Gene Expression During Tracheal Morphogenesis Manoj Ambalavanan 1
Gene Expression During Tracheal Morphogenesis Manoj Ambalavanan 1, John Snowball 3, Jeffrey Whitsett 2, 3, Debora Sinner 2, 3 1 University Of Cincinnati; 2 University of Cincinnati College of Medicine; 3 Cincinnati Children’s Hospital Medical Center Introduction Goal Tracheomalacia is a congenital disorder in which the supportive cartilage rings of the trachea are either severely weakened or missing. As a result, patients usually experience difficulty breathing, decreased air flow and in some cases death. Furthermore, tracheomalacia is commonly misdiagnosed as asthma, which can lead to further complications when implementing a treatment plan. Therefore, by understanding the molecular mechanisms underlying tracheal cartilage development, better treatments and diagnostic procedures can be implemented. To identify genes regulating tracheal morphogenesis. Methods 1. Transgenic mouse model Wls. Shh. Cre. 2. Polymerase chain reaction to genotype mice. 3. Alcian blue staining to visualize cartilage. 4. Whole Mount In-Situ Hybridization: In this procedure an antisense RNA probe is synthesized. The antisense probe then binds to the complementary sequence in the tissue of interest. After binding occurs, a colorimetric reaction takes place as detailed in the figure below. The colorimetric reaction indicates the sites of gene expression. Figure 2: Tomato. Shh. Cre mice, shown on the right, distinguishes the epithelium (green) from the mesenchyme (red) in the developing trachea and lungs. Cartilaginous rings are formed in the mesenchyme. Mesenchymal Expression E 15. 5 Hox. A 5 E 15. 5 Hox. A 3 Epithelial Expression The Wntless gene (Wls) and its associated Wnt pathways are critical in understanding the embryonic development, including the patterning of the upper airways. This gene encodes for a cargo receptor, which then facilitates the secretion of Wnt proteins to the cell surface of the producing cell. Once at the surface, Wnt growth factors are spread to the receiving cell in order to trigger cellular responses. In the present study, we sought to identify Wnt regulated genes involved in formation of tracheal cartilage in the fetal mouse. Expression of Hox, Sox and OSR genes were assessed because of their known role in cartilage formation. Results E 12. 5 E 14. 5 E 18. 5 E 12. 5 E 11. 5 E 13. 5 Osr 2 Osr 1 E 11. 5 E 13. 5 Figure 3: Expression pattern of candidate m. RNAs during normal tracheal development was determined by in situ hybridization. Shh sense probe served as negative control. E 14. 5 Hox. A 1 E 14. 5 HSPG Lipoprotein particles . Control Wnt Sox 10 Wls Wnt Canonical Pathway: Cell fate determination Wnt producing cell Non. Canonical Pathway: Tissue polarity, cell movement Wnt responsive/receiving cell Shh Sense Conclusions and future directions Wls. Shh. Cre Figure 1: Alcian blue staining shows that cartilaginous rings are visualized at E 14 in control and are absent in Wls. Shh. Cre embryos. 1. Deletion of Wls in developing tracheal epithelium blocks cartilage formation. 2. Tracheal cartilage is detected after E 14. 3. Hox. A 3 and Hox. A 5 are expressed in the tracheal mesenchyme, while Osr 1, Osr 2, Hox. A 1 and Sox 10 are expressed in tracheal epithelium. 4. Future studies will define specific roles of these genes in tracheal cartilage morphogenesis.
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