We have identified a robust regulatory circuit in which epithelial to mesenchymal FGF signaling regulates the expression of a Wnt ligand and the level of WNT-β-Catenin signaling, and in which mesenchymal WNT-β-Catenin signaling regulates the expression of FGF receptors and the level of mesenchymal FGF signaling (). The only external (non-mesenchymal) factor that has thus far been identified that can modulate this signaling system is FGF9, which is produced by the mesothelial lining of the lung and by airway epithelium, and potentially WNT7b, which is produced in airway epithelium. The primary output of this system appears to be regulation of mesenchymal proliferation, but clearly other mesenchymal factors that regulate lung epithelial proliferation, survival and differentiation must also act downstream of FGF and/or β-Catenin.
Fig. 9 Molecular pathways by which WNT-β-Catenin and FGF regulate lung mesenchymal development. (1) At early stages of lung development (E10–E12), FGF9 is produced by lung mesothelium and epithelium and signals to mesenchymal FGFR1c and FGFR2c (more ...)
Two predictions can be derived from the FGF–WNT interaction model (). First: down-regulation or inhibition of any component of this system should diminish the output of the entire system. Second: up-regulation of any component of the system should reinforce the entire system. In vitro
organ culture experiments and in vivo
genetic experiments support the circular and self-sustaining nature of this signaling network. For example, lung tissue lacking mesenchymal β-Catenin was predicted and shown to down-regulate mesenchymal responsiveness to FGF9. Similarly, loss of mesenchymal FGF signaling resulted in diminished WNT-β-Catenin signaling, loss of Fgfr
expression and inability of the tissue to be rescued by treatment with FGF9. However, phenotypic differences resulting from loss of either mesenchymal β-Catenin or mesenchymal FGFR1 and FGFR2 indicate that other factors must also feed into this signaling circuit to sustain mesenchymal viability. Apparent differences could also be due to variation in the timing or efficiency of Cre-mediated inactivation of β-Catenin and Fgfrs 1
, versus complete loss of mesenchymal FGF signaling in Fgf9−/−
lungs. In FGF loss of function models (Fgf9−/−
), the lung becomes atrophic by birth but does not exhibit widespread cell death (Colvin et al., 2001
; White et al., 2006
). By contrast, β-CateninDermo1
lungs exhibit widespread apoptosis by E18.5. These observations provide further evidence that these two pathways are not equivalent and that β-Catenin signaling may have a more important role in mesenchymal cell survival. Furthermore, some intrinsic or extrinsic signal(s) likely maintains low-level mesenchymal β-Catenin signaling and cell viability in the absence of FGF signaling. This is supported by the observed reduction, but not elimination, of WNT-β-Catenin activity (Lef1
expression) in Fgf9−/−
lung mesenchyme. However, besides FGF9 and WNT7b, factors that regulate mesenchymal WNT-β-Catenin signaling have not been identified.
is expressed in lung epithelium (Pepicelli et al., 1998
; Weidenfeld et al., 2002
embryos have reduced lung mesenchymal proliferation and defects in lung vascular smooth muscle (Shu et al., 2002
). It is, therefore, likely that WNT7b signals directly to adjacent sub-epithelial mesenchyme and acts synergistically or redundantly with WNT2a (). Wang et al. (2005)
have shown that WNT7b can activate FZD1, 4 and 7 and that these WNT receptors are expressed in lung mesenchyme and in vascular smooth muscle precursors; however, direct targets of WNT-β-Catenin signaling were not examined in lung mesenchyme of Wnt7b−/−
mice. Supporting the idea that WNT7b could regulate mesenchymal WNT-β-Catenin activity independent of FGF9, we showed that the expression of Wnt7b
was increased in Fgf9−/−
lungs. Thus, WNT7b is likely to be a second independent external factor that can modulate the mesenchymal WNT-β-Catenin–FGFR regulatory circuit.
, a target of WNT-β-Catenin signaling, appears to be uniformly expressed throughout distal mesenchyme. This suggests that Wnt2a
, which is expressed in sub-mesothelial mesenchyme, might support both autocrine signaling to sub-mesothelial mesenchyme and paracrine signaling to sub-epithelial mesenchyme. Although a lung mesenchymal phenotype was not reported for Wnt2a−/−
mice (Monkley et al., 1996
) and the original knockout line was not saved, a second, recently constructed line of Wnt2a−/−
mice shows decreased lung mesenchymal proliferation (E. Morrisey, personal communication). Additionally, Wnt7b−/−
mice show decreased lung mesenchymal proliferation (Shu et al., 2002
). These data support a potential role for these ligands acting together in the regulation of mesenchymal WNT-β-Catenin signaling. To demonstrate this in vivo
, future epistasis studies will be required in which Wnt2a
double heterozygous mice are examined.
To address the contribution of WNT-β-Catenin signaling in lung epithelium, several labs have conditionally inactivated β-Catenin in epithelium or overexpressed the WNT antagonist, DKK, in epithelium (De Langhe et al., 2005
; Mucenski et al., 2003
; Shu et al., 2005
). These studies showed that WNT-β-Catenin signaling regulates the expression of N-Myc
in lung epithelium and regulates the extent of branching morphogenesis. Down-regulation of epithelial Fgfr2b
could account for the observed defects in branching morphogenesis in these mice. Importantly, the identity of the WNT ligand(s) that regulates epithelial WNT-β-Catenin signaling is not known; however, in addition to activating FZD1, 4 and 7 in lung mesenchyme, WNT7b can activate FZD10, which is expressed in lung epithelium (Wang et al., 2005
). A possible autocrine role for WNT7b in epithelium is supported by observed defective epithelial differentiation in Wnt7b−/−
lung tissue (Shu et al., 2002
In summary, FGF9, and most likely WNT7b, are two ligands that can independently signal from mesothelial (FGF9) and epithelial (FGF9 and WNT7b) cells to lung mesenchyme to regulate growth. The positive feedback loop within the mesenchymal compartment (Wnt2a, FGFR1, FGFR2 and β-Catenin) allows input from both FGF and WNT signaling systems to modulate the output of the entire system, thus providing a mechanism to tightly regulate mesenchymal lung development and, indirectly, epithelial morphogenesis.