162
H. A. Marquez and F. Chen
Aldh1a2
−/− embryos show Nkx2.1 expression at the posterior ventral foregut, indicating that RA is not required for lung specification. However, these embryos do not
form lung buds both in vivo or in foregut explant culture (Desai et al. 2006). Culturing foregut explants from Aldh1a2
−/− mutants in RA- or RARβ agonist-containing
medium rescues primary lung budding (Desai et al. 2006). Together, the data suggest
that RA signaling is necessary for the initiation of bud morphogenesis.
During branching morphogenesis, RA is still synthesized and active in specific
regions of the mouse lung as demonstrated by the ongoing expression of Aldh1a1 in
the proximal epithelium, Aldh1a2 in the pleura, and RARE-lacZ expression in both
(Chazaud et al. 2003; Malpel et al. 2000). The purpose of ongoing RAR activity in the
proximal epithelium may be to stabilize the airway by preventing ectopic branching
(Chazaud et al. 2003). In contrast, later, in the distal epithelium, RA signaling is
downregulated for proper lung growth and patterning (Chazaud et al. 2003; Malpel
et al. 2000). Indeed, treatment of embryonic lung explants with high concentrations
of RA inhibits branching and blocks the formation of distal epithelial structures
(Malpel et al. 2000).
Targeting RARs
In an embryonic lung undergoing branching morphogenesis (pseudoglandular
phase), Rarβ is expressed specifically within the lung proximal airway epithelium
and the subepithelial mesenchyme, whereas Rarγ is expressed within the distal
bud epithelium and tracheal mesenchyme. Rarα, on the other hand, is ubiquitously
expressed in all cell types in the lung during branching morphogenesis (Chazaud
et al. 2003). To investigate the function of the different isoforms of RARs, both pharmacologic approaches (that utilize pan-RAR and RAR isoform-specific agonists and
antagonists) and genetic approaches (knockout or overexpression of RAR genes or
tissue-selective expression of a dominant negative RAR) have been used.
Culture of E8.0 mouse embryos (36 h before lung bud morphogenesis) or E8.5
mouse foregut explants in BMS493, a pan-RAR inverse agonist, for 48 h, resulted
in lung hypoplasia or complete abrogation of primary lung bud formation (Desai
et al. 2004; Mollard et al. 2000), confirming the indispensability of RA signaling for
primary lung bud formation. Interestingly, RAR inhibition by BMS493 at E8.5 does
not prevent lung specification, as Nkx2.1 is still expressed in the presumptive lung
field of the foregut endoderm. However, continued treatment of the mouse foregut
explant with BMS493 eventually leads to the disappearance of the Nkx2.1
+ cells
in the lung field. These data suggest that RA, though not required for specification
of the lung, is important for lung progenitor survival. Moreover, the effect of RAR
inhibition on primary lung bud morphogenesis appears to be limited to a narrow
temporal window, as treatment of E8.5 foregut explants with BMS493 beyond the
15-somite stage does not block primary lung bud formation (Desai et al. 2004).
At the pseudoglandular, phase, RA inhibits distal bud formation and instead,
promotes formation of conducting airways. This has been demonstrated by treating
H. A. Marquez and F. Chen
Aldh1a2
−/− embryos show Nkx2.1 expression at the posterior ventral foregut, indicating that RA is not required for lung specification. However, these embryos do not
form lung buds both in vivo or in foregut explant culture (Desai et al. 2006). Culturing foregut explants from Aldh1a2
−/− mutants in RA- or RARβ agonist-containing
medium rescues primary lung budding (Desai et al. 2006). Together, the data suggest
that RA signaling is necessary for the initiation of bud morphogenesis.
During branching morphogenesis, RA is still synthesized and active in specific
regions of the mouse lung as demonstrated by the ongoing expression of Aldh1a1 in
the proximal epithelium, Aldh1a2 in the pleura, and RARE-lacZ expression in both
(Chazaud et al. 2003; Malpel et al. 2000). The purpose of ongoing RAR activity in the
proximal epithelium may be to stabilize the airway by preventing ectopic branching
(Chazaud et al. 2003). In contrast, later, in the distal epithelium, RA signaling is
downregulated for proper lung growth and patterning (Chazaud et al. 2003; Malpel
et al. 2000). Indeed, treatment of embryonic lung explants with high concentrations
of RA inhibits branching and blocks the formation of distal epithelial structures
(Malpel et al. 2000).
Targeting RARs
In an embryonic lung undergoing branching morphogenesis (pseudoglandular
phase), Rarβ is expressed specifically within the lung proximal airway epithelium
and the subepithelial mesenchyme, whereas Rarγ is expressed within the distal
bud epithelium and tracheal mesenchyme. Rarα, on the other hand, is ubiquitously
expressed in all cell types in the lung during branching morphogenesis (Chazaud
et al. 2003). To investigate the function of the different isoforms of RARs, both pharmacologic approaches (that utilize pan-RAR and RAR isoform-specific agonists and
antagonists) and genetic approaches (knockout or overexpression of RAR genes or
tissue-selective expression of a dominant negative RAR) have been used.
Culture of E8.0 mouse embryos (36 h before lung bud morphogenesis) or E8.5
mouse foregut explants in BMS493, a pan-RAR inverse agonist, for 48 h, resulted
in lung hypoplasia or complete abrogation of primary lung bud formation (Desai
et al. 2004; Mollard et al. 2000), confirming the indispensability of RA signaling for
primary lung bud formation. Interestingly, RAR inhibition by BMS493 at E8.5 does
not prevent lung specification, as Nkx2.1 is still expressed in the presumptive lung
field of the foregut endoderm. However, continued treatment of the mouse foregut
explant with BMS493 eventually leads to the disappearance of the Nkx2.1
+ cells
in the lung field. These data suggest that RA, though not required for specification
of the lung, is important for lung progenitor survival. Moreover, the effect of RAR
inhibition on primary lung bud morphogenesis appears to be limited to a narrow
temporal window, as treatment of E8.5 foregut explants with BMS493 beyond the
15-somite stage does not block primary lung bud formation (Desai et al. 2004).
At the pseudoglandular, phase, RA inhibits distal bud formation and instead,
promotes formation of conducting airways. This has been demonstrated by treating