β-Catenin signaling is essential for mammalian larynx ...and esophagus with reduced and...

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RESEARCH ARTICLE β-Catenin signaling is essential for mammalian larynx recanalization and the establishment of vocal fold progenitor cells Vlasta Lungova 1 , Jamie M. Verheyden 2, *, Xin Sun 2, * , and Susan L. Thibeault 1, ABSTRACT Congenital laryngeal webs result from failure of vocal fold separation during development in utero. Infants present with life-threatening respiratory problems at birth, and extensive lifelong difficulties in breathing and voicing. The molecular mechanisms that instruct vocal fold formation are rarely studied. Here, we show, for the first time, that conditional inactivation of the gene encoding β-catenin in the primitive laryngopharyngeal epithelium leads to failure in separation of the vocal folds, which approximates the gross phenotype of laryngeal webbing. These defects can be traced to a series of morphogenesis defects, including delayed fusion of the epithelial lamina and formation of the laryngeal cecum, failed separation of the larynx and esophagus with reduced and disorganized cartilages and muscles. Parallel to these morphogenesis defects, inactivation of β-catenin disrupts stratification of epithelial cells and establishment of p63 + basal progenitors. These findings provide the first line of evidence that links β-catenin function to the cell proliferation and progenitor establishment during larynx and vocal fold development. KEY WORDS: β-Catenin, Epithelial lamina, Laryngeal web, Vocal folds INTRODUCTION Laryngeal webs are congenital malformations related to the incomplete recanalization of the laryngotracheal tube, when the vocal folds (VFs) fail to separate and obstruct the entrance of lower airway structures. These anomalies result from different degrees of failure of the epithelial lamina resorption during intrauterine development and they may present at the moment of birth with life-threating respiratory problems requiring immediate attention (Hartnick and Cotton, 2000; Wyatt et al., 2005). They can cause difficulties in breathing, swallowing and voicing across the lifespan. Webs range from thin and membranous to thicker, high-grade webs, with associated mesenchymal abnormalities, such as aberrant shape of the cricoid cartilage (Cohen, 1985; Hartnick and Cotton, 2000; Ahmad and Soliman, 2007). Thickness and size of webbing determines the severity of the breathing complications (Cohen, 1985; Ahmad and Soliman, 2007) (Fig. S1). Prevalence of laryngeal webbing at birth is 1 in 10,000 (Sacca et al., 2017). There is an association between anterior laryngeal webbing and velocardiofacial syndrome where laryngeal anomalies range from webs (Fokstuen et al., 1997) to complete atresia (Lipson et al., 1991) with a worldwide incidence estimated at 1 in 2000 to 1 in 4000 live births (Sacca et al., 2017). Despite the importance of a complete VF separation, little is known about how this is achieved during development. Several studies have described laryngeal and VF embryonic development in humans (Sanudo and Domenech-Matteu, 1990; Zaw-Tun and Burdi, 1985; Hartnick and Cotton, 2000; Wyatt et al., 2005; Ahmad and Soliman, 2007) and rodent models (Lobcko et al., 1979; Henick, 1993). We have recently extended these findings and delineated five principal morphogenetic events that occur during murine VF development and provided gene expression pattern analysis that could serve as the foundation for studying mechanisms of normal and aberrant VF formation (Lungova et al., 2015). These developmental events include: (1) initiation of the larynx and VFs with apposition of the lateral walls of the primitive laryngopharynx (LPh) at E (embryonic day) 10.5; (2) establishment of the epithelial lamina (EL) with fusion of the lateral walls of the primitive LPh (E11.5); (3) EL recanalization and separation of VFs that is synchronized with (4) stratification of VF epithelium and development of laryngeal cartilages and muscles in the lamina propria (LP) (E13.5-18.5); and last, (5) maturation of VF epithelium and LP during postnatal stages (Fig. S2). Based on these steps, we hypothesize that precisely controlled EL formation together with proper specification of VF epithelial progenitors are prerequisites for the accurate final VF separation. The β-catenin signaling pathway plays a crucial role during development and adult stem cell maintenance of many organs (Stevens et al., 2003; Dessimoz et al., 2005; Apte et al., 2007), including lungs and trachea (Mucenski et al., 2003; Goss et al., 2009; Harris-Johnson et al., 2009; Woo et al., 2011). It regulates diverse cellular processes such as cell proliferation, apoptosis or cell fate determination. In the ventral anterior foregut endoderm, β-catenin is required for the initiation of the respiratory lineage and its inactivation results in the absence of both trachea and lungs (Goss et al., 2009; Harris-Johnson et al., 2009). After lung specification, inactivation of β-catenin in the lung epithelium leads to aberrant branching and proximal-distal patterning (Mucenski et al., 2003; Shu et al., 2005). Finally, inactivation of β-catenin in the developing lung mesenchyme leads to decreased mesenchymal growth and disrupted endothelial differentiation (Yin et al., 2008; Li et al., 2008). In addition to its central role in Wnt signaling, β-catenin is a component of adherence junctions connecting classical cadherins through α-catenin to the actin cytoskeleton (Bienz, 2004; Lyashenko et al., 2011; Perez-Moreno et al., 2003). β-Catenin-mediated cell adhesion is particularly important for neuroepithelial and endoderm formation in embryoid bodies (Lyashenko et al., 2011), for directing coordinated cellular organization and movements within epithelia, and for transmitting information from the environment to the interior of cells (Perez- Moreno et al., 2003). Received 25 July 2017; Accepted 18 January 2018 1 Department of Surgery, University of Wisconsin-Madison, 5107 WIMR, 1111 Highland Avenue, Madison, WI 53705, USA. 2 Laboratory of Genetics, Biotechnology Center, University of Wisconsin-Madison, 425G Henry Mall, Madison, WI 53706, USA. *Present address: Department of Pediatrics, University of California, San Diego, CA 92093, USA. Authors for correspondence ([email protected]; [email protected]) X.S., 0000-0001-8387-4966; S.L.T., 0000-0002-9046-4356 1 © 2018. Published by The Company of Biologists Ltd | Development (2018) 145, dev157677. doi:10.1242/dev.157677 DEVELOPMENT

Transcript of β-Catenin signaling is essential for mammalian larynx ...and esophagus with reduced and...

Page 1: β-Catenin signaling is essential for mammalian larynx ...and esophagus with reduced and disorganized cartilages and muscles. Parallel to these morphogenesis defects, inactivation

RESEARCH ARTICLE

β-Catenin signaling is essential for mammalian larynxrecanalization and the establishment of vocal fold progenitor cellsVlasta Lungova1, Jamie M. Verheyden2,*, Xin Sun2,*,‡ and Susan L. Thibeault1,‡

ABSTRACTCongenital laryngeal webs result from failure of vocal fold separationduring development in utero. Infants present with life-threateningrespiratory problems at birth, and extensive lifelong difficulties inbreathing and voicing. The molecular mechanisms that instruct vocalfold formation are rarely studied. Here, we show, for the first time, thatconditional inactivation of the gene encoding β-catenin in the primitivelaryngopharyngeal epithelium leads to failure in separation of thevocal folds, which approximates the gross phenotype of laryngealwebbing. These defects can be traced to a series of morphogenesisdefects, including delayed fusion of the epithelial lamina andformation of the laryngeal cecum, failed separation of the larynxand esophagus with reduced and disorganized cartilages andmuscles. Parallel to these morphogenesis defects, inactivation ofβ-catenin disrupts stratification of epithelial cells and establishmentof p63+ basal progenitors. These findings provide the first line ofevidence that links β-catenin function to the cell proliferation andprogenitor establishment during larynx and vocal fold development.

KEY WORDS: β-Catenin, Epithelial lamina, Laryngeal web,Vocal folds

INTRODUCTIONLaryngeal webs are congenital malformations related to theincomplete recanalization of the laryngotracheal tube, when thevocal folds (VFs) fail to separate and obstruct the entrance of lowerairway structures. These anomalies result from different degrees offailure of the epithelial lamina resorption during intrauterinedevelopment and they may present at the moment of birth withlife-threating respiratory problems requiring immediate attention(Hartnick and Cotton, 2000; Wyatt et al., 2005). They can causedifficulties in breathing, swallowing and voicing across the lifespan.Webs range from thin and membranous to thicker, high-grade webs,with associated mesenchymal abnormalities, such as aberrant shapeof the cricoid cartilage (Cohen, 1985; Hartnick and Cotton, 2000;Ahmad and Soliman, 2007). Thickness and size of webbingdetermines the severity of the breathing complications (Cohen,1985; Ahmad and Soliman, 2007) (Fig. S1). Prevalence of laryngealwebbing at birth is 1 in 10,000 (Sacca et al., 2017). There is anassociation between anterior laryngeal webbing and velocardiofacial

syndrome where laryngeal anomalies range from webs (Fokstuenet al., 1997) to complete atresia (Lipson et al., 1991) with aworldwideincidence estimated at 1 in 2000 to 1 in 4000 live births (Sacca et al.,2017). Despite the importance of a complete VF separation, little isknown about how this is achieved during development.

Several studies have described laryngeal and VF embryonicdevelopment in humans (Sanudo and Domenech-Matteu, 1990;Zaw-Tun and Burdi, 1985; Hartnick and Cotton, 2000; Wyatt et al.,2005; Ahmad and Soliman, 2007) and rodent models (Lobcko et al.,1979; Henick, 1993). We have recently extended these findings anddelineated five principal morphogenetic events that occur duringmurine VF development and provided gene expression patternanalysis that could serve as the foundation for studying mechanismsof normal and aberrant VF formation (Lungova et al., 2015). Thesedevelopmental events include: (1) initiation of the larynx and VFswith apposition of the lateral walls of the primitive laryngopharynx(LPh) at E (embryonic day) 10.5; (2) establishment of the epitheliallamina (EL) with fusion of the lateral walls of the primitiveLPh (E11.5); (3) EL recanalization and separation of VFs thatis synchronized with (4) stratification of VF epithelium anddevelopment of laryngeal cartilages and muscles in the laminapropria (LP) (E13.5-18.5); and last, (5) maturation of VF epitheliumand LP during postnatal stages (Fig. S2). Based on these steps, wehypothesize that precisely controlled EL formation together withproper specification of VF epithelial progenitors are prerequisitesfor the accurate final VF separation.

The β-catenin signaling pathway plays a crucial role duringdevelopment and adult stem cell maintenance of many organs(Stevens et al., 2003; Dessimoz et al., 2005; Apte et al., 2007),including lungs and trachea (Mucenski et al., 2003; Goss et al.,2009; Harris-Johnson et al., 2009; Woo et al., 2011). It regulatesdiverse cellular processes such as cell proliferation, apoptosis or cellfate determination. In the ventral anterior foregut endoderm,β-catenin is required for the initiation of the respiratory lineageand its inactivation results in the absence of both trachea and lungs(Goss et al., 2009; Harris-Johnson et al., 2009). After lungspecification, inactivation of β-catenin in the lung epitheliumleads to aberrant branching and proximal-distal patterning(Mucenski et al., 2003; Shu et al., 2005). Finally, inactivation ofβ-catenin in the developing lung mesenchyme leads to decreasedmesenchymal growth and disrupted endothelial differentiation (Yinet al., 2008; Li et al., 2008). In addition to its central role in Wntsignaling, β-catenin is a component of adherence junctionsconnecting classical cadherins through α-catenin to the actincytoskeleton (Bienz, 2004; Lyashenko et al., 2011; Perez-Morenoet al., 2003). β-Catenin-mediated cell adhesion is particularlyimportant for neuroepithelial and endoderm formation in embryoidbodies (Lyashenko et al., 2011), for directing coordinated cellularorganization and movements within epithelia, and for transmittinginformation from the environment to the interior of cells (Perez-Moreno et al., 2003).Received 25 July 2017; Accepted 18 January 2018

1Department of Surgery, University of Wisconsin-Madison, 5107 WIMR,1111 Highland Avenue, Madison, WI 53705, USA. 2Laboratory of Genetics,Biotechnology Center, University of Wisconsin-Madison, 425G Henry Mall,Madison, WI 53706, USA.*Present address: Department of Pediatrics, University of California, San Diego,CA 92093, USA.

‡Authors for correspondence ([email protected]; [email protected])

X.S., 0000-0001-8387-4966; S.L.T., 0000-0002-9046-4356

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In the present study, we report, for the first time, the requirementof β-catenin function in VF and larynx morphogenesis. Conditionalablation of β-catenin in the primitive LPh epithelium during ELformation leads to failed VF separation that resembles humanlaryngeal webbing on a gross tissue level. The failure in VFseparation is preceded by delayed EL fusion and disruptedestablishment of VF basal epithelial progenitors. Delayed ELfusion may be caused by cell cycle arrest in the LPh epithelium andsurrounding mesenchyme, consistent with the role of β-catenin incell proliferation (Huang et al., 2007; Shutman et al., 1999;Rowlands et al., 2003). In more severe cases, inactivation ofβ-catenin affected the EL integrity.Mesenchymal cells filled the gapbetween the EL fragments and precluded VF separation. Besidesreduced cell proliferation, loss of β-catenin also disrupted initialdifferentiation of VF basal progenitors into p63-expressing cells,consistent with its role in promoting progenitor identity (Goss et al.,2009; Harris-Johnson et al., 2009). Specification of basallypositioned VF progenitors is necessary for their terminalconversion into functional basal cells giving rise to a suprabasallayer that disintegrates during VF separation. In summary, thisstudy provides the first evidence linking β-catenin function to VFmorphogenesis by regulating proliferation and cell fatedetermination of epithelial and mesenchymal cell lineages duringthe EL formation and recanalization, shedding light on the etiologyof poorly understood congenital laryngeal malformations.

RESULTSInactivation of β-catenin in developing VFsTo determine whether β-catenin signaling is active in developingVFs, we examined the expression of Axin2, a reporter gene forWNT/ β-catenin activity. We found that during EL formation, Axin2was expressed in the EL as well as in the surrounding LP (Fig. 1A).To investigate the requirements for β-catenin signaling in VF andlarynx morphogenesis, we disrupted β-catenin function in the EL byconditional gene inactivation using ShhCre (Harris-Johnson et al.,2009; Harfe et al., 2004). By mating ShhCre mice to TdTomatoreporter mice, we found that ShhCre was robust at the EL during ELformation (at E11.0), the site of prospective VFs. This is in contrastto the dorsal compartment of the primitive LPh, which gives rise tothe esophagus (Fig. 1B-D). ShhCrewas also robust in epithelial cellsin more advanced stages of VF formation during EL recanalizationat E14.5 (Fig. 1E,J). ShhCre lineaged cells were found at the EL, thelaryngeal cecum (LC) and the pharyngoglottic duct (PD) (Fig. 1E-J).Moreover, the ShhCre lineage tracing analysis revealed that duringEL formation ShhCre lineage signal extended into adjacent,cytokeratin (K) 8 negative mesenchyme (Fig. 1C-F). By RNAin situ hybridization, we found that Shh expression remainedprimarily in the epithelium (Lungova et al., 2015). These datasuggest that the mesenchymal lineaged cells either arise from rareShh expressing cells beyond the sensitivity of RNA in situ detection,or from epithelial cells that have undergone epithelial-mesenchymaltransition (EMT) and delaminated into mesenchyme of the LP(Fig. 1G-J). These cells intermix with SOX9-expressingchondrocytes or surround MF-20-expressing myoblasts (Fig. 1G-J).By mating Shhcre mice to mice carrying a conditional knockoutallele of β-catenin (Ctnnb1tm2Kem), we generated Shhcre/+;Ctnnb1tm2Kem/tm2Kem (hereafter referred to as β-Catcko, forconditional knockout) mutant embryos. In the epithelial cells ofE11.5 embryos, we found that β-catenin was severely reduced in theEL, at the site of prospective VFs, whereas it remained present in thedorsal compartment of the primitive LPh when compared withcontrol embryos (Fig. 1K,L). As Shhcre activity in the mesenchyme is

