Six1 is expressed in distal epithelium and mesenchyme of embryonic lung
Semi-quantitative RT-PCR analysis demonstrated that levels of Six1
transcripts are highest during initiation and branching phases of lung development (E9.5–E11.5 and E11.5–E17.5; respectively in mouse) then decline significantly during the differentiation phase (after E18.5; ). Six1
transcripts were detected in the lung bud from E9.5 () and also in the pharyngeal region and ear ( and Xu et al., 2002
; Zheng et al., 2003
). Six1 protein was more specifically expressed in distal lung mesenchymal and epithelial cells from around E12.5 to E18.5 (). The specificity of Six1 antibody was confirmed by Western blotting of lung proteins where it detected a proper expected protein band of 32 kDa, which corresponds to the size of Six1 protein (data not shown).
Six1 expression during lung development
It has been reported that LacZ
expression in Fgf10LacZ/+
mouse lung can be used as a reporter for Fgf10
expression (Mailleux et al., 2005
). Interestingly, X-gal staining of E14 Fgf10LacZ/+
lungs and Six1 antibody staining show striking similarity in the focal expression pattern of Fgf10
and Six1 in the distal mesenchyme adjacent to distal tips of branching tubules (). Double staining of X-gal-stained Fgf10LacZ/+
lungs with Six1 antibody further confirmed the expression of both Six1 and Fgf10
in the distal mesenchyme ().
Six1−/− embryos die quickly after birth due to respiratory failure and display severe lung hypoplasia
mutant embryos die quickly after birth, have defects in several organs and are relatively smaller in size than wildtype littermates (Xu et al., 2002
; Laclef et al., 2003
). Observation of the newborn pups revealed that the 25% of them, which were genotyped as Six1−/−
mice, gasped for breath and appeared cyanotic. When compared with the lungs of wildtype littermates the lungs of E14.5 and E18.5 Six1−/−
embryos were significantly smaller in appearance () but had normal initial lobation. No Six1 protein expression was evident in Six1−/−
lungs (Figure S1 G in the Supplemental Data
), and both histological and marker analyses showed that heterozygous lungs have no apparent lung defects in mouse embryos, which continue to grow, breathe and breed normally after birth (data not shown).
Severely hypoplastic lung phenotype in Six1−/− embryos
Histological analysis of hematoxylin-eosin-stained sections from wildtype versus Six1−/− mice showed that, as expected, E18.5 wildtype lungs were properly fluid inflated (). However, Six1−/− lungs were not fluid inflated and had a smaller, collapsed appearance (). This severe lung phenotype was also observed in E14.5 Six1−/− embryos (). Furthermore, Six1−/− lungs were severely hypoplastic: morphogenesis of E18.5 mutant lungs appeared to be arrested in the pseudoglandular stage of development (). Six1−/− lungs also showed an increase in the ratio of interstitial mesenchyme to epithelial tubules, while expansion of epithelial tubules did occur, but was greatly reduced versus wildtype lungs (compare ). Also, primitive pre-alveoli were greatly reduced in Six1−/− lungs (), with different degrees of severity (compare with an with ). Thus, E18.5 Six1−/− mutant lungs depicted the histopathological features of pulmonary hypoplasia: small sized lungs, greatly reduced branching morphogenesis, narrow bronchi, arrested expansion of epithelial tubules and dense mesenchymal cellularity as well as apparent failure of normal lung maturation (interstitial condensation), which should occur in late lung development (). Moreover, Six1−/− mice exhibited congestion of large blood vessels (). These data suggest that Six1 −/− mice die quickly after birth because of respiratory failure and the lungs of these mice display several severe hypoplastic defects, including lack of inflation and pulmonary hemorrhage.
Increased epithelial cell differentiation in Six1−/− lungs
Reduced expression of distal epithelial progenitor markers in Six1−/− lungs
Six1−/− embryos and neonates exhibit blood vessel congestions
Next, to determine the timing of onset of phenotypic abnormalities during Six1−/−
lung organogenesis we examined lung development at E10–10.5 (Figure S1 in the Supplemental Data
). In Six1−/−
lungs, very early branching and development were comparable with control littermate lungs, wherein two primary buds arose in the ventrolateral foregut at E9.5 and underwent stereotypic rounds of branching and outgrowth in order to give rise to an early tree like organ (Figs. S1A–B, D–E
). However, both whole-mount and sections at E13–14.5 revealed that lung hypoplasia was noticeable as early as E13–13.5 of development and was more severe from E14.5 ( and data not shown for E13–13.5). Consequently, E13–13.5, E14.5 and E18.5 were used as the developmental stage of choice to analyze the Six1−/−
lung phenotype in this study. Taken together, these data suggest that Six1
is unlikely to be involved directly in primary lung bud induction and is thus not required for the initiation of early mammalian lung organogenesis. Thus the major impact of Six1
deletion is on lung branching morphogenesis subsequent to the first round(s) of domain branching.
