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Smooth muscle in the lung is thought to derive from the developing lung mesenchyme. Smooth muscle formation relies upon coordination of both autocrine and paracrine signaling between the budding epithelium and adjacent mesenchyme to govern its proliferation and differentiation. However, the pathways initiating the earliest aspects of smooth muscle specification and differentiation in the lung are poorly understood. Here, we identify the Wnt2 ligand as a critical regulator of the earliest aspects of lung airway smooth muscle development. Using Wnt2 loss and gain of function models, we show that Wnt2 signaling is necessary and sufficient for activation of a transcriptional and signaling network critical for smooth muscle specification and differentiation including myocardin/Mrtf-B and the signaling factor Fgf10. These studies place Wnt2 high in a hierarchy of signaling molecules that promote the earliest aspects of lung airway smooth muscle development.
Disruption to lung smooth muscle development and function in humans can lead to chronic health complications including asthma and pulmonary hypertension (Camoretti-Mercado et al., 2003; Halayko et al., 1998; Halayko and Stephens, 1994; Humbert et al., 2004; Solway et al., 1998). Understanding the signaling pathways coordinating the various specification and differentiation processes occurring during smooth muscle development are critical to a complete understanding of lung development as well as pathogenesis in the postnatal lung. In the early lung bud, the condensing lung mesenchyme is thought to generate several cell lineages including smooth muscle cells, endothelial cells, and resident fibroblasts such as myofibroblasts, lipofibroblasts, and stromal fibroblasts (reviewed in (Morrisey and Hogan, 2010). At the level of the mainstem bronchi, smooth muscle develops partially around the airway on the dorsal surface with cartilaginous rings forming on the ventral surface. Slightly more posterior in the proximal airways, smooth muscle forms around the entire epithelium while the most distal portion of the respiratory tree lacks smooth muscle (Tollet et al., 2001, 2002). Airway smooth muscle development (ASM) closely follows the growth and differentiation of the airway epithelium temporally and spatially. At E11.5 in the mouse, a smooth muscle layer starts to envelop the trachea and proximal bronchi and continues to develop around the proximal airway tubules as lung development progresses (Badri et al., 2008). Current theories suggest that ASM originates from a distal pool of Fgf10-expressing mesenchymal cells that surrounds the tip of the branching airway epithelium (Mailleux et al., 2005; Ramasamy et al., 2007). These distal cells proliferate and actively or passively translocate to surround the proximal airways undergoing epithelial tube outgrowth and branching.
Several signaling pathways are known to be critical in lung smooth muscle development including FGF, BMP, Shh, and Wnt signaling. Fgf10 signaling is important for ASM development and mediates this process in part, through signaling interactions with the epithelium. Fgf10 signaling from the mesenchyme activates a gene expression program in the adjacent developing epithelium, which signals in a reciprocal fashion back to the mesenchyme to direct smooth muscle differentiation (Mailleux et al., 2005). The pro-myogenic signals expressed in the developing airway epithelium include Shh and Bmp4 and these factors direct smooth muscle differentiation along the branching epithelial tubule as Fgf10-expressing cells condense around the primitive airway (Mailleux et al., 2005). Fgf9 signaling from the mesothelium is also reported to mediate the development of immature smooth muscle cells in the distal mesenchyme by limiting the differentiation response of the mesenchyme to Shh signaling (Colvin et al., 2001).
Published data indicate that Wnt/β-catenin signaling is active and plays a role during smooth muscle development in the lung. From E14.5 to E18.5, Wnt/β-catenin signaling activity is observed in the developing ASM (Shu et al., 2005). Wnt7b is a canonical Wnt ligand expressed in the developing epithelium, and Wnt7b knockout mice display perinatal pulmonary hemorrhaging due to disrupted vascular smooth muscle (VSM) development (Shu et al., 2002). Further investigation into the mechanisms of Wnt7b signaling revealed that Wnt7b broadly regulates the development of smooth muscle cells in the early lung bud mesenchyme (Cohen et al., 2009). Additionally, treating lung explants with the canonical Wnt signaling inhibitor Dickkopf-1 (Dkk-1) impairs smooth muscle gene expression (De Langhe et al., 2005).
