BACKGROUND
organogenesis in the lung requires the sequential development of the large airways, bronchioles and alveoli.
the lung begins as a simple structure with increasing complexity that is necessary for efficient air exchange at maturity.
recently, several laboratories have demonstrated that this complex process involves the promotion of mechanical stretch  <cit> .
specifically, muscle contractions of the large airways compress amniotic fluid in the fetal lung generating a pressure gradient that is converted to biochemical signals necessary for cell differentiation.
mechanical force modulation of biochemical processes is a well known phenomenon that is mediated by multimeric proteins  <cit> .
mechanicosensing and the genes involved, however, are less well known.
sensing molecules such as integrins, ion channels, and kinase-linked receptors have been implicated in changes in gene expression related to stretch  <cit> .
most of the evidence from these studies implicates vascular responses to stretch in hemodynamic stasis, although little is known about these sensors in fetal organogenesis.
recently, control of muscle contractions via the rho kinase-dependent and independent pathways was shown to be important in lung development  <cit> .
in particular, the cystic fibrosis transmembrane conductance regulator  was found to influence muscle contractions in lung development via a rho kinase-independent mechanism involving atp and calcium  <cit> .
in other work, we and others demonstrated that the canonical wnt/β-catenin pathway plays crucial roles in lung organogenesis  <cit> .
these studies utilized our laboratory's transient in utero gene transfer technique  <cit>  with sense and antisense constructs to modulate gene expression  <cit> .
this technique has the advantage in lung development studies by transiently targeting the developing respiratory epithelium for temporal modulation of gene expression without any inflammatory consequences and no premature expression leakage.
in the study reported here the role of cftr in downstream regulation of lung differentiation effectors was examined.
specifically, the relationship between cftr function and wnt/β-catenin signaling in lung development was examined.
previous studies have suggested that wnt signaling via a stretch-induced mechanism involving parathyroid hormone-related peptide  is required for normal lung development  <cit> .
therefore, we used our experimental system to determine if altering cftr expression has downstream effects on wnt and pthrp which would provide further evidence of cftr involvement in mechanicosensing necessary for normal lung development.
RESULTS
cftr influences wnt/β-catenin signaling
bat-gal reporter mice express β-galactosidase under the control of wnt//β-catenin-responsive elements  <cit> .
to evaluate the role of cftr in modulating the wnt signaling cascade, fetuses from time pregnant bat-gal mice were injected at e <dig>  e <dig>  and e <dig> via the amniotic fluid using our established techniques  <cit> .
recombinant adenoviruses with either egfp , cftr , or antisense cftr , and β-galactosidase enzyme and total protein assays performed and the enzymatic activity was expressed as units/μg protein.
bat-gal expression during normal lung development was found to be highly regulated.
at e <dig> little if any wnt/β-catenin-dependent β-galactosidase activity was detected in control tissues .
enzyme activity peaked at e <dig> and then decreased by e <dig>  cftr over expression resulted in a small but significant  increase in bat-gal expression at e <dig>  at e <dig> there was a non-significant decrease in activity in the cftr-treated animals, and by e <dig> there was no difference between control and cftr-treated animals.
in contrast, antisense knockdown of cftr showed highly significant  increases in bat-gal activity at e <dig>  e <dig>  and e <dig> when compared to either control or cftr-treated animals consistent with a relationship between cftr expression and canonical wnt signaling between e16–e <dig> in the mouse lung.
the distribution by fluorescent immunohistochemistry of β-galactosidase expression was significantly different in fetuses treated at e <dig> and examined at e <dig> , adcmvegfp, control, lungs showed little or no expression as would be expected from the enzyme assay data presented in fig.
