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Six1 is a member of the six-homeodomain family of transcription factors. Six1 is expressed in multiple embryonic cell types and plays important roles in proliferation, differentiation and survival of precursor cells of different organs, yet its function during lung development was hitherto unknown. Herein we show that Six1−/− lungs are severely hypoplastic with greatly reduced epithelial branching and increased mesenchymal cellularity. Six1 is expressed at the distal epithelial tips of branching tubules as well as in the surrounding distal mesenchyme. Six1−/− lung epithelial cells show increased expression of differentiation markers, but loss of progenitor cell markers. Six1 overexpression in MLE15 lung epithelial cells in vitro inhibited cell differentiation, but increases the expression of progenitor cell markers. In addition, Six1−/− embryos and newborn mice exhibit mesenchymal overproliferation, decreased Fgf10 expression and severe defects in the smooth muscle component of the bronchi and major pulmonary vessels. These defects lead to rupture of major vessels in mutant lungs after birth. Treatment of Six1−/− epithelial explants in culture with recombinant Fgf10 protein restores epithelial branching. As Shh expression is abnormally increased in Six1−/− lungs, we also treated mutant mesenchymal explants with recombinant Shh protein and found that these explants were competent to respond to Shh and continued to grow in culture. Furthermore, inhibition of Shh signaling with cyclopamine stimulated Six1−/− lungs to grow and branch in culture. This study provides the first evidence for the requirement of Six1 in coordinating Shh-Fgf10 signaling in embryonic lung to ensure proper levels of proliferation and differentiation along the proximodistal axis of epithelial, mesenchymal and endothelial cells. These findings uncover novel and essential functions for Six1 as a critical coordinator of Shh- Fgf10 signaling during embryonic lung development. We propose that Six1 is hence critical for coordination of proper lung epithelial, mesenchymal and vascular development.
Mammalian lung development is initiated by the formation of two primary epithelial buds, which arise from the laryngotracheal groove in the ventral floor of the primitive foregut endoderm and extend into the surrounding splanchnic mesenchyme. These buds undergo stereotypic rounds of branching and outgrowth to give rise to a tree-like respiratory organ, which contains different specialized epithelial cell types organized along the proximodistal axis (Cardoso, 2000; Metzger et al, 2009; Warburton et al., 2000, 2008, 2010). Controlled and rapid proliferation of lung epithelial progenitors, in association with the outgrowth and branching of the epithelial tubes, is essential for producing an alveolar gas diffusion surface sufficient to sustain extra-uterine life. The undifferentiated epithelial cells leave the progenitor pool to give rise first to lineage-committed precursor cells and eventually to fully differentiated cell lineages (Cardoso and Lü, 2006; Rawlins and Hogan, 2006). Disruption of normal lung developmental processes can result in neonatal respiratory failure or distress (i.e. pulmonary hypoplasia, a hallmark of congenital lung diseases) if lung formation is severely affected or susceptibility to lung diseases during later life if milder changes occur in the developing lung (Warburton et al., 2006).
Normal lung morphogenesis and patterning depend critically on interactive signaling between the endodermal epithelium and mesenchyme derived from splanchnic mesoderm. The pulmonary mesenchyme gives rise to several different cell types, including smooth muscle of the upper airways during branching morphogenesis. Moreover, the mesenchyme produces many important growth factors and signaling molecules required for airway epithelial branching and development, e.g. members of the fibroblast growth factor (Fgf) family (reviewed by Warburton et al., 2000, 2005, Morrisey and Hogan, 2010). In turn, the epithelium produces signaling molecules essential for mesenchymal proliferation and differentiation including sonic hedgehog (Shh) and bone morphogenetic protein 4 (BMP4) (Bellusci et al., 1996, 1997a,b; Litingtung et al., 1998; Pepicelli et al., 1998).