Wnt2 is a ligand robustly expressed throughout the lung mesenchyme during the course of lung development. Wnt2 is first expressed in the mesoderm surrounding the anterior foregut endoderm from E9.0–E9.5 during the period when the lung is specified, and Wnt2 continues to be expressed in the mesoderm condensing around the early lung bud to form the primitive lung mesenchyme with expression persisting up to perinatal timepoints (Goss et al., 2009; Monkley et al., 1996). The expression pattern of Wnt2 suggests that it may play a role in the development of mesenchymal-derived lineages in the lung. Previous work has shown that Wnt2, in cooperation with Wnt2b, is essential for specification of the respiratory lineage in the anterior foregut endoderm (Goss et al., 2009). However, whether Wnt2 plays a role in regulating mesenchymal lineage differentiation in the lung is unclear. In this report, we show that a loss of Wnt2 leads to a severe deficiency in ASM development in the lung. We show that Wnt2 acts upstream of Fgf10 and the critical transcription factors myocardin and Mrtf-B to regulate early ASM differentiation in the multipotent lung mesenchyme. Moreover, Wnt2 acts in a paracrine manner to promote Wnt7b expression in the endoderm which in turn promotes lung smooth muscle development. Thus, Wnt2 plays an important role in the lung mesenchyme by promoting the earliest stages of ASM development in addition to its role in specifying the respiratory endoderm fate.
Wnt2 is robustly expressed in the developing lung mesenchyme throughout lung development (Goss et al., 2009). To investigate whether Wnt2 regulates mesenchymal development, E18.5 wild type control and Wnt2−/− null mutant embryos were examined for smooth muscle defects. Immunostaining for SM22α expression, a marker of smooth muscle cells, revealed a reduction in smooth muscle surrounding the proximal and distal bronchiolar airways (Fig. 1A and B and data not shown). Higher magnification of Wnt2−/− null mutant lung cross-sections showed sporadic SM22α-expressing cells around the airways, resulting in significant gaps in the sub-epithelial ASM layer (Fig. 1C and D). Interestingly, SM22α expression in the smooth muscle layer of adjacent pulmonary arteries appeared unchanged (Fig. 1A and B, arrows).
The Wnt/β-catenin transgenic reporter mice BAT-GAL and TOP-GAL reveal Wnt/β-catenin signaling activity in the developing lung smooth muscle (Okubo and Hogan, 2004; Shu et al., 2005), and Wnt2 is reported to signal canonically through the Wnt/β-catenin pathway in the lung (Goss et al., 2009). To determine if Wnt2 is required for Wnt/β-catenin signaling activity in the developing ASM, Wnt2+/− mutants were crossed into the transgenic BAT-GAL reporter background to yield Wnt2−/−:BAT-GAL embryos (Fig. 1E and F). In E14.5 control BAT-GAL embryos, lacZ-positive cells were labeled in the developing epithelium and in the presumptive ASM layer (Fig. 1E). In Wnt2−/−:BAT-GAL mutants, there was a reduction in the number of lacZ-positive cells in the developing ASM layer, indicating a loss of Wnt/β-catenin activity in these cells (Fig. 1F). BAT-GAL activity was also reduced in Wnt2−/−:BAT-GAL mutant lung epithelium as previously reported (Fig. 1F and (Goss et al., 2009). Additionally, immunostaining for Axin2, a Wnt/β-catenin transcriptional target (Jho et al., 2002), showed reduced expression in the presumptive ASM layer of E14.5 Wnt2−/− null lungs (Fig. 1G and H). Quantification of Axin2 gene expression in E14.5 control and Wnt2−/− null lungs confirmed a significant reduction in the level of Axin2 expression in the absence of Wnt2 signaling (Fig. 1I). Together, these data suggest that Wnt2 signals through the β-catenin dependent canonical pathway in the lung mesenchyme to mediate proper ASM development.
We hypothesized that the loss of mature ASM in Wnt2−/− null mutants was due to an early loss of SMC development in the primitive mesenchyme. SM22α is a marker of both primitive and mature SMCs (Solway et al., 1995). In E11.5 wild type embryos, SM22α expression was observed in a sub-epithelial population of cells forming around the developing proximal airway (Fig. 2A). In Wnt2−/− null embryos, this expression was reduced indicating a loss of SMC differentiation in the absence of Wnt2 signaling (Fig. 2B). Quantitative PCR (Q-PCR) on E11.5 control and Wnt2−/− null lung buds confirmed an approximate 50% reduction of SM22α expression in Wnt2−/− null lung buds (Fig. 2I). At E14.5, a thick and contiguous layer of SM22α–expressing cells was observed in the developing ASM of wild type embryos (Fig. 2C). In contrast, fewer SM22α expressing cells were labeled in Wnt2−/− null lungs and the loss results in a thinned layer of ASM with pronounced gaps along the epithelium-mesenchyme border (Fig. 2D).
