Notum attenuates Wnt/β–catenin signaling to promote tracheal cartilage patterning

Abstract

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 Wls;Shh 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/β-catenin target and inhibitor. Notum is induced by Wnt/β-catenin in developing trachea. Deletion of Notum activated mesenchymal Wnt/β-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.

Disruption of a Hedgehog-Foxf1-Rspo2 Signaling Axis Leads to Tracheomalacia and a Loss of Sox9+ Tracheal Chondrocytes

“…We performed RNAscope in situ hybridization on E11.5 embryos to validate the RNA-seq analysis and examine which cell populations exhibited a change in Rspo2, Notum and Wnt4 expression. In controls, Rspo2 and Notum were strongly expressed in the ventral tracheal mesoderm, whereas Wnt4 was weakly expressed in the mesoderm surrounding the esophagus and trachea as well as the epithelium ( Figures 6A and 6B, Supplemental Figure 3E), all consistent with previous publications (Bell et al, 2008;Caprioli et al, 2015;Gerhardt et al, 2018). In both Foxg1Cre;Gli3T Flag/+ and Foxg1Cre;Foxf1 f/f mutants, Rspo2 and Notum were dramatically downregulated, while there was only a modest reduction of Wnt4 levels in half the embryos ( Figures 6A and 6B, Supplemental Figure 3E).…”

“…Rspo2, a secreted protein that interacts with Lgr4/5/6 and Lrp6 receptor complexes to potentiate Wnt/b-catenin signaling, was one of the most downregulated transcripts at both E10.5 and 11.5 (-1.86 and -2.73 Log2FC, respectively) (Bell et al, 2008;Carmon et al, 2011;de Lau et al, 2011;Gong et al, 2012;Kazanskaya et al, 2004;Kim et al, 2008;Lebensohn and Rohatgi, 2018;Ruffner et al, 2012). Wnt4 was modestly downregulated in the E10.5 foregut (-1.54 Log2FC), whereas Notum, a known Wnt target gene and Wnt feedback inhibitor was reduced about two-fold in the E11.5 Gli3T trachea ( Figure 5B-C, Supplemental Figure 3C) (Gerhardt et al, 2018). These data demonstrate that HH/Gli signaling transcriptionally regulate components of the canonical Wnt pathway, which are known to activate Sox9 expression in the tracheal mesenchyme (Snowball et al, 2015).…”

“…In addition to Sox9 and Foxf1, this identified Rspo2, Wnt4, and Notum, all key regulators of the canonical Wnt pathway ( Figure 5A-C and Supplemental Figure 3D-E) and all of which exhibit impaired tracheal chondrogenesis when mutated in mice (Bell et al, 2008;Caprioli et al, 2015;Gerhardt et al, 2018). Focusing on the Wnt pathway, we additionally observed reduced expression of Wnt11 (Supplemental Figure 3C), whose role in tracheal development has not yet been identified but is known to support Sox9+ chondrocyte maturation in other tissues (Liu et al, 2014;Tada and Smith, 2000).…”

“…Transcriptional profiling of Foxg1Cre;Gli3T Flag/+ foregut tissue reveals that in addition to loss of Foxf1 and Sox9, there is a dramatic reduction in expression of Rspo2, a secreted ligand known to potentiate Wnt signaling which is required for cartilage development (Bell et al, 2008). In situ hybridization confirmed reduced expression of Rspo2 as well as the Wnt response gene Notum in the ventral tracheal mesenchyme (Gerhardt et al, 2018). Re-analysis of published ChIP-seq data suggests that Rspo2 is a direct transcriptional target of Foxf1.…”

notum1, acting downstream of pitx2, is essential for proper eye and craniofacial development

“…One line found Notum-/-mice to display dentin dysplasia and reduced viability, with one quarter also displaying kidney agenesis (Vogel et al, 2016). Another line confirmed Notum homozygous perinatal lethality due to abnormal kidney development while also demonstrating that Notum was essential for proper patterning of tracheal mesenchyme (Gerhardt et al, 2018).…”