The sine oculis (SIX) proteins belong to a family of six homeodomain transcription factors, which are homologues of Drosophila sine oculis. In mammals, Six (Six1–6) genes and Eya are co-expressed and exhibit synergistic genetic interactions to regulate the development of multiple organs by controlling cell cycle regulators and inhibition of apoptosis (Kawakami, et al., 2000, Xu et al., 1997b; Ford et al., 1998; Coletta et al., 2004). In addition, Six1 and Six4 play important roles in the formation of various organs such as olfactory epithelium, cranial ganglia, inner ear, kidney, skeletal muscle and skeleton (reviewed by Kawakami et al., 2000; Kumar, 2009). Six1−/− mouse embryos have defects in the proliferation and survival of the precursor cells of multiple organs, and die at birth (Xu et al., 2002; Laclef et al., 2003; Li et al., 2003). Mutations in the Six protein family (Six1 or Six5) or in Eya1 protein are responsible for Branchio-Oto-Renal syndrome in humans (Abdelhak et al., 1997; Ruf et al., 2004; Hoskins, et al., 2007; Krug et al., 2010).
The Eya1 gene encodes a transcriptional co-activator that acts with Six1 to control the development of different organs (Zou et al., 2004, 2006a; Grifone et al., 2004). We recently reported a severely hypoplastic lung phenotype in Eya1−/− embryos (El-Hashash et al., 2011). Yet the specific functions of Six1 in lung development are unknown.
Herein, we found that Six1 deletion causes severe lung hypoplasia, increased epithelial differentiation, pulmonary smooth muscle defects and disruption of Shh-Fgf10 signalling, which suggest that Six1 is necessary for proper coordination of epithelial, mesenchymal and endothelial tissues during lung development.
Six1 knockout (KO) mice on the 129 background, Fgf10LacZ/+, and Fgf10LacZ/− hypomorph mice have been published previously (Kelly et al., 2001; Laclef et al., 2003; Xu et al., 2002, 2003; Mailleux et al., 2005). Genotyping of mice and embryos was performed as previously described (Kelly et al., 2001; Laclef et al., 2003; Xu et al., 2002, Perl et al., 2002). Wildtype littermates were used as controls.
Histological staining and ISH using Six1 probe were performed as previously described (Xu et al., 1997a; De Langhe et al., 2006). Real-time PCR and semi-Quantitative PCR were performed in triplicate as described before (Del Moral et al. 2006a,b). PCR primers used in this study were described in Table 1 in the Supplemental Data.
Immunohistochemical/X-gal staining on paraffin sections or fixed MLE-15 cells and bromodeoxyuridine (BrdU) labeling were performed using standard protocols as described (Tefft et al., 2005; El-Hashash et al., 2005; Del Moral et al. 2006a,b). Antibodies used in this study are described in Table 2 in the Supplemental Data. Staining was performed in triplicate. For all immunohistochemistry, mutant sections were mounted on the same slides of wildtype control sections and processed for antibody staining under the same conditions.
Lungs from E12.5 embryos were treated with dispase (BD Biosciences) for 5 min at 4 °C and then transferred to Dulbecco’s Modified Eagle’s Medium: Nutrient Mixture F-12 (DMEM:F12) with 10% fetal bovine serum (FBS; Invitrogen) to block the enzymatic reaction. The epithelial buds were separated from the mesenchyme, embedded in 200 μl of Matrigel diluted 1:1 with DMEM:F12 serum-free media containing 250 ng/ml recombinant Fgf10 (R & D Systems), and then grown in culture as described (Carraro et al., 2009). Isolated mesenchymal explants were grown on Matrigel and treated with 500 ng/ml recombinant Shh (R & D Systems) as previously described (Weaver et al., 2003; Del Moral et al., 2006a).
Embryonic lungs were removed at E11.5 grown in culture and treated with cyclopamine as described (Mailleux et al., 2005). Briefly, lungs were placed on Nuclepore filters in 30 μl of a 1:1 mixture of Matrigel and culture medium (DMEM/F12 medium containing 0.1% heat-inactivated fetal bovine serum and penicillin/streptomycin) and kept for 30 minutes at 37°C. After Matrigel polymerization, these filters were laid on the surface of 500μl culture medium containing cyclopamine (TRC Biomedical Research Chemicals, Canada) in Nunclon dishes. Concentrations of 2, 5 and 10 μmol/l cyclopamine were tested and cyclopamine proved toxic at 10 μmol/l as previously described (Yao et al., 2002; Mailleux et al., 2005). Lungs were cultured for 2 days in the presence of cyclopamine and then fixed in 4% PFA.