Pdgfrα and Pdgfrβ are receptor tyrosine kinases through which members of the platelet-derived growth factor family (Pdgf) of ligands signal to regulate proliferation, migration, and differentiation of embryonic mesenchyme (reviewed in (Betsholtz, 2004). Pdgfrα and Pdgfrβ are broadly expressed in the multipotent mesenchymal cells of the early lung bud and are important in the development of smooth muscle cells from the lung mesenchyme (Bostrom et al., 1996; Hellstrom et al., 1999). Both receptors continue to be expressed in the embryonic lung in both ASM and VSM (Cohen et al., 2009).
In E11.5 wild type embryos, expression of Pdgfrβ was observed in mesenchymal cells that are coalescing around the developing airways and vasculature (Fig. 2E). However, in Wnt2−/− null embryos, Pdgfrβ expression was reduced throughout the mesenchyme (Fig. 2F). Q-PCR on E11.5 control and Wnt2−/− null lung buds demonstrated a significant reduction in Pdgfrβ expression (Fig. 2I). In E14.5 wild type embryos, Pdgfrβ expression was observed in the sub-epithelial mesenchyme as well as the developing smooth muscle encompassing the vasculature (Fig. 2G, arrowhead). In Wnt2−/− null embryos, Pdgfrβ expression in the mesenchyme surrounding the airways was reduced whereas Pdgfrβ expression in the VSM did not appear to be effected (Fig. 2H, arrowhead). At E14.5 Pdgfrα was also found to be robustly expressed in the presumptive ASM of wild type embryos and loss of Wnt2 signaling led to a reduction in Pdgfrα expression in the mesenchyme surrounding the airways (Supplemental Fig. 1). Together, this data suggests that loss of Wnt2 function disrupts the development of a SM22α+;Pdgfrα/β+ population of immature SMCs in the early lung mesenchyme, which leads to a loss of mature ASM.
To assess whether the earliest stages of smooth muscle cell differentiation are activated in Wnt2−/− null lungs, expression levels of the transcription factors myocardin and Mrtf-B were examined. Myocardin and Mrtf-B are members of the myocardin related transcription factor (Mrtf) family of serum response factor (SRF) co-factors and expressed in the developing and mature smooth muscle of several tissues including the lung (reviewed in (Wang and Olson, 2004). Myocardin is a critical upstream regulator of myogenesis and in primitive smooth muscle cells, myocardin transactivates numerous smooth muscle differentiation genes including SM22α and smooth muscle α-actin (SMA) (Wang et al., 2001; Wang et al., 2003). At E11.5, in situ hybridizations on wild type embryos showed myocardin expression around the proximal airway of the primary lung bud (Figure 3A, arrowheads). In Wnt2−/− null mutant embryos at both E11.5 and E14.5, there was a reduction in myocardin expression surrounding the developing airway (Fig. 3A–D). Quantification of myocardin expression levels in E11.5 control and Wnt2−/− null lung buds demonstrated an approximate 80% reduction in myocardin expression in Wnt2−/− null lung buds (Fig. 3E). Additionally, quantification of Mrtf-B expression was also significantly reduced in E11.5 Wnt2−/− null lung buds as compared to control lung buds (Fig. 3E). The loss of myocardin and Mrtf-B expression in Wnt2−/− null lung buds indicates the down-regulation of an early myogenic transcriptional program in the lung, suggesting that Wnt2 is required for initiation of the smooth muscle gene program in the developing lung.
Multiple cell types are hypothesized to derive from the developing lung mesenchyme including endothelial and lymphatic cell lineages (Chinoy, 2003). Moreover, lung mesenchyme could provide a necessary source of signaling factors, including Wnt2, required for proper investment of vascular endothelium from outside the lung. Thus, we sought to determine whether differentiation of other mesenchymal lineages including vascular and lymphatic endothelial cell lineages were affected in Wnt2−/− mutants.
