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Tracheal cartilage provides structural support to the airway during breathing. Abnormal formation and patterning of ventrally located tracheal cartilage block the airway and cause life-threatening conditions, such as tracheomalacia and congenital tracheal stenosis. Although uncommon, these conditions are often associated with high morbidity and mortality in humans (Faust et al., 1998; Hasaniya et al., 2006; Rutter et al., 2006). Despite such clinical significances, the developmental process of tracheal cartilage and the pathogenesis of abnormal tracheal cartilage development remain unknown.
In mouse embryos, the trachea becomes surrounded by mesenchymal cells derived from the splanchnic mesoderm following the partitioning of the foregut into ventral trachea and dorsal esophagus on embryonic day (E) 11.5 along the dorso-ventral axis (Perl et al., 2002). Tracheal cartilage development begins with the commitment of these tracheal mesenchymal cells on the ventral trachea to the chondrocytic lineage followed by a sequence of chondrogenic events (McAteer, 1984). These chondrocyte-committed mesenchymal cells proliferate, aggregate, and condense into pre-cartilaginous nodules, which subsequently differentiate into chondrocytes and form cartilage (Tacchetti et al., 1992; de Crombrugghe et al., 2001). Sox9, a transcriptional factor containing the high-mobility-group (HMG) DNA-binding domain, is known to be required for each step of chondrogenesis. Inhibition of Sox9 prior to mesenchymal condensation completely inhibits cartilage formation, while Sox9 inhibition after mesenchymal condensation prevents chondrocyte differentiation of the condensed mesenchymal cells (Akiyama et al., 2002). The dosage of Sox9 has also been shown to be important for cartilage formation, as haploinsufficiency causes defective formation of cartilage primordia, leading to hypoplastic cartilage formation, including tracheal cartilage (Bi et al., 2001). Furthermore, Sox9 has been suggested as an important factor in specifying spatiotemporal patterning of tracheal cartilage primordia in early tracheal development (Elluru and Whitsett, 2004).
Sonic Hedgehog (Shh), a secreted glycoprotein expressed in the notochord and floor plate, is suggested to be a potential regulator of Sox9 in cartilage development. Presomitic mesoderm culture has shown that Shh promotes chondrogenesis of mesenchymal cells by inducing the expression of Sox9 (Zeng et al., 2002). Transgenic mice overexpressing Shh, specifically in chondrocytes, die at birth due to severe skeletal defects involving ribs, sternum, and limbs. These mice display excessive up-regulation of Sox9 expression in cartilage elements, leading to a delay in hypertrophic chondrocyte differentiation, which precedes bone formation. (Tavella et al., 2004). The role of Shh signalling and its regulation of Sox9 during chondrogenesis leading to tracheal cartilage formation, however, have not been clearly defined. More specifically, the molecular and cellular mechanisms that lead to normal patterning and formation of tracheal cartilage, and, in particular, regulatory interactions between Shh signalling and Sox9 in tracheal cartilage formation, are yet to be determined.
Shh transduces its signal by binding to its receptor protein, Patched-1 (Ptch-1) or Patched-2 (Ptch-2). The binding of Shh to Ptch activates transmembrane receptor complex Smoothened (Smo), which in turn leads to the activation of downstream Shh transcription factors Gli genes (Gli1, Gli2, and Gli3) (Villavicencio et al., 2000). Shh along with Gli genes play an essential role in growth and differentiation of foregut. Embryos with mutations in Shh and Gli genes exhibit foregut malformations ranging from development of hypoplastic lung and tracheo-esophageal tube, to the complete absence of trachea, esophagus, and lung (Litingtung et al., 1998). Despite these detailed characterizations of foregut and lung malformations including persistent embryonic tracheoesophageal tube (Litingtung et al., 1998), studies on tracheal cartilage development to date have been limited to gross morphologic descriptions of hypoplastic tracheal cartilage observed in Gli2−/− and Gli2−/−Gli3+/− mice (Motoyama et al., 1998). The essential requirement for Shh signalling in tracheal cartilage development is further supported by a recent report describing complete lack of cartilaginous rings in the foregut tube of Shh−/− embryos (Miller et al., 2004).
Here, we demonstrate that Shh signalling controls the patterning and formation of tracheal cartilage by spatiotemporal regulation of a chondrogenic gene, Sox9. We show that in Shh−/− embryos, the three sequential steps of chondrogenesis—proliferation, condensation, and chondrocyte differentiation—fail to occur, preventing tracheal cartilage development. The loss of Sox9 expression, a gene required for all chondrogenic events in Shh−/− embryos, results in defective chondrogenesis. Shh is essential for spatial and temporal restriction and maintenance of Sox9 expression during tracheal cartilage development as observed by altered timing and patterns of Sox9 expression in Shh−/− embryos. The blockage of hedgehog signalling using Shh inhibitor cyclopamine in in vitro tracheal culture leads to the absence of tracheal cartilage formation. In contrast, adding exogenous Shh peptide to in vitro foregut culture from Shh−/− embryos results in a rescue of both the formation and patterning of tracheal cartilage. Interestingly, treatment of a Shh−/− foregut and wild-type tracheal cultures with either exogenous Bmp4 or Noggin, known downstream regulators of chondrogenesis, partially rescues cartilage development or causes excessive tracheal cartilage formation, respectively. This was accompanied by the induction of Bmp4 and Sox9 expression, clearly implicating Shh signalling in the regulation of downstream chondrogenic genes during tracheal cartilage development. Taken together, we conclude that Shh signalling is a key initiator and critical regulator of tracheal cartilage formation and patterning during foregut development.