rather minor compared with that in the epithelium, expression ofβ-catenin in mesenchymal cells remained strong (Fig. 1L). These dataindicate that Shhcre is an effective tool for β-catenin inactivation in theepithelium of prospective VFs.

β-Catcko mutants exhibit incomplete recanalization of thelaryngotracheal tubeβ-Catcko mutants die at birth with multiple defects, includingagenesis of the lung (Harris-Johnson et al., 2009). We first analyzedthe VF phenotype at E18.5, shortly before birth. β-Catcko mutantsfailed to completely disintegrate the EL, thereby resembling thephenotype described in laryngeal webbing at a gross tissue level.Unlike in control embryos (Fig. 2A,B), unseparated mutant VFsobstruct the entrance into the trachea in both the milder (Fig. 2D,E)and more severe (Fig. 2G,H) cases. Upon close examination, wefound, that, in milder cases, mutant VFs, at the site of EL persistentfusion, are mostly lined with a single epithelial cell layer on eachside. In contrast, there are two cell layers on each side in fullyseparated VFs in control embryos (Fig. 2B,E, insets). Our previouscharacterization of the wild-type (WT) VFs suggests that transitionfrom one layer into two layers precedes successful VF separation(Lungova et al., 2015). In more severe cases, a large proportion ofthe EL is replaced by mesenchymal cells that have migrated into thespace between VFs, suggesting that VFs have completely growntogether (Fig. 2H). Ventral to the VFs is the LC (Fig. 2E,H), whichin control embryos becomes a part of the glottis once VFs separate(Fig. 2B). The dorsal region opens into the posterior glottis(Fig. 2C). In the mutant, the dorsal extent of the glottis is not asclearly defined, owing to the absence of the septum (Fig. 2F,I). Inmore severe cases, unseparated VFs occluded more than 50% of theglottal region, and the posterior glottis was significantly narrowed,when compared with milder cases (Fig. 2F,I). These phenotypes inthe severe cases are consistent with high-grade thick laryngeal websin human (Wyatt et al., 2005; Ahmad and Soliman, 2007). Wesought to further characterize the cause for the phenotypes in miceto understand the etiology and progression of laryngeal webbing.

The β-Catckomutant laryngeal phenotype can be traced to anearly defect of incomplete EL fusionWe found that aberrant VF development in β-Catcko mutants wasalready apparent at E11.5 during EL formation. In control embryosat E11.5, lateral walls of the primitive LPh obliterated the ventrallumen and formed the EL (Fig. 3A,B). More caudally, the tracheawas already separated from the esophagus (Fig. 3C). In β-Catcko

mutant embryos at E11.5, although the cranial part of the primitiveLPh fused normally to establish the EL (Fig. 3D), the caudal regionwith the prospective VFs remained open (Fig. 3E). More caudally,the trachea was not separated from the esophagus (Fig. 3F). At thecranial level of the larynx and esophagus in the control, dorsal andventral patterning was delineated by SOX2 expression dorsally andNKX2-1 expression ventrally (Fig. S3A). In the mutant, the SOX2expression domain was extended ventrally at the expense of NKX2-1 (Fig. S3B). This is similar to failed patterning at the more caudaltrachea and esophagus level shown previously (Minoo et al., 1999;Que et al., 2007; Domyan et al., 2011).

Two days later at E13.5, in control embryos, three morphogeneticevents occurred. First, the LC appeared as a T-shaped lumenextending caudally along the ventral border of the EL, indicating thestart of EL recanalization, whereas remaining epithelial cells at theEL were still fused (Fig. 3G,H). Second, a septum developedbetween the esophagus and VFs, and the cricoid cartilage formed inthis space (Fig. 3G,I). Third, in the fused EL near the cricoid, a

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cavity could be detected, indicating the start of recanalization intothe PD (Fig. 3G,I). In β-Catcko mutants at E13.5, part of the lateralwalls finally fused to establish a shortened EL (Fig. 3J,K). However,all three subsequent morphogenetic events were disrupted. First,there was a delay in LC expansion (Fig. 3J,K). Second, no septum orcricoid cartilage separated the esophagus and future larynx withVFs (Fig. 3J,L). Third, because there was no septum delineating thedorsal region of the EL, there was no apparent PD (Fig. 3J,L).At E16.5, in control embryos, with continuing dilation of the LC

and PD (Fig. 3M,N), the EL gradually disintegrates to open thelaryngotracheal tube by E18.5. Meanwhile, epithelial cells liningthe developing VFs acquired a two-cell layer morphology (Fig. 3N),suggesting that these events may precede final VF separation atE18.5. In β-Catcko mutants at E16.5, the LC finally expands(Fig. 3O-R). However, in milder mutants, EL is lined by a singleepithelial cell layer that fails to stratify, and a longer region of the ELremain fused compared with control (Fig. 3P). In more severemutants, the ventral and dorsal EL are interrupted by mesenchymalcells that run through (Fig. 3Q,R). Staining for β-catenin indicatesthat the mesenchymal cells that disrupt the EL are a mixtureof β-catenin+ and β-catenin− cells (Fig. S3E,F). As the overallβ-catenin expression is low at this stage, the β-catenin− cells could

still be wild type in genotype. This more severe gross phenotyperesembles severe cases of human laryngeal webs.

To elucidate the origin of severe laryngeal webbing, we stainedepithelial cells with cytokeratin (K) 8. In a subset of the more severeβ-Catckomutants, at E13.5, the ventral tip of the EL pinched off fromthe remaining cells of the EL, in contrast to controls (Fig. 4A-D),and the LC was formed in this small cluster of pinched off epithelialcells (Fig. 4D). At E16.5, in the control, the LC gradually expandsand may drive the separation of the remainder of the EL as they arestill connected (Fig. 4E,F). In β-Catcko mutants, however, thedelayed LC expansion cannot drive the separation of the remainderof the EL, as the two epithelial regions are now separated bymesenchymal cells (Fig. 4G,H). Our findings demonstrate that thedefects in EL formation preceded the failure of VF separation.

Cellular mechanism underlying aberrant EL formationTo address whether β-catenin plays a role in cell survival and/orproliferation, we examined embryos at E11.5 using TUNEL assayfor cell death and EdU incorporation in S-phase for cellproliferation. We found no increased cell death in the mutant ELor adjacent LP when compared with control mice (Fig. S4A,B).In contrast, there was a statistically significant decline of cell

Fig. 1. Inactivation of β-catenin in the primitive LPh leads to a failure in VF separation. (A) Axin2 expression, as determined by RNA in situ hybridization, invibratome transverse sections at the level of developing VFs at E11.5. (B) Anti-TdTom (red) immunofluorescent staining in ShhCre; R26R embryos at E11.0during EL formation. (C-F) Double immunofluorescent staining for anti-TdTom (red) and anti-cytokeratin K8 (green) in ShhCre; R26R embryos at E11.0 (C,D) andE14.5 (E,F). Boxed region in C ismagnified in D. (G,H) Double immunofluorescent staining for anti-TdTom (red) and anti-SOX9 (green) in ShhCre; R26Rembryos atE14.5. (I,J) Double immunofluorescent staining for anti-TdTom (red) and anti-MF-20 (green) in ShhCre; R26R embryos at E14.5. Boxed regions in E,G,I aremagnified in F,H,J, respectively. (K,L) Immunofluorescent anti-β-catenin staining in transverse sections of the EL at E11.5, when the EL is established. Arrowin L indicates diminished β-catenin activity in the EL of the β-Catcko mutants. For each experiment, at least three different individuals were collected for analysis. Foreach individual, at least two transverse sections from cranial and caudal VF regions were characterized. The experiment was replicated twice. EL, epithelial lamina;LC, laryngeal cecum; LP, lamina propria; LPh, laryngopharynx; PD, pharyngoglottic duct; CC, cricoid cartilage; dLM, dorsal laryngeal muscles.

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proliferation in the EL and LP, in the caudal, but not cranial, regionof the developing larynx in β-Catcko mutants (EL caudal region,P=0.0001; LP caudal region, P=0.0320) (Fig. S4C-G). Thisreduction may contribute to the failed epithelial morphogenesisthat follows, such as the expansion of the LC.Previous studies have shown that β-catenin regulates cell

proliferation via its interaction with cyclin D1 (Shutman et al.,1999; Rowlands et al., 2003; Atanasoski et al., 2001). Cyclin D1also interacts with cell cycle inhibitors, such as p27Kip and p21Cip

genes, that control the exit of cells from the cell cycle (Geng et al.,2001; Lee et al., 2006). Immunofluorescent staining revealed thatcyclin D1 was strikingly absent in the mutant EL and was alsoreduced in surrounding LP when compared with control (Fig. 5A-F).In contrast, expression of p27Kip increased in both the EL and LPduring EL formation (Fig. 5G–L). Low cyclin D1 levels and highp27Kip expression is consistent with the outcome that a majority ofcells have withdrawn from the cell cycle and are arrested at a G1checkpoint. Statistically significant differences in cyclin D1 and p27expression levels were also confirmed by quantitative analysis usingStudent’s t-test (EL cyclin D1, P=0.0009; LP cyclin D1, P=0.0428;EL p27, P=0.0002; LP p27, P=0.0008) (Fig. 5M,N).To elucidate whether reduced cell proliferation is responsible for

the mutant phenotypes, we stimulated proliferation by introducingp27Kip mutation into the β-catenin mutant (Shh-Cre/+; Ctmnb1F/F;p27−/−) and addressed whether it rescues EL fusion in the mutant.

At E13.5, inactivation of p27Kip gene from the genotype of β-Catcko

mutants rescued a septum between the larynx and esophagus, the ELventral to this appears fused and is connected to the LC (Fig. 5O-R).However, the size of the EL remains shorter than in control embryos,as confirmed by K8 staining (Fig. 5P,R), possibly owing to theexpansion of the mesenchymal cells between the esophagus and thelarynx (Fig. 5O,Q). This ectopic expansion of the mesenchyme maybe due to global knockout of p27Kip, which led to increasedproliferation in the mesenchyme independently of its interactionwith β-catenin in the epithelium. These results demonstrate thatinactivation of p27Kip led to a partial rescue of the β-catenin mutantmorphogenesis defect, suggesting that epithelial cell proliferationdefects contribute to, but are not the sole reason for, themorphogenesis phenotypes in the mutant.

β-Catenin inactivation causes defects in differentiation ofmesenchymal cells in the LPAbnormalities of mesenchyme-derived structures, such as cartilageand muscle, are common features in high-grade laryngeal webs(Hartnick and Cotton, 2000; Ahmad and Soliman, 2007).Accompanying epithelium defects, we also found that β-Catcko

mutants exhibited defects of the cartilaginous and muscularstructures in the mesenchyme (Fig. 2D-I). The middle region ofthe thyroid cartilage and the shape of arytenoids were also altered(Fig. 2A,D,G), affecting the attachment of the thyroarytenoid (TA)

Fig. 2. Inactivation of β-catenin in the primitive LPh leads to laryngeal webs. (A-C) Hematoxylin and Eosin staining in transverse sections demonstrating themorphology of the VFs in control embryos at E18.5. Boxed regions in A are magnified in B and C. Boxed region in B is magnified in the inset. A solid blackarrow indicates the two-layered VFepithelium in control embryos. (D-F) Hematoxylin and Eosin-stained transverse sections demonstratingmorphology of the VFsin β-Catcko mutants at E18.5 in milder cases. Boxed regions in D are magnified in E and F. Boxed region in E is magnified in the inset. A dashed blackarrow indicates aberrant stratification of VF epithelium in mutant VFs. (G-I) Hematoxylin and Eosin-stained transverse sections demonstrating morphology of theVFs in β-Catcko mutants at E18.5 in severe cases. Boxed regions in G are magnified in H and I. For each experimental group, at least three different individualswere collected for analysis. For each individual, at least two transverse sections from cranial and caudal VF regions were characterized. The experimentwas replicated twice. AC, arytenoid cartilage; CC, cricoid cartilage; EL, epithelial lamina; Es, esophagus; dLM, dorsal laryngeal muscles; G, glottis; LC, laryngealcecum; LL, lateral laminae; PG, posterior glottis; spt, septum; TA, thyroarytenoid muscle; TC, thyroid cartilage; VF, vocal fold.

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muscle (Fig. 2B,E,H). To more precisely define the mesenchymaldefects revealed by histological observation, we outlined cell typeswith anti-SOX9 antibody for laryngeal cartilage and with anti-myosin heavy chain MF-20 antibody for developing myoblasts. Incontrol embryos at E11.5, there were intensely stained SOX9-expressing cells subjacent to the ventral tip, and these will give rise tothe middle region of the thyroid cartilage (intermediate lamina, IL)(Fig. 6A,B). Intensely stained cells lateral to the larynx will give riseto lateral laminae (LL) of the thyroid cartilage (Fig. 6A). Lightlystained cells subjacent to the dorsal EL (at the site of the arytenoidswelling) will give rise to the arytenoids (Fig. 6A). In β-Catcko

mutants at E11.5, SOX9-expressing cells were detected principally atthe sites of the arytenoid swellings and developing lateral lamina,whereas SOX9 expression around the ventral tip of the EL was muchreduced (Fig. 6C,D). In β-Catcko mutants at E13.5, the cluster ofSOX9-expressing cells ventrally from the ELwasmuch smaller whencompared with controls, resulting in an underdeveloped intermediatelamina (Fig. 6E,F). Although arytenoids formed, the cricoid did notdevelop, likely owing to absence of a septum (Fig. 6F).