Q-PCR was performed on E12.5 control and Wnt2−/− null lung buds to assess endothelial marker gene expression. Pecam1 expression was not significantly reduced in Wnt2−/− null lung buds whereas Flk1 was slightly reduced to 85% of wild-type expression (Fig. 4B). Immunostaining confirmed that PECAM1 (CD31) expression was unaltered in Wnt2−/− mutants (Supplemental Fig. 2). Members of the Vegf family of molecules are important for regulating vascular and lymphatic development in multiple tissue types (Lohela et al., 2009). Vegf-A, Vegf-C, and Vegf-D are expressed in the lung during development and Vegf-A and Vegf-D are critical for proper vascular endothelial development while Vegf-C is necessary for lymphatic endothelial development (Karkkainen et al., 2004; Thebaud, 2007). Q-PCR revealed comparable expression levels of Vegf-A and Vegf-D between control and Wnt2−/− null lung buds (Fig. 4C and D). Interestingly, Vegf-C expression was significantly reduced in Wnt2−/− null lung buds (Fig. 4E). Vegf-C signals predominantly through Vegfr-3 and is critical for early development of the lymphatic vasculature (Veikkola et al., 2001). Expression of Vegfr-3 was also analyzed in E12.5 control and Wnt2−/− null lung buds and Q-PCR showed a slight but not significant reduction in Vegfr-3 expression in the absence of Wnt2 function (Fig. 4F). Expression of Lyve-1, another marker of lymphatic endothelium (Banerji et al., 1999), was unchanged in Wnt2−/− null lungs at E12.5 (Fig. 4G). Together, these data indicate that the loss of Wnt2 signaling in the embryonic lung does not appreciably effect expression of most early endothelial marker genes with the exception of minor decreases in Flk-1 and Vegf-C expression.
Several signaling pathways are critical for smooth muscle development in the mouse lung including the Shh, Bmp, Wnt7b, and Fgf10 signaling pathways (reviewed in (Morrisey and Hogan, 2010). Wnt2 is expressed in the primitive mesenchyme that is condensing around the tracheal bifurcation and presumptive lung bud (Goss et al., 2009), and Wnt2 signaling may regulate smooth muscle development up or down-stream of any of these pathways. In situ hybridization and Q-PCR on wild type and Wnt2−/− null mutant embryos showed that Wnt7b expression is reduced in Wnt2−/− null mutant foreguts (Fig. 5A–C). Fgf10 is a ligand that is expressed in the developing lung mesenchyme and mediates lung bud outgrowth and ASM development and has been shown to be a direct downstream target of canonical Wnt/β-catenin signaling (Cohen et al., 2007). Fgf10 expression in the mesenchyme of Wnt2−/− null mutant lungs is reduced (Fig. 5D–F). Shh and Bmp4 are well-established epithelial-mesenchymal regulators of smooth muscle development in the lung during branching morphogenesis (Bellusci et al., 1997a; Bellusci et al., 1997b; Weaver et al., 2000; Weaver et al., 1999). In Shh−/− null mutant lungs there is a dramatic loss of smooth muscle development (Bellusci et al., 1997a; Pepicelli et al., 1998), and activation of Bmp4 signaling been shown to be downstream of Fgf10 signaling (Mailleux et al., 2005). In situ hybridization and Q-PCR show that Shh expression is not changed in Wnt2−/− null mutant lungs (Fig. 5G–I). Expression of Bmp4 is also unaltered in Wnt2−/− lungs (Fig. 5J–L). Taken together, these data suggests that Wnt2 signaling acts upstream of Wnt7b and Fgf10 expression, but not Shh or Bmp4, in the primitive lung mesenchyme.
Although Wnt2−/− mutants display severe defects in ASM development, it is unclear whether Wnt2 signaling is sufficient to promote smooth muscle differentiation and development in the lung mesenchyme. To test whether Wnt2 signaling was sufficient to activate the smooth muscle differentiation program in mesenchymal cells, we treated the mesenchymal cell line 10T1/2, a cell line known to differentiate into smooth muscle cells under certain culture conditions (Darland and D'Amore, 2001; Lockman et al., 2007; Wang et al., 2007b), with recombinant Wnt2 (rWnt2). Culturing 10T1/2 cells in the presence of rWnt2 for 48 hours led to increased expression of several smooth muscle related genes including SM22α, SMA, Pdgfrα, and Pdgfrβ, as assessed by Q-PCR (Fig. 6A). Expression of the canonical Wnt target gene Axin2 was also significantly upregulated in the presence of rWnt2, indicating activation of Wnt/β-catenin signaling in 10T1/2 cells by this recombinant ligand (Fig. 6A).