Tracheal Cartilage Formation Is Absent in Shh−/− Embryos
Tracheal cartilage is formed on the ventral side of trachea as a C-shaped ring and arranged in a segmented pattern along the anterior-posterior body axis (Fig. 1G,H,I). Alcian blue-PAS (AB-PAS) staining of wild-type tracheal sections showed that the trachea and esophagus are completely separated by E11.5 (Perl et al., 2002) (Fig. 1A), and semi-circular tracheal cartilage is formed by E15.5 (Fig. 1E, arrow). Mice embryos lacking Shh genes display persistent embryonic foregut tracheo-esophageal tube (Litingtung et al., 1998) (Fig. 1B,D,F). Whole-mount AB staining of the E16.5 Shh−/− tracheo-esophageal tube showed the absence of any tracheal cartilaginous ring formation other than an occasional isolated nest of cartilaginous cells identified by AB (Fig. 1J). These results are consistent with the previously suggested role of Shh in tracheal cartilage formation (Miller et al., 2004). The lack of tracheal cartilage in Shh−/− embryos was further confirmed by our AB-PAS-stained transverse section of E15.5 Shh−/− embryo (Fig. 1F). Thus, we conclude that Shh signalling is necessary for tracheal cartilage development.
Cellular Events Leading to the Absence of Chondrogenesis in Shh−/− Trachea
We next examined tracheal chondrogenesis in three well-known sequential phases in wild-type and Shh−/− embryos: proliferation of chondrocyte-committed mesenchymal cells, mesenchymal condensation, and cartilage differentiation. Proliferation of chondrocyte-committed mesenchymal cells was examined by double staining tracheal sections with BrdU-Sox9, an early marker of mesenchymal commitment to the chondrocyte lineage (Fig. 2A–D). In wild-type embryos, strong expression of Sox9 was observed in ventral tracheal mesenchyme at E13.5 and E14.5 (Fig. 2A,B). The continuous expression of Sox9 along the tracheal tube at E13.5 then became segmented by E14.5 (Fig. 2B). Concurrently, the segmentation of mesenchymal proliferation occurred at E14.5 (Fig. 2B). Proliferating Sox9-positive cells in the segmented pattern along the anterior-posterior axis mark the sites where tracheal cartilage forms in wild-type. In contrast, Sox9 expression was significantly reduced and only weakly expressed in the mesenchymal cells of E13.5 Shh−/− tracheo-esophageal tube (arrow, Fig. 2C) and expression of Sox9 was completely abolished by E14.5 (Fig.2D). Both the reduction in mesenchymal proliferation and loss of its segmented patterning were observed in mesenchyme of E14.5 Shh−/− tracheo-esophageal tubes (Fig. 2D). Therefore, Shh signalling regulates both proliferation of mesenchymal cells on the ventral side of future trachea and specific patterning along the anterior-posterior axis by modulating the number of mesenchymal cells that are committed to become chondrocytes.
To determine if the process of condensation of mesenchymal cells and the formation of a precartilaginous nodule are affected in the absence of Shh, we next examined the embryos using peanut agglutinin (PNA) staining, a lectin molecule that stains condensing mesenchymal cells (Gillotte et al., 2003). Sagittal and transverse sections of E14.5 wild-type embryos showed PNA-positive signals in the segments of tracheal cartilage primordia and in ventral tracheal mesenchyme, respectively (Fig. 2E,F). PNA signal was, however, no longer detectable at E15.5 (data not shown), indicating that mesenchymal condensation is completed prior to E15.5 in wild-type. Condensed mesenchymal cells differentiated into chondrocytes and formed tracheal cartilage, as shown by AB-PAS staining of E15.5 wild-type embryos (Fig. 2J). Neither mesenchymal condensation (Fig. 2G,H) nor chondrocyte differentiation (Fig. 2L) was detected in the Shh−/− embryonic tracheo-esophageal tube, consistent with the absence of tracheal cartilage formation. Therefore, Shh signalling is required for all three stages of chondrogenesis in tracheal cartilage development.
Chondrogenic Genes Require Shh Signalling for Their Expressions
To determine if the failure to form cartilage in the tracheosophageal tube of Shh−/− embryos was due to downregulation of chondrogenic genes, we next examined the expression of Sox9 and Col2a1 genes, which have been previously shown to be required for cartilage formation and patterning in tracheal cartilage (Krengel et al., 1996; Akiyama et al., 2002; Elluru and Whitsett, 2004). Since Sox9 expression in Shh−/− tracheo-esophageal tube was either unclear or not detectable at E13.5 and E14.5 (Fig. 2C,D), we questioned whether the absence of Shh signaling in Shh−/− tracheo-esophageal tube completely reduced the expression of Sox9 or if Sox9 expression was initially expressed but abolished at the time of chondrogenesis. To answer this question, the expressions of Sox9 and Col2a1 in both wild-type and Shh−/− tracheas were examined on E11.5, E13.5, and E15.5 by in situ hybridization.