Concurrent with differentiation of laryngeal cartilages, othermesenchymal cells initiated differentiation into laryngeal muscles.At E11.5 and E13.5, in β-Catckomutants, accompanying a decrease inSOX9 expression, there were slightly more MF20-expressingmesenchymal cells, especially in the ventral laryngeal region whencompared with controls (Fig. 6A-F). At E16.5 in the control, MF20-expressing cells have given rise to three principal groups of laryngealmuscles. Ventrally there is the thyroarytenoid muscle that is a pairedmuscle attached to the intermediate lamina (Fig. 6G,H). Dorsallythere are the laryngeal muscles, which consist of the interarytenoidmuscle connecting the posterior surfaces of arytenoid cartilages, andposterior cricoarytenoid muscles that connect arytenoid cartilageswith the cricoid (Fig. 6G,I). The thyroarytenoid muscle functions inphonation and acts as a sphincter that tightens and narrows thelaryngeal inlet during swallowing. The muscles are brought closertogether in parallel to close the glottis by the contraction ofinterarytenoid muscles. On the other hand, the posteriorcricoarytenoid muscle opens VFs for breathing. Both muscles,interarytenoid and posterior cricoarytenoid, are located in the septum

Fig. 3. Inactivation of β-catenin inthe primitive LPh leads to a delayedand aberrant EL formation andrecanalization. (A-L) Hematoxylinand Eosin-stained transversesections demonstrating morphology ofthe VFs in control embryos andβ-Catcko mutants at E11.5 and atE13.5. Boxed regions in G,J aremagnified in H,K and I,L, respectively.(M-R) Hematoxylin and Eosin-stainedtransverse sections demonstratingmorphology of the VFs in controlembryos (M,N), and in β-Catcko

mutants at E16.5, in milder cases(O,P) and severe cases (Q,R). Boxedregions in the M,O,Q are magnified inN,P,R, respectively. Black solidarrows in N denote expansion of theLC and PD, respectively; anarrowhead indicates the two-layeredVF epithelium. A black solid arrow in Pindicates expansion of the LC.Arrowheads in P,R indicate a flatsingle layer of VF basal cells in milder(P) and severe (R) cases of laryngealwebbing. For each experimentalgroup, at least three differentindividuals were collected for analysis.For each individual, at least twotransverse sections from cranial andcaudal VF regions werecharacterized. The experiment wasreplicated twice. AC, arytenoidcartilage; CC, cricoid cartilage; EL,epithelial lamina; Es, esophagus;dLM, dorsal laryngeal muscles; LC,laryngeal cecum; LPh, primitivelaryngopharynx; PD, pharyngoglotticduct; PG, posterior glottis; spt,septum; TA, thyroarytenoid muscle;TC, thyroid cartilage; Tr, trachea; VF,vocal fold.

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(here referred to for simplicity as dorsal laryngeal muscles) (Fig. 6G,I).β-Catenin ablation in developing VF epithelium disrupts theattachment and function of all these muscles. In β-Catcko mutants,the thyroarytenoidmuscle inserts into the lateral laminae instead of theintermediate lamina of the thyroid, and its mass seems to be reduced(Fig. 6J,K). This defect may lead to insufficient glottal closure, whichsignificantly affects swallowing and voicing. Concurrently, the dorsallaryngeal muscles are disconnected due to the absence of the septumand cricoid cartilage (Fig. 6J,L); they cannot function together toapproximate theVFs or abduct them. These data suggest that epithelialcells interact with the surrounding mesenchyme starting from earlystages of VF morphogenesis during EL formation. Primarily, loss ofβ-catenin in the epithelium leads to profound disorganization of theadjacent mesenchymal cells.

Inactivation of β-catenin affects remodeling of VF basementmembraneNext, we characterized changes in shape and cellular organization ofVF, including remodeling of their basement membrane during ELformation. Using images from transmission electron microscopyand expression analysis of laminin α 5 (Lam 5), we confirmed thatin control embryos, during EL fusion at E11.5, prospective basalcells had a well-defined basement membrane (Fig. S5A-J). Cellsthat lost contact with the lumen, e.g. cells at the ventral tip of the EL,

also lost apical characteristics such as filopodia (Fig. S5). Cells atthe EL tip formed basal protrusions that pointed towards thesurrounding mesenchyme (Fig. S5C,E). In β-Catckomutants, cells atthe ventral tip of the EL retained the original elongated cell shapewith apical filopodia and tight junctions (Fig. S5G-I, arrowheads).Their Lam 5 (Fig. S5F) positive basement membrane was smoothand without protrusions (Fig. S5H,J). During EL recanalization atE16.5, compared with the continuous Lam 5-positive basementmembrane in control embryos (Fig. S5K), there is fragmentation ofLam 5 expression between the interrupted EL segments (Fig. S5L,M),suggesting a breakdown of basement membrane. Despite thesechanges, E-cadherin expression remains robust in the remainder ofthe EL in the mutant (Fig. S5N,O), suggesting that β-catenin loss doesnot have a direct effect on adherence junctions.

β-Catenin inactivation in the EL disrupts establishment of VFepithelial basal progenitorsTo understand whether β-catenin inactivation affects specification ofprospective VF basal progenitors and their subsequent differentiation,we assayed for the expression of p63 and cytokeratins K8 andK5. p63is a nuclear marker of basal cells, and at E11.5 it was expressed in allepithelial cells alongwith a simple epithelial cellmarkerK8 (Fig. 7A-D).p63+ K8+ cells were cuboidal and perfectly organized in two single-cell layers that were fused along the midline (Fig. 7A-D). In the β-Catcko mutant, in epithelial layers that have not yet fused, K8 wasexpressed in all cells whereas p63 was detected in a mosaic fashion(Fig. 7E-H). Interestingly, even though p63+ cells are cuboidal as inthe control, p63− cells were slightly taller (Fig. 7E). Negative stainingfor Alcian Blue shows that p63-negative cells in β-Catcko mutants didnot adopt a different fate (Fig. S3C,D). At E13.5 in control embryos,p63+ cells increased in number along the EL midline (Fig. 7I,J). K8expression in these cells became weaker than in cells lining the PD orLC (Fig. 7K). These p63+ cells with downregulated K8 expressionrepresent basal cell progenitors for the stratified squamous VFepithelium. In β-Catckomutants, the prospective LC cells in the ventraltip of the EL remained p63− K8+, whereas cells at the EL expressedp63 and K8 (Fig. 7L-N). More dorsally, there is a large domain ofp63− cells expressing K8, which are columnar (Fig. 7M,N). At E18.5,after EL separation in the wild type and control, expression ofcytokeratin 5 (K5), a stratified epithelial marker, begins to be detected(Lungova et al., 2015). The epithelium is composed of two cell layers,the basal p63+, K5+ and K8-low cell layer and an apical p63-low, K5+

and K8+ cell layer (Fig. 7O-S). This expression of cytokeratins isunique for VFs within the foregut (Lungova et al., 2015). Incomparison, in β-Catcko mutant mice, fused VFs were lined with asingle layer of p63+ andK8+ epithelial cells, most ofwhich, but not all,also expressed K5 (Fig. 7T-X). A few basal cells were p63− and K8+,suggesting that these cells either did not initiate p63 expression or didnot maintain it (Fig. 7V,W). In the dorsal compartment near the PDregion, the previously observed columnar cells remain, andwere p63−,K5− and K8+ (data not shown). In summary, these data suggest thatinactivation β-catenin disrupts the normal formation of p63+, K5+ andK8− basal progenitor cells.