To further investigate the effects of rWnt2 on smooth muscle development, E11.5 wild type lung buds were dissected and cultured for 48 hours with either control PBS or rWnt2 in a lung explant assay. Explants were collected and examined for changes in smooth muscle development by histology and Q-PCR. In lung bud explants cultured in the presence of rWnt2, SM22α, myocardin, MRTFB, Fgf10, Pdgfrβ, and Pdgfrα expression were all significantly increased (Fig. 6B). This increase in smooth muscle gene expression was accompanied by increased smooth muscle cell development around the developing airways in the rWnt2 treated explants compared to control explants (Fig. 6C and D). Bmp4 and Wnt7b expression were also examined by Q-PCR to determine whether rWnt2 activated expression of these pro-myogenic epithelial expressed factors. Interestingly in the presence of rWnt2, expression of Wnt7b was significantly upregulated whereas Bmp4 expression was not significantly increased when compared to control wild type untreated lung bud explants (Fig. 6E). This data in conjunction with the decreased Wnt7b expression in Wnt2−/− null mutants suggests that Wnt2 signaling coordinates smooth muscle development in part through activated Wnt7b signaling.
The whole lung bud explant assay does not permit the assessment of Wnt2 autocrine signaling on activation of smooth muscle gene expression. To assess the role of autocrine Wnt2 signaling in the lung mesenchyme in the absence of endoderm, primary cultures of isolated E11.5 wild type lung mesenchyme were cultured in the absence or presence of rWnt2 for 48 hours. In the presence of rWnt2, the expression of several smooth muscle genes were significantly upregulated including myocardin, MRTFB, Fgf10, SM22α, Pdgfrα, and SMA (Figure 6F). Surprisingly Fgf10 expression was increased 5-fold in the presence of rWnt2, suggesting that Fgf10 signaling can be activated via autocrine Wnt2 signaling in the primitive lung mesenchyme. Although activation of smooth muscle gene expression occurred in primary lung mesenchyme cultures treated with rWnt2, the overall magnitude was less compared to whole lung bud explant cultures. These data suggest that Wnt2 mediated paracrine signaling also contributes to smooth muscle gene expression and development in the lung.
Genetic models have shown that Fgf10 signaling is necessary for ASM development in the lung (Mailleux et al., 2005; Ramasamy et al., 2007). Our data show that loss of Wnt2 expression leads to decreased Fgf10 expression (Fig. 5D–F). However it is unclear whether Wnt2 simply acts upstream of Fgf10 signaling to promote smooth muscle development or whether Fgf10 also cooperates to increase Wnt2 expression in a feed-forward loop. To test whether Fgf10 signaling modulates Wnt2 expression, E11.5 wild type lung bud explants were treated with either PBS or recombinant Fgf10 (rFgf10) and expression of Wnt2 was analyzed by Q-PCR. Wnt2 gene expression levels were unaffected by exogenous rFgf10 in wild type lung bud explants, suggesting that Fgf10 signaling neither activates nor inhibits Wnt2 expression (Fig. 7A). Treatment of lung bud explants with rFgf10 did result in a significant upregulation of several smooth muscle marker genes including SM22α, SMA, and Pdgfrα but not myocardin or Mrtf-B (Fig. 7A). This upregulation in smooth muscle gene expression was confirmed by SM22α immunostaining showing increased ASM development in rFgf10 treated wild type explants (Fig. 7B and C). Previous reports examining Fgf10 hypomorphic models suggested that Fgf10 regulates smooth muscle development through epithelial intermediates including Bmp4 (Mailleux et al. 2005). However, we did not observe a significant increase in Bmp4 and Wnt7b expression in the presence of rFgf10 (Supplemental Fig. 3).
