Study On The Mechanism Of Cleft Palate In Loxl3 Knockout Mice

Lysyl oxidase is an extracellular copper-dependent amino acid oxidase.Lysine oxidase can promote the cross-linking of collagen and elastin in humans and many animals,and these two are the major extracellular matrix structural proteins.LOXL3 is one of the members of the Lysyl oxidase(LOX)family.Previous work in our laboratory showed that mice with a systemic knockout of the Loxl3 gene died during perinatal period,accompanied by severe cleft palate,spinal deformity,decreased lung volume and weight,reduced cystic space,and deformed and smaller thoracic cavity.However,the mechanism of cleft palate in Loxl3 knockout mice remains unclear.Based on the above findings,this project attempts to reveal the mechanism of cleft palate in Loxl3 knockout mice and hopes to provide a theoretical solution to the clinical treatment of cleft palate.Firstly,we determined that the Loxl3 knockout mice began to delay the palatal uplifting 14 days after the embryonic period by paraffin sectioning and HE staining,so that the palatal process could not be fully fused and eventually resulted in cleft palate.And immunofluorescence results showed that in the embryonic 14-day mouse,Loxl3 was expressed in the palate.We detected the expression of palatal hyaluronic acid in embryonic day 14 of Loxl3 knockout mice by Alcian blue staining and found no abnormalities.We also detected the proliferation of palatal cells in Loxl3 knockout mice at the same time by Brdu.We found that the proliferation of palatal cells was reduced,so we preliminary concluded that the delayed rise of the palate in Loxl3 knockout mice is due to reduced cell proliferation.We detected a series of palatal development-related genes at the RNA level by qPCR and found that Shh and its downstream gene,Osr2,were reduced at the RNA level.The results of in situ hybridization more intuitively reflect this problem.We also examined the levels of SHH and OSR2 protein by Western Blot and found that the results were in agreement with the qPCR results.SHH and OSR2 also decreased at the protein level.We used the neutral protease to separate the palatal epithelial cells and mesenchymal cells,and detected the RNA levels of Shh in epithelial cells and Osr2 in mesenchymal cells by qPCR,and found that the epithelial cells in Loxl3 knockout mice in mesenchymal cells,shh and Osr2 were reduced at the RNA level,and the protein levels of OSR2 in SHH and mesenchymal cells were detected by Western Blot.The results were consistent with qPCR results.We found that the hydroxyproline content was reduced in the mesenchymal cells of Loxl3 knockout mice.Therefore,we conclude that knockdown of Loxl3 results in a decrease in collagen content and a decrease in Shh expression,which leads to a decrease in the expression of downstream gene Osr2,a decrease in Osr2 that leads to a decrease in cell proliferation,a delay in the palatal process,and eventually a failure to complete the fusion and result in cleft palate.