DISCUSSIONThe larynx is a vitally important organ, sitting at the crossroadsbetween the gastrointestinal and respiratory tracts, therebyorchestrating swallowing, breathing, coughing and, in humans,vocalization. Loss of a functioning airway is life-threatening, as isloss of airway protection from ingested food and drink. Impairedvoice production holds significant implications for individual healthand wellness, social and occupational function, and societal

Fig. 4. Inactivation of β-catenin affects the EL integrity. (A-H) Anti-K8staining of cells at the EL in green at 13.5 (A-D) and E16.5 (E-H). (A,B) Anti-K8staining in control embryos. (C,D) Anti-K8 staining in β-Catcko mutants. Awhitedashed arrow in D indicates the process of separation of the tip of the EL fromthe remaining cells at the EL. (E-H) Anti-K8 staining of cells at the LC, EL andPD in green at E16.5 in control embryos (E,F) and β-Catcko mutants (G,H).Boxed regions in A,C,E,G are magnified in B,D,F,H. For each experimentalgroup, at least three different individuals were collected for analysis. For eachindividual, at least two transverse sections from cranial and caudal VF regionswere characterized. The experiment was replicated twice. EL, epitheliallamina; Es, esophagus; LC, laryngeal cecum; PD, pharyngoglottic duct; VF,vocal fold.

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productivity. Molecular mechanisms that cause congenital laryngealwebbing have not been elucidated and there is also little histologicaldata that further characterize tissue from patients. In humans withlaryngeal webbing, fibrous tissue covers the larynx (Cohen, 1985;Hartnick and Cotton, 2000; Wyatt et al., 2005; Ahmad and Soliman,2007). This gross phenotype has beenmodeled for the first time in theβ-Catckomutant. The described phenotype in β-Catckomutants rangesfrom milder to severe laryngeal webs that occlude more than 50% ofthe glottis; this range on a gross tissue level is similar with clinicalobservations (Cohen, 1985; Ahmad and Soliman, 2007). In thismouse model, we traced the morphological defects to surprisinglyearly events of VF morphogenesis, when epithelial cells fuse toestablish the EL and initiate their differentiation into VF basalepithelial progenitors. Our finding illustrates that a thoroughunderstanding of the origin of defects, as demonstrated throughanimal models, can provide important insights into the etiology ofhuman congenital laryngeal malformations.Genetic evidence from β-Catckomutants establishes β-catenin as a

key player involved in early larynx and VF morphogenesis. This isconsistent with the dual role of β-catenin in regulating expression of

genes involved in cell proliferation, likely mediated by cyclin D1,and, simultaneously, genes that are involved in specification of VFbasal epithelial progenitors. Moreover, as a part of cell adherentjunctions, β-catenin can also affect cell adhesion and EL integrity.Inactivation of β-catenin in the epithelium and/or in a small numberof cells in the mesenchyme also disrupts normal patterning oflaryngeal cartilages and muscles.

Our data stipulate a combination of mechanical and geneticcontrols that drive EL recanalization and VF separation (Fig. S2).This mechanism is unique to the VFs and has not been described inany other tissues. Mechanical forces are generated within theepithelium, as the cavities, the LC and PD, expand and exertpressure on the epithelial walls, and in the mesenchyme by initialcontraction of the dorsal laryngeal muscles that pull on the VFs(Fig. S2). Recent research has shown that the epithelial wall adaptsto enable the expansion of the lumen (Andrew and Ewald, 2010;Hoijman et al., 2015). Besides cell division, which increases thenumber of cells in the epithelial layer, mitotically active epithelialcells remodel their shape and contract apicobasally, as shown duringDrosophila tracheal or zebrafish inner ear morphogenesis (Nelson

Fig. 5. Inactivation of β-catenin in the EL leads todownregulation of cyclin D1 and upregulation of p27Kip.(A-F) Red anti-cyclin D1 staining in control embryos (A-C) andin β-Catcko mutants (D-F) at E11.5. Boxed regions in A and Daremagnified in B and E, respectively.White dashed arrows inE,F indicate cyclin D1-negative cells in the EL. (G-L) Red anti-p27Kip staining in control embryos (G-I) and β-Catcko mutants(J-L) at E11.5. White solid arrows in K,L indicate p27-positivecells at the EL. (M,N) Statistical analysis of quantitativedistribution of cyclin D1- (M) and p27- (N) positive cells.Student’s t-test was performed to compare cyclin D1 and p27expression at the EL and LP in control embryos and in β-Catcko

mutants. For each experimental group, at least three differentindividuals were collected for analysis. For each individual, atleast two transverse sections from cranial and caudal VFregions were characterized. The experiment was replicatedtwice. (O,Q) Hematoxylin and Eosin-stained transversesections demonstrating morphology of VFs in control (O) anddouble mutant (Q) embryos at E13.5. (P,R) Anti-K8 greenstaining in control embryos (P) and double mutants (R). Boxedregions in O,Q are magnified in P,R, respectively, showing thesize of the EL. For a double-mutant embryo, only twoindividuals were collected for analysis due to the lowfrequency of double mutant production. For each individual, atleast two transverse sections from cranial and caudal VFregions were characterized. The experiment was replicatedtwice. EL, epithelial lamina; Es, esophagus; LC, laryngealcecum; LP, lamina propria; LPh, laryngopharynx; PD,pharyngoglottic duct; spt, septum.

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et al., 2012; Hoijman et al., 2015). Both mechanisms contribute tolumen expansion and simultaneously determine its shape (Hoijmanet al., 2015). Therefore, cell cycle arrest in β-Catcko mutantsmay delay the LC expansion and thus affect EL recanalization(Fig. 8A,B). Our results also indicate that the cells at the ventral tipof the EL may guide the LC expansion. Prior to the LC expansion,these cells send finger-like projections into the surroundingmesenchyme. These projections of the basement membrane canhave endocytic or exocytic properties (Hexige et al., 2015) or canwork as mechanosensors (Murphy and Courtneidge, 2011).However, because the LC appears in this region 2 days later, wehypothesize that these basal cell progenitors may coordinateextracellular matrix degradation with the basal cell membraneextension, as the LC expands. Absence of these protrusions in themutant EL can be associated with the delay in LC extension inβ-Catcko mutants. In the more severe cases of failed EL separation,when the EL loses its integrity and mesenchymal cells fill the spacein between, they block the EL separation, despite the pressureexerted from LC expansion (Fig. 8C,D).The β-catenin mutant also exhibits a number of mesenchymal

defects, including cartilage and muscle malformation. While ShhCre

activity is detected in a few of the mesenchymal cells in addition torobust signal in the epithelium, the mesenchymal cells do not makemajor contribution to either the cartilage or muscle. This suggeststhat the cartilage and muscle defects are not due to direct loss ofβ-catenin in these cells. Secondary causes could include earliercellular defects. For example, failed formation of the laryngealseptum would lead to the lack of cricoid cartilage. The

mesenchymal defects could also be due to altered signals fromthe epithelium following inactivation of β-catenin in the epithelium.Prior research has shown that changes in signals such as SHH andBMP would affect cartilage or muscle formation (Murtaugh et al.,1999; Zeng et al., 2002). The defects in mesenchyme could alsofeedback to regulate epithelial formation. For example, dorsallaryngeal muscles connect arytenoid cartilages and arytenoids withthe cricoid cartilage, and together they contribute mechanical forceto EL recanalization. In the β-Catcko mutant, the altered attachmentof the dorsal laryngeal muscles, misshapen thyroid and arytenoidcartilages, and absence of cricoid may significantly reduce the forcenecessary to pull VFs apart.