The data thus far suggests that Wnt2 signaling mediates ASM development in part, through activation of Fgf10 signaling. To determine whether exogenous Fgf10 could rescue the loss of smooth muscle development in Wnt2−/− null mutant lungs, E11.5 lung buds were dissected from Wnt2+/− heterozygous mutant crosses for lung explant rescue assays with rFgf10. Lung bud explants were individually cultured in the presence of either PBS or rFgf10 for 48 hours, and subsequently harvested for histological sectioning and Q-PCR. rFgf10 treatment of Wnt2−/− null lung bud explants resulted in increased levels of SM22α, Pdgfrβ, and SMA (Fig. 7D). Expression of myocardin, Mrtf-B, and Pdgfrα was not statistically changed (Fig. 7D). However, there was a less than significant rescue in Wnt7b expression with addition of Fgf10 (Fig. 7D). Immunostaining for SM22α expression also showed increased smooth muscle development in rFgf10 treated Wnt2−/− null lung bud explants supporting the Q-PCR findings (Fig. 7E–G). Overall, this data suggests that rFgf10 acts downstream of Wnt2 to promote the expansion and differentiation of smooth muscle cells in the lung that have already initiated expression of myocardin and Mrtf-B (Fig. 8).
Our data indicate that Wnt2 signaling is necessary and sufficient to promote the differentiation of ASM from the multipotent lung mesenchyme through direct or indirect activation of a myogenic regulatory network including myocardin and Mrtf-B. These data show that Wnt2 signaling activates Fgf10 signaling in the mesenchyme, which further drives proliferation and smooth muscle differentiation into mature ASM. Our results also suggest that Wnt2 signaling activates Wnt7b signaling in the adjacent epithelium, which is known to signal in a paracrine manner to the sup-epithelial mesenchyme to promote smooth muscle differentiation.
Smooth muscle development in the lung is poorly understood in part because the origins of the various smooth muscle lineages are still unclear and the pathways regulating proliferation, migration, and differentiation of primitive smooth muscle cells are complex and unresolved. Recent genetic evidence demonstrates that Wnt/β-catenin signaling lies upstream of signals governing smooth muscle development in the mouse lung. Loss of the canonical ligand Wnt7b in the embryonic lung epithelium leads to severe VSM defects, demonstrating a critical role for paracrine Wnt/β-catenin signaling between the developing epithelium and mesenchyme (Cohen et al., 2009; Shu et al., 2002). Additional studies demonstrate that genetic deletion of β-catenin in the primitive lung mesenchyme also disrupts ASM development (De Langhe et al., 2008), implicating a potential function for other canonical Wnt ligands expressed in the lung for proper ASM development. In this study, we identify Wnt2 as a critical Wnt ligand required in the lung mesenchyme to mediate ASM development. We show that Wnt2 is required for BAT-GAL activity in ASM indicating that it acts through the β-catenin dependent canonical pathway to regulate ASM development.
While defects in ASM are observed Wnt2−/− null mutants, the VSM appears grossly normal. Wnt7b signaling from the lung epithelium has been shown to regulate VSM development (Cohen et al., 2009; Shu et al., 2002), and analysis of Wnt2−/− null mutant lungs demonstrates that Wnt7b is expressed at about 50% of normal levels in Wnt2−/− null mutants. Additionally, Wnt2 expression is reduced in Wnt7b−/− null lungs (unpublished observations), suggesting there is signaling interplay between epithelial and mesenchymal expressed Wnt ligands in the lung. If Wnt2 signaling is important for VSM development, the potential effects from loss of Wnt2 signaling may be compensated by the continued expression of Wnt7b or Wnt2b, which is also expressed in the lung mesenchyme and could function redundantly with Wnt2 signaling in VSM development (Goss et al., 2009). The distinct molecular pathways that regulate VSM versus ASM development in the lung are poorly understood. Moreover, it is unclear whether ASM and VSM have a common or distinct developmental origins. Disruption of other pathways including Shh result in defects in both ASM and VSM (Miller et al., 2004). Thus, additional fate-mapping as well as developmental studies will be required to determine the similarities and differences in VSM and ASM origins and cellular phenotypes.
A recent study describing conditional mutants in β-catenin signaling in the developing lung mesenchyme suggested a role for Wnt/β-catenin in the differentiation of the endothelial lineage (De Langhe et al., 2008). Wnt2−/− null embryoid bodies are also unable to effectively differentiate into endothelial cells (Wang et al., 2007a). Our data suggests that either Wnt2 is not required for lung endothelial development or that it acts redundantly with other Wnt ligands to regulate endothelial development in the lung.
Wnt2 is expressed during the earliest stages of lung morphogenesis suggesting the perinatal ASM deficiency in Wnt2−/− null mutants could be the result of an early defect in the development of smooth muscle cells in the distal mesenchyme. Analysis of Wnt2−/− null mutants during the early stages of myogenesis indicated reduced expression of the genes regulating the earliest aspects of smooth muscle cell differentiation from the primitive mesenchyme including myocardin, Mrtf-B, and Pdgfrβ These data indicate that Wnt2 signaling regulates the development of the multipotent mesenchymal into immature smooth muscle cells in the mouse lung.
