditional Knock Out embryos largely phenocopy Pitx1(-/-)/Pitx2(-/-) double knockout embryos, suggesting that ss-catenin signaling in the mesenchyme depends mostly on the PITX family of transcription factors. We have dissected this relationship further in the developing lungs and find that mesenchymal deletion of beta-catenin differentially affects two major mesenchymal lineages. The amplification but not differentiation of Fgf10-expressing parabronchial smooth muscle progenitor cells is drastically reduced. In the angioblast-endothelial lineage, however, only differentiation into mature endothelial cells is impaired.

Conclusion: Taken together these findings reveal a hierarchy of gene activity involving ss-catenin and PITX, as important regulators of mesenchymal cell proliferation and differentiation.

Figure 1. Decreased branching, partial right isomerization and epithelial proximalization in CKO lungs.

(a–d) β-gal staining of Dermo1^Cre/+/Rosa26R WT lungs at E11.5 and E13.5 reports Cre recombination activity in the mesenchyme of the embryonic lung. (a–b) Whole mount pictures of β-gal staining. (c) Vibratome section through E13.5 lung. (d) Paraffin section through E13.5 β-gal stained lung and counterstained with eosin. (e–h) (e) E12.5 WT and (f) CKO lungs which show decreased branching. (g) E14.5 WT lung and (h) CKO lung which shows partial right isomerization, astereotypic branching and a grape like structure, and absence of trachea. (i–j) H&E staining of sections through the left lobe of (i) E14.5 WT and (j) CKO lungs. Left lobe of CKO lung features proximalized airways and partial lobular septation. (k–l) Immunostaining for β-catenin on sections of (k) E14.5 WT and (l) CKO lungs show a mosaic deletion of β-catenin throughout the lung mesenchyme.

Figure 2. Expression pattern of PITX members in the developing wild type and mutant embryonic E13.5 lungs.

(a–h) Immunostaining for PITX family members and α-SMA. (a,b) PITX1 expression in wild type lung is detected throughout the distal epithelium. (b) Expression of PITX1 in the CKO lung is not changed compared to WT. (c) PITX2 expression in wild type lung is detected throughout the epithelium as well as in the mesenchyme at the exception of the mesenchyme directly surrounding the proximal epithelium (arrows). (d) In CKO lungs, PITX2 expression is drastically reduced in the epithelium and mesenchyme. (e) In wild type lungs, PITX3 expression is present in the differentiated PSMCs adjacent to the bronchi. (f) In CKO lungs, PITX3 expression is no longer found as a continuous layer around the bronchi likely reflecting a defect in the formation of the PSMC. (g, h) SMA expression on the same section as (e) and (f).

Figure 3. Reduced Fgf10, Spry2, Spry4 and Pitx2 expression in CKO lungs.

Gene expression in E13.5 lungs by WMISH and LacZ staining in WT and CKO lungs (n = 3 for each probe). (a–b) Fgf10 expression is reduced in the sub-mesothelial mesenchyme of CKO lungs. Inset: vibratome section through the distal left lobes. (c–d) Spry2 expression is reduced in the epithelium of CKO lungs. (e–f) No difference in Shh or (g–h) Ptch expression levels in CKO lungs compared to WT lungs. Inset : vibratome section through the distal right lobes. (i–j) Spry4 expression is reduced in the distal mesenchyme of CKO lungs illustrating reduced mesenchymal FGF9 signaling. (k–l) β-gal staining of TOPGAL lungs shows similar levels of TOPGAL activity in WT and CKO lungs illustrating the specificity of the β-catenin deletion throughout the mesenchyme. (o–n) Pitx2 is completely ablated in CKO lungs at E13.5. (o–p) Pitx2 expression is still present in E11.5 CKO embryos even though the Pitx2^−/− phenotype is already apparent indicating that interaction of PITX2 with β-catenin is necessary for β-catenin signaling.

Figure 4. Reduced FGFR expression, P-ERK and proliferation in CKO mesenchyme.