In wild-type embryo, Sox9 expression was strongly detected in the ventral tracheal mesenchyme beginning at E11.5 (Fig. 3A) and this ventral-specific expression of Sox9 persisted through E15.5, when the chondrocyte differentiation and cartilage formation were completed (Fig. 3C,E). Sox 9 expression was readily detectable in the tracheal cartilage well after chondrogenesis on E17.5 (see Supp. Fig. S1A, which is available online). Sagittal sections of wild-type embryos showed continuous Sox9 expression along the anterior-posterior axis only in the ventral tracheal mesenchyme at E12.5 (Fig. 3G), which then became segmented on E13.5 (Fig. 3H). In the mesenchyme of Shh−/− tracheo-esophageal tube, Sox9 was transiently expressed from E11.5 to E13.5 (Fig. 3B,D); its expression was completely lost by E15.5 (Fig. 3F) and was not detectable at E17.5 (Supp. Fig. S1B). Although Sox9 expression was weakly detectable at E11.5 and E13.5, the ventral restriction of Sox9 expression pattern was disrupted and instead a circumferential expression pattern on both the dorsal and ventral sides of the tracheo-esophageal tube in Shh−/− embryos was observed (arrows, Fig. 3B,D). Transient early expression and expression patterning of Sox9 in Shh−/− tracheo-esophageal tubes during chondrogenesis suggest that Shh signalling is not only required for initiation of Sox9 expression but is necessary for its maintenance and the ventral-specific expression of Sox9.
The Col2a1 expression pattern overlapped with the expression of Sox9 in developing wild-type trachea as previously described (Elluru and Whitsett, 2004), (Fig. 3I–P). Col2a1 expression is known to be essential for differentiation of condensed mesenchymal cells into chondrocytes and subsequent formation of the cartilage during the later stage of chondrogenesis (Barbieri et al., 2003). In the mutant tracheo-esophageal tube, unlike Sox9 expression, Col2a1 expression was not detectable between E11.5 and E15.5 (Fig. 3J,L,N). Therefore, the lack of Col2a1 expression, together with the loss of Sox9 expression in the Shh−/− tracheo-esophageal tube, account for the failure of both chondrogenesis and subsequent tracheal cartilage formation observed in Shh−/− embryos.
Increased Apoptosis and Reduced Proliferation of Sox9-Expressing Cells and Decrease in Gli1 Expression Are Attributable to the Loss of Sox9 Expression in Shh−/− Foregut
Shh signalling promotes cellular proliferation (Dahmane et al., 2001; Sanchez et al., 2004) and inhibits apoptosis (Thibert et al., 2003). To determine whether the absence of chondrogenesis due to the lack of Sox9-positive mesenchymal cells in Shh−/− embryos is due to lack of proliferation or excessive loss of the committed cells, we examined mesenchymal proliferation and apoptosis using BrdU and TUNEL assays. Our BrdU and TUNEL assays show that there is a significant reduction in proliferation of mesenchymal cells and an increase in the number of apoptotic cells in the Shh−/− tracheo-esophageal mesenchyme compared to the wild-type (Fig. 4). We then asked whether such a decrease in proliferation and increase in apoptosis in the Sox9-expressing mesenchymal cells accounted for the loss of Sox9 expression observed in the Shh−/− tracheo-esophageal mesenchyme. BrdU-Sox9 double-staining of wild-type embryos showed a number of Sox9-expressing mesenchymal cells that were proliferating in the ventral mesenchyme of trachea (yellow arrow, Fig. 4A). However, in Shh−/− embryos, although weak Sox9 expression was circumferentially found in the tracheo-esophageal mesenchyme, the majority of these cells were not proliferating (Fig. 4B). In addition, our TUNEL-Sox9 double-staining showed that in wild-type embryos, none of the Sox9-expressing cells were undergoing detectable apoptosis (Fig. 4C). In contrast, many of the Sox9-expressing mesenchymal cells were undergoing cell-programmed death in Shh−/− embryos (arrow, Fig. 4D). Taken together, the loss of Shh signalling results in increased cell death and reduced proliferation of Sox9-expressing cells, which contributes to the failure of maintaining Sox9 expression past its initial expression.
Gli1, one of the downstream transcriptional factors of Shh, up-regulates Sox9 expression by directly binding to a putative binding site within Sox9cre1, a cis-acting regulatory element 1.1 Mb upstream of Sox9 (Bien-Willner et al., 2007). We have shown that Sox9 expression was detected at E11.5 but was lost beyond E13.5 in the Shh−/− tracheo-esophageal tubes (Fig. 3B,D,F). To determine whether the Gli1 expression level in Shh−/− embryos correlates with the apparent reduction in Sox9 expression, we measured the expression of Gli1 in E11.5 and E13.5 wild-type trachea and the Shh−/− tracheoesophageal tube using real-time PCR. Interestingly, compared to Gli1 expression in wild-type at E11.5 and E13.5, there was a 50% reduction of Gli1 expression at E11.5, and up to 90% reduction of Gli1 expression was noted at E13.5 in the Shh−/− tracheoesophageal tube (Fig. 4E). This result suggests that Sox9 expression during tracheal cartilage development requires Gli1 and this regulatory interaction may be dose-dependent.
