The Expression Patterns Of Lhx9 And Lhx4 In Zebrafish Embryogenesis And Their Functions In The Retinal Development

Many human genetic diseases are related to LHX(LIM homeobox)gene.At present,the research on the function of Lhx9 and Lhx4 in the vertebrates embryogenesis is fragmentary,which is mainly due to the lack of the whole expression pattern of Lhx9and Lhx4 in the embryo development.In addition,more than 80%of Lhx were found to be related to the retinal development.However,the expression pattern and function of Lhx9 and Lhx4 in zebrafish retina have not been reported.In this study,the expression patterns of lhx9 and lhx4 in zebrafish embryogenesis and retinal development were detected using whole-mount in situ hybridization and section in situ hybridization.The results showed that the expression of lhx9 and lhx4changed dynamically with the zebrafish embryogenesis from 24hpf to 5dpf.lhx9 not only expressed in the brain,but also expressed in the development of the inner of the retinal inner nuclear layer(INL),heart and pectoral fin for a long time;lhx4 not only expressed in the embryo brain and spinal cord,but also expressed instantaneously in the early stage of auditory capsule development,and continuously expressed in the outer nuclear layer(ONL)and the outer of the INL in the later stage of retinal development.A zebrafish model with lhx9 or lhx4 knockdown was successfully constructed by using morpholino(MO)technique in this study.The results showed that lhx9knockdown by MO did not affect the differentiation and development of different types of cells,structure and function in the zebrafish retina.lhx4 knockdown by MO caused that zebrafish embryos exhibited phenotypes similar to human genetic pituitary diseases(such as combined pituitary hormone deficiency),indicating that this model cannot be used to study the function of Lhx4 in the retinal development.Then,a zebrafish model of lhx4 knockdown in the retina was successfully constructed by using vivo-MO.The results showed that lhx4 knockdown in the retina by vivo-MO inhibited the differentiation of Rhodopsin~+RP in the ONL and Parvalbumin~+AC in the INL,these decreased retinal cells may be transformed into excessive ON cone BC though the high expression of the retinal vsx2,thus damaging the visual function of zebrafish.This study not only fully demonstrated the expression patterns of lhx9 and lhx4 in the zebrafish embryogenesis and retinal development for the first time,which helps us to further study the important functions of these two transcription factors in vertebrate embryo and retina development,but also studied the role of lhx9 and lhx4 in the zebrafish retinal development by MO knockdown technology for the first time,which provides a solid experimental foundation for the regulation mechanism of the vertebrate retinal development.Therefore,this study contributes to our comprehensive understanding the expression patterns and regulatory functions of two LIM-HD transcription factors,Lhx9 and LHX4,in the vertebrate embryogenesis and retinal development.