Lung epithelial cells MLE15, a gift from Jeffrey Whitsett (Cincinnati University), were grown in culture and treated as well as processed for transfection as described (Tefft et al., 2002). Briefly, for transfection MLE15 cells were seeded in eight chamber slides (BD Biosciences) at 50 % confluency. The next day they were transfected with 200 ng of expression vector for mouse Six1 or a luciferase-expressing vector (control) using lipofectamine LTX (Invitrogen). Fresh medium was added 6 hours later. Cells were fixed with 4%PFA three days after transfection as above. We used an expression vector encoding a VP16 fusion protein and the transfection efficiency was monitored by fluorescence staining using anti-VP16 antibody.
Data are presented as means ± SD. Statistical significance between wildtype control and mutant lungs was calculated using Student’s t-test. For counting PHH3-, Caspase-3, SP-B, SP-C or CC10- positive cells, labeled epithelial or mesenchymal cells and total cell number per section were counted. Three random fields were chosen (200–350 cells per field) from three different embryos of each indicated genotype. The percent of labeled cells to the total cell number was reported. Analyses were done using GraphPad Prism software (GraphPad Software, Inc., San Diego, http://www.graphpad.com). Densitometry analysis was produced by the software Image J.
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; Fig. 1A). Six1 transcripts were detected in the lung bud from E9.5 (Fig. 1D,E) and also in the pharyngeal region and ear (Fig. 1D,E 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 (Fig. 1B,C,F,G,K,L). 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).
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 (Fig. 1H,I). Double staining of X-gal-stained Fgf10LacZ/+ lungs with Six1 antibody further confirmed the expression of both Six1 and Fgf10 in the distal mesenchyme (Fig. 1J).
The Six1 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 (Fig. 2A,D) 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).
Histological analysis of hematoxylin-eosin-stained sections from wildtype versus Six1−/− mice showed that, as expected, E18.5 wildtype lungs were properly fluid inflated (Fig. 2D,E,I). However, Six1−/− lungs were not fluid inflated and had a smaller, collapsed appearance (Fig. 2D,F,J). This severe lung phenotype was also observed in E14.5 Six1−/− embryos (Fig. 2A–C, G,H). Furthermore, Six1−/− lungs were severely hypoplastic: morphogenesis of E18.5 mutant lungs appeared to be arrested in the pseudoglandular stage of development (Fig. 2E,F,I,J). 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 Fig. 2F,J with E,I). Also, primitive pre-alveoli were greatly reduced in Six1−/− lungs (Fig. 2J), with different degrees of severity (compare Fig. 2F,J with Fig. 4G an Fig. 2C,H with Fig. 6B,F). 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 (Fig. 2F,J). Moreover, Six1−/− mice exhibited congestion of large blood vessels (Fig. 8B,F). 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.
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 (Fig. 2A–C,G,H 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.
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 (Fig. 3C,D). 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=3, p< 0.05; Fig. 3A,B,G). 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 (Fig. 3J,K).
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 (Fig. 3E,F). Caspase-3 staining showed no obvious increase in cell death in E13.5, E14.5 and E18.5 mutant lungs (Fig. 3H,I, and data not shown). Together, these data suggest that Six1 is a critical regulator of cell proliferation in the embryonic lung.
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, 2005, 2008). 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 in Fig. 4J).
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 (Fig. 4A,F,K). 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 (Fig. 4,B,G,L). 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; Fig. 4E). 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; Fig. 4E). 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 Fig. 4D,I,N with C,H,M). The increase of both SP-B and SP-C expression was also evident in Six1−/− lungs early during the pseudoglandular stage at E14.5 (Fig. 5B,E).
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 (Figs. 5C, 6C,D,G,H and data not shown). As shown in Fig. 5C, 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.
