Potential role of Sox9 in patterning tracheal cartilage ring formation in an embryonic mouse model

Objective: To identify genes expressed early in the formation of the mouse trachea that control patterning of tracheal cartilaginous rings. Design: The mouse larynx and trachea begin as an out-pouching from the ventral foregut endoderm at embryonic day (E) 9. Digoxigenin-labeled RNA probes to putative tracheal patterning genes were generated by in vitro transcription. Embryos ranging in age from E9 to E16 were then subjected to whole-mount in situ hybridization using these labeled RNA probes. The RNA probes were then localized using antidigoxigenin antibodies tagged with a reporter molecule. In this manner, the 3-dimensional spatial and temporal expression of putative tracheal patterning genes was examined. Subjects: F/VBN mice. Results: In the developing mouse trachea, the expression of Sox9 messenger RNA preceded cartilage ring formation. Sox9 was expressed as 2 distinct longitudinal stripes along the posterolateral aspect of the trachea as early as E9, when the developing trachea is first identified. Collagen 2A1, a cartilage-specific protein, was subsequently expressed in the same longitudinal pattern as Sox9, consistent with the early commitment of Sox9-expressing cells to the cartilage program. As cartilage rings formed, Sox9 and collagen 2A1 was expressed over the lateral and anterior aspects of the trachea. Conclusions: We have developed a system to study the early expression of genes that may pattern the formation of the trachea. We have identified a gene (Sox9) with a known role in chondrocyte differentiation that is expressed in a highly specific temporal and spatial pattern in the developing upper respiratory tract.