Shh Signalling Regulates Ventralization of Sox9 Expression
Chondrocyte-committed tracheal mesenchymal cells show ventral-restriction of Sox9 expression prior to chondrogenesis (Fig. 3A). Our in situ result, however, showed ectopic expression pattern of Sox9, which was found circumferentially around the Shh−/− tracheo-esophageal tube both at E11.5 (Fig. 3B) and E13.5 (Fig. 3D). Therefore, we questioned whether adjacent respiratory epithelial identity expressing Shh regulates the ventral restriction of Sox9 expression, thus determining the location of tracheal cartilage formation. First, we examined whether circumferential Sox9 expression detected around the mutant foregut tube was due to the disrupted epithelial identity in persistent embryonic tracheoesophageal tube in Shh−/− embryos. We, therefore, double-stained E13.5 wild-type and Shh−/− tracheal sections with Sox9 and TTF-1, a respiratory epithelium marker. Thyroid transcription factor-1 (TTF-1), also known as Nkx2.1, is a homeodomain transcriptional factor that is expressed in tracheal epithelium and in lungs (Minoo et al., 1999). The double-labelling in the wild-type section showed circumferential expression of TTF-1 in the tracheal epithelium (arrow), while Sox9 expression was confined to the ventral and lateral mesenchyme of the wild-type trachea (arrowhead), marking the place where tracheal cartilage forms at E13.5 (Fig. 5A). In contrast, TTF-1 expression was detected on both ventral and lateral sides of the tracheo-esophageal epithelium, but its expression was not completely circumferential in Shh−/− embryos (arrow, Fig. 5B), indicating that the tube is comprised of both trachea (TTF-1-positive) and esophagus (TTF-1-negative). Subsequent Sox9 immunostaining on a TTF-1-stained section showed that Sox9 expression was extended into the area where TTF-1 expression was absent (TTF-1 negative: yellow arrow, Fig. 5C). Thus, Sox9 was expressed (black arrow, Fig. 5C) in mesenchyme of presumptive esophagus. In wild-type, Sox9 expression was not detected in the esophageal mesenchyme (Fig. 5A). Therefore, in the absence of Shh signalling, ventral restriction of Sox9 expression was altered and Sox9 expression was found in the non-respiratory area of the tracheo-esophageal tube. These results suggest that Shh signalling regulates the patterning of tracheal cartilage by restricting ventral-specific expression of Sox9 in the tracheal mesenchyme.
Shh Signalling Is Required for Induction of Tracheal Cartilage Formation In Vitro
To verify the direct requirement of Shh signalling in tracheal cartilage development, we used an in vitro tracheal culture system where we inhibited hedgehog signalling using cyclopamine (a plant alkaloid that interferes with Hh signaling) (Chen et al., 2002; Nagase et al., 2005). We cultured E10.5 wild-type tracheal-lung explants with either cyclopamine or tomatidine control, an alkaloid structurally related to cyclopamine but without the inhibitory effect on Hh signalling (Cooper et al., 1998), for 7 days. AB-TTF-1 staining of the cyclopamine-treated E10.5 wild-type tracheal explant showed a complete absence of tracheal cartilage (Fig. 6B), while the tomatidine-treated explant formed tracheal cartilage in the ventral tracheal mesenchyme (arrow, Fig. 6A). This result is consistent with our in vivo results showing that in the absence of Shh signalling, tracheal cartilage formation does not occur.
We next asked whether treatment with exogenous Shh peptide would rescue tracheal cartilage phenotype observed in Shh−/− embryos. After 7 days of culturing E10.5 Shh−/− tracheal explants with or without Shh peptide, the presence of tracheal cartilage was studied using AB staining. Treating the tracheal explant with Shh peptide resulted in the rescuing of tracheal cartilage formation (arrow, Fig. 6D), while untreated samples did not form any tracheal cartilage (Fig. 6C). We further stained the sections with TTF-1 to investigate whether spatial patterning of tracheal cartilage was also restored. Interestingly, TTF-1 staining showed that the foregut tube from Shh−/− embryos partitioned into esophagus and trachea in the presence of exogenous Shh, and tracheal cartilage formation was preferentially induced around and adjacent to the TTF-1-positive tracheal tube (arrowhead), indicating a spatial restriction of chondrogenesis induced by exogenous Shh (Fig. 6D). Although the tracheal cartilage formed was not semicircular as it is in vivo, tracheal cartilage formation was rescued, verifying a direct role for Shh signalling in inducing and regulating tracheal cartilage formation.
Exogenous Bmp4 and Noggin Rescue Tracheal Cartilage Formation in the Absence of Shh Signalling In Vitro
Bone morphogenetic proteins (BMPs) and their antagonist, Noggin, have been implicated in regulating cartilage and foregut development. Bmp4 is known as a pro-chondrogenic gene, while Noggin is known as an anti-chondrogenic gene acting as an antagonist to Bmp4 (Weaver et al., 1999, 2003; Que et al., 2006). We, therefore, examined the expression of these two genes in transverse tracheal sections of E11.5 wild-type and Shh−/− embryos to study how their expression is affected in the absence of Shh signalling. Bmp4 and Noggin expression was detected in ventral and lateral mesenchyme and dorsal and lateral mesenchyme of wild-type trachea, respectively, while expression of both genes was significantly reduced, if not absent, in the Shh−/− foregut tube (Supp. Fig. S2).
