Lung morphogenesis is controlled by reciprocal signaling between all cell layers: the epithelium, mesenchyme (including developing blood vessels), and the mesothelium (reviewed in, Cardoso and Lu, 2006; Morrisey and Hogan, 2010). Multiple interacting signaling pathways and transcription factors regulate the proliferation of lung progenitor cells (Fig. 6). The direct targets of most of the genes implicated in embryonic lung progenitor proliferation have not been identified. This makes it hard to determine if the effect observed, for example proliferation, is directly downstream of the gene/pathway under investigation.
Epithelial Progenitor Regulation
The distal lung epithelial progenitors express a repertoire of transcription factors. Nmyc is both necessary and sufficient for the division of multipotent lung epithelial progenitors and likely promotes self-renewal (Okubo et al., 2005). Similarly, the forkhead genes Foxp1 and Foxp2 are enriched in the multipotent epithelial progenitors and Foxp2−/−; Foxp1+/− mutants have smaller lungs with decreased levels of proliferation and Nmyc expression, but normal proximal–distal patterning (Shu et al., 2007). Sox9 is a useful marker for the multipotent epithelial progenitors, but lung epithelial specific deletion did not result in a phenotype (Perl et al., 2005).
Fgfr signaling mediates branching morphogenesis of the lung. In vitro FGF10 is both a chemoattractant and a mitogen for the lung epithelium during branching (Bellusci et al., 1997b; Park et al., 1998). Fgf10 hypomorphs have decreased levels of epithelial proliferation during the pseudoglandular stage and this is associated with decreased levels of distal epithelial phosphorylated-ERK MAPK and decreased expression of a canonical Wnt pathway reporter (Ramasamy et al., 2007). By contrast, overexpression of FGF10 in the developing lung apparently arrests epithelial progenitors in an undifferentiated state (Nyeng et al., 2008). These results are consistent with a role for mesenchymal FGF10 in promoting self-renewal, and inhibiting differentiation, of the multipotent lung epithelial progenitors. The transcription factors Etv4/5 and Elf5 mediate some of the effects of Fgfr signaling in these cells (Liu et al., 2003; Metzger et al., 2007).
Wnt7b is expressed in the distal tips of the developing lung epithelium and acts by means of a canonical signaling pathway to promote both epithelial and mesenchymal proliferation (Rajagopal et al., 2008). Wnt7b null embryos have a normal overall body size, but severely hypoplastic lungs. Lung progenitor proliferation is significantly decreased, particularly in the distal tip epithelium and subepithelial mesenchyme, but the number of Sox9+ multipotent progenitor cells per tip is normal and differentiation proceeds at the normal rate (Rajagopal et al., 2008). The effects of Wnt7b in the epithelium are possibly mediated through loss of distal epithelial Bmp4 and Id2 expression (Rajagopal et al., 2008). However, Id2 null lungs have normal rates of BrdU incorporation during the pseudoglandular stage (E.L.R., unpublished data). Wnt2 is expressed in the mesenchyme surrounding the epithelial buds and also promotes lung progenitor proliferation. Similar to Wnt7b null animals, Wnt2 null mice have smaller lungs with both epithelial and mesenchymal proliferation defects, but normal overall patterning (Goss et al., 2009). Wnt5a is highly expressed in the distal lung epithelium and surrounding mesenchyme. The null phenotype suggests that it may act antagonistically to Wnt7b and Wnt2 and repress epithelial and mesenchymal progenitor proliferation (Li et al., 2002). Wnt signaling has also been implicated in proximal–distal epithelial patterning and differentiation during lung development and the roles of other Wnt ligands remain obscure (Shu et al., 2005; Zhang et al., 2008). Interestingly, Wnt signaling is also absolutely required for the initial specification of the respiratory epithelial lineage from the foregut endoderm (Goss et al., 2009; Harris-Johnson et al., 2009; Chen et al., 2010).