Changes of cell proliferation of both epithelial and mesenchymal tissues in Six1−/− lungs
Our observations about the abnormal mesenchymal phenotype of the Six1−/−
lungs further suggest that Six1
abrogation results in increased mesenchymal cell proliferation and/or cell survival. To test this hypothesis, we stained for Phospho-histone 3 (PHH3), which is an immunomarker specific for cells undergoing mitoses and is a reliable method for identifying mitotic figures and determining mitotic index (Colman et al., 2006
). When lung hypoplasia became noticeable at E13.5, PHH3 staining showed a marked increase of PHH3-positives cells in the mutant mesenchyme, but a clear decrease in epithelial cell proliferation in Six1−/−
lungs (). At E14.5, a stage when the lung phenotype became severe, PHH3 staining demonstrated a 1.7-fold decrease in the number of mitotic cells in the epithelium, but a 1.6-fold increase in the number of mitotic cells per total cell number in the mesenchyme of Six1−/−
lungs (55.0±2.2% versus 31.0±3.0% for the epithelium, and 26.0±2.0% versus 43.0±2.8 for the mesenchyme, n
< 0.05; ). On the contrary, cell proliferation analysis using PHH3 staining as a proliferation marker at E12.5 showed that cell proliferation appears comparable for both epithelial and mesenchymal compartments between Six1−/−
and control lungs ().
Cell proliferation and apoptosis in Six1−/− lung epithelium/mesenchyme
At later stages of development, cell proliferation was further examined by exposing E18.5 embryos in utero to BrdU for 1 hour. The relative proportion of cells that had entered or passed through S phase was then determined by immunocytochemistry of lung sections, which showed a dramatic increase of BrdU-positive proliferative cells in Six1−/− lungs (). Caspase-3 staining showed no obvious increase in cell death in E13.5, E14.5 and E18.5 mutant lungs (, and data not shown). Together, these data suggest that Six1 is a critical regulator of cell proliferation in the embryonic lung.
Six1 deletion increases epithelial differentiation in embryonic lungs
Lung epithelial cells differentiate during mid to late gestation of embryonic development, resulting in distinct cell lineages being distributed along a proximodistal axis (Warburton et al., 2000
). Since Six1
negatively regulates cell differentiation in other cell types (e.g. placodal neuronal progenitors; Schlosser et al., 2008
), we next determined whether lung epithelial cell differentiation had occurred properly after Six1
deletion by examining the expression of markers for proximal (CC10) and distal (SP-A, SP-B, and SP-C) epithelial differentiation at E18.5. Transcripts for these differentiation markers were increased in E18.5 Six1−/−
lungs compared to wildtype littermates (example is shown for SP-B
During lung development, late differentiation markers SP-C and SP-B are expressed in alveolar type II (AEC-2) cells of the distal airways, whereas CC10 expression is normally confined to non-ciliated Clara epithelial cells of the proximal airways in wildtype lungs (). Immunohistochemistry of mutant and control sections, which were mounted on the same slides and developed for staining under the same conditions, showed an apparent increase in the relative number of epithelial cells positive for SP-B, SP-C and CC10 proteins in Six1−/− lungs (). In particular, the percent of cells positive for SP-C showed an increase, which ranged from 1.7–2.4 fold, in Six1−/− mutant versus control lungs (positive cells were counted in 200× per field, n=3; ). A similar increase was shown also for SP-B- or CC10-positive cells (positive cells were counts in 200× per field, n = 3, p< 0.05; ). Interestingly, while more cells are expressing these differentiation markers, the level of protein expression is not increased on a per cell basis in Eya1−/− lungs compared to control lungs (compare ). The increase of both SP-B and SP-C expression was also evident in Six1−/− lungs early during the pseudoglandular stage at E14.5 ().