(a–b) Immunohistochemistry. Reduced expression for FGFR2 in E13.5 CKO lung mesenchyme. Expression in the CKO epithelium is unaffected. (c–d) Immunofluorescence for phospho-ERK (P-ERK) in green (arrows) and β-catenin in red, DAPI (Blue). (e–f) Immunofluorescence for phospho-HistonH3 (PH3) in green and β-catenin in red, DAPI (Blue) (g–j) H&E stained sections through E12.5 WT and CKO lungs cultured for 48h in vitro in the presence or absence of 200 ng/ml FGF9. (g,i) WT lungs grown in the presence of FGF9 (i) show decreased branching, dilation of the epithelium and overproliferation of the distal mesenchyme compared to untreated lungs (g). (h,j) CKO lungs grown in the presence of FGF9 (j) only show an epithelial effect and dilation of the epithelium while proliferation of the distal mesenchyme remains absent. (k) Upper part: western blot analysis on primary culture of WT and CKO lung mesenchyme treated or not with FGF9 with P-ERK, total-ERK, PITX2 and FGFR2 antibodies. Lower part: Co-Immunoprecipitation of PITX2 with β-catenin from primary culture of wild type lung mesenchyme cultured in the presence of 10 mM LiCl. Absence of co-immunoprecipitation of PITX3 with β-catenin from primary culture of wild type lung mesenchyme. (l) Relative β-catenin, Fgfr2 and Pitx2 expression levels in primary cultures of mesenchyme treated with siRNA to β-catenin (top) and Pitx2 (bottom) analyzed by real time PCR.

Figure 5. Reduced c-Myc expression and lack of PSMC progenitor amplification in CKO lungs.

(a-b) Section RISH for c-Myc on E14.5 WT and CKO lungs. Expression of c-Myc is lost in CKO lung mesenchyme were β-catenin is completely deleted but remains the same were β-catenin expression is unaltered. These sections are adjacent to the ones represented in Fig. 1k,l illustrating mosaic β-catenin deletion. (c-d) Immunofluorescence for β-catenin (green) and α-SMA (red) on sections through E13.5 WT and CKO lungs. Absence of β-catenin in CKO lung mesenchyme and patchy α-SMA expression around the bronchi (d) and high magnification inset in (d). (e–f) Untreated primary cultures of mesenchyme from both WT and CKO lungs spontaneously differentiate into smooth muscle cells in vitro. (g–h) Primary cultures from WT lungs treated with FGF9 fail to differentiate into smooth muscle cells (g) while primary cultures from CKO lungs (h) are not affected in their differentiation after FGF9 treatment. (i–l) Immunofluorescence for β-catenin (green) and P-ERK (red) on primary culture of WT and CKO lung mesenchyme treated or not with FGF9. Upon FGF9 treatment, Note the drastic increase in P-ERK expression in WT cells in comparison to CKO cells.

Figure 6. Lack of PSMC progenitor amplification and failure of endothelial progenitor cell differentiation.

(a–b') β-gal staining on WT and CKO lungs crossed with the Fgf10^LacZ reporter line. β-gal staining in the CKO lung (b) is severely reduced and the presence of single progenitor cells are apparent. (a'–b') Close up on the accessory lobe shows the presence of single Fgf10^LacZ positive PSMC progenitor cells in the distal mesenchyme of the CKO lung. Lineage tracing of the Fgf10^LacZ positive PSMC progenitor cells in CKO lungs (b') shows fewer cells are relocating around the bronchi compared to the WT lungs (a'). (c–d) IHC for β-catenin (brown staining) on paraffin sections of WT and CKO lungs crossed with the Fgf10^LacZ reporter. (d) Presence of β-gal staining in the distal mesenchyme in the absence of β-catenin expression (arrow). (e–f) β-gal staining on WT and CKO lungs crossed with the Flk1^LacZ reporter line. High magnification of E13.5 left lobes show an increase in Flk1^LacZ expression in the CKO lung compared to WT lungs. Arrowheads illustrate the reduction in size of the sub-mesothelial mesenchymal domain containing the Fgf10 expressing PSCM progenitors and in which no Flk1-positive cells are present. (g–h) Immunofluorescence staining for PECAM on E14.5 WT and CKO lungs. Absence of PECAM in CKO lungs (f). (i–j) Immunofluorescence staining for endothelial-Claudin5 on E14.5 WT and CKO lungs. Absence of endothelial-Claudin5 in CKO lungs (f). (k–l) β-gal staining on E13.5 WT and CKO embryos crossed with the Flk1^LacZ reporter line. CKO embryos (l) show and increased expansion of Flk1^LacZ positive angioblasts throughout the embryonic mesenchyme compared to WT embryos (k). (m–n) Intracardiac India ink injection of E13.5 WT and CKO embryos. CKO embryos show defects in vasculogenesis and leakage of India ink from premature blood vessels is apparent (n) compared to WT embryos (m).

Figure 7. Model for lineage differentiation of the PSMCs.

Fgf10 and Pitx2 expressing PSMC progenitors are located in the submesothelial mesenchyme and respond to FGF9 and β-catenin signaling to amplify and remain undifferentiated , , , . As the epithelium grows out, the PSMC progenitors come in contact with BMP4 secreted by the epithelium. These cells then stop expressing Fgf10 and get committed to the PSCM lineage . When these cells eventually spread out on the epithelial basement membrane containing Fibronectin , they differentiate into mature PSMC and start to express α-SMA and switch from PITX2 to PITX3 expression.