Our data indicate that Wnt2 signaling is necessary and sufficient to promote the differentiation of ASM from the multipotent lung mesenchyme through direct or indirect activation of a myogenic regulatory network including myocardin and Mrtf-B. These data show that Wnt2 signaling activates Fgf10 signaling in the mesenchyme, which further drives proliferation and smooth muscle differentiation into mature ASM. Our results also suggest that Wnt2 signaling activates Wnt7b signaling in the adjacent epithelium, which is known to signal in a paracrine manner to the sup-epithelial mesenchyme to promote smooth muscle differentiation.
Smooth muscle development in the lung is poorly understood in part because the origins of the various smooth muscle lineages are still unclear and the pathways regulating proliferation, migration, and differentiation of primitive smooth muscle cells are complex and unresolved. Recent genetic evidence demonstrates that Wnt/β-catenin signaling lies upstream of signals governing smooth muscle development in the mouse lung. Loss of the canonical ligand Wnt7b in the embryonic lung epithelium leads to severe VSM defects, demonstrating a critical role for paracrine Wnt/β-catenin signaling between the developing epithelium and mesenchyme (Cohen et al., 2009
; Shu et al., 2002
). Additional studies demonstrate that genetic deletion of β-catenin in the primitive lung mesenchyme also disrupts ASM development (De Langhe et al., 2008
), implicating a potential function for other canonical Wnt ligands expressed in the lung for proper ASM development. In this study, we identify Wnt2 as a critical Wnt ligand required in the lung mesenchyme to mediate ASM development. We show that Wnt2 is required for BAT-GAL
activity in ASM indicating that it acts through the β-catenin dependent canonical pathway to regulate ASM development.
While defects in ASM are observed Wnt2−/−
null mutants, the VSM appears grossly normal. Wnt7b signaling from the lung epithelium has been shown to regulate VSM development (Cohen et al., 2009
; Shu et al., 2002
), and analysis of Wnt2−/−
null mutant lungs demonstrates that Wnt7b is expressed at about 50% of normal levels in Wnt2−/−
null mutants. Additionally, Wnt2
expression is reduced in Wnt7b−/−
null lungs (unpublished observations), suggesting there is signaling interplay between epithelial and mesenchymal expressed Wnt ligands in the lung. If Wnt2 signaling is important for VSM development, the potential effects from loss of Wnt2 signaling may be compensated by the continued expression of Wnt7b or Wnt2b, which is also expressed in the lung mesenchyme and could function redundantly with Wnt2 signaling in VSM development (Goss et al., 2009
). The distinct molecular pathways that regulate VSM versus ASM development in the lung are poorly understood. Moreover, it is unclear whether ASM and VSM have a common or distinct developmental origins. Disruption of other pathways including Shh result in defects in both ASM and VSM (Miller et al., 2004
). Thus, additional fate-mapping as well as developmental studies will be required to determine the similarities and differences in VSM and ASM origins and cellular phenotypes.
A recent study describing conditional mutants in β-catenin signaling in the developing lung mesenchyme suggested a role for Wnt/β-catenin in the differentiation of the endothelial lineage (De Langhe et al., 2008
null embryoid bodies are also unable to effectively differentiate into endothelial cells (Wang et al., 2007a
). Our data suggests that either Wnt2 is not required for lung endothelial development or that it acts redundantly with other Wnt ligands to regulate endothelial development in the lung.
Wnt2 is expressed during the earliest stages of lung morphogenesis suggesting the perinatal ASM deficiency in Wnt2−/− null mutants could be the result of an early defect in the development of smooth muscle cells in the distal mesenchyme. Analysis of Wnt2−/− null mutants during the early stages of myogenesis indicated reduced expression of the genes regulating the earliest aspects of smooth muscle cell differentiation from the primitive mesenchyme including myocardin, Mrtf-B, and Pdgfrβ These data indicate that Wnt2 signaling regulates the development of the multipotent mesenchymal into immature smooth muscle cells in the mouse lung.
A Wnt-Fgf regulatory network is thought to govern development of the lung mesenchyme (Yin et al., 2008), and mice carrying hypomorphic or null alleles of Fgf10
, the cognate receptor of Fgf10, exhibit ASM deficiencies (De Langhe et al., 2006
; Ramasamy et al., 2007
). In Wnt2−/−
null mutant lungs, both Fgf10
expression and Fgfr2
expression are reduced, implicating a Wnt2-Fgf10 signaling network in the coordination of ASM development. We show that Fgf10 signaling promotes ASM development through upregulation of several mature smooth muscle markers but not the early smooth muscle differentiation factors myocardin or Mrtf-B. These findings suggest that Fgf10 signaling mediates the expansion and differentiation of immature smooth muscle cell already committed to the ASM lineage.
In summary, these studies demonstrate that Wnt2 signaling is necessary for ASM development in the mouse lung. Wnt2 expression during early lung morphogenesis is required for activation of the smooth muscle gene expression program including myocardin, Mrtf-B, and Fgf10. Exogenous Fgf10 can partly rescue the loss of Wnt2 by promoting differentiation of cells already committed to the ASM lineage. Thus, these results define a Wnt2-Fgf10 signaling axis governing early ASM development in the lung.