Title: Localization of Retinoic Acid in Palate and Manidible Formation
Monal Patel (Presenter)
University Of Louisville School of Dentistry
Objectives: Could the cleft palate be due to loss of RA in palate? Is there any RA signaling in developing palate? Can we detect any problems in
mandible? What is etiology of choanal stenosis in Rdh10 mutant embryos?
Methods: Embryonic specimens at 12.5 (E12.5) had been stained for β-galactosidase, via the RARE-lacZ gene, to detect RA signaling. They were dehydrated, mounted in paraffin, sliced, and counter-stained with Nuclear Fast Red. For defects in mandible, paraffin sections from mutants and control embryos were Immunostained with SOX9, blocked with antibodies and DAPI, marking cartilage precursors, and were viewed/recorded under fluorescent. For etiology of choanal stenosis, paraffin sections from E13.5/E14.5 Rdh10 mutant embryos and controls were examined for abnormal fusion of epithelium between the nasal septum and palate shelves (PS).
Results: Specimens with the RARE-lacZ gene, had RA signaling activity was detected in the Anterior-palate, particular in the Sub-Nares Anterior Zone (“SNAZ”). Elevated levels of SOX9 were detected in Meckel’s cartilage (MC) in mutant mandibles compared to control and changes in shape and size of each individual cartilage precursor cell. Control embryos had cartilage precursor cells with nuclei that are round and uniformly distributed, while mutant cartilage precursors were abnormally shaped and packed. After examining paraffin sections from different mutants and controls at E13.5 we found that 7/10 mutant embryos had Ectopic Fusion (EF), abnormal PS fusion with nasal septum within that SNAZ region. At E14.5, 1/4 of mutant embryos had EF.
Conclusions: RA signaling was active on the PS in the anterior-palate epithelial tissue in E12.5 mice embryos, which hasn’t been seen before. We believe this SNAZ region is where RA is working to help fuse the PS together. With SOX9 immunostaining we detected abnormalities in development of MC in Rdh10 mutants, indicating RA may be required for normal mandible formation, and that RA defects here may contribute to formation of cleft palate. Finally, we observed EF of the nasal septum to PS tissue in Rdh10 mutant embryos, suggesting Rdh10 and RA may be needed to prevent epithelium fusion during normal choanal development.