6 Retinoic Acid Signaling and Development of the Respiratory System
163
E11.75 and E12.5 lung explants with BMS493 which downregulates RA activity
and stimulates branching and the formation of distal buds. Treatment of E11.75 and
E12.5 lung explants with exogenous RA, on the other hand, decreases branching
morphogenesis (Mollard et al. 2000). These data suggest that RA stabilizes the
proximal respiratory tubules.
In concurrence with this result, ligand-independent constitutive activation of
RARα (RARα-VP16) under the control of the lung epitheium-specific Sftpc promoter in vivo results in lungs that arrest in the pseudoglandular phase. These lungs
do not form saccules or type I alveolar cells nor do they express late distal lung
markers, such as surfactant proteins B or C. Down-regulation of RARα signaling,
therefore, is required to allow for the differentiation of the distal lung at the later
phases of lung development (Wongtrakool et al. 2003).
Interestingly, knocking out individual RARs in mice does not lead to significant
defects in the formation of the respiratory tract. This is believed to be due to the
functional redundancy of RAR isotypes (Mendelsohn et al. 1994). However, some
RAR isotype-specific effects are observed later during the formation of alveoli that
continues after birth in mice. For example, Rarα
−/− mouse lung development is
similar to controls at PN14 (toward the end of alveolar stage), but at PN50, the
number of alveoli and the alveolar surface area in Rarα-deficient lung are reduced
compared to the control lungs, suggesting that RARα has a role in maintaining
alveolar integrity (Massaro et al. 2003).
The role of RARβ on alveoli formation is less clear, as genetic deletion of Rarβ
yielded conflicting data on lung septation and alveolarization. One study showed that
Rarβ
−/− animals septate earlier and faster than controls (Massaro et al. 2000), while
analysis of a different Rarβ—null mutant revealed a reduction in the gas-exchange
surface area per lung volume (Snyder et al. 2005).
In compound mutants, in which one allele of Rarγ was deleted, the whole lung
elastic tissue and the number of alveoli were decreased, while the alveolar space
was increased. Elastin transcription in lipofibroblasts was reduced at PN14 in these
mutants, indicating Rarγ is a positive regulator of alveolar septation (McGowan et al.
2000).
Double Rarα
−/− /Rarβ2
−/− mutants, on the other hand, exhibited dramatic phenotypes in the respiratory system early in its development, with the two lungs hypoplastic or one absent and the other one hypoplastic (Mendelsohn et al. 1994). These RAR
double mutants invariably died either in utero or shortly after birth and presented a
number of congenital abnormalities comparable to VAD embryos (Mendelsohn et al.
1994; Wilson et al. 1953).
To overcome the limitations of single RAR knockout (compensation by other
RARs) and double RAR knockout (premature death before onset of the alveolar phase
of lung development), Yang and colleagues developed mouse lines in which a dominant negative RAR was expressed under the control of lung epithelium-specific promoters in mice only when they are given the antibiotic doxycycline. The authors gave
the neonatal mice doxycycline to overexpress dominant negative RAR in the lung
epithelium between PN1 and PN21, a critical time period for alveoli formation. These
mice exhibited an emphysema-like phenotype characterized by increased airspaces,
163
E11.75 and E12.5 lung explants with BMS493 which downregulates RA activity
and stimulates branching and the formation of distal buds. Treatment of E11.75 and
E12.5 lung explants with exogenous RA, on the other hand, decreases branching
morphogenesis (Mollard et al. 2000). These data suggest that RA stabilizes the
proximal respiratory tubules.
In concurrence with this result, ligand-independent constitutive activation of
RARα (RARα-VP16) under the control of the lung epitheium-specific Sftpc promoter in vivo results in lungs that arrest in the pseudoglandular phase. These lungs
do not form saccules or type I alveolar cells nor do they express late distal lung
markers, such as surfactant proteins B or C. Down-regulation of RARα signaling,
therefore, is required to allow for the differentiation of the distal lung at the later
phases of lung development (Wongtrakool et al. 2003).
Interestingly, knocking out individual RARs in mice does not lead to significant
defects in the formation of the respiratory tract. This is believed to be due to the
functional redundancy of RAR isotypes (Mendelsohn et al. 1994). However, some
RAR isotype-specific effects are observed later during the formation of alveoli that
continues after birth in mice. For example, Rarα
−/− mouse lung development is
similar to controls at PN14 (toward the end of alveolar stage), but at PN50, the
number of alveoli and the alveolar surface area in Rarα-deficient lung are reduced
compared to the control lungs, suggesting that RARα has a role in maintaining
alveolar integrity (Massaro et al. 2003).
The role of RARβ on alveoli formation is less clear, as genetic deletion of Rarβ
yielded conflicting data on lung septation and alveolarization. One study showed that
Rarβ
−/− animals septate earlier and faster than controls (Massaro et al. 2000), while
analysis of a different Rarβ—null mutant revealed a reduction in the gas-exchange
surface area per lung volume (Snyder et al. 2005).
In compound mutants, in which one allele of Rarγ was deleted, the whole lung
elastic tissue and the number of alveoli were decreased, while the alveolar space
was increased. Elastin transcription in lipofibroblasts was reduced at PN14 in these
mutants, indicating Rarγ is a positive regulator of alveolar septation (McGowan et al.
2000).
Double Rarα
−/− /Rarβ2
−/− mutants, on the other hand, exhibited dramatic phenotypes in the respiratory system early in its development, with the two lungs hypoplastic or one absent and the other one hypoplastic (Mendelsohn et al. 1994). These RAR
double mutants invariably died either in utero or shortly after birth and presented a
number of congenital abnormalities comparable to VAD embryos (Mendelsohn et al.
1994; Wilson et al. 1953).
To overcome the limitations of single RAR knockout (compensation by other
RARs) and double RAR knockout (premature death before onset of the alveolar phase
of lung development), Yang and colleagues developed mouse lines in which a dominant negative RAR was expressed under the control of lung epithelium-specific promoters in mice only when they are given the antibiotic doxycycline. The authors gave
the neonatal mice doxycycline to overexpress dominant negative RAR in the lung
epithelium between PN1 and PN21, a critical time period for alveoli formation. These
mice exhibited an emphysema-like phenotype characterized by increased airspaces,