To investigate whether the loss of Bmp4 or Noggin expression plays a role in the failure of tracheal cartilage formation in the Shh−/− tracheoesophageal tube, we attempted to rescue the cartilage phenotype in Shh−/− foregut explants by treating the culture explants with either exogenous Bmp4 or Noggin peptide. After 7 days, the explants were sectioned and stained with AB and TTF-1 antibody to study both formation and patterning of tracheal cartilage. Interestingly, both culture explants treated with either Bmp4 (Fig. 7B) or Noggin (Fig. 7C) resulted in a partial rescue of tracheal cartilage phenotype (arrows, Fig. 7B,C). In both cases, however, tracheal cartilage formation occurred in TTF-1-negative areas (arrowheads). This suggests that although exogenous Bmp4 and Noggin are sufficient to partially induce chondrogenesis in the absence of Shh, Shh is required for both the formation and patterning of tracheal cartilage.
Since exogenous Noggin had unexpectedly rescued tracheal cartilage formation, we questioned whether treating wild-type tracheal explants with exogenous Noggin would likewise result in ectopic tracheal cartilage formation. After 7 days of culturing, our AB-TTF-1 double-staining showed that, compared to the control (Fig. 7D), treating the trachea with exogenous Bmp4 had led to excessive tracheal cartilage formation (Fig. 7E). Specifically, cartilage formation occurred circumferentially around the trachea with the Bmp4 treatment (Fig. 7E), as compared to ventrally-restricted cartilage formation in the control trachea (Fig. 7D). Noggin treatment also caused excessive tracheal cartilage development (Fig. 7F) in a similar manner to tracheal explants treated with Bmp4 peptide. In order to confirm the pro-chondrogenic effects of exogenous Bmp4 and Noggin peptides on the tracheal cultures, we examined the expressions of two chondrogenic genes, Sox9 and Bmp4, in wild-type tracheal cultures. As expected from the AB staining, Sox9 expression was clearly detected in tracheal cartilage to a similar extent in all three conditions (arrows, Fig. 7G,H,I). Interestingly, in all three conditions, Bmp4 was clearly expressed in tracheal cartilage (arrows, Fig. 7J,K,L), confirming the pro-chondrogenic effects of not only Bmp4, but also Noggin peptides in in vitro tracheal cultures. Taken together, these results indicate that both Bmp4 and Noggin genes downstream of Shh promote cartilage formation in trachea through regulation of chondrogenic genes, Bmp4 and Sox9.
In this study, we hypothesized that Shh signalling plays an essential role in tracheal cartilage development. We examined how cellular and genetic events control tracheal cartilage development and how they are disrupted in the absence of Shh signalling, leading to the absence of tracheal cartilage formation. We showed that spatial and temporal restriction of Sox9 expression correlating with Shh transcription factor Gli1, accompanied by corresponding cellular changes such as reduced proliferation and increased apoptosis, account for the absence of tracheal cartilage in Shh−/− embryos. Inhibiting Shh signalling in vitro with Shh inhibitor cyclopamine resulted in complete inhibition of tracheal cartilage formation in wild-type trachea mimicking the in vivo phenotype, while exogenous Shh peptide rescued the cartilage phenotype in the Shh−/− tracheo-esophageal tube. Moreover, exogenous Bmp4 and Noggin induced chondrogenesis in the absence of Shh without restoring patterning of chondrogenesis. Based on our findings, we conclude that Shh signaling is required for both formation and patterning of tracheal cartilage.
Shh Signalling Is Essential for the Formation of Tracheal Cartilage
Absence of tracheal cartilage in Shh−/− embryos, the failure of tracheal cartilage development in the cyclopamine-treated wild-type tracheal explants, and rescue of tracheal cartilage defect in Shh−/− tracheal explants by exogenous Shh indicate the requisite role of Shh in tracheal cartilage development. In the absence of Shh signaling, all three stages of chondrogenesis were affected and this was accompanied by corresponding lack of Sox9 expression (Fig. 2D). Sox9 has been previously shown to be required for chondrogenesis and without its expression, both mesenchymal condensation and chondrocyte differentiation do not occur (Akiyama et al., 2002). Therefore, the observed defective chondrogenic processes in persistent tracheo-esophageal tube in Shh−/− embryo are likely due to the absence of Sox9 expression during chondrogenesis and Shh signalling regulates tracheal cartilage formation by maintaining Sox9 expression. The loss of Sox9 expression was confirmed by our in situ results. Given that Sox9 directly interacts with and up-regulates Col2a1 (Bell et al., 1997), the lack of sufficient Sox9 expression may also account for the undetectable Col2a1 expression in E11.5 Shh−/− tracheo-esophageal tube (Fig. 3J). The lack of expression of Col2a1, which is required for the formation of cartilage cells (Barbieri et al., 2003), contributed further to the absence of tracheal cartilage formation in Shh−/− embryos.