Six1 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 (Fig. 6A,E,I; Okubo et al., 2005; Rawlins, 2008), wherein Six1 was also highly expressed (Fig. 1B,C). Sox9 transcription factor was specifically expressed in the distal epithelial progenitors from E11.5 to E16.5 (Figs. 6A, 7A,B) 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 (Fig. 7D,H and data not shown), a stage when lung hypoplasia became noticeable, and prematurely abolished in the distal epithelium at E14.5 (Fig. 6B,F,J). In addition, real-time PCR analysis of E14.5 mutant lungs showed markedly reduced expression of Sox9 at the gene levels (Fig. 6K).
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 Fig. 7, 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 (Fig. 7A,C,E,G). 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 (Fig. 6C,D,G,H,L).
Taken together, these data suggest that Six1−/− embryos display lung epithelial defects that correlate with increased epithelial differentiation and decreased epithelial progenitors.
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 (Figs. 8A,B,C,D). 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 (Fig. 8E,F). In addition, caspase-3-positive apoptotic cells were few and did not significantly change within the pulmonary vasculature of Six1−/− and control lungs (Fig. 8I).
In addition, many Six1−/− pulmonary vessels were either small/collapsed (compare Fig. 9A with 9B,D) or showing rupture of the vascular smooth muscle wall, which was thin (one layer; compare Fig. 9C and 9D) with herniation of the endothelial lining (Fig. 9B, 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.
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 (Fig. 9A,E). 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 (Fig. 9B,D). Similarly, α–SMA expression was hardly detectable in Six1−/− bronchial smooth muscles (Fig. 9F), although muscle cells appeared to morphologically exist with their characteristic spindle shape (Nie et al., 2010) surrounding pulmonary vasculature and large bronchial airways (Fig. 9D,H), as compared to control lungs (Fig. 9C,G). 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 (Fig. 8I). 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 (Fig. 9I,J, n = 3 for each genotype), suggesting that vascular endothelium and capillary network are formed 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 (Fig. 1), 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 (Fig. 10L) and in Shh protein expression in both E14.5 and E18.5 Six1−/− lungs (Fig. 10B,D versus A,C). 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 (Fig. 10B,D,L and data not shown at P0, n = 3 for each genotype).
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 (Fig. 10E–H, J–K). Beginning around E16.5 of embryonic development, Shh, Ptc and Gli1/Gli2 RNA levels are normally declining in wildtype lungs, and Shh overexpression results in increased levels of both Ptc and Gli 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 (Fig. 10F,H,K versus E,G,J). 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 Fig. 10 L, Fgf10 expression was markedly decreased in parallel to increased Shh expression levels in Six1−/− lungs, as measured by real-time PCR (Fig. 10L). 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,b). 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 Fig. 10I).
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 Fig. 11, all isolated mesenchymal explants from either control wildtype lungs (Fig. 11A,E; n= 8) or Six1−/− lungs (Fig. 11C,G; 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 (Fig. 11B,F; n=8) and Six1−/− (Fig. 11D,H; n=8) mesenchymal explants was observed, associated with many elongated mesenchymal cells invading into the Matrigel at the periphery.
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; Fig. 11I,M,Q,K,O,S) as previously described for wildtype lungs (Bellusci et al., 1997a,b). 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; Fig. 11L,P,T versus J,N,R). 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−/− epithelium.
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; Fig. 12). Without the addition of cyclopamine, all tested wildtype control lungs showed an increasing number of distal end buds (Fig. 12A,E,I), while all Six1−/− mutant lungs did not grow well in culture and showed a delay in their development compared to control lungs (Fig. 12C,G,K; 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 (Fig. 12D,H,L; n=9) and control lungs (Fig. 12B,F,J). 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 (Fig. 13). Thus, both Shh and SP-B expression levels was downregulated to control levels (Fig. 13A–C,G–I), while Sox9 expression and PHH-3 positive cells increased in mutant lungs treated with 5 μmol/l cyclopamine (Fig. 13D–F, J–M).
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.
We have demonstrated an important, seemingly complex role for Six1 transcription factor in a signaling network coordinating several different integrated processes required for correct lung development.
