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

De Langhe SP, Carraro G, Tefft D, Li C, Xu X, Chai Y, Minoo P, Hajihosseini MK, Drouin J, Kaartinen V, Bellusci S - PLoS ONE (2008)

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

View Article: PubMed Central - PubMed

Affiliation: Developmental Biology Program, Department of Surgery, Saban Research Institute of Childrens Hospital Los Angeles, Los Angeles, California, USA.

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.

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Related in: MedlinePlus

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.
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pone-0001516-g004: 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.

Mentions: The observed reduction in Fgf10 and Spry4 expression, indicative of reduced FGFR2C signaling in CKO lung mesenchyme, led us to investigate its mechanism. Shu et al., (2005) reported that inactivation of β-catenin in the distal lung epithelium, leads to a down-regulation of Fgfr2 receptor in the epithelium. By inactivating β-catenin in the lung mesenchyme, we might expect to observe a similar down-regulation of Fgfr2 in the mesenchyme. Using immunohistochemistry, we found that indeed FGFR2 expression is reduced in the mesenchyme but not epithelium of CKO lungs (Fig. 4a,b). Interestingly, FGFR2 expression is also reduced in Pitx2−/− lungs (supplemental Fig. S2). In this case, however, FGFR2 expression was also reduced in the epithelium, as Pitx2 deletion was not mesenchyme specific. To further address how ß-catenin signaling in the mesenchyme regulates Fgfr2 expression, we silenced β-catenin and Pitx2 expression using siRNA in primary cultures of WT mesenchyme and monitored using real time PCR the levels of Fgfr2 expression. Using siRNA in primary cultures of lung mesenchyme, a downregulation in β-catenin expression of 33%±8 (n = 3, P = 0.03) compared to scrambled led to a corresponding downregulation in Fgfr2 expression of 40%±4 (n = 3, P = 0.003) (Fig 4l). Similarly, a downregulation in Pitx2 expression of 50%±8 (n = 3, P = 0.01) compared to scrambled led to a corresponding downregulation in Fgfr2 expression of 40%±6 (n = 3, P = 0.009) (Fig. 4l).


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

De Langhe SP, Carraro G, Tefft D, Li C, Xu X, Chai Y, Minoo P, Hajihosseini MK, Drouin J, Kaartinen V, Bellusci S - PLoS ONE (2008)

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.
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pone-0001516-g004: 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.
Mentions: The observed reduction in Fgf10 and Spry4 expression, indicative of reduced FGFR2C signaling in CKO lung mesenchyme, led us to investigate its mechanism. Shu et al., (2005) reported that inactivation of β-catenin in the distal lung epithelium, leads to a down-regulation of Fgfr2 receptor in the epithelium. By inactivating β-catenin in the lung mesenchyme, we might expect to observe a similar down-regulation of Fgfr2 in the mesenchyme. Using immunohistochemistry, we found that indeed FGFR2 expression is reduced in the mesenchyme but not epithelium of CKO lungs (Fig. 4a,b). Interestingly, FGFR2 expression is also reduced in Pitx2−/− lungs (supplemental Fig. S2). In this case, however, FGFR2 expression was also reduced in the epithelium, as Pitx2 deletion was not mesenchyme specific. To further address how ß-catenin signaling in the mesenchyme regulates Fgfr2 expression, we silenced β-catenin and Pitx2 expression using siRNA in primary cultures of WT mesenchyme and monitored using real time PCR the levels of Fgfr2 expression. Using siRNA in primary cultures of lung mesenchyme, a downregulation in β-catenin expression of 33%±8 (n = 3, P = 0.03) compared to scrambled led to a corresponding downregulation in Fgfr2 expression of 40%±4 (n = 3, P = 0.003) (Fig 4l). Similarly, a downregulation in Pitx2 expression of 50%±8 (n = 3, P = 0.01) compared to scrambled led to a corresponding downregulation in Fgfr2 expression of 40%±6 (n = 3, P = 0.009) (Fig. 4l).

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

View Article: PubMed Central - PubMed

Affiliation: Developmental Biology Program, Department of Surgery, Saban Research Institute of Childrens Hospital Los Angeles, Los Angeles, California, USA.

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.

Show MeSH
Related in: MedlinePlus