6 Retinoic Acid Signaling and Development of the Respiratory System
165
Current State of the Field
Historically, studies have focused on the analysis of the morphologic consequences
of targeting RA signaling. Now, most studies focus on determining how VA/RA
regulate lung development at the molecular level.
RA-Regulated Pathways During Lung Development
Several studies demonstrated that lung development requires complex pathways
(FGF, SHH, BMP, TGFβ, and WNT) that operate as a gene regulatory network
(Bellusci et al. 1997; Desai et al. 2004; Domyan et al. 2011; Goss et al. 2009; HarrisJohnson et al. 2009; Lebeche et al. 1999; Rankin et al. 2016). These pathways are
regulated by RA at different phases of lung development, pointing to the role of RA
as a regulator of key signals that drive lung formation and differentiation
FGF10 is a growth factor critical for the induction of lung bud outgrowth (Bellusci
et al. 1997; Desai et al. 2004, 2006). Indeed, FGF10 is active in isolated lung mesenchymal cells in culture (Lebeche et al. 1999). However, in Fgf10-null mice, lung
progenitor cells are specified but cannot expand to undergo primary bud morphogenesis (Min et al. 1998; Sekine et al. 1999). Several studies using a combination of
genetic, pharmacological and mouse explant culture approaches demonstrated that
RA regulates Fgf10 expression at the time of primary lung bud formation (Desai
et al. 2004, 2006). Specifically, activation of RARβ using synthetic agonist induces
the expression of Fgf10 in the lung field and primary bud morphogenesis (Desai
et al. 2006). Finally replacing FGF10 in RA-deficient foregut ex vivo restored lung
budding. Note that although RA is required for FGF10 induction during the embryonic phase, diffuse RA activation appears to block FGF10 expression in the lung
and prevents normal branching during the pseudoglandular stage (Bellusci et al.
1997; Weaver et al. 1999). This is consistent with other experiments showing that
down-regulation of RA activity in the distal lung is required for normal branching.
Later, Cardoso’s laboratory (Chen et al. 2007) performed a global gene expression analysis of E8.5 foregut explants from wild-type mice cultured with or without
BMS493 and E8.5 foregut explants from Aldh1a2
−/− mice cultured with or without
RA. In RA deficient foreguts, they found upregulation of a large number of TGFβ
targets simultaneous with downregulation of Fgf10. Interestingly, preventing activation of endogenous TGFβ signaling with a pan-specific TGFβ-blocking antibody
restored bud formation and FGF10 expression. In contrast, hyperactivating TGFβ signaling with exogenous TGFβ1 in RA-sufficient foreguts reproduced the lung defects
observed upon RA deficiency. Moreover, RA treatment of the RA deficient foregut
cultures down regulated TGFβ and its targets. All these data supported a novel mechanism of RA-TGFβ-FGF10 interactions in the developing foregut, in which endogenous RA maintains low levels of TGFβ activity to allow FGF10 expression and
induction of lung buds.
165
Current State of the Field
Historically, studies have focused on the analysis of the morphologic consequences
of targeting RA signaling. Now, most studies focus on determining how VA/RA
regulate lung development at the molecular level.
RA-Regulated Pathways During Lung Development
Several studies demonstrated that lung development requires complex pathways
(FGF, SHH, BMP, TGFβ, and WNT) that operate as a gene regulatory network
(Bellusci et al. 1997; Desai et al. 2004; Domyan et al. 2011; Goss et al. 2009; HarrisJohnson et al. 2009; Lebeche et al. 1999; Rankin et al. 2016). These pathways are
regulated by RA at different phases of lung development, pointing to the role of RA
as a regulator of key signals that drive lung formation and differentiation
FGF10 is a growth factor critical for the induction of lung bud outgrowth (Bellusci
et al. 1997; Desai et al. 2004, 2006). Indeed, FGF10 is active in isolated lung mesenchymal cells in culture (Lebeche et al. 1999). However, in Fgf10-null mice, lung
progenitor cells are specified but cannot expand to undergo primary bud morphogenesis (Min et al. 1998; Sekine et al. 1999). Several studies using a combination of
genetic, pharmacological and mouse explant culture approaches demonstrated that
RA regulates Fgf10 expression at the time of primary lung bud formation (Desai
et al. 2004, 2006). Specifically, activation of RARβ using synthetic agonist induces
the expression of Fgf10 in the lung field and primary bud morphogenesis (Desai
et al. 2006). Finally replacing FGF10 in RA-deficient foregut ex vivo restored lung
budding. Note that although RA is required for FGF10 induction during the embryonic phase, diffuse RA activation appears to block FGF10 expression in the lung
and prevents normal branching during the pseudoglandular stage (Bellusci et al.
1997; Weaver et al. 1999). This is consistent with other experiments showing that
down-regulation of RA activity in the distal lung is required for normal branching.
Later, Cardoso’s laboratory (Chen et al. 2007) performed a global gene expression analysis of E8.5 foregut explants from wild-type mice cultured with or without
BMS493 and E8.5 foregut explants from Aldh1a2
−/− mice cultured with or without
RA. In RA deficient foreguts, they found upregulation of a large number of TGFβ
targets simultaneous with downregulation of Fgf10. Interestingly, preventing activation of endogenous TGFβ signaling with a pan-specific TGFβ-blocking antibody
restored bud formation and FGF10 expression. In contrast, hyperactivating TGFβ signaling with exogenous TGFβ1 in RA-sufficient foreguts reproduced the lung defects
observed upon RA deficiency. Moreover, RA treatment of the RA deficient foregut
cultures down regulated TGFβ and its targets. All these data supported a novel mechanism of RA-TGFβ-FGF10 interactions in the developing foregut, in which endogenous RA maintains low levels of TGFβ activity to allow FGF10 expression and
induction of lung buds.