Epithelial differentiation increases during pseudoglandular phase in Six1−/− lungs
Next, we further investigated Six1 effects on lung epithelial cell differentiation in vitro using MLE15 lung epithelial cells, which express endogenous Six1 as well as different epithelial differentiation and progenitor cell markers (, and data not shown). As shown in , MLE15 cells transfected with Six1 expression vector showed a decrease in the expression levels of several lung epithelial differentiation markers, which further supports the inhibitory function of Six1 on lung epithelial differentiation.
Distal epithelial progenitors are severely reduced in the Six1−/− embryonic lungs
is a critical regulator of embryonic cell maintenance/survival, and Six1−/−
mouse embryos have defects in the proliferation and survival of the progenitors of several organs (Xu et al., 2002
; Laclef et al., 2003
; Li et al., 2003
). Therefore, we next determined the effects of Six1
on lung epithelial progenitors using antibodies against Sox9, Id2 and N-myc, which were highly expressed in lung distal epithelial progenitors (; Okubo et al., 2005
; Rawlins, 2008
), wherein Six1 was also highly expressed (). Sox9 transcription factor was specifically expressed in the distal epithelial progenitors from E11.5 to E16.5 (, ) and becomes undetectable by E18.5 (Liu and Hogan, 2002
). In Six1−/−
lungs, signals of Sox9, Id2 and N-myc transcription factors were markedly reduced at E13.5 ( and data not shown), a stage when lung hypoplasia became noticeable, and prematurely abolished in the distal epithelium at E14.5 (). In addition, real-time PCR analysis of E14.5 mutant lungs showed markedly reduced expression of Sox9
at the gene levels ().
Normal expression of distal epithelial progenitor markers in Six1−/− lungs before E13–13.5 of development
Next, to determine whether normal embryonic epithelial progenitors have ever formed in Six1−/− lungs, we stained for progenitor cell markers at early embryonic stages. As shown in , Six1−/− distal epithelial tips stained positively for Sox9 and Id2 and no apparent change was observed in the expression levels of these markers at E12–12.5 compared to wildtype littermates (). This suggests that distal epithelial progenitors have formed normally but become rapidly depleted at the pseudoglandular phase of Six1−/− lungs. Therefore, Six1 must be a critical regulator of the maintenance of lung epithelial progenitors and of the correct expression of progenitor cell markers. This hypothesis was further supported by our in vitro studies using MLE15 epithelial cells. Thus, Six1 overexpression in MLE15 lung epithelial cells stimulated a marked increase in Sox9 and Id2-expressing cells as well as increased mitotic cells in culture ().
Taken together, these data suggest that Six1−/− embryos display lung epithelial defects that correlate with increased epithelial differentiation and decreased epithelial progenitors.
Six1−/− embryos display pulmonary vascular defects
At E18.5 and at birth (P0), close examination of Six1−/− embryos showed congestion of the large pulmonary vessels, suggesting a pulmonary vascular defect in these embryos (). Notably, this congestion of blood vessels was not observed in any other region of the embryos or neonates (data not shown), suggesting a specific defect in the lung vasculature. Moreover, no apparent change in the number of PHH3-positive mitotic smooth muscle or endothelial cells was observed in the large pulmonary blood vessels (). In addition, caspase-3-positive apoptotic cells were few and did not significantly change within the pulmonary vasculature of Six1−/− and control lungs ().
In addition, many Six1−/− pulmonary vessels were either small/collapsed (compare ) or showing rupture of the vascular smooth muscle wall, which was thin (one layer; compare ) with herniation of the endothelial lining (, and data not shown). These results showed that Six1−/− lungs of late stage embryos and neonates exhibited vascular smooth muscle-specific defects and congested blood vessels. Thus, in Six1−/− embryos weakened pulmonary blood vessels could rupture at birth, possibly due to the increased pulmonary blood flow or internal mechanical strain on the lungs, which occurs in association with the transition to air breathing at birth, particularly when the lung is dysplastic.
