| The lateral line system of zebrafish is an important mechanosensory organ located on its body surface.It can sense the changes of water flow and water pressure,and transmit the signals to the central nervous system.The lateral line system of zebrafish can be divided into two main parts:an anterior lateral line(aLL)that extends on the head,the neuromasts of aLL are located in the ear,mandible,head,upper and lower orbit,etc.The other is the posterior lateral line(pLL)that located in the trunk and tail.pLL is formed by the migration of posterior lateral line primordium(pLLP).Meanwhile,pLLP migrates along the trunk at a fixed speed toward the tail.As the basic functional unit of the lateral system,the neuromast consists of three kinds of cells:hair cells in the center,supporting cells and the mantle cells.Among them,the hair cells perform the main functions.Meanwhile,They can sense the mechanical stimulation of the surrounding environment through the cilia and transmit it to the nerve centerStudies have shown that the hair cells in zebrafish neuromast are similar to human inner ear in both structure and function,but the difference is that human hair cells can not regenerate after being damaged by various factors,which will lead to permanent hearing loss.However,the hair cells in zebrafish neuromast can regenerate spontaneously after being damaged.Because of this,the lateral line system of zebrafish is often used for hearing and deafness research.There are two main hair cell regeneration ways of zebrafish,the new hair cells are directly differentiated from the supporting cells or the supporting cells are transformed into hair cells after proliferationAiml(absent in melanoma 1),whose ??-crystallin domain can bind to calcium and inhibit melanoma,is considered as a new tumor suppressor gene.It is found that Aiml can inhibit cell migration and proliferation in prostate cancer epithelial cells.Moreover,Aim1a and Aim1b are two kinds of genes in zebrafish.In our previous results,we found that Aimla expressed specifically in zebrafish lateral line supporting cells by whole-mount in situ hybridization and immunofluorescence co-localization experiment Considