BMP and WNT signaling are involved in tracheal cartilage development in chicken embryos

The respiratory system consists of the tracheal tube which extends from the back of the mouth, down the neck and branches into the bronchial tubes of the lungs, which are the major sites of respiration. Cartilaginous rings are present along the length of the trachea and are essential for keeping the airways open. Understanding trachea and cartilage development is crucial to better understanding respiratory disorders and diseases. We analyzed cartilage development across different chicken embryonic stages using a combination of cellular and molecular biology tools. We found that chondrogenic condensations are barely visible histologically from HH35 (E8.5) while differentiated cartilage rings are distinct by HH36 (E10). In situ hybridisation data shows that Bmp2 is expressed in a ring-like pattern along the length of the tracheal tube from HH36. We further show that BMP2, but not BMP4, colocalises with COL2A1. qPCR data shows that genes such as COL2A1, and WNT are substantially upregulated between HH34 to HH37, while other genes (e.g. SHH, TBX4/5) are downregulated. Within this three to four day period, we detected a reduction in SHH and WNT during cartilage condensation formation and an upregulation of COL2A1 and WNT during overt cartilage differentiation. We next used a known Bmp2 type 1 receptor inhibitor (DMH1) to attempt to disrupt tracheal cartilage development. Following inhibition, we observed a widening and lengthening of the tracheal cartilage rings with some narrowing of the spaces between the rings. Our gene expression analysis reveals significant increases in COL2A1, WNT, and BMP4 following BMP receptor blocking compared to sham controls and indicates that these genes are important for the molecular response that results in patterning the tracheal cartilage ring morphology. This study provides a framework to further understand development of these important airway cartilages in the avian model.