Our data demonstrate that Six1 plays an essential role in proliferation of both the lung epithelium and mesenchyme. This observation is noticeable during the pseudoglandular phase where cell proliferation at distal epithelial tips that express Six1 is reduced from E13.5 onwards in Six1−/− lungs. In contrast, mesenchymal proliferation is somewhat unexpectedly greatly increased in the face of Six1 deletion in Six1−/− lungs at E13.5 and later in development.
Six1 transcription factor plays critical roles in the regulation of precursor cell cycle and proliferation in different organs (Li et al., 2003; Kumar, 2009). Loss of Six1 greatly reduces cell proliferation of otic epithelium (Zheng et al., 2003) and of both muscle and kidney (Li et al., 2003). Conversely, high levels of Six1 expand the pool of proliferative neuronal progenitors (Schlosser et al., 2008). Thus, whereas lack of Six1 expression in Six1−/− lung distal epithelium may account for reduced cell proliferation at the distal tips, the increased mesenchymal proliferation in Six1−/− lungs is less likely to be a direct effect of Six1 deletion. Instead, Six1 must act indirectly on mesenchymal proliferation by affecting other key regulators of mesenchymal cell proliferation such as Shh signaling, which regulates cell proliferation of lung mesenchyme (Litingtung et al., 1998; Pepicelli et al., 1998).
In this current study we propose that the severe Six1−/− hypoplastic lung phenotype described herein (Fig. 2) may be explained by abnormal persistence of Shh expression/activity, occasioned by absence of Six1 activity, for several reasons. Firstly, Shh acts as a mitogen for lung mesenchyme and thus controls epithelial branching indirectly during lung development (Bellusci et al., 1997b). Interestingly, embryonic Six1−/− lungs show increased mesenchymal cellularity with reduced branching and exhibit failure of interstitial condensation. The later is an important feature of prenatal fetal lung maturation, an abnormal phenotype that is most likely to be mediated through abnormally persistent high-level expression and activity of Shh (Bellusci et al., 1997b). Indeed, Shh expression and activity are high at E18.5 and at birth in Six1−/− lungs (i.e. after E16.5, a time point where Shh expression should start to decrease progressively in wildtype lungs (Bellusci et al., 1997b). Secondly, the phenotype of the Shh over-expressing transgenic lung bears striking similarities to the Six1−/− lung phenotype described herein (Fig. 2 and Bellusci et al., 1997b). Thirdly, both Six1 and Shh are highly expressed at distal epithelial tips of branching tubules (Fig. 1B,C, and Bellusci et al., 1997b) as well as being involved in controlling branching morphogenesis through regulation of mesenchymal development (Fig. 3, Bellusci et al., 1997b; Litingtung et al., 1998; Pepicelli et al., 1998).
Finally, our findings that Six1−/− lungs treated with the Shh-inhibitor cyclopamine continue to grow and branch in culture similar to wildtype lung (Figs. 12,,13)13) provide strong confirmatory evidence for the negative modulatory effect of Six1 on Shh signaling activity to ensure proper embryonic lung development. Moreover, since Shh null mice also have pulmonary hypoplasia (Litingtung et al., 1998; Pepicelli et al., 1998), it is clear that levels of Shh expression must be tightly regulated in order for normal lung development to occur. Thus our findings are crucial for understanding how Shh expression/activity is indeed tightly regulated and for identifying Six1 as a potential regulator of Shh expression and activity, which ensures appropriate mesenchymal/epithelial proliferation and differentiation in the embryonic lung. Our recent findings that deletion of Eya1, which acts as a transcriptional coactivator of Six1 during the development of different organs (Jemc and Rebay, 2007), also results in an ectopic increase of Shh activity and a hypoplastic lung phenotype (El-Hashash et al., 2011) similar to the herein reported Six1−/− lung phenotype, further supports the importance of the Six1-Eya1 pathway as a potential key regulator of Shh expression level/activity in the lung.