The role of Bmp signaling in lung epithelial progenitor regulation has been difficult to define. In vitro and overexpression studies gave conflicting results, suggesting that the responses of the different progenitor populations to Bmp signaling are highly dose-specific (for example, Bellusci et al., 1996; Weaver et al., 2000). Bmp4 is expressed in the distal epithelial tip cells, as well as the mesenchyme surrounding the developing bronchioles, whereas Bmpr1a is found throughout the epithelium and mesenchyme (Weaver et al., 2000, 2003). Lung epithelial-specific deletion of either Bmpr1a or Bmp4 results in hypoplastic lungs with significantly reduced rates of epithelial proliferation and a decrease in the levels of Shh and Nmyc (Eblaghie et al., 2006). Analysis of the phenotype suggested that Bmp signaling may be important for the self-renewal of the distal epithelial progenitor cells, but that it also affects cell morphology and survival. The specific roles of other Bmp signaling components have not been determined. Tgfβ signaling has been implicated in lung epithelial progenitor proliferation in vitro, perhaps acting antagonistically to FGF10 in the multipotent progenitors through effects on Pten transcription (Xing et al., 2008). The lung epithelial-specific disruption of individual transforming growth factor-beta (TGFβ) superfamily ligands, receptors and signaling components is in process, but specific effects on progenitor populations have yet to be identified (for example, Chen et al., 2005). Deletion of Pten throughout the developing lung epithelium led to epithelial hyperplasia and changes in cell fate allocation (Tiozzo et al., 2009). However, Pten expression is widespread in the lung and it is not clear in which specific embryonic progenitor populations its function is required.
The phenotypes of transgenic overexpression or targeted deletion of the miR17-92 microRNA cluster are consistent with a normal role in promoting self-renewal, and inhibiting differentiation, of the multipotent lung epithelial progenitors (Lu et al., 2007; Ventura et al., 2008). Direct targets of the miR17 family in the lung include Rbl2, Mapk14, and Stat3 mRNAs (Lu et al., 2007; Carraro et al., 2009).
Mesenchymal Progenitor Regulation
Specific transcription factors expressed in the different mesenchymal progenitor cell populations have not been identified, even for the well-established parabronchial smooth muscle progenitors. Nevertheless, tremendous progress has been made in identifying molecules which regulate overall lung embryonic mesenchymal proliferation, rather than specific progenitor populations. FGF9 is highly expressed in both the mesothelium and the distal endoderm during the pseudoglandular stage of lung development. Fgf9 null lungs have a smaller mesenchymal compartment than wild-type and, as a consequence, branching morphogenesis is reduced (Colvin et al., 2001). This initial observation has lead to the elucidation of a complex signaling loop, involving the FGF, Shh, and Wnt pathways, which regulates the proliferation and differentiation of mesenchymal progenitor cell populations in the pseudoglandular stage lung. The cross-talk between these pathways is in the process of being extensively characterized. However, the detailed consequences of signaling for specific mesenchymal progenitor populations continues to lag behind. It is clear that the different progenitor populations respond differently to the same signaling molecules, even though they are developmentally related and in close proximity. For example, an increase in the levels of FGF9 protein inhibits the development of bronchiolar smooth muscle, but not vascular smooth muscle (Weaver et al., 2003; White et al., 2006). The cell biology underlying these varying responses is likely to include, first, the different developmental histories of the progenitor populations (different transcription factors, signaling pathway components, epigenetic marks). Second, slight differences in progenitor location within the organ resulting in varying levels of signaling, or a different combinatorial pattern of signals.