Defective smooth muscle integrity in Six1−/− embryonic lungs
Severe reduction of vascular and bronchial smooth muscle α-actin expression in Six1−/− lungs
Our hypothesis is that the weakened and congested state of the pulmonary blood vessels observed in the lungs of Six1−/−
embryos/neonates is largely due to defects in vascular smooth muscle cell (VSMC) differentiation. To test this hypothesis, we determine whether smooth muscle had differentiated properly in Six1−/−
embryos/neonates by immunostaining for smooth muscle α-actin (α–SMA), which is normally mainly detected in cells surrounding blood vessels and the conducting airways (Mitchell et al., 1990
; Low and White, 1998
). At E18.5, differentiated smooth muscle cells are normally seen surrounding all the major pulmonary vasculature and the large upper bronchial airways in late development in control wildtype lungs (). In contrast, most of the blood vessels of E18.5 Six1−/−
lungs contained noticeably thin and less differentiated smooth muscle with frank breaches in the vessel wall, while some had almost undetectable smooth muscle α-actin staining (). Similarly, α–SMA expression was hardly detectable in Six1−/−
bronchial smooth muscles (), although muscle cells appeared to morphologically exist with their characteristic spindle shape (Nie et al., 2010
) surrounding pulmonary vasculature and large bronchial airways (), as compared to control lungs (). These defects in vascular and bronchial smooth muscle were not correlated with increased smooth muscle cell death in E18.5 Six1−/−
lungs, as indicated by caspase-3 staining of blood vessels and bronchi (). These data suggest that Six1
is critical for the differentiation of both pulmonary bronchial and vascular smooth muscle and hence the integrity of the lung vasculature during lung development.
We next stained Six1−/− lungs with an antibody to platelet endothelial adhesion molecule (PECAM) to determine whether the smaller vessels in the lung, which lack vascular smooth muscle, were affected in Six1−/− embryos. Signals for PECAM, a marker for endothelial cells, were observed in E18.5 Six1−/− and control lungs (, n = 3 for each genotype), suggesting that vascular endothelium and capillary network are formed in Six1−/− lungs.
Increased Shh signaling activity, but decreased Fgf10 expression in Six1−/− lungs
To determine the possible mechanisms by which absence of Six1
results in the phenotype described above, we examined the expression of Shh. Two lines of reasoning led us to examine Shh expression and its relationship with Six1
in the lung. Firstly, similar to Six1 (), Shh
expression is most intense in the distal epithelial tips of the lung and is downregulated during the differentiation phase of murine lung development (from E16.5; Urase et al., 1996
; Bellusci et al., 1997b
). Secondly, ectopic lung-specific overexpression of Shh
from an Sp-c
promoter/enhancer construct (Sp-c:Shh
) in transgenic mice yields a phenotype that is very similar to the Six1−/−
lung phenotype (see above; and Bellusci et al., 1997b
). In addition, both real-time PCR and immunohistochemistry showed a dramatic increase (5-fold) in Shh
gene expression in E18–18.5 Six1−/−
versus control lungs () and in Shh protein expression in both E14.5 and E18.5 Six1−/−
lungs (). Thus Shh expression continued to increase at both gene and protein levels in Six1−/−
lungs after E16.5, a time point where Shh
expression should start to decrease progressively in wildtype lungs (Bellusci et al., 1997b
), until birth ( and data not shown at P0, n
= 3 for each genotype).
Increased Shh signaling activity, but decreased Fgf10 expression in Six1−/− lungs
Hedgehog signaling competence is reflected by measuring Shh receptor patched-1 (Ptc-1) and Gli1 expression levels, which are expressed in lung distal mesenchyme and are mediators of Shh activity (Grindley et al., 1997
, Lees et al., 2005
; Madison et al., 2005
). Therefore, we next investigated the expression of Ptc-1 and Gli1 (). Beginning around E16.5 of embryonic development, Shh, Ptc
RNA levels are normally declining in wildtype lungs, and Shh
overexpression results in increased levels of both Ptc
mRNAs in the lung (Grindley et al., 1997
; Bellusci et al., 1997b
). In Six1−/−
lungs, the expression levels of Ptc-1 and Gli proteins were dramatically increased in the mesenchyme at E14.5 and E18.5 compared to wildtype control lungs (). Together, these data suggest that Six1
absence causes a significant increase in Shh expression and activity and that Six1
is necessary for normal down-regulation of Shh signaling activity starting from E16.5 in the lung.
Since ectopic Shh
overexpression inhibits Fgf10
expression in the lung (Bellusci et al., 1997b
), we next examined changes in Fgf10
expression in mutant versus wildtype lungs. As shown in L, Fgf10
expression was markedly decreased in parallel to increased Shh
expression levels in Six1−/−
lungs, as measured by real-time PCR (). This suggests that Six1
acts upstream of Fgf10 signaling, which is active in the mesenchyme at the very distal tips of branching tubules and is critical for lung bud formation and epithelial branching (Bellusci et al., 1997a
). This conclusion was further supported by analysis of Six1
expression in Fgf10
hypomorphic lungs where no change in Six1
expression was apparently evident at different stages of lung development (example is shown in ).
