Guanine Nucleotide Exchange Factor Trio Regulates Neural Crest Cell Migration And Craniofacial Development

Objective: zebrafish model of trio gene knockout and mouse model of neural crest cell(NCC)specific Trio knockout constructed in vivo,NCC isolated and cultured in vitro were used to explore the effect of Trio on NCC cellular function and craniofacial development.Methods: In vivo,trio was knocked down by injection of morpholino(MO)in zebrafish(trio MO),the development of mandibular and craniofacial cartilage were separately observed under microscope directly and by Alcian blue staining.The effect of trio on NCC migration path of zebrafish was observed by using the neural crest cell specific labeling technique of transgenic zebrafish(sox10: EGFP)by means of Timelapse.Further more,Mouse model of NCC-specific Trio knockout(CKO)was constructed.Mineralization of craniofacial bone in 3 weeks old mice was observed by skeleton staining and Micro-CT scanning,the original expression pattern of Trio was determined by In Situ Hybridization,Immunohistochemistry and Immunofluorescence.The cellular function of NCC in mice was observed by Immunofluorescence assay,and the mineralization of mandibular was examined through Von Kossa staining,Total Collagen staining and Immunofluorescence staining for Collagen I(COL I).In vitro,Isolation,NCC was cultured and knocked down of Trio(sh Trio),wound healing assay and Transwell were performed to detect NCC migration,Immunofluorescence was performed to observe cytoskeleton dynamic.The role of Trio and its potential downstream in NCC migration was explored through pull down assay and PLA.Results: In zebrafish and mouse models,Trio knockdown leads to NCC migration inhibition and NCC derivatives impairment,resulting in craniofacial developmental defects such as mandibular retrusion.In addition,Trio regulated the activities of Rac1 and Cdc42,and affected the nuclear transport of β-catenin and NCC cell polarity,respectively.It is worth noting that the injection of ca-Rac1 or ca-Cdc42 m RNA into zebrafish can partially rescue the craniofacial abnormalities caused by Trio deficiency.Conclusion: Trio plays an important role in craniofacial development as a key regulator of NCC migration.