<dig>  antisense cftr treated lungs, which had the largest increase in β-galactosidase enzyme activity, exhibited expression in randomly distributed individual cells .
in contrast, cftr over expression, which showed a smaller but significant increase in enzyme activity, exhibited increased clusters of β-galactosidase expressing cells .
in fetuses treated at e <dig> and examined at e <dig>  normal, control, and cftr over expression , β-galactosidase was seen around small airways with some expression in the parenchyma consistent with cftr accelerating the normal developmental wnt/β-catenin signaling.
in contrast, inhibition of cftr expression at e <dig> resulted in further expansion of β-galactosidase positive cell populations to the parenchyma .
thus, consistent with the results of fig.
<dig>  wnt/β-catenin signaling patterns are dependent upon cftr expression levels.
over expression facilitated the normal pattern for wnt/β-catenin and antisense inhibition of cftr resulted in an abnormal increase and distribution in foci of β-galactosidase positive cells.
developmental programming of pthrp expression by cftr
based on the previously demonstrated relationship between mechanical stretch, wnt signaling and pthrp expression  <cit>  and our previous work on cftr that has shown that it modulates stretch to influence gene regulation in the developing lung  <cit> , we investigated the relationship between cftr-dependent stretch and pthrp levels.
immunohistochemistry was used to visualize pthrp expression at e <dig>  the time when cftr expression had the greatest effect on bat-gal reporter expression .
in control animals low levels of pthrp were detected in the developing parenchyma .
in marked contrast, down-regulation of cftr with the antisense construct resulted in a dramatic decrease in pthrp immunostaining , whereas cftr over expression  resulted in a significant increase in pthrp expression in comparison to both control- and antisense cftr-treated lungs .
quantitative evaluation of pthrp showed statistically significant differences between control-, antisense cftr-, and cftr-treated lungs .
thus, there was a direct correlation between cftr expression and pthrp levels in the developing lung at e <dig>
discussion
cftr is expressed at high levels during specific periods of lung organogenesis and expression subsequently decreases by  <dig> fold as the lung matures  <cit> .
this pattern of expression suggests a role for this gene in normal lung development.
the finding that cftr has a role in mechanical stretch which is necessary for lung organogenesis  <cit>  provides a possible mechanism for this gene in lung development.
specifically, our findings of a direct correlation between cftr expression and pthrp levels  in developing lung and the inverse correlation with wnt/β-catenin signaling  are consistent with the mechanical stretch-dependent development of surfactant production <cit> .
these data collectively reveal a possible mechanism for how a chloride channel, cftr, could have a global effect on lung development.
by directly effecting mechanical stretch necessary for generating lung complexity this chloride channel could have an essential role in normal lung organogenesis.
in the normal bat-gal mouse lung wnt expression peaked at a specific time  in lung development that may be defined by the total induced stretch .
if cftr increases stretch as shown previously  <cit>  and illustrated in fig.
<dig>  then one would predict that the slope of the change in stretch would be increased.
thus, wnt expression would peak earlier for a shorter time and lower level.
antisense cftr, however, would lower the slope of the stretch effect, prolonging wnt activation and increasing its activity.
thus, the changes observed in fig.
<dig> can be explained by a change in cftr-dependent, stretch-induced regulation of wnt.
this interpretation is consistent with the previous work by the torday and rehan group showing the relationship between stretch and wnt  <cit>
our previous studies examining the effects of in utero cftr gene transfer on lung growth and development showed that over expression of cftr resulted in increased bronchial cell differentiation and proliferation, decreased alveolar complexity, and increased stretch  <cit> .
in this paper we demonstrated that cftr over expression increases pthrp ; and decreased cftr expression dramatically increased wnt signaling .
if one examines all these cftr-dependent effects both previously published  <cit>  and current data provided here, cftr, a chloride channel, has many, seemingly independent, affects on lung organogenesis.
however, stretch was previously shown by torday and rehan  <cit>  to correlate directly with pthrp which controls a cascade in which lower pthrp  increases wnt activation that leads to increased bronchial cell differentiation and proliferation and decreased alveolar complexity.
