Temporal And Spatial Expression Analysis Of Tks4 Protein During Mouse Brain Development And Construction Of Tks4 Knockout Mouse

The development of the mammalian cerebral cortex is the basis for the establishment of the structure and function of the nervous system.It is the result of a series of biological events.This process includes the proliferation,and differentiation of neural precursor cells,neuronal migration,axon projection and synapses connections.Among these events,the correct migration of neurons is an important prerequisite for the proper development of the cerebral cortex.Neuronal migration works synergistically with multiple signaling pathways and related signaling molecules.Reelin signaling pathway plays a pivotal role in neuronal migration,which contributes to cortical lamination.of cerebral cortex.Tks4(Tyrosine kinase substrate with four SH3 domains)is a protein product of SH3PXD2 B,the pathogenic gene of a recessive genetic disease-FTHS syndrome(Frank-Ter Haar syndrome).Tks4 has one PX domain and four SH3 domains.Existing studies have shown that Tks4 mainly plays a role in the formation of podosomes and invadopodia,remodel of actin cytoskeleton,generation of reactive oxygen species,and the occurrence and differentiation of adipose tissue.Some clinical data also showed that some patients showed different degrees of mental defects,accompanied by nervous system-related diseases.Previous work in our laboratory showed that knocking down Tks4 in projection neurons significantly delayed neuronal migration.This phenomenon is related to the activity of Reelin signaling pathway in cortical neurons.However,the roles of Tks4 in the brain development are still unclear.Therefore,we studied the spatiotemporal expression pattern of Tks4 in mouse cerebral cortex.We found that the expression of Tks4 is consistently high during the embryonic stages and gradually decreased after birth,with a minimal expression in adulthood.In immunofluorescence staining experiments of rat hippocampal neurons,we found that Tks4 showed a cytoplasmic distribution,which is similar to the subcellular distribution results in other cell lines.Further studies found that Tks4 was widely distributed in mouse brain during E14.5-E18.5,and relatively high expression was observed in CP,VZ/SVZ,insular cortex and piriform cortex.To further study the effect of Tks4 deletion on the mouse brain development,we constructed a Tks4 knock-out mouse model using CRIPSR/Cas9 technology.Tks4 knockout mice were significantly smaller in body and brain size when comparing to their wild-type littermates.Most interestingly,the olfactory bulbs was extremely small and had an irregular organization.Our study revealed the temporal and spatial distribution and expression of Tks4 in mouse cortex,and found that Tks4 is critical for the proper formation of brain.These data provided important evidence for further dissection of Tks4 function in brain development.