Genetic evidence suggests that FGF9 is itself a mitogen for the subepithelial mesenchyme where it signals through Fgfr1 and Fgfr2. The submesothelial mesenchyme was absent in the Fgf9 null lungs and expanded when Fgf9 was overexpressed. Moreover, adding FGF9 to cultured lungs caused a preferential increase in submesothelial mesenchymal proliferation (White et al., 2006). By contrast, the effects on proliferation of the subepithelial mesenchyme in these conditions were much weaker and most likely mediated by Shh. Shh expression was decreased in the Fgf9 null lungs. Moreover, simultaneously adding FGF9 and the Shh pathway inhibitor cyclopamine to cultured lungs blocked the effects of FGF9 on the subepithelial mesenchyme (White et al., 2006). Consistent with a role for Shh in subepithelial mesenchymal proliferation, it is highly expressed in the distal epithelial endoderm budding tips and its receptor, Patched1 (Ptch1), is highly expressed throughout the distal mesenchyme (Weaver et al., 2003). In vitro and in vivo overexpression experiments also support the idea that Shh functions as a mitogen for the mesenchymal progenitors (Bellusci et al., 1997a; Weaver et al., 2003). There is evidence that TGFβ signaling negatively regulates the expression of Shh signaling components in the lung mesenchyme (Li et al., 2008).
Subsequent studies identified an FGF9–Wnt regulatory loop that also promotes mesenchymal proliferation. FGF9 in the mesothelium induces Wnt2a expression in the submesothelial mesenchyme. Blocking Wnt signaling throughout the mesenchyme and mesothelium by deleting a floxed β-catenin allele with Dermo1-Cre resulted in lower levels of proliferation, decreased mesenchymal levels of Fgfr1 and 2, and was sufficient to prevent the mesenchyme from responding to exogenous FGF9 (De Langhe et al., 2008; Yin et al., 2008). The model suggested by Yin et al. is that Wnt2 (downstream of FGF9 and previously known as Wnt2a) functions to mediate submesothelial mesenchyme proliferation. Whereas, Wnt7b (which is expressed independently of FGF9) in the distal epithelium acts on the subepithelial mesenchyme (Shu et al., 2002; Rajagopal et al., 2008; Yin et al., 2008). Loss of function studies have also shown that Wnt7b signaling from the epithelium by means of the canonical β-catenin–mediated pathway is required for smooth muscle precursor cell proliferation (Cohen et al., 2009).
FGF10+ cells in the distal mesenchyme at the pseudoglandular stage are parabronchial smooth muscle progenitors and moreover, Fgf10 hypomorphic lungs have a smaller number of smooth muscle cells (Mailleux et al., 2005). However, the hypomorphs do not display changes in proliferation or apoptosis of the mesenchymal progenitors and FGF10 elicited no direct response (neither activation of Erk or Akt) on primary lung mesenchymal cells (Mailleux et al., 2005). These data suggest that, unlike the epithelial progenitors, the function of FGF10 is not to promote progenitor self-renewal, rather it is required for smooth muscle specification. Similarly, no requirement for canonical Notch signaling in proliferation of the mesenchymal progenitor populations has been identified (Morimoto et al., 2010). The roles of other pathways, including Egfr, Bmp, cytokine, in the different populations of lung mesenchymal progenitors remain to be tested.
Vascular Progenitor Regulation
In addition to their roles in mesenchymal proliferation and differentiation, FGF9 and Shh also affect vascular development in the lung. Fgf9 null lungs have a smaller capillary network around the budding epithelial tips at E11.5. This could be partially phenocopied by deletion of Fgfr1 and Fgfr2 throughout the developing mesenchyme and mesothelium, but not by endothelial-specific Fgfr deletion (White et al., 2007). The effects of FGF9 on the developing vasculature are therefore indirect and the authors show that it most likely regulates mesenchymal Vegfa expression. Loss of Shh in the lung epithelium resulted in a simplification of the lung vascular network (Miller et al., 2004). Similarly, lung-specific deletion of the Shh receptor, Smoothened, also resulted in a decreased vascular network (White et al., 2007). However, Shh and FGF9 were unable to compensate for one another in vitro, suggesting that they act independently in the embryonic lung mesenchyme to regulate vascular development (White et al., 2007).