In the β-catenin mutant, failure in VF separation leading to anteriorlaryngeal webbing is preceded by delayed EL fusion and aberrantspecification of VF basal epithelial progenitors (Fig. 8). Our dataobtained from β-Catcko mutants supported previous observations thatEL fusion coincides with intensive cell proliferation (Lobsko et al.,1979; Müller et al., 1985; Lungova et al., 2015). In β-Catcko mutants,low cyclin D1 levels and high p27Kip expression in epithelial andmesenchymal cells in the LP show that during early stages of VFmorphogenesis, the majority of cells withdraw from the cell cycle andarrest at a G1 checkpoint. The lateral walls remain closely juxtaposedand fail to fuse at the midline at the appropriate time. Eventually, theventral part of the EL fuses (Fig. 8B). In the control, during ELformation, the juxtaposed epithelium is a single layer where all cells arep63+ K8+. Two days later, they start to convert into two layers: a basalp63+ K8− VF progenitor layer, and a suprabasal p63−K8+ cell layer.The basal layer represents a reservoir of stem cells for epithelial

Fig. 6. Inactivation of β-catenin at the EL leads to defectivedifferentiation of mesenchymal cells. (A-D) Green anti-SOX9 and red anti-myosin MF-20 staining in transversesections of control embryos (A,B) and β-Catckomutants (C,D) atE11.5. Boxed regions in A,C aremagnified in B,D, respectively.(E,F) Green anti-SOX9 and red anti-myosin MF-20 staining intransverse sections of control embryos (E) and β-Catcko

mutants (F) at E13.5. (G,J) Hematoxylin and Eosin transversesections demonstrating morphology of the VFs at E16.5 incontrol embryos (G) and β-Catcko mutants (J). Boxed regionsare magnified in H,K (H, control; K, β-Catckomutants) and I,L (I,control embryos; L, β-Catckomutants), respectively. Green anti-SOX9 staining shows differentiation of cells into cartilage; redanti-MF-20 staining shows differentiation of mesenchymal cellsinto muscle. For each experimental group, at least threedifferent individuals were collected for analysis. For eachindividual, at least two transverse sections from cranial andcaudal VF regions were characterized. The experiment wasreplicated twice. AC, arytenoid cartilage; AS, arytenoidswelling; CC, cricoid cartilage; EL, epithelial lamina; Es,esophagus; dLM, dorsal laryngeal muscles; IL, intermediatelamina; LC, laryngeal cecum; LL, lateral lamina; LPh,laryngopharynx; PD, pharyngoglottic duct; spt, septum; TA,thyroarytenoid muscle.

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regeneration and tissue repair (Yu et al., 2005; Mou et al., 2016). In themutant, large patches of the epithelium are p63− K8+, suggesting thatthese cellsmay have failed to establish their basal cell state, and becameK8 suprabasal only. For the p63+ cells that are present, they failed totransition into the bilayer structure with P63+ K8− mature progenitorsin the basal layer. Future studies may focus on the role of β-catenin inregulation of VF basal cell differentiation during development and/orpostnatally during VF epithelial regeneration.Advances in the understanding of morphogenetic processes

during VF embryonic development will promote greater insight intothe pathophysiology of vocal fold congenital disorders. We haveshown, for the first time, that loss of β-catenin during early stages ofVF morphogenesis is associated with congenital laryngeal

malformations resembling laryngeal webbing. Characterization ofthe regulatory mechanisms controlling the establishment andexpansion of VF progenitors will help in devising therapeuticstrategies for targeting these disorders and optimizing surgicaltechniques for their corrections. The suggested role of β-catenin indifferentiation of the VF basal cell population also opens a newavenues for researching the function of β-catenin in postnatal VFepithelial regeneration and tissue repair.

MATERIALS AND METHODSGeneration of β-catenin mutant mice and embryo isolationMice carrying a conditional loss-of-function allele of β-catenin(Ctnnb1tm2Kem) were mated to mice carrying a ShhCre allele to generate

Fig. 7. Inactivation of β-catenin at the EL leadsto defective specification of VF basalprogenitors. (A-H) Red anti-p63 and green anti-K8 staining in control embryos (A-D) and mutants(E-H) at E11.5. Boxed region in E is magnified inthe inset. Boxed regions in B and Faremagnified inC,D and G,H, respectively. White solid arrows inB,C,F,G indicate p63+ cells; white dashed arrowsin F,G indicate p63− cells. (I-K) Red anti-p63 andgreen anti-K8 staining in control embryos at E13.5.A solid arrow in J indicates p63+ cells and a dashedwhite arrow in K indicates K8 downregulation in theEL. (L-N) Red anti-p63 and green anti-K8 stainingin mutants at E13.5. Boxed regions in L aremagnified in M and N, with dorsal and ventralregions magnified in the insets. Dashed arrowsindicate p63− cells (M), solid arrows indicate K8+

expression in mutants (N). (O,T) Hematoxylin andEosin staining of fully separated VFs in controlembryos (O) and fused VFs in mutants in mildercases (T). Boxed region in O is magnified in P-S.(P,Q) Red anti-p63 staining, (P,R) green anti-K8staining and (S) green anti-K5 staining. Boxedregion in T is magnified in U-X. (U-W) Anti-p63(red) and anti-K8 (green) staining. (X) Green anti-K5 staining. Arrowheads in V and X indicate p63−

and K5− cells, respectively. For each experimentalgroup, at least three different individuals werecollected for analysis. For each individual, at leasttwo transverse sections from cranial and caudal VFregions were characterized. The experiment wasreplicated twice. AC, arytenoids; CC, cricoid; EL,epithelial lamina; Es, esophagus; dLM, dorsallaryngeal muscles; G, glottis; LC, laryngeal cecum;LPh, laryngopharynx; PD, pharyngoglottic duct;TC, thyroid cartilage; TA, thyroarytenoid muscle;VF, vocal fold.

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Shhcre/+; Ctnnb1tm2Kem/tm2Kem (β-Catcko, for conditional knockout) (Harris-Johnson et al., 2009; Harfe et al., 2004; Brault et al., 2001). Offspring weregenotyped using the following primer pairs: for Cre, 5′-TGATGAGGTT-CGCAAGAACC-3′ and 5′-CCATGAGTGAACGAACCTGG-3′, productsize 420 bp; for Ctnnb1tm2Kem, 5′-AAGGTAGAGTGATGAAAGTTGTT-3′and 5′-CACCATGTCCTCTGTCTATTC-3′, product sizes 324 bp from theCtnnb1tm2Kem allele and 221 bp from the wild-type allele. Timed pregnantfemales were dissected, and mutant and control animals studied at theindicated embryonic stages, following the regulations of protocols approvedby the University of Wisconsin Animal Care and Use Committee. Shhcre/+;Ctnnb1tm2Kme/− embryos were used as controls.

Phenotype analyses of β-Catcko mutantsTo assay for Cre activity through TdTom expression, the R26R reporter linewas introduced into the background of the ShhCre line (Harris-Johnson et al.,2009; Muzumdar et al., 2007). TdTom activity was detected using standardimmunofluorescence staining protocol on paraffin wax-embeddedtransverse sections using anti-red fluorescent protein at E11.0 and E14.5.

Whole-mount in situ hybridization with digoxigenin-labeled probes forAxin2 was performed on transverse 120 µm vibratome sections at the levelof developing larynx and vocal folds at embryonic stage E11.5, as describedby Neubuser et al. (1997).

Generation of β-Catcko; p27−/− double mutants and embryoisolationShhcre/+; Ctnnb1tm2Kem/+; Cdkln1btm1Mlf/+ males were crossed toCtnnb1tm2Kem/tm2Kem; Cdkln1btm1Mlf/+ females to generate Shhcre/+;Ctnnb1tm2Kem/tm2Kem; Cdkln1btm1Mlf/tm1Mlf double mutants (here referred toas β-Catcko; p27−/− double mutants). Offspring were genotyped using thefollowing primer pairs for Cre (as mentioned above): Ctnnb1tm2Kem (asmentioned above); andCdkln1btm1Mlf, 5′-GATGGACGCCAGACAAGC and5′-CTCCTGCCATTCGTATCTGC for the wild-type allele (product size 1-90 bp), and 5′-CTTGGGTGGAGAGGCTATTC and 5′-AGGTGAGATG-ACAGGAGATC for the mutant allele (product size 280 bp). Timed-matedpregnant females were dissected, and mutant and control animals studied atthe indicated embryonic stages.

Tissue collection for immunofluorescent stainingPregnant females were sacrificed at E11.5, E13.5, E16.5 and E18.5 (forβ-Catckomutants), at E11 and E14.5 (for ShhCre lineage tracing experiment),and at E13.5 (for double β-Catcko p27−/− mutants) following regulations ofprotocols approved by the University of Wisconsin Animal Care and UseCommittee. For each experimental group, at least three different mouseembryos were collected for analysis. We included nine wild-type embryos forcharacterization of Axin2 expression and lineage-tracing experiments (E11and E14.5); 18 control mouse embryos and 18 β-Cat ckomutants were used forcharacterization of the β-Catcko mutant phenotype. In double-mutant mice,only two embryos were collected for analysis due to the low frequency of thedouble mutants (1:16) (total number of mouse embryos=47). Mouse neckregions were dissected and immediately fixed in 4% paraformaldehyde inphosphate-buffered saline at 4°C overnight, dehydrated in a gradient series ofethanol, treated with xylene and embedded in paraffin wax. Paraffin waxblocks were cut into serial sections (5 µm), dewaxed and rehydrated, heated toboiling in 10 mM citrate buffer (pH 6) for antigen retrieval and treated with0.5% triton in PBS for cell membrane permeabilization. Sections were thenstained using a standard immunofluorescent protocol (Lungova et al., 2015).For each individual at stage E11.5 and E13.5, two transverse sections (onecranial and one caudal section) of developing VFs were characterized,accounting for a very small size of the larynx during early stages of VFformation. In more advanced stages of VF development, four transversesections (two cranial and two caudal) were characterized. Each experimentwas replicated twice in the laboratory. Primary antibodies used are listed inTable S1. They were applied overnight at 4°C. Secondary antibodies used arelisted in the Table S1, they were applied for 1 h and 30 min at roomtemperature. Slides were mounted using Vectashield with DAPI (VectorLaboratories). Images were taken with a Nikon Eclipse E600 with a cameraOlympusDP71, imageswere adjusted for brightness using the installedDP 71software. Figure panels were created using Powerpoint 2010. Alcian Bluestainingwas performed using the AlcianBlue 1%2.5 pH stain kit (NewcomerSupply, number 9102A), according the manufacturer’s protocol.

TUNEL assayTdT-mediated dUTP-biotin nick end labeling (TUNEL, Chemicon-Millipore) was employed to detect apoptosis. After rehydration, sectionedsamples were treated with proteinase K at 20 μg/ml at room temperature for15 min (EMD Millipore). After equilibration buffer, the reaction mixturewas prepared as per manufacturer instructions and applied at 37°C for50 min. After an anti-digoxigenin-peroxidase reaction for 30 min at roomtemperature, positive cells were visualized using the chromogen substratediaminobenzidine (DAB kit, Vector Laboratories) and slides werecounterstained with Hematoxylin.

Cell proliferation assay, cell quantification and statisticalanalysisPregnant females received an intraperitoneal injection of 100 µg EdU (Sigma-Aldrich) per gram of bodyweight 1 h before sacrifice. Embryos were fixed

Fig. 8. Schematic illustration demonstrating the EL fusion andrecanalization in control embryos and β-Catcko mutants, in milder andmore severe cases of laryngeal webbing. (A1) Formation of the EL in controlembryos at E11.5. (A2) Formation of LC (red cells) and PD (yellow cells) incontrol embryos at E13.5. Epithelial cells in the EL express p63 (gray nuclei),proliferate and accumulate along the EL midline. Meanwhile mesenchymalcells differentiate into laryngeal cartilages. (B1) Inactivation of β-catenin inβ-Catcko mutants leads to the failure in the EL fusion at E11.5 and disruptedinitial differentiation of VF basal progenitors. Some VF epithelial cells remaincolumnar and fail to upregulate p63 (green nuclei). (B2) The delay in the ELfusion leads to the delay in LC expansion (in red) and the absence of the PDand the septum with the cricoid cartilage. (C) Failure in specification of basallypositioned of VF progenitors versus a suprabasal cell layer may prevent ELrecanalization in milder web cases. (D) In more severe cases of laryngealwebs, inactivation of β-catenin in the epithelium affects the EL integrity. Afterdelayed EL fusion, cells at the tip of the EL can pinch off from the remaining EL,enabling mesenchymal cells to migrate into the space in between. It is alsopossible that some epithelial cells undergo aberrant epithelial-mesenchymaltransition and contribute to laryngeal web formation. AS, arytenoid swelling;AC, arytenoid cartilage; CC, cricoid cartilage; EL, epithelial lamina; IL,intermediate lamina; LC, laryngeal cecum; PD, pharyngoglottic duct.

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and processed to produce paraffin wax-embedded sections. EdU detectionwas performed according to the manufacturer’s protocol (Invitrogen).Immunofluorescently labeled cells in the EL and LP were manuallycounted using ImageJ. For each experiment at E11.5, three separate mutant-control littermate pairs were analyzed. For each mutant and control sample,two sections were quantified – cranial and caudal sections (three controlembryos, n=6 sections and three mutant embryos, n=6 sections). Thepercentages of EdU+ nuclei were compared using General Linear Modelprocedure with RepeatedMeasures Analysis of Variance. Results are reportedas mean±s.d., and were considered statistically significant at P≤0.05.

Quantification of cyclin D1 and p27 expression and statisticalanalysisSimilar to quantification of EdU-positive cells, cyclin D1 and p27immunofluorescently labeled cells were manually counted using ImageJ.For each experiment at E11.5, three separate mutant-control littermate pairswere analyzed. For each mutant and control sample, one section wasquantified at the caudal region of the developing VFs (three controlembryos, n=3 sections and three mutant embryos, n=3 sections). Thepercentage of cyclin D1+ or p27+ nuclei were compared using the Student’st-test. Results are reported as mean±s.d., and were considered statisticallysignificant at P≤0.05.

AcknowledgementsWe gratefully acknowledge Drew Ronnenberg and Sierra Raglin for their expertassistance with the tissue sample preparation for this study. We also gratefullyacknowledge Glen Leverson for providing the statistical analysis.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: V.L., X.S., S.L.T.; Methodology: V.L., J.M.V., X.S., S.L.T.; Formalanalysis: V.L., J.M.V., X.S., S.L.T.; Writing - original draft: V.L., X.S., S.L.T.; Writing -review & editing: V.L., J.M.V., X.S., S.L.T.; Supervision: X.S., S.L.T.; Projectadministration: S.L.T.; Funding acquisition: S.L.T.

FundingThis work was supported by National Institutes of Health grants (NIDCD R01DC004336 and R01 DC012773). Deposited in PMC for release after 12 months.

Supplementary informationSupplementary information available online athttp://dev.biologists.org/lookup/doi/10.1242/dev.157677.supplemental

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