We showed a reduction in the expression level of Gli1 in the E11.5 and E13.5 Shh−/− tracheo-esophageal fistula by 50 and 90%, respectively. This result suggests that, in the absence of Shh, Gli1expression is temporally regulated. A significant decrease in the Gli1 mRNA expression level at E13.5 correlated to the changes we saw in Sox9 expression, which also was lost beyond E13.5. This result is in agreement with the previously known function of Gli1, which directly interacts with Sox9 to up-regulate its expression (Bien-Willner et al., 2007). Therefore, the maintenance of Sox9 expression during tracheal cartilage formation likely requires both continuous activation by Gli1 and a dose-dependent response by Sox9 expression to promote cartilage formation in trachea. Expression of Gli1 at E11.5, although reduced by 50%, also opens up the possibility of other potential activators of Gli1 other than Shh itself, which is yet to be identified. In fact, Gli1 expression in Shh−/− embryo has previously been reported (Bai et al., 2002). Gli1 expression, although weaker than in wild-type, is detected in Shh−/− gut including foregut at E10.5, and Indian hedgehog (Ihh) is suggested to be a potential alternate activator of Gli1 expression in the absence of Shh signaling (Bai et al., 2002). However, our immunostaining of Ihh expression indicates the absence of Ihh expression in Shh−/− foregut (data not shown), further implicating additional potential regulators other than the Hedgehog (Hh) family genes.
The Shh signalling pathway has previously been suggested to regulate cellular apoptosis and proliferation. Treating proliferating brain and prostate tumor cells with cyclopamine inhibited the mitogenic effects of Shh signalling (Dahmane et al., 2001; Sanchez et al., 2004). Moreover, Shh signalling was shown to inhibit apoptosis by blocking Ptch-induced apoptosis in the chick spinal cord development (Thibert et al., 2003). Likewise, our results confirmed both pro-proliferative and anti-apoptotic roles of Shh signalling, which ultimately culminates in producing a sufficient number of mesenchymal cells committed to develop tracheal cartilage in wild-type. In the absence of Shh signaling, however, both of these processes were affected, and by E13.5 the number of mesenchymal cells was significantly reduced (data not shown), impeding tracheal cartilage development. Therefore, Shh signalling appears to directly regulate Sox9 expression through Gli1 and, secondarily, through its pro-proliferative and anti-apoptotic roles, which ultimately increases the number of Sox9-expressing cells.
Shh Signalling Regulates Ventralization and Segmentation of Tracheal Cartilage
Shh signalling has been implicated in the patterning of many organs during embryogenesis, such as limbs (Lopez-Martinez et al., 1995), central nervous system (Schell-Apacik et al., 2003), and hair follicles (Mill et al., 2003). Here, we investigated the role of Shh signalling in segmentation and ventral specification of tracheal cartilage.
Proliferation in ventral tracheal mesenchyme became segmented in wild-type embryos, prior to tracheal cartilage formation (Fig. 2B), indicating the potential role of cellular proliferation in segmental patterning of tracheal cartilage. On the other hand, TUNEL staining of wild-type sagittal tracheal sections from E13.5 to E15.5 did not show notable apoptotic cells in tracheal mesenchyme (data not shown). This suggests that apoptosis may not play a direct or dominant role in segmentation of wild-type tracheal cartilage. In Shh−/− embryos, however, although much reduced, mesenchymal proliferation in the tracheo-esophageal tube was still detectable at E14.5, while ventral-specific and segmented patterning of proliferation was lost and increased apoptosis was detected in a non-specific pattern (data not shown). These results suggest that Shh signalling may regulate tracheal cartilage patterning, in part, by controlling the specific patterning of mesenchymal proliferation in trachea.
We demonstrated that Shh signalling controls patterning of tracheal cartilage by regulating the expression pattern of Sox9 expression. Our in situ results showed that Sox9 expression was detected in ventral tracheal mesenchyme and became segmented at the onset of chondrogenesis. Such expression sites coincide with the sites where tracheal cartilage forms. In Shh−/− embryos, ventral-specific expression of Sox9 was disrupted (Fig. 3D) and was found in non-respiratory area of tracheo-esophageal tube (Fig. 5C). We could not assess whether segmentation of Sox9 expression was also disrupted in Shh−/− embryos since Sox9 expression was already significantly reduced by E13.5, when Sox9 expression became segmented. Thus, our results suggest that Shh signalling controls the ventral specification of tracheal cartilage through regulation of the expression pattern of the prechondrogenic gene, Sox9.