Shh protein stimulates Six1−/− mesenchymal growth, while Fgf10 protein induces Six1−/− epithelial branching morphogenesis in culture
Having established that Shh-Fgf10 signaling is altered in Six1−/−
lungs, we next investigated whether Shh and Fgf10 protens affect Six1−/−
lungs in vitro. To test this possibility, the distal mesenchyme and epithelium of E12.5 Six1−/−
lungs were separated and individually cultured in Matrigel-based three-dimensional scaffold in the presence or absence of Shh or Fgf10 recombinant protein, respectively. As shown in , all isolated mesenchymal explants from either control wildtype lungs (; n=
8) or Six1−/−
lungs (; n=
8), which were cultured for 48 hr in control media, condensed and died. In cultures treated with 500 ng/ml Shh protein (Weaver et al., 2003
; Li et al., 2005
), a remarkable increase in the size of all tested
control (; n
=8) and Six1−/−
=8) mesenchymal explants was observed, associated with many elongated mesenchymal cells invading into the Matrigel at the periphery.
Effect of exogenous Shh and Fgf10 proteins on isolated distal lung mesenchyme and epithelium; respectively
Next, we tested the hypothesis that excessive Shh
-mediated reduction of Fgf10
expression resulted in reduction of epithelial branching morphogenesis by using epithelial culture of Six1−/−
lungs. E12.5 Six1−/−
epithelial explants were grown for 48 hr in culture in Matrigel in the presence of 250 ng/ml Fgf10 (n
=8). In the absence of Fgf10, neither the wildtype nor the Six1−/−
lung isolated airways showed signs of branching and died (n
=6; ) as previously described for wildtype lungs (Bellusci et al., 1997a
). But notably, addition of exogenous Fgf10 rescued Six1−/−
branching defects so that most mutant epithelial explants (7 out of 8 explants) continued to grow and branch to form new branching tips in the presence of Fgf10 protein in a similar fashion to control explants (8 out of 8 explants grew) from early culture times (8–16hr of culture; ). The inductive effect of Fgf10 protein on branching was even more pronounced after 24 hr of culture (data not shown). These data suggest that Six1−/−
distal epithelial tip cells remain fully competent to respond to inductive signals from lung mesenchyme and that Six1−/−
branching defects are therefore not an intrinsic property of the Six1−/−
Cyclopamine inhibition of the Shh pathway induces Six1−/− lung growth in culture
To test the effect of inhibition of the biological effect of Shh signaling on Six1−/−
lung growth and branching in vitro, we carried out cultures of whole lungs isolated at E12–E12.5 over a period of 42 hr in the presence or absence of the specific Shh antagonist, cyclopamine (Yao et al., 2002
; ). Without the addition of cyclopamine, all tested
wildtype control lungs showed an increasing number of distal end buds (), while all Six1−/−
mutant lungs did not grow well in culture and showed a delay in their development compared to control lungs (; n
=9). By contrast, in the presence of 5 μmol/l cyclopamine a notable increase of lung size and epithelial branching after 42 hr of culture associated with a dilation of the distal epithelium was observed in all tested Six1−/−
lungs (; n
=9) and control lungs (). These data suggest that inhibition of Shh signaling activity can, at least partially, rescue Six1−/−
lung developmental defects. This conclusion was further supported by developmental marker analyses at 26 hr of culture (). Thus, both Shh and SP-B expression levels was downregulated to control levels (), while Sox9 expression and PHH-3 positive cells increased in mutant lungs treated with 5 μmol/l cyclopamine ().
Fig. 12 Effect of cyclopamine-mediated inhibition of the Shh pathway on Six1−/− lung growth in culture for 42 hr. (A,E,I) control E12–12.5 lungs grow and form new branches in culture in the absence of cyclopamine. (B,F,J) control E12–12.5 (more ...)
Developmental changes of Six1−/− lung growing in culture for 26 hr after treatment with cyclopamine
Taken together, our results provide the first direct evidence for the requirement of Six1 in coordinating Shh-Fgf10 signaling during lung development and that the Six1-Shh-Fgf10 signaling pathway is therefore critical for lung epithelial branching morphogenesis.