The large FGF family plays an important role in the regulation of cell differentiation, proliferation and development, including lung branching [50]. Several studies have identified FGFs implicated in the bidirectional signaling between epithelium and mesenchyme during lung development [44,50,51]. For example, FGF10 is expressed in the distal submesothelial mesenchyme and activates FGF receptor 2b (FGFR2b) in the adjacent epithelial cells (Figure 3) to induce lung budding, epithelial cell expansion and migration, and ECM organization [52,53,54]. Indeed, in Fgf10^−/− mice, the lung does not develop below the trachea [55,56]. Moreover, Bellusci et al. have identified, in mice, a subtype of Axin2+/FGF10+-resident mesenchymal alveolar niche cells that are close to alveolar type 2 (AT2) stem cells and that control the proliferation and differentiation of AT2 cells [57].

FGF9 is another FGF family member that is expressed in the mesothelium from the pseudo-glandular stage (week 5 to 17) onwards, regulating the local activation of Wnt signaling to promote mesenchymal cell proliferation [35,44] (Figure 3). FGF9 also plays a critical role in lung development, as indicated by the finding that Fgf9^−/− mice die at the neonatal stage due to lung hypoplasia caused by very reduced mesenchymal cell expansion [58].

BMP4 belongs to the transforming growth factor superfamily. Bmp4^−/− mice die early during development, mainly due to an absence of mesoderm differentiation [59]. During lung development, BMP4 expression is detected in the distal epithelium buds and in the adjacent mesenchyme already at the pseudo-glandular stage (week 5 to 17) [60,61]. Conversely, BMP type I receptor (BMPR1) is expressed in both epithelium and mesenchyme (Figure 2B) [62]. In association with sonic hedgehog (SHH), BMP4 antagonizes FGF10 that is expressed in the surrounding mesenchyme [63]. Conditional knock-out of Bmpr 1a, the gene encoding the BMP4 receptor in the epithelium, leads to abnormal lung development with reduced cell proliferation, increased apoptosis and abnormal lung morphogenesis. This indicates that BMP4 plays important roles in lung development [64]. Moreover [65], in cultured mouse embryonic lung, reduction of gremlin expression, a BMP4 antagonist, using antisense oligonucleotides promotes epithelial cell proliferation and abnormal lung formation/function. Finally, BMP4 overexpression in the distal bud tips leads to lung hypoplasia, reduction of AT2 cells, and enlargement of the terminal buds [62]. However, the exact role of this signaling pathway during lung development remains debated, because mathematical models to mimic the FGF10-SHH interaction accurately model bronchial branching independently of BMP4 expression [66,67].

SHH also is part of a key developmental signaling pathway. It is implicated in central nervous system patterning, and limb, digit and lung development [68]. In Shh^−/− mice, a single lobe, lung hypoplasia with absence of left and right asymmetry, enhanced cell death and decreased lung mesenchymal cell proliferation are observed [69]. SHH is expressed with BMP4 in the distal bud epithelium during lung development. It binds to and activates its receptor Patched 1 (PTCH1) that is localized in the adjacent mesenchyme. Patched 1 activation downregulates FGF10 expression [44,52,70]. Indeed, in Shh^−/− mice, FGF10 expression in the mesenchyme immediately adjacent to the epithelium is increased [71]. In addition, BMP4 is overexpressed and wingless-related integration site family member 2 (WNT2) is downregulated in the mesenchyme. In 2013, Peng et al. [72] identified a cardiopulmonary mesoderm progenitor population that is defined by the expression of WNT2, glioma-associated oncogene 1 (Gli1) and Islet 1, and gives rise to the lung mesenchyme and cardiac lineage in the mouse. This population is regulated by Hedgehog signaling because Gli proteins are the main transcriptional effectors of this pathway, and start to be expressed at the lung organogenesis step [72]. This suggests that SHH is broadly involved in mesenchymal signaling in the developing lung.

EGF and its tyrosine kinase receptor EGF-R are expressed in the epithelial and mesenchymal compartments during lung development. EGF stimulates lung branching in fetal mice [40]. In agreement, in Egfr^−/− mice, neonatal lethality is high and epithelial cell development is impaired in several organs, including the lung [73]. Importantly, lung branching is reduced and alveolarization and septation are deficient in Egfr^−/− mice [74]. Similarly, mouse lung branching can be inhibited in ex-vivo cultures using antisense oligonucleotides against EGF [40,75]. Furthermore, the interplay between retinoid acid (RA) and EGFR during fetal lung development stimulates lung branching [76].