Whether Shh signalling directly or indirectly regulates the ventralization of Sox9 still needs to be answered. It is likely that Shh signalling regulates ventralization of Sox9 expression through induction of bone morphogenetic protein 4 (Bmp4). Bmp4 and Shh are known to regulate each other through a feedback loop. In mouse lung mesenchyme, Bmp4 clearly is activated by Shh signaling (Weaver et al., 2003) and Bmp4 expression is down-regulated in Shh−/− (Litingtung et al., 1998). Bmp4 also plays a ventralizing role in development (Que et al., 2006). Therefore, Shh signalling likely activates Bmp4, which then controls ventral restriction of Sox9 expression in developing trachea. The fact that the region of Bmp4 expression in wild-type tracheal mesenchyme at E11.5 overlaps with Sox9 expression site (Supp. Fig. S2A), indicates a likely interaction between the two genes. This is further supported by the significant loss of Bmp4 expression in the Shh−/− tracheo-esophageal tube (Supp. Fig. S2B) where ventral restriction of Sox9 expression is also disrupted. The expression of Noggin in dorsal mesenchyme of wild-type trachea at E11.5 was also greatly reduced, if present, in the absence of Shh signalling (Supp. Fig. S2D). Considering the dorsalizing role of Noggin (Que et al., 2006), dorsal restriction of Noggin expression in conjunction with ventralization of Bmp4 expression ensures the absence of Bmp4 expression in the dorsal mesenchyme of trachea and corresponding ventral restriction of Sox9 expression. Taken together, these data suggest that, while Shh signalling may directly up-regulate Sox9 expression through its downstream gene, Gli1, it may indirectly induce ventralization of Sox9 expression through activation of Bmp4. In addition to its ventralizing role, Bmp4 is also known to up-regulate Sox9 expression (Shum et al., 2003) and, thus, Bmp4 activation by Shh may serve as an indirect means to ensure adequate expression of Sox9. And the loss of such Bmp4 expression in the absence of Shh signalling is likely a contributing factor in the failure of maintaining Sox9 expression in the Shh−/− tracheo-esophageal tube.
Bmp4 and Noggin in Tracheal Cartilage Development
Bmp4 and its coordinated expression with Noggin are suggested to be required for proper cartilage development (Nifuji and Noda, 1999; Tsumaki et al., 2002). Bmp4 activates Sox9, which subsequently turns on Noggin. Noggin then inhibits Bmp4, ultimately limiting the size and position of developing cartilage (Capdevila and Johnson, 1998; Pathi et al., 1999; Zehentner et al., 2002). To examine the relative contribution of Noggin and Bmp4 in the absence of Shh in tracheal cartilage development, we attempted to rescue the tracheal cartilage phenotype by supplementing Shh−/− tracheal explants with exogenous Bmp4 or Noggin peptide. In this study, we studied the role of these two genes in tracheal cartilage development. Surprisingly, both exogenous Bmp4 and Noggin rescued tracheal cartilage formation in Shh−/− tracheal explants but in non-respiratory areas. Treatment of wild-type tracheal explants with Bmp4 induced excessive and nearly circumferential formation of tracheal cartilage compared to the control. This is consistent with the previously known function of Bmp4, which was to enhance chondrogenesis and promote cartilage generation (Duprez et al., 1996; Pizette and Niswander, 2000). Unexpectedly, exogenous Noggin also induced cartilage formation, and the cartilage formation was spatially unrestricted in a circumferential manner. Noggin has previously been known for its anti-chondrogenic role. For example, adding Noggin to limb explant also inhibited the level of chondrogenesis (Nifuji et al., 2004). Despite such anti-chondrogenic functions of Noggin, an increase in Bmp4 level upon induction of Noggin misexpression has previously been shown in chick limbs (Pathi et al., 1999). It was suggested that Bmp4 undergoes an autoregulatory negative feedback loop, which was then possibly eliminated by Noggin misexpression, thus partially rescuing the inhibitory effect of Noggin on Bmp4 (Pathi et al., 1999). Likewise, it is possible that exogenous Noggin in Shh−/− tracheal explant eliminated the autoregulatory negative feedback of Bmp4, and partially restored Bmp4 expression, and rescued tracheal cartilage formation. Such mechanisms may also be responsible for the excessive cartilage formation observed in Noggin-treated wild-type culture. In order to check this possibility, we stained wild-type tracheal culture sections with Bmp4. Bmp4 immunostaining clearly showed Bmp4 expressions in cartilages of all three conditions (control, Bmp4- and Noggin-treated cultures) to the similar extent (Fig. 7J,K,L). This result strengthens the modulating role of exogenous Noggin in an interfering autoregulatory feedback loop of Bmp4, which compensates for the inhibition of Bmp4 expression by Noggin. However, the exact genetic interaction of Noggin with Shh, Bmp4, and Sox9 in chondrogenesis, which leads to tracheal cartilage formation, is yet to be determined.
In conclusion, we show that Shh signalling is necessary and critical in the formation and patterning of tracheal cartilage by maintaining the Sox9 expression level and by regulating the expression sites of Sox9. The regulation of Sox9 expression is done both directly through Gli1 and indirectly possibly through interaction with another chondrogenic gene such as Bmp4. Proper patterning and formation of tracheal cartilage are critical for the formation of a functional airway system, which otherwise would result in serious clinical complications. We believe that our findings shed light on tracheal cartilage repair and regeneration studies by demonstrating that Shh signalling is a key gene that is responsible for the ventralization and segmentation of tracheal cartilage and its formation.
Shhtm1Amc mice (Shh+/−), purchased from Jackson Laboratories (Bar Harbor, ME), were time mated. The genotypes of heterozygous embryos were determined using polymerase chain reaction (PCR), while the homozygous embryos were determined by phenotype.
Embryos were fixed in 4% paraformaldehyde, dehydrated with increasing ethanol concentration, and embedded in paraffin wax. Paraffin sections (6–7 μm) were dewaxed, rehydrated, and stained with either 50% hematoxylin and 0.5% eosin in 70% ethanol, or 1% Alcian blue (pH 2.5) in 3% glacial acetic acid and then with 0.5% periodic acid, followed by treatment with Schiff's reagent for Alcian-blue/PAS staining.