A Wnt-Fgf regulatory network is thought to govern development of the lung mesenchyme (Yin et al., 2008), and mice carrying hypomorphic or null alleles of Fgf10 and Fgfr2, the cognate receptor of Fgf10, exhibit ASM deficiencies (De Langhe et al., 2006; Ramasamy et al., 2007). In Wnt2−/− null mutant lungs, both Fgf10 expression and Fgfr2 expression are reduced, implicating a Wnt2-Fgf10 signaling network in the coordination of ASM development. We show that Fgf10 signaling promotes ASM development through upregulation of several mature smooth muscle markers but not the early smooth muscle differentiation factors myocardin or Mrtf-B. These findings suggest that Fgf10 signaling mediates the expansion and differentiation of immature smooth muscle cell already committed to the ASM lineage.
In summary, these studies demonstrate that Wnt2 signaling is necessary for ASM development in the mouse lung. Wnt2 expression during early lung morphogenesis is required for activation of the smooth muscle gene expression program including myocardin, Mrtf-B, and Fgf10. Exogenous Fgf10 can partly rescue the loss of Wnt2 by promoting differentiation of cells already committed to the ASM lineage. Thus, these results define a Wnt2-Fgf10 signaling axis governing early ASM development in the lung.
Generation and genotyping information on the Wnt2−/− mutant mice has been described (Goss et al., 2009). Genotyping of the BAT-GAL mice has been described (Maretto et al., 2003). The University of Pennsylvania Institutional Animal Care and Use Committee approved all animal protocols.
Embryos were fixed in 4% paraformaldehyde for 24 hours, dehydrated in a series of ethanol washes, and then embedded in paraffin for tissue sectioning. Dissected embryonic lung buds and foreguts were fixed in 4% paraformaldehyde overnight, dehydrated in a series of methanol washes and blocked a 10% normal goat serum/PBS solution before wholemount immunostaining. Radioactive in situ hybridization and immunohistochemistry were performed as previously described (Shu et al., 2001). Tissue sections were stained with the following antibodies and dilutions: anti-Axin2 (Abcam; 1:100), anti-Pdgfrβ (Cell Signaling Technology Inc.; 1:50), anti-PECAM-1 (PharMingen; 1:500), and anti-SM22α (Abcam; 1:500). β-galactosidase histochemical staining of embryos was performed as previously described by our laboratory (Shu et al., 2002).
Total RNA was isolated from lung tissue at the indicated time points using Trizol reagent, reverse transcribed using SuperScript First Strand Synthesis System (Invitrogen), and used in quantitative real time PCR (Q-PCR) analysis using the oligonucleotides listed in Supplemental Table 1. RNA was isolated from at least 5 lung bud tissue samples corresponding to each genotype.
Lung buds were dissected from E11.5 embryos and cultured in BGjb media (Invitrogen) supplemented with 0.1mg/ml ascorbic acid for 48 hours as previously described (Zhang et al., 2008). Explants were cultured on a 0.4μm membrane filter (BD Falcon). For mesenchymal cultures, whole lung buds were dissected at E11.5 and subjected to collagenase digestion to give rise to single cells and mesenchymal cells were isolated via differential adhesion. For rescue experiments, embryos were collected from wild-type females or generated from Wnt2+/− heterozygous mutant crosses. Exogenous recombinant human Wnt2 protein (Novus Biologicals) was added to the lung explant media at a concentration of 0.15ug/ml. Exogenous recombinant human Fgf10 (R & D Systems, Inc.) was added to the lung explant media at a concentration of 0.2ug/ml. At least 8 explants were used for each experimental condition and treatment.
10T1/2 cells were cultured in Eagle's Basal medium with Earle's BSS, 2 mM L-glutamine, 1.5 g/L sodium bicarbonate, and 10% heat-inactivated fetal bovine serum as suggested by ATCC. Recombinant human Wnt2 protein was added to the 10T1/2 media at a concentration of 0.15ug/ml, and cultured for 48 hours before harvesting for Q-PCR analysis.
These studies were supported by funding from the NIH (HL087825 and HL100405 to E.E.M.) and a Predoctoral Fellowship from the American Heart Association to A.M.G..
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