Wnt signaling regulates ion channel expression to promote smooth muscle and cartilage formation in developing mouse trachea.

Ion channels play critical roles in the physiology and function of the nervous system and contractile tissue; however, their role in noncontractile tissue and embryonic development has yet to be understood. Tracheobronchomalacia (TBM) and complete tracheal rings (CTR) are disorders affecting the muscle and cartilage of the trachea and bronchi, whose etiology remains poorly understood. We demonstrated that trachealis muscle organization and polarity are disrupted after epithelial ablation of Wntless (Wls), a cargo receptor critical for the Wnt signaling pathway, in developing trachea. The phenotype resembles the anomalous trachealis muscle observed after deletion of ion channel encoding genes in developing mouse trachea. We sought to investigate whether and how the deletion of Wls affects ion channels during tracheal development. We hypothesize that Wnt signaling influences the expression of ion channels to promote trachealis muscle cell assembly and patterning. Deleting Wls in developing trachea causes differential regulation of genes mediating actin binding, cytoskeleton organization, and potassium ion channel activity. Wnt signaling regulates the expression of Kcnj13, Kcnd3, Kcnj8, and Abcc9 as demonstrated by in vitro studies and in vivo analysis in Wnt5a and ß-catenin-deficient tracheas. Pharmacological inhibition of potassium ion channels and Wnt signaling impaired contractility of developing trachealis smooth muscle and formation of cartilaginous mesenchymal condensation. Thus, in mice, epithelial-induced Wnt/ß-catenin signaling mediates trachealis muscle and cartilage development via modulation of ion channel expression, promoting trachealis muscle architecture, contractility, and cartilaginous extracellular matrix. In turn, ion channel activity may influence tracheal morphogenesis underlying TBM and CTR.NEW & NOTEWORTHY Ion channels play critical roles in the physiology and function of the nervous system and contractile tissue; however, their role in noncontractile tissue and embryonic development has yet to be understood. In this study, we focused on the role of ion channels in the differentiation and patterning of the large airways of the developing respiratory tract. We identify a mechanism by which Wnt-beta-catenin signaling controls levels of ion channel-encoding genes to promote tracheal differentiation.

BMP4 and Wnt signaling interact to promote mouse tracheal mesenchyme morphogenesis.

Tracheobronchomalacia and complete tracheal rings are congenital malformations of the trachea associated with morbidity and mortality for which the etiology remains poorly understood. Epithelial expression of Wls (a cargo receptor mediating Wnt ligand secretion) by tracheal cells is essential for patterning the embryonic mouse trachea's cartilage and muscle. RNA sequencing indicated that Wls differentially modulated the expression of BMP signaling molecules. We tested whether BMP signaling, induced by epithelial Wnt ligands, mediates cartilage formation. Deletion of Bmp4 from respiratory tract mesenchyme impaired tracheal cartilage formation that was replaced by ectopic smooth muscle, recapitulating the phenotype observed after epithelial deletion of Wls in the embryonic trachea. Ectopic muscle was caused in part by anomalous differentiation and proliferation of smooth muscle progenitors rather than tracheal cartilage progenitors. Mesenchymal deletion of Bmp4 impaired expression of Wnt/ß-catenin target genes, including targets of WNT signaling: Notum and Axin2. In vitro, recombinant (r)BMP4 rescued the expression of Notum in Bmp4-deficient tracheal mesenchymal cells and induced Notum promoter activity via SMAD1/5. RNA sequencing of Bmp4-deficient tracheas identified genes essential for chondrogenesis and muscle development coregulated by BMP and WNT signaling. During tracheal morphogenesis, WNT signaling induces Bmp4 in mesenchymal progenitors to promote cartilage differentiation and restrict trachealis muscle. In turn, Bmp4 differentially regulates the expression of Wnt/ß-catenin targets to attenuate mesenchymal WNT signaling and to further support chondrogenesis.

Notum attenuates Wnt/ß-catenin signaling to promote tracheal cartilage patterning.