thus, it is possible to correlate all the effects seen independently by altering cftr levels in the fetal lung with a direct effect of cftr on stretch that then modifies the torday-rehan pthrp-wnt pathway for terminal differentiation of the lung.
initially, mechanical stretch promotes proximal airway development but with growth, distal airway differentiation results in decreased pressure and wnt-dependent alveolar differentiation and complexity.
our results correlated with those of torday and rehan  <cit>  suggest that cftr could play a central role in this process along with other genes that affect mechanical stretch including rho kinase  <cit>  and enac  <cit> .
delineation of the exact role of cftr in this process requires further investigation.
however, several recent findings may suggest a mechanism.
treharne et al.
<cit>  recently showed that cftr function was linked to casein kinase  <dig>  a rather promiscuous protein kinase.
in addition, cftr function is known to be associated with actin filaments  <cit>  and recent work has shown that a camp/protein kinase a-dependent annexin  <dig> s100a <dig> complex with cftr also affects its function  <cit> .
these observations provide increasing evidence that the cftr gene product plays important roles beyond that of a chloride channel and that it is part of a complex of molecules within the cytoskeleton of the cell that have a high potential for kinase-mediated gene regulation.
therefore, we speculate that stretch of the cytoskeleton could be the signal to modulate cftr function and mechanical stretch.
thus, cftr could function as a mechanicosensor essential for lung organogenesis.
CONCLUSIONS
during late lung development cftr probably acting through a mechanicosensory pathway regulates wnt/β-catenin signaling.

 organogenesis in the lung requires the sequential development of the large airways, bronchioles and alveoli. the lung begins as a simple structure with increasing complexity that is necessary for efficient air exchange at maturity. recently, several laboratories have demonstrated that this complex process involves the promotion of mechanical stretch. specifically, muscle contractions of the large airways compress amniotic fluid in the fetal lung generating a pressure gradient that is converted to biochemical signals necessary for cell differentiation. mechanical force modulation of biochemical processes is a well known phenomenon that is mediated by multimeric proteins. mechanicosensing and the genes involved, however, are less well known. sensing molecules such as integrins, ion channels, and kinase-linked receptors have been implicated in changes in gene expression related to stretch. most of the evidence from these studies implicates vascular responses to stretch in hemodynamic stasis, although little is known about these sensors in fetal organogenesis. recently, control of muscle contractions via the rho kinase-dependent and independent pathways was shown to be important in lung development. in particular, the cystic fibrosis transmembrane conductance regulator was found to influence muscle contractions in lung development via a rho kinase-independent mechanism involving atp and calcium. in other work, we and others demonstrated that the canonical wnt/β-catenin pathway plays crucial roles in lung organogenesis. these studies utilized our laboratory's transient in utero gene transfer technique with sense and antisense constructs to modulate gene expression. this technique has the advantage in lung development studies by transiently targeting the developing respiratory epithelium for temporal modulation of gene expression without any inflammatory consequences and no premature expression leakage. in the study reported here the role of cftr in downstream regulation of lung differentiation effectors was examined. specifically, the relationship between cftr function and wnt/β-catenin signaling in lung development was examined. previous studies have suggested that wnt signaling via a stretch-induced mechanism involving parathyroid hormone-related peptide is required for normal lung development. therefore, we used our experimental system to determine if altering cftr expression has downstream effects on wnt and pthrp which would provide further evidence of cftr involvement in mechanicosensing necessary for normal lung development.  