Lung induction involves the interplay between epithelial Shh and mesenchymal Fgf10. Our data show that Six1−/− embryonic lungs are characterized by a significant decrease in Fgf10 expression, which is combined with increased Shh expression during the pseudoglandular stage and later in development (Fig. 10L). This suggests that Six1 potentially promotes Fgf10 signaling pathway by negatively modulating Shh signaling, which in turn is a key inhibitor of Fgf10 signaling. This is internally consistent with the known function of Shh as a negative regulator of Fgf10 signaling to induce lung bud formation/epithelial branching (Bellusci et al., 1997a,b; Pepicelli et al, 1998).
It should be noted that Six1 abrogation negatively impinges on lung branching morphogenesis subsequent to the first round(s) of domain branching (Fig. S1) somewhat similarly to Shh−/− lungs (Pepicelli et al, 1998). This occurs in parallel to increased Shh activity and reduced Fgf10 expression in Six1−/− lungs. Thus, we propose that critical interactions between these three molecules occur in a novel Six1-Shh-Fgf10 pathway, which regulates subsequent rounds of epithelial branching. This conclusion is further supported by the findings that inhibiting Shh function in culture increases epithelial branching of Six1−/− lungs and treatment of Six1−/− epithelium in vitro with Fgf10 protein stimulates branching of the isolated mesenchyme-free airways (Figs. 11–13).
Other lines of evidence to support our novel hypothesis that Six1 is a potential critical regulator of the Shh-controlled Fgf10 signaling in the lung come from knockout mice. Thus, similar to Six1 functions reported herein, Fgf10 signaling is essential for the maintenance of epithelial progenitor cell proliferation (Ramasamy et al., 2007) and positively regulates the expression of epithelial progenitor marker Sox9 (Abler et al., 2009). In addition, Fgf10 signaling also inhibits differentiation of the lung epithelium (Nyeng et al., 2008) similar to Six1 differentiation inhibitory functions (this study and Schlosser et al., 2008). Furthermore, both epithelial branching (Abler et al., 2009) and bronchial smooth muscle cells (Mailleux et al., 2005, Ramasamy et al., 2007) are severely defective in Fgf10 hypomorphic or Fgf10/Fgfr2b inactivated lungs, which are very similar to the Six1−/− lung phenotype (Figs. 2, ,9).9). Moreover, Six1 is required for the maintenance of Fgf10 expression during inner ear development (Zheng et al., 2003).
It is noteworthy that Shh signaling facilitates proper Fgf10 localization during distal tip branching by inhibiting Fgf10 expression mainly in the inter-bud regions after mesenchymal Fgf10 expression has been initiated. However, Shh expression is also upregulated at new sites of branching, which is associated with higher levels of Fgf10 expression. This creates an apparent paradox as to how Fgf10 expression could be maintained in the presence of high levels of Shh expression (Chuang and McMahon, 2003). Based on our data about the Six1 expression pattern, which co-expresses with Fgf10 near the distal epithelium and inhibits the activity of Shh signaling in distal epithelium, wherein Six1 is also expressed (Figs.1B,C,H–J, ,10),10), we now propose a model in which Six1 is a potential key regulator/coordinator of Shh/Fgf10 expression pattern and activity during lung branching. We propose that Six1 focally expressed in distal mesenchyme inhibits Ptc1 receptor expression and thus maintains focal Fgf10 expression by inhibiting local Shh activity. The response of distal tip epithelial cells to Fgf10 is further proposed to be facilitated because epithelial Six1 is also potentially necessary to inhibit Shh expression and/or autocrine activity in these cells. Indeed, Shh has been shown recently to exhibit autocrine functions during the organogenesis of multiple organs (McGlinn and Tabin, 2006; Yang et al., 2008; Handrigan and Richman, 2009) and Shh autocrine activities in the lung epithelium are under investigation. Whether Six1 controls other coordinators of Shh-Fgf10 signaling such as Hedgehog-binding protein (Hhip), BMP4 and Fgf9 will be the subjects of future study.
