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[Preprint]. 2023 Aug 24:2023.01.10.523309.
doi: 10.1101/2023.01.10.523309.

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

Nicholas X Russell  1 , Kaulini Burra  2 , Ronak Shah  3 , Natalia Bottasso-Arias  4 , Megha Mohanakrishnan  1 , John Snowball  5 , Harshavardhana H Ediga  6 , Satish K Madala  6 , Debora Sinner  7
Affiliations

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

Nicholas X Russell et al. bioRxiv. .

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Abstract

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 β-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/β-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.

Keywords: Potassium channels; Wnt; cartilage; trachea; trachealis muscle.

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Figures

Figure 1:
Figure 1:. Canonical and Non-canonical Wnt signaling play distinct roles in trachealis smooth muscle cell patterning and organization.
A) Whole mount images of E13.5 Wlsf/f (control) trachea-lung are shown, inset depicts a higher magnification of the dorsal view of the trachea. A’) Cross section of an E13.5 control trachea demonstrating ventral localization of chondroblasts (Sox9+ cells) and dorsal localization of trachealis muscle cells. B) Epithelial deletion of Wls disrupts tracheal smooth muscle cell organization and morphology. In ShhCre;Wlsf/f tracheas, muscle is ectopic and oriented parallel to the tracheal elongation axis (Compare insets in A and B). Sox9 is seldom detected on Wls deficient tracheal mesenchyme as determined by immunofluorescence of cross section (B’) and whole mount tissue staining. Double-headed arrows in insets A’ and B’ indicate orientation of the tracheal elongation axis. C) Heat map depicting changes in expression of genes influencing cytoskeleton organization detected in E13.5 Wls deficient tracheas. D) RNA in situ hybridization depicts localization of transcripts for Nkx2.1, Col2a1, Wnt4, Wnt5a and Wnt7b in transverse tracheas at E13.5. Note the epithelial localization of Wnt7b and Nkx2.1. Wnt4 transcripts are enriched are primarily observed to the dorsal aspect of the tracheal epithelium, while Wnt5a transcripts are observed in the ventral tracheal mesenchyme overlapping with the localization of Col2a1. E) Whole mount stainings of E13.5 tracheas depicting the abnormal organization of the smooth muscle after deletion of Wnt5a and β-catenin in tracheal mesenchyme. Note that after deletion of β-catenin smooth muscle cells are ectopic and abnormally oriented parallel to the elongation axis reminiscent of the Wls deficient phenotype F) Cross section staining of E13.5 tracheas depicting the abnormal shape, patterning, and lack of organization of the trachealis smooth muscle cells after deletion of Wls, Wnt5a, Ror2, or β-catenin. While Wnt5a or Ror2 deficient tracheas have altered shape and assembly of smooth muscle cells, only deletion of Wls and β-catenin caused ectopic localization of smooth muscle.
Figure 2:
Figure 2:. Epithelial deletion of Wls alters expression of potassium ion channels in developing trachea.
A) Heat map depicting potassium ion channel encoding genes differentially regulated in response to Wnt signaling. B) GO term enrichment analysis identified pathways associated with ion channels upregulated or downregulated after the deletion of Wls in embryonic trachea. C) Heat map of ion channel encoding genes demonstrates differential expression patterns among epithelial, muscle, and cartilaginous cells of the trachea at E13.5. Expression of Nkx2.1, Sox9, and Acta2 confirmed the epithelial, cartilaginous, and muscle identity of the cell isolation respectively. Kcnc2 is expressed in chondrogenic cells, Kcnj8 and Abcc9 are present in trachealis muscle cells, while Kcnj13 and Cftr are expressed in epithelial cells. D) RNA scope in situ hybridization depicting differential localization and expression of ion channel transcripts and related molecules in epithelium or mesenchyme of E13.5 esophagus and trachea. E) RT-PCR analysis demonstrates differential levels of transcripts in tracheas and lungs at E13.5. Abcc9, Kcnc2, Kcnip1, and Prss8 are relatively more abundant in trachea than lungs, while Ano4 is more abundant in lungs. N=3.
Figure 3:
Figure 3:. Decreased Wnt/β-catenin activity is associated with increased potassium ion channel expression.
A) RT-PCR performed on E13.5 Wlsf/f and ShhCre;Wlsf/f tracheas detected decreased expression of Ano4 and Notum (a direct target of Wnt/β-catenin), and increased Kcnd3 expression. T-test: *p<0.05 **p<0.01 N=5. B) RNA scope in situ hybridization on E13.5 tracheal cross sections depicting localization of Prss8 in Wlsf/f tracheal epithelium. Kcnd3, Kcnj8, and Abcc9 are strongly detected in mesenchyme surrounding the esophagus and at lower levels in the tracheal tissue. In ShhCre;Wlsf/f trachea, Prss8, Abcc9, Kcnj8, and Kcnd3 RNA levels were increased. Abcc9 and Kcnj8 transcripts were ectopically located in the tracheal mesenchyme and epithelium. C) RNA scope in situ hybridization demonstrates increased Kcnd3, Prss8, Abcc9, and Kcjn8 RNAs in β-catenin deficient tracheas; Mesenchymal deletion of Wnt5a did not affect expression of these genes. D) RT-PCR analysis performed on E13.5 Foxg1Cre;β-cateninf/f tracheas support the RNA scope findings. Abcc9, Kcnj8, and Kcnd3 are increased after mesenchymal deletion of β-catenin, while Notum is downregulated. Despite the increased number of smooth muscle cells, Kcnj8 RNA was increased in Foxg1Cre;β-catenin f/f trachea as determined after normalizing Kcnj8 transcript levels to Myocd. Ttest: *p<0.05 **p<0.01, ****p<0.001 N=5
Figure 4:
Figure 4:. Abnormal ion channel activity and deficient Wnt signaling impair the organization and contractility of the developing trachealis muscle ex vivo.
A) Wls-regulated ion channels influence muscle contractility. Diagram depicting functional relationships of differentially regulated ion channels on muscle contractility was generated using RNA seq data from E13.5 ShhCre;Wlsf/f tracheas. Red boxes indicate genes which are upregulated, and blue boxes indicate those which are downregulated. The size of the boxes represents fold change expression after epithelial deletion of Wls. B) Deletion of Wls impairs contractility of tracheal mesenchymal cells in free floating collagen assay. Bright field images depict the extent of the contraction in Wlsf/f primary cells determined by the reduction in the size of the surface of the collagen gel. ShhCre;Wlsf/f cells did not contract and the gel surface remained almost unchanged throughout the experiment. Graph denotes contractility of primary tracheal mesenchymal cells determined by measurement of collagen gel surface T-test ****p<0.0005 N=7. C) Inhibition of Wnt signaling by pharmacological treatment with WntC59 impaired contractility of cells. No significant effect on contractility was observed when Wnt/β-catenin independent signaling was pharmacologically inhibited with JNK inhibitor II. Two-way ANOVA, *p< 0.05 WntC59 vs No addition, JNKinhII, DMSO, **p<0.01vs WntC59 vs No Addition, DMSO, #p<0.05 vs JNK inhII, ###p<0.001 WntC59 vs JNK inhII N=3 D) Activation of Kcnj8 channel with Minoxidil or Diazoxide, did not affect the contractility of primary tracheal mesenchymal cells, while WntC59 impaired contraction. Two-way ANOVA *p<0.05 WntC59 vs DMSO, MS25 (Minoxidil Sulfate 25uM), MS50 (Minoxidil Sulfate 50uM), D25 (Diazoxide 25uM), D50 (Diazoxide 50uM) N=4. E) Trachea lung explants isolated at E12.5 were cultured in the air-liquid-interface (ALI) over 72 hours. Tissue treated with WntC59 or VU590, displayed disorganized trachealis smooth muscle cells lacking cell-cell adhesion (arrows) and disrupted cell orientation. Minoxidil sulfate treatment did not alter smooth muscle organization while compared to tissue treated with DMSO. Representative images are shown.
Figure 5:
Figure 5:. Mesenchymal condensation is altered by inhibition or activation of inwardly rectifying ion channels.
A) Trachea-lung explants isolated from E12.5 Sox9KIeGFP embryos were cultured in ALI for 72 hours. Mesenchymal condensations required for cartilage formation were present in the DMSO tissue after 72 hr incubation (arrow). Inhibition of Wnt signaling with WntC59 or inhibition of potassium ion channel with VU590 blocked mesenchymal condensations. Corresponding low magnification bright field images of the cultures are shown to the right of fluorescent images. B) Activation of KATP ion channels delayed mesenchymal condensations without substantially impacting the growth of the trachea and lung branching as determined by bright field images. C) Primary tracheal mesenchymal cells were seeded at high concentration in micromasses. Treatment with rBMP4 produced extracellular matrix as determined by the Alcian blue staining (compare to DMSO). Combined treatment with rBMP4 and VU590 or rBMP4 and Minoxidil sulfate severely impaired the secretion of extracellular matrix as determined by the lack of Alcian blue staining. Pretreatment of primary tracheal mesenchymal cells with VU590 before seeding in micromasses prevented secretion of cartilaginous extracellular matrix as determined by the lack of Alcian blue staining in micromasses. Pretreatment with Minoxidil sulfate, an activator of KATP channels, did not significantly affect production of extracellular matrix in micromasses.
Figure 6:
Figure 6:. Model
Transverse section of a E13.5 mouse trachea depicting trachealis smooth muscle (αSMA) and ventrolateral cartilage (Sox9) is shown. Epithelial Wls-induced Wnt signaling, modulates expression of ion channels including Kcnj8-Abcc9 and Kcnj13. We propose that ion channels facilitate the rearrangement of the smooth muscle cell fibers, smooth muscle cell contractility and adhesion, and influences cartilage formation.
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