cftr influences wnt/β-catenin signaling bat-gal reporter mice express β-galactosidase under the control of wnt//β-catenin-responsive elements. to evaluate the role of cftr in modulating the wnt signaling cascade, fetuses from time pregnant bat-gal mice were injected at e e and e via the amniotic fluid using our established techniques. recombinant adenoviruses with either egfp, cftr, or antisense cftr, and β-galactosidase enzyme and total protein assays performed and the enzymatic activity was expressed as units/μg protein. bat-gal expression during normal lung development was found to be highly regulated. at e little if any wnt/β-catenin-dependent β-galactosidase activity was detected in control tissues. enzyme activity peaked at e and then decreased by e cftr over expression resulted in a small but significant increase in bat-gal expression at e at e there was a non-significant decrease in activity in the cftr-treated animals, and by e there was no difference between control and cftr-treated animals. in contrast, antisense knockdown of cftr showed highly significant increases in bat-gal activity at e e and e when compared to either control or cftr-treated animals consistent with a relationship between cftr expression and canonical wnt signaling between e16–e in the mouse lung. the distribution by fluorescent immunohistochemistry of β-galactosidase expression was significantly different in fetuses treated at e and examined at e, adcmvegfp, control, lungs showed little or no expression as would be expected from the enzyme assay data presented in fig. antisense cftr treated lungs, which had the largest increase in β-galactosidase enzyme activity, exhibited expression in randomly distributed individual cells. in contrast, cftr over expression, which showed a smaller but significant increase in enzyme activity, exhibited increased clusters of β-galactosidase expressing cells. in fetuses treated at e and examined at e normal, control, and cftr over expression, β-galactosidase was seen around small airways with some expression in the parenchyma consistent with cftr accelerating the normal developmental wnt/β-catenin signaling. in contrast, inhibition of cftr expression at e resulted in further expansion of β-galactosidase positive cell populations to the parenchyma. thus, consistent with the results of fig. wnt/β-catenin signaling patterns are dependent upon cftr expression levels. over expression facilitated the normal pattern for wnt/β-catenin and antisense inhibition of cftr resulted in an abnormal increase and distribution in foci of β-galactosidase positive cells. developmental programming of pthrp expression by cftr based on the previously demonstrated relationship between mechanical stretch, wnt signaling and pthrp expression and our previous work on cftr that has shown that it modulates stretch to influence gene regulation in the developing lung, we investigated the relationship between cftr-dependent stretch and pthrp levels. immunohistochemistry was used to visualize pthrp expression at e the time when cftr expression had the greatest effect on bat-gal reporter expression. in control animals low levels of pthrp were detected in the developing parenchyma. in marked contrast, down-regulation of cftr with the antisense construct resulted in a dramatic decrease in pthrp immunostaining, whereas cftr over expression resulted in a significant increase in pthrp expression in comparison to both control- and antisense cftr-treated lungs. quantitative evaluation of pthrp showed statistically significant differences between control-, antisense cftr-, and cftr-treated lungs. thus, there was a direct correlation between cftr expression and pthrp levels in the developing lung at e discussion cftr is expressed at high levels during specific periods of lung organogenesis and expression subsequently decreases by fold as the lung matures. this pattern of expression suggests a role for this gene in normal lung development. the finding that cftr has a role in mechanical stretch which is necessary for lung organogenesis provides a possible mechanism for this gene in lung development. specifically, our findings of a direct correlation between cftr expression and pthrp levels in developing lung and the inverse correlation with wnt/β-catenin signaling are consistent with the mechanical stretch-dependent development of surfactant production. these data collectively reveal a possible mechanism for how a chloride channel, cftr, could have a global effect on lung development. by directly effecting mechanical stretch necessary for generating lung complexity this chloride channel could have an essential role in normal lung organogenesis. in the normal bat-gal mouse lung wnt expression peaked at a specific time in lung development that may be defined by the total induced stretch. if cftr increases stretch as shown previously and illustrated in fig. then one would predict that the slope of the change in stretch would be increased. thus, wnt expression would peak earlier for a shorter time and lower level. antisense cftr, however, would lower the slope of the stretch effect, prolonging wnt activation and increasing its activity. thus, the changes observed in fig. can be explained by a change in cftr-dependent, stretch-induced regulation of wnt. this interpretation is consistent