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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

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 Fgf10LacZ 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 Fgf10LacZ positive PSMC progenitor cells in the distal mesenchyme of the CKO lung. Lineage tracing of the Fgf10LacZ 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 Fgf10LacZ 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 Flk1LacZ reporter line. High magnification of E13.5 left lobes show an increase in Flk1LacZ 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 Flk1LacZ reporter line. CKO embryos (l) show and increased expansion of Flk1LacZ 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).
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pone-0001516-g006: 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 Fgf10LacZ 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 Fgf10LacZ positive PSMC progenitor cells in the distal mesenchyme of the CKO lung. Lineage tracing of the Fgf10LacZ 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 Fgf10LacZ 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 Flk1LacZ reporter line. High magnification of E13.5 left lobes show an increase in Flk1LacZ 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 Flk1LacZ reporter line. CKO embryos (l) show and increased expansion of Flk1LacZ 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).

Mentions: So far, we have provided experimental evidence suggesting that CKO deletion of β-catenin in the lung mesenchyme perturbs the amplification but not the differentiation of the PSMC progenitors into smooth muscle cells. To directly visualize the fate of the Fgf10-expressing progenitors, we crossed our mutant mice with a previously published Fgf10LacZ enhancer-trap line [38]. Due to the stability of the LacZ protein, this line can be used to lineage trace transiently the Fgf10 expressing PSMC progenitors [9]. CKO lungs showed a marked reduction in Fgf10/LacZ expressing progenitors in the distal mesenchyme at E13.5 vs. WT lungs (Fig. 6b,a). Close analysis of the accessory lobe further illustrated the presence of single progenitor cells in the distal mesenchyme of the CKO lung (Fig 6b',a' arrowhead) and patchy LacZ expression around the bronchi compared to WT lungs (Fig. 6b',c' arrows). Immunhistochemistry for β-catenin on paraffin sections of CKO lungs crossed with the Fgf10LacZ reporter reveals that expression of Fgf10 in the distal mesenchyme of CKO lungs is not due to the lack of recombination of the ß-cateninflox allele (Fig. 6c,d). This confirms our previous observation that ß-catenin signaling in the lung mesenchyme is important for the amplification of the PSMC progenitors or transient amplifying cells. We then examined whether the loss of ß-catenin signaling affects the differentiation of the lung mesenchyme into endothelial cells. For this, CKO lungs were generated in a Flk1LacZ reporter background [39], which expresses LacZ under the control of the endogenous Flk1 promoter. Flk1 is an early marker of angioblast and its expression is maintained in mature endothelial cells [39]. Interestingly, Flk1 expression was highly upregulated throughout the entire CKO embryo (Fig. 6k,l) including the lungs (Fig. 6e,f). Ablation of mesenchymal ß-catenin signaling therefore did not seem to interfere with the specification and amplification of the angioblast. However, PECAM and endothelial-Claudin5 staining on CKO lungs vs. WT lungs revealed an impaired differentiation of the angioblasts into mature endothelial cells and blood vessels in the CKO lungs vs. WT lungs (Fig. 6g–j). We also examined the pattern of vasculature by injecting India ink in the left ventricle of the CKO and WT hearts, only to find a clear defect in vasculogenesis throughout the CKO embryo (Fig. 6m,n). Leakage of India ink from abnormal and immature blood vessels could be observed throughout the CKO embryo. A similar underdeveloped vascular system was also observed in Pitx2−/− embryos after Intracardiac India ink injection (supplemental Fig. S2). However, PECAM staining could still be detected in endothelial cells of Pitx2−/− embryos (data not shown) indicating a possible redundancy with other PITX or LEF1/TCF transcription factors. Our results indicate that inactivation of β-catenin in the mesenchyme inhibits the differentiation of angioblasts into mature endothelial cells. However, it is important to note that ablation of β-catenin in mature endothelial cells using the Tie2Cre driver line did not affect vasculogenesis and angiogenesis, or PECAM expression [40]. Tie2 expression starts later during endothelial cell differentiation and so our data suggests that ß-catenin signaling is an important regulator of early endothelial cell development [41], [42].