The lung contains different specialized epithelial cell types organized along the proximodistal axis. Yet little is known about the mechanisms that control epithelial progenitor cell self-renewal and cell fate determination in the lung. Most recently, we have shown that Eya1, which is transcriptional coactivator for Six1 during embryogenesis (Zou et al., 2004; Schlosser et al., 2008; Jemc and Rebay, 2007), is required for controlling the balance between epithelial progenitor self-renewal and differentiation in the embryonic lung (El-Hashash et al., 2011). The current study suggests that Six1 is likely to play a similar role in the self-renewal and determination of epithelial progenitor cell fate, based on its expression in distal lung epithelial cell populations that contain progenitor cells (Okubo et al., 2005; Rawlins et al., 2009) and decreased epithelial progenitors but increased epithelial differentiation after interfering with Six1 function in vivo or in vitro (Figs. 1,,44–7). Indeed, Six1 and Eya1 genes play critical roles in the proper expression of neuronal determination and differentiation genes in the olfactory, otic and epibranchial placodes (Zou et al., 2004; Friedman et al., 2005; Bricaud and Collazo, 2006; Ikeda et al., 2007) as well as in the proliferation and survival of sensory and neuronal progenitors (Li et al., 2003; Zheng et al., 2003; Bricaud and Collazo, 2006; Zou et al., 2006b; Kriebel et al., 2007). In addition, Six1 promotes cell fate of murine pre-placodal ectoderm and inner ear hair cells as well as leach ectodermal teloblast cells (Brugmann et al., 2004; Bricaud and Collazo, 2006 Quigley et al., 2010).
Most recently, it has been shown that Six1 is essential for smooth muscle cell formation in other branching organs, e.g. kidney (Nie et al., 2010). Herein we have extended these observations to the embryonic lung to demonstrate that Six1 is critical for controlling late development of smooth muscle as it differentiates from the mesenchyme because Six1−/− embryos show reduced expression of vascular α-SMA in pulmonary VSMCs. Nonetheless, the undetectable expression level of Six1 in pulmonary VSMCs (data not shown) suggests several possibilities as to its actions in the regulation of VSMC development. First, Six1 may be necessary for the expression of other auto- or paracrine factors in epithelial or mesenchymal cell lineages required for VSMC differentiation/growth. For instance, Six1 deletion causes downregulation of the expression levels of Fgf10 (Fig. 10L), which regulates the growth of aortic VSMC in vitro (Onda et al., 2003). A second possible mechanism is that loss of Six1 could result in defective differentiation of VSMCs, which could lead to degradation of the smooth muscle component of the vessel wall. Interestingly, the pulmonary vascular defects of Six1−/− embryos seem less likely to be directly linked to lung hypoplasia because other mouse models that have severe lung hypoplasia, e.g. Fgf9−/− mice, exhibit normal vascular development (Colvin et al., 2001). Conversely, Six1 is likely to play a role during the early events of pulmonary VSMC differentiation, because Six1 expression levels decrease during late gestation in the lung after E18.5 (Fig. 1A). Since Eya1 deletion has similar effects on pulmonary VSMCs (El-Hashash et al., 2011), we therefore propose that the pathway of Six1-Eya1, which are expressed together in the most distal mesenchyme, may initiate a mesenchymal differentiation program that propagates from the distal to proximal regions as the lung grows, resulting in the proper differentiation of vascular smooth muscle, which occurs reciprocally to distal-proximal progression of mesenchymal differentiation.
In conclusion, the novel data presented herein provide evidence that Six1 is important for the co-ordination of the Shh-Fgf10 signaling pathway, which regulates and coordinates key interlocking aspects of epithelial branching and both mesenchymal and vascular development during embryonic lung organogenesis.
Cross-sections from E10–E10.5 Six1−/− embryos show apparently normal formation, and early branching of lung buds (A,B; arrows) compared to control littermates (D,D; arrowheads). (C,F) Cross-sections show severe lung phenotype in Six1−/− embryos (C) compared to wildtype littermates (F) at E16.5. (G) Immunoperoxidase staining with anti-Six1 antibody (counterstained with hematoxylin) shows undetectable Six1 expression in E14.5 Six1−/− lungs. b; bronchus. Scale bars: 50 μm.
We thank C. Wigfall for editing the manuscript, This study was funded by NIH-NHLBI P01 HL 60231, HL 44060, HL44977 and GM096195 grants to DW as well as California Institute of Regenerative Medicine (CIRM) stem cell grants to DW and AHE.
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