RA is essential for normal embryo development, including lung development [77]. In the mouse, retinoic acid receptor (RAR) double knock-out (RARα-/-/RARβ2-/-) leads to agenesis of the left lung and hypoplasia of the right lung [78]. RA is produced by the mesenchyme surrounding the lung primordia, and also by the epithelial compartment of the proximal bronchi [79]. Importantly, a RA-transforming growth factor (TGF)-β-FGF10 interaction has been described during lung bud induction, where RA downregulates TGF-β to allow FGF10 expression [80].

TGF-β, a pleiotropic growth factor and a key EMT inducer, is another modulator of epithelium–mesenchyme interactions in the developing lung [81]. Its three isoforms play a crucial role during lung organogenesis [82]. Their expression is well characterized in the mouse where TGF-β1 is expressed in the mesenchyme, TGF-β2 in the distal epithelium, and TGF-β3 in the proximal mesenchyme and pulmonary mesothelium [83,84]. In Tgf-β2^−/− mice, the distal airways are collapsed and the proximal airways are dilated [82], whereas in Tgf-β3^−/− mice lung development is delayed, causing their death [85]. Conversely, Tgfr-β2 ablation in mesodermal tissue results in abnormal lung branching and lung development, while its ablation in epithelial cells that produce surfactant protein C results in a decrease of alveolar type I (AT1) epithelial cells during post-natal alveolarization [86]. TGF-β also plays a role in FGF10 regulation by RA [80] as illustrated by the finding that ectopic TGF-β1 expression inhibits FGF10-induced lung morphogenesis in cultured embryonic lung endodermal explants [87].

The Hippo pathway and its downstream targets Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) play major roles in tissue homeostasis, organ development, and organ size [88,89] by regulating various processes, such as cell proliferation/survival, the response to mechanical stress and cell geometry. This pathway involves a kinase cascade and adaptors that ultimately regulate YAP/TAZ activities. The name comes from the Drosophila kinase Hippo, the mammalian orthologs of which are the kinases MST1/2. Upon activation of the Hippo signaling pathway, MST1/2 phosphorylates LATS1/2, thereby activate these kinases that in turn phosphorylate YAP and TAZ. Phosphorylated YAP and TAZ are retained in the cytoplasm or degraded by the ubiquitin system. Conversely, when the Hippo pathway is inactive, YAP and TAZ shuttle to the nucleus where they bind to TEA domain (TEAD) transcription factors and regulates the transcription of many downstream genes [89]. Taz knockout mice show abnormal alveolarization, leading to airway enlargement that mimics human emphysema [90,91]. Conditional Yap knockout in lung epithelium causes disruption of bronchial morphogenesis [91]. During alveologenesis, YAP/TAZ inactivation by cell crowding orients NKX2.1 activity towards AT2 cells, a mechanism thought to be a negative feedback to limit AT1 cell expansion [92]. In bronchia, YAP is activated in distal airways, and its induction prevents multi-ciliated cell differentiation [93]. These observations suggest a general role of YAP/TAZ in favoring the stem cell compartment at the expenses of terminal differentiation. In line, YAP overexpression in adult tracheal cells results in basal cell hyperplasia and stratification [94]. Interestingly, injury in airway epithelial cells leads to downregulation of hippo signaling that increases the concentration of nuclear YAP [38], and induces FGF10 secretion by the adjacent mesenchymal cells [95]. Moreover, in the absence of Yap, epithelial progenitors cannot respond to local TGF-β signaling [93]. Overall, the Hippo pathway plays a critical role in lung development and response to injury directly in epithelial cells or indirectly through epithelium/mesenchyme signaling.

Other factors also are involved in lung development, such as components of the vascular endothelial growth factor (VEGF) and WNT signaling pathways. Their expression is highly regulated in space and time, allowing optimal lung development, at least partly through epithelium-mesenchyme interactions. Single-cell transcriptomic analyses will help to identify all the factors involved in lung development.