with the previous work by the torday and rehan group showing the relationship between stretch and wnt our previous studies examining the effects of in utero cftr gene transfer on lung growth and development showed that over expression of cftr resulted in increased bronchial cell differentiation and proliferation, decreased alveolar complexity, and increased stretch. in this paper we demonstrated that cftr over expression increases pthrp; and decreased cftr expression dramatically increased wnt signaling. if one examines all these cftr-dependent effects both previously published and current data provided here, cftr, a chloride channel, has many, seemingly independent, affects on lung organogenesis. however, stretch was previously shown by torday and rehan to correlate directly with pthrp which controls a cascade in which lower pthrp increases wnt activation that leads to increased bronchial cell differentiation and proliferation and decreased alveolar complexity. thus, it is possible to correlate all the effects seen independently by altering cftr levels in the fetal lung with a direct effect of cftr on stretch that then modifies the torday-rehan pthrp-wnt pathway for terminal differentiation of the lung. initially, mechanical stretch promotes proximal airway development but with growth, distal airway differentiation results in decreased pressure and wnt-dependent alveolar differentiation and complexity. our results correlated with those of torday and rehan suggest that cftr could play a central role in this process along with other genes that affect mechanical stretch including rho kinase and enac. delineation of the exact role of cftr in this process requires further investigation. however, several recent findings may suggest a mechanism. treharne et al. recently showed that cftr function was linked to casein kinase a rather promiscuous protein kinase. in addition, cftr function is known to be associated with actin filaments and recent work has shown that a camp/protein kinase a-dependent annexin s100a complex with cftr also affects its function. these observations provide increasing evidence that the cftr gene product plays important roles beyond that of a chloride channel and that it is part of a complex of molecules within the cytoskeleton of the cell that have a high potential for kinase-mediated gene regulation. therefore, we speculate that stretch of the cytoskeleton could be the signal to modulate cftr function and mechanical stretch. thus, cftr could function as a mechanicosensor essential for lung organogenesis.  during late lung development cftr probably acting through a mechanicosensory pathway regulates wnt/β-catenin signaling. 
adenoviruses containing full-length cftr, a short anti-sense cftr gene fragment, or a reporter gene as control were used in an intra-amniotic gene therapy procedure to transiently modify cftr expression in the fetal lung. the mechanism for cftr modulation of lung development was examined by in utero gene transfer of either a sense or antisense construct to alter cftr expression levels. the bat-gal transgenic reporter mouse line, expressing β-galactosidase under a canonical wnt/β-catenin-responsive promoter, was used to assess the relative roles of cftr, wnt, and parathyroid hormone-related peptide in lung organogenesis. these data are consistent with cftr participating in the mechanicosensory process essential to regulate wnt/β-catenin signaling required for lung organogenesis. a direct correlation between cftr expression levels and pthrp levels was found. an inverse correlation between cftr and wnt signaling activities was demonstrated. cystic fibrosis transmembrane conductance regulator was shown previously to modify stretch induced differentiation in the lung.
 cystic fibrosis transmembrane conductance regulator was shown previously to modify stretch induced differentiation in the lung. the mechanism for cftr modulation of lung development was examined by in utero gene transfer of either a sense or antisense construct to alter cftr expression levels. the bat-gal transgenic reporter mouse line, expressing β-galactosidase under a canonical wnt/β-catenin-responsive promoter, was used to assess the relative roles of cftr, wnt, and parathyroid hormone-related peptide in lung organogenesis. adenoviruses containing full-length cftr, a short anti-sense cftr gene fragment, or a reporter gene as control were used in an intra-amniotic gene therapy procedure to transiently modify cftr expression in the fetal lung.  a direct correlation between cftr expression levels and pthrp levels was found. an inverse correlation between cftr and wnt signaling activities was demonstrated.  these data are consistent with cftr participating in the mechanicosensory process essential to regulate wnt/β-catenin signaling required for lung organogenesis. 