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)

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 Fgf10LacZ 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 Fgf10LacZ positive PSMC progenitor cells in the distal mesenchyme of the CKO lung. Lineage tracing of the Fgf10LacZ 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 Fgf10LacZ 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 Flk1LacZ reporter line. High magnification of E13.5 left lobes show an increase in Flk1LacZ 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 Flk1LacZ reporter line. CKO embryos (l) show and increased expansion of Flk1LacZ 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).
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Related In: Results  -  Collection

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pone-0001516-g006: 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 Fgf10LacZ 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 Fgf10LacZ positive PSMC progenitor cells in the distal mesenchyme of the CKO lung. Lineage tracing of the Fgf10LacZ 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 Fgf10LacZ 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 Flk1LacZ reporter line. High magnification of E13.5 left lobes show an increase in Flk1LacZ 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 Flk1LacZ reporter line. CKO embryos (l) show and increased expansion of Flk1LacZ 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).
Mentions: So far, we have provided experimental evidence suggesting that CKO deletion of β-catenin in the lung mesenchyme perturbs the amplification but not the differentiation of the PSMC progenitors into smooth muscle cells. To directly visualize the fate of the Fgf10-expressing progenitors, we crossed our mutant mice with a previously published Fgf10LacZ enhancer-trap line [38]. Due to the stability of the LacZ protein, this line can be used to lineage trace transiently the Fgf10 expressing PSMC progenitors [9]. CKO lungs showed a marked reduction in Fgf10/LacZ expressing progenitors in the distal mesenchyme at E13.5 vs. WT lungs (Fig. 6b,a). Close analysis of the accessory lobe further illustrated the presence of single progenitor cells in the distal mesenchyme of the CKO lung (Fig 6b',a' arrowhead) and patchy LacZ expression around the bronchi compared to WT lungs (Fig. 6b',c' arrows). Immunhistochemistry for β-catenin on paraffin sections of CKO lungs crossed with the Fgf10LacZ reporter reveals that expression of Fgf10 in the distal mesenchyme of CKO lungs is not due to the lack of recombination of the ß-cateninflox allele (Fig. 6c,d). This confirms our previous observation that ß-catenin signaling in the lung mesenchyme is important for the amplification of the PSMC progenitors or transient amplifying cells. We then examined whether the loss of ß-catenin signaling affects the differentiation of the lung mesenchyme into endothelial cells. For this, CKO lungs were generated in a Flk1LacZ reporter background [39], which expresses LacZ under the control of the endogenous Flk1 promoter. Flk1 is an early marker of angioblast and its expression is maintained in mature endothelial cells [39]. Interestingly, Flk1 expression was highly upregulated throughout the entire CKO embryo (Fig. 6k,l) including the lungs (Fig. 6e,f). Ablation of mesenchymal ß-catenin signaling therefore did not seem to interfere with the specification and amplification of the angioblast. However, PECAM and endothelial-Claudin5 staining on CKO lungs vs. WT lungs revealed an impaired differentiation of the angioblasts into mature endothelial cells and blood vessels in the CKO lungs vs. WT lungs (Fig. 6g–j). We also examined the pattern of vasculature by injecting India ink in the left ventricle of the CKO and WT hearts, only to find a clear defect in vasculogenesis throughout the CKO embryo (Fig. 6m,n). Leakage of India ink from abnormal and immature blood vessels could be observed throughout the CKO embryo. A similar underdeveloped vascular system was also observed in Pitx2−/− embryos after Intracardiac India ink injection (supplemental Fig. S2). However, PECAM staining could still be detected in endothelial cells of Pitx2−/− embryos (data not shown) indicating a possible redundancy with other PITX or LEF1/TCF transcription factors. Our results indicate that inactivation of β-catenin in the mesenchyme inhibits the differentiation of angioblasts into mature endothelial cells. However, it is important to note that ablation of β-catenin in mature endothelial cells using the Tie2Cre driver line did not affect vasculogenesis and angiogenesis, or PECAM expression [40]. Tie2 expression starts later during endothelial cell differentiation and so our data suggests that ß-catenin signaling is an important regulator of early endothelial cell development [41], [42].

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