Role And Mechanism Of LIS1 In Lung And Limb Development

LIS1(Lissencephaly 1,also known as PAFAH1B1)is necessary for Dynein-mediated intracellular material transport and correct localization of Golgi apparatus,intracellular body,lysosome and mitochondria.Lisl gene is also the first gene confirmed to be related to neuronal migration diseases.Its heterozygous deletion in human leads to severe cerebral malformation diseases,which are mainly manifested as loss of gyrus,increase of gray matter volume and decrease of white matter volume,thus being called Lissencephaly diseases.In recent years,the functional research of Lisl gene has mainly focused on the development of the cerebral nervous system and the pathogenesis of Lissencephaly diseases,while the role of Lisl in other organs development remains elusive.In order to explore the functions of Lisl gene in the development of tissues and organs.We have respectively established a lung epithelial cell Lis1 gene specific knockout mouse model and a limb mesenchymal cell Lisl gene specific knockout mouse model.Through phenotypic analysis of mouse embryos,we found that lung and limbs of Lisl KO mouse embryos were basically absent during development.TUNEL and pHH3 staining were performed on mouse embryonic lung and limbs revealed that there was a large number of apoptosis in Lis1 KO mouse embryonic lung epithelial cells and limb mesenchymal cells,while cell proliferation was not different from that of control mice.Further,through in situ hybridization analysis on mouse embryos,we found that the expression pattern of target genes downstream of FGF signaling pathway in lung and limbs of Lis1 KO mouse embryos was abnormal,and the expression level of these genes was also lower than that of control mice.In addition,RNA-Seq analysis of Lisl KO mice forelimb in E10.5 showed that the knockout of Lis1 gene did affect the correct transduction of FGF signaling pathway.At the same time,by utilizing krasLSL-Gl2D/+transgenic mouse model to activate MAPK signaling pathway,we found the developmental defects of mouse embryo lung and limbs are partially rescued in ShhCre;Lislf/f;KrasLSL-Gl2D/+ and Prxl Cre;Lislf/f;KrasLSL-Gl2D/+ mice.This result further indicates that FGF signaling pathway is inhibited in lung and limbs of Lisl KO mice.In order to study how Lisl gene regulates FGF signaling pathway at cellular and molecular levels,we isolated and established immortalized Lislf/f MEF(Mouse Embryonic Fibroblast)cells.We found that the transduction of FGF signaling pathway is blocked when the Lisl gene is knocked out in MEF cells.Through immunoprecipitation assay,we found that LIS1 and its interaction protein NDE1/NDEL1 have certain interaction with FGF receptor in vivo.In addition,immunofluorescence and Western Blotting results also showed that LIS1 regulate the intracellular localization of FGF receptor and its protein stability.It is worth noting that activation of FGF signaling pathway can also promote tyrosine phosphorylation of NDEl,which in turn leads to dissociation of LIS1/NDE1 complex.Therefore,our research indicates that LIS1 is crucial to the normal development of mouse embryo lung and limbs.In addition,our research revealed that the LIS1/NDE1 complex is an important regulator of FGF signaling pathway,which provides new insights into the bidirectional regulation of FGF receptor in intracellular transportation mechanism and endocytosis.More importantly,our research also provides a new direction for elucidating the molecular mechanism of related genetic diseases caused by LIS1/NDE1 mutation.