Quenching of the endogenous peroxidases in 3% H2O2 in 10% methanol was followed by antigen retrieval in antigen unmasking solution (H-3300, Vector Laboratories, Burlingame, CA). The sections were blocked with blocking reagent (Roche, Nutley, NJ) for 1 hr and incubated with the following primary antibodies at 4°C overnight: rabbit anti-Sox9 (1:200, a kind gift from Dr. Wegner), mouse anti-TTF-1 (1:50, Lab Vision, Fremont, CA), rabbit anti-Bmp4 (1:200, Abcam, Cambridge, MA), and goat anti-Noggin (1:100, Santa Cruz Biotechnology, Santa Cruz, CA). Slides were incubated with secondary antibodies at 1:200 dilutions. The reaction product was visualized by either incubating with avidin-biotin-peroxidase complex (ABC) solution followed by substrate diaminobenzidine (DAB) staining, or by observing slides under a fluorescence microscope after mounting the slides with DAPI-containing mounting media (Vector Laboratories).
For peanut agglutinin staining, the slides were incubated with FITC-conjugated peanut agglutinin (1:150 in PBS) (Sigma, St. Louis, MO) for 1 hr at RT and then mounted with DAPI-containing mounting media and observed under a fluorescent microscope.
In Situ Hybridization
In situ hybridization was performed as described (Hui and Joyner, 1993). The slides were fixed in 4% paraformaldehyde, permeabilized with proteinase K in 1×PBS (0.02 mg/ml) and refixed in 4% paraformaldehyde. The slides were further treated in 0.2M HCl and then in 0.1M triethanolamine (TEA) with 0.025 ml acetic anhydride/liter of TEA. The slides were then hybridized with DIG-labeled RNA probes (DIG labelling mix, Roche) at 55°C overnight. The next day, the slides were washed in 5× SSC and 2× SSC/50% formamide solution. Following treatment of slides with RNAse A, the slides were washed in 2× SSC and 0.2× SSC. The slides were then blocked with 1× blocking reagent (Roche) for 1 hr and treated with anti-DIG alkaline phosphatase antibody (1:2,000, Roche) for 1 hr. BM Purple AP substrate (Roche) was used for the development of colour. The following cloned plasmids were used as templates for generating DIG-labeled RNA probes: Sox9 was kindly provided by Dr. V. Vidal and Col2a1 by Dr. B. de Crombrugghe.
Proliferation and Apoptosis Assay
5-bromo-2′deoxyuridine (BrdU) was injected into a pregnant mouse (100 μM BrdU/g of animal weight). The sections were treated with anti-BrdU IgG1 antibody (1:20, Becton Dickinson, Franklin Lakes, NJ), and then with an appropriate secondary antibody. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling (TUNEL) assay was used to study cellular apoptosis on paraffin sections using DeadEnd™ Fluorometric TUNEL kit (Promega, Madison, WI).
Sox9-BrdU double-immunostaining was performed simultaneously. Slides were incubated with a mixture of Sox9 (1:200) and BrdU (1:20), followed by incubation with a mixture of appropriate secondary antibodies. Sox9-TUNEL double-immunostaining was performed sequentially. Following Sox9 immunostaining, the slides were further treated according to TUNEL staining protocol.
RNA Extraction, cDNA Synthesis, and qPCR
Total RNA from tracheal cultures was extracted using an RNeasy Mini Kit (Qiagen, Valencia, CA) according to the instructions provided with the kit. cDNA was then synthesized with 1 μg of RNA using a SuperScript™ II First-Strand Synthesis Kit (Invitrogen, Carlsbad, CA). It was then purified utilizing a QIAquick PCR Purification Kit (Qiagen), respectively. A quantitative polymerase chain reaction (qPCR) of Gli1 was performed using a self-designed primer and the following conditions: 5′GAAGGAATT CGTGTGCCATT3′, 5′GCAACCTTC TTGCTCACACA3′, T: 51.1°C. The results were then analyzed using Pfaffl's methods (Pfaffl, 2001).
Tracheal-Lung Organ Culture
Lung and trachea were dissected out from E10.5 wild-type embryos and cultured on cell culture inserts (1.0-μm pore size, Falcon, Colorado Springs, CO) in serum- free BGJb Medium (Gibco, Gaithersburg, MD). The culture medium was supplemented with transferrin (1 μg/ml), sodium ascorbate (1 mg/ml, Sigma), and antibiotics. Tracheal-lung explants were treated with either cyclopamine, a Shh inhibitor (10 μg/ml diluted in DMSO, Toronto Research Chemicals, North York, Ontario, Canada), or tomatidine (10 μg/ml diluted in DMSO, Sigma), and incubated in 95% air, 5% CO2 at 37°C. The medium was changed every other day. This culture protocol was modified from Litingtung et al. (1998). In the rescue experiments, tracheal explants were treated with Shh (1.0 μg/ml, R&D Systems, Minneapolis, MN), Bmp4 (300 ng/ml, R&D Systems), or Noggin peptide (300 ng/ml, R&D Systems).
Jinhyung Park is a recipient of a RESTRACOMP (Research Training Competition).