Tracheobronchomalacia (TBM) is a common congenital disorder in which the cartilaginous rings of the trachea are weakened or missing. Despite the high prevalence and clinical issues associated with TBM, the etiology is largely unknown. Our previous studies demonstrated that Wntless (Wls) and its associated Wnt pathways are critical for patterning of the upper airways. Deletion of Wls in respiratory endoderm caused TBM and ectopic trachealis muscle. To understand mechanisms by which Wls mediates tracheal patterning, we performed RNA sequencing in prechondrogenic tracheal tissue of Wlsf/f;ShhCre/wt embryos. Chondrogenic Bmp4, and Sox9 were decreased, while expression of myogenic genes was increased. We identified Notum, a deacylase that inactivates Wnt ligands, as a target of Wls induced Wnt signaling. Notum's mesenchymal ventral expression in prechondrogenic trachea overlaps with expression of Axin2, a Wnt/ß-catenin target and inhibitor. Notum is induced by Wnt/ß-catenin in developing trachea. Deletion of Notum activated mesenchymal Wnt/ß-catenin and caused tracheal mispatterning of trachealis muscle and cartilage as well as tracheal stenosis. Notum is required for tracheal morphogenesis, influencing mesenchymal condensations critical for patterning of tracheal cartilage and muscle. We propose that Notum influences mesenchymal cell differentiation by generating a barrier for Wnt ligands produced and secreted by airway epithelial cells to attenuate Wnt signaling.

The aryl hydrocarbon receptor differentially modulates the expression profile of antibody isotypes in a human B-cell line.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental contaminant and high affinity ligand for the aryl hydrocarbon receptor (AhR). In animal models, AhR activation by TCDD generally inhibits antibody secretion. However, it is less clear if this translates to human antibody production. Using a human Burkitt lymphoma B-cell line (CL-01) that can be stimulated to secrete Ig and undergo class switch recombination to other Ig isotypes, the current study evaluated the effects of AhR activation or antagonism on the human Ig isotypic expression profile with CD40L+IL-4 stimulation. Our results suggest that AhR agonists (TCDD and indirubin) have little to no effect on IgM or IgA secretion, which were also not induced with stimulation. However, AhR activation significantly inhibited stimulation-induced IgG secretion, an effect reversed by the AhR antagonist CH223191. Evaluation of Ig heavy chain (IgH) constant region gene expression (ie Cµ, Cγ1-4, Cα1-2, and Cε that encode for IgM, IgG1-4, IgA1-2, and IgE, respectively) demonstrated differential effects. While Cµ and Cα2 transcripts were unaffected by stimulation or AhR agonists, AhR activation significantly inhibited stimulation-induced Cγ2-4 and Cε mRNA transcripts, which was reversed by AhR antagonism. Notably, AhR antagonism in the absence of exogenous AhR ligands significantly increased IgG and IgA secretion as well as the expression of Cγ2-4 and Cε. These results suggest that modulation of AhR activity differentially alters the IgH isotypic expression profile and antibody secretion that may be partly dependent on cellular stimulation. Since a variety of chemicals from anthropogenic, industrial, pharmaceutical, dietary, and bacterial sources bind the AhR, the ability of environmental exposures to alter AhR activity (i.e. activate or inhibit) may have a direct influence on immune function and antibody-relevant disease conditions.

Wnt5a and Notum Influence the Temporal Dynamics of Cartilaginous Mesenchymal Condensations in Developing Trachea.

The trachea is essential for proper airflow to the lungs for gas exchange. Frequent congenital tracheal malformations affect the cartilage, causing the collapse of the central airway during the respiratory cycle. We have shown that Notum, a Wnt ligand de-acylase that attenuates the canonical branch of the Wnt signaling pathway, is necessary for cartilaginous mesenchymal condensations. In Notum deficient tracheas, chondrogenesis is delayed, and the tracheal lumen is narrowed. It is unknown if Notum attenuates non-canonical Wnt signaling. Notably, we observed premature tracheal chondrogenesis after mesenchymal deletion of the non-canonical Wnt5a ligand. We hypothesize that Notum and Wnt5a are required to mediate the timely formation of mesenchymal condensations, giving rise to the tracheal cartilage. Ex vivo culture of tracheal tissue shows that chemical inhibition of the Wnt non-canonical pathway promotes earlier condensations, while Notum inhibition presents delayed condensations. Furthermore, non-canonical Wnt induction prevents the formation of cartilaginous mesenchymal condensations. On the other hand, cell-cell interactions among chondroblasts increase in the absence of mesenchymal Wnt5a. By performing an unbiased analysis of the gene expression in Wnt5a and Notum deficient tracheas, we detect that mRNA of genes essential for chondrogenesis and extracellular matrix formation are upregulated by E11.5 in Wnt5a mutants. The expression profile supports the premature and delayed chondrogenesis observed in Wnt5a and Notum deficient tracheas, respectively. We conclude that Notum and Wnt5a are necessary for proper tracheal cartilage patterning by coordinating timely chondrogenesis. Thus, these studies shed light on molecular mechanisms underlying congenital anomalies of the trachea.

Wnt signaling regulates ion channel expression to promote smooth muscle and cartilage formation in developing mouse trachea.

Ion channels play critical roles in the physiology and function of the nervous system and contractile tissue; however, their role in non-contractile tissue and embryonic development has yet to be understood. Tracheobronchomalacia (TBM) and complete tracheal rings (CTR) are disorders affecting the muscle and cartilage of the trachea and bronchi, whose etiology remains poorly understood. We demonstrated that trachealis muscle organization and polarity are disrupted after epithelial ablation of Wls, a cargo receptor critical for the Wnt signaling pathway, in developing trachea. The phenotype resembles the anomalous trachealis muscle observed after deletion of ion channel encoding genes in developing mouse trachea. We sought to investigate whether and how the deletion of Wls affects ion channels during tracheal development. We hypothesize that Wnt signaling influences the expression of ion channels to promote trachealis muscle cell assembly and patterning. Deleting Wls in developing trachea causes differential regulation of genes mediating actin binding, cytoskeleton organization, and potassium ion channel activity. Wnt signaling regulated expression of Kcnj13, Kcnd3, Kcnj8, and Abcc9 as demonstrated by in vitro studies and in vivo analysis in Wnt5a and ß-catenin deficient tracheas. Pharmacological inhibition of potassium ion channels and Wnt signaling impaired contractility of developing trachealis smooth muscle and formation of cartilaginous mesenchymal condensation. Thus, in mice, epithelial-induced Wnt/ß-catenin signaling mediates trachealis muscle and cartilage development via modulation of ion channel expression, promoting trachealis muscle architecture, contractility, and cartilaginous extracellular matrix. In turn, ion channel activity may influence tracheal morphogenesis underlying TBM and CTR.

Establishing an Endoscopic Chronic Subglottic Stenosis Rabbit Model.

OBJECTIVES/HYPOTHESIS: To develop a reproducible and consistent chronic subglottic stenosis (SGS) in an endoscopic animal model. STUDY DESIGN: Prospective study. METHODS: We conducted a prospective study using New Zealand white rabbits. Chronic SGS was induced endoscopically by Bugbee electrocautery to 50% to 75% of the subglottic area's circumference, followed by 4-hour endotracheal intubation. The rabbit airways were endoscopically assessed and sized with uncuffed endotracheal tubes (ETTs) before the injury, during follow-up, and at the endpoints. There were four endpoints: 2, 4, 6, and 8 weeks post SGS induction. Animals were humanely euthanized for histopathological examination of the subglottic injury site and microscopic measurement of the cricoid lumen. RESULTS: Twenty-two rabbits reached the endpoints, and 18 rabbits developed chronic SGS. ETT size significantly decreased by 0.5 from preinjury to the endpoint in all groups, P < .001. Control median cricoid lumen measurements were 20.48 mm2 , the median cricoid lumen measurement for the 2 weeks endpoint was 14.3 mm2 , 4 weeks 11.69 mm2 , 6 weeks 16.03 mm2 , and 8 weeks endpoint median was 16.33 mm2 . Histopathological examination showed chronic scar tissue and new cartilage formation at the cricoid level, mainly at the posterior subglottic injury site starting from 4 weeks postinjury. Collagen staining revealed substantial amounts of organized collagen and different collagen orientation starting 4 weeks postinjury lasting until 8 weeks postinjury. CONCLUSION: We developed an animal model to study chronic SGS. This model will be utilized to compare different endoscopic treatment interventions in acute SGS versus chronic SGS and further define the molecular basis of SGS. LEVEL OF EVIDENCE: NA Laryngoscope, 132:1909-1915, 2022.