Unveiling the Temporal Dynamics of Mesenchymal Condensations in Tracheal Development through Non-Canonical Wnt Signaling and Notum

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 trachea is stenotic. It is unknown if Notum attenuates non-canonical Wnt signaling. Notably, after mesenchymal deletion of the non-canonical Wnt5a ligand, we observed premature tracheal chondrogenesis. 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 increased in the absence of mesenchymal Wnt5a. By performing an unbiased analysis of the gene expression in Wnt5a and Notum deficient tracheas, we detected 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. 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. E11.5 tracheas were isolated from Wnt5a f/f and Dermo1Cre;Wnt5a f/f embryos. E13.5, Chondroblasts, Epithelial cells, and Myoblasts were isolated by FACS selection from, Notum 300/300; gammaSMAeGFP and Notum 150/150 gammaSMAeGFP mouse trachea using APC-Epcam stain to select epithelial cells and GFP to isolate muscle cells. Double negative cells (Apc-, gfp-) were collected and represented the chondroblast population. RNA was isolated from tissue or cells and submitted for RNA sequencing.气管对于肺部完成气体交换所需的正常气流至关重要。常见的先天性气管畸形会累及软骨组织，导致呼吸周期中中央气道塌陷。我们此前证实，Notum作为一种可抑制Wnt信号通路经典分支的Wnt配体去酰化酶，对软骨间充质凝聚（cartilaginous mesenchymal condensations）是必需的。在Notum缺陷的气管中，软骨发生（chondrogenesis）进程延迟，气管出现狭窄。目前尚不清楚Notum是否可抑制非经典Wnt信号通路。值得注意的是，在间充质中敲除非经典Wnt5a配体后，我们观察到气管软骨发生提前。我们提出假说：Notum与Wnt5a共同介导间充质凝聚的适时形成，进而形成气管软骨。气管组织的体外培养实验显示，化学抑制Wnt非经典通路可促进凝聚提前发生，而抑制Notum则会导致凝聚进程延迟。此外，激活非经典Wnt信号会阻碍软骨间充质凝聚的形成。另一方面，在间充质缺失Wnt5a的情况下，软骨母细胞（chondroblasts）间的细胞互作增强。通过对Wnt5a与Notum缺陷气管的基因表达进行无偏分析，我们发现：在Wnt5a突变体中，与软骨发生及细胞外基质形成相关的关键基因的mRNA在胚胎发育第11.5天（E11.5）即已上调。该表达谱与在Wnt5a与Notum缺陷气管中观察到的软骨发生提前及延迟现象一致。我们得出结论：Notum与Wnt5a通过协调软骨发生的时序，对正常气管软骨模式形成至关重要。因此，本研究揭示了先天性气管畸形背后的分子机制。 从Wnt5a f/f与Dermo1Cre;Wnt5a f/f胚胎中分离得到胚胎发育第11.5天的气管组织。从Notum 300/300; gammaSMAeGFP及Notum 150/150 gammaSMAeGFP小鼠的气管组织中，通过荧光激活细胞分选（Fluorescence-Activated Cell Sorting, FACS）分离得到胚胎发育第13.5天的软骨母细胞、上皮细胞（Epithelial cells）与肌母细胞（Myoblasts）：以APC-Epcam染色标记上皮细胞，利用GFP荧光分选肌肉细胞。收集双阴性细胞（APC阴性、GFP阴性），该群体即为软骨母细胞群。从组织或细胞中提取RNA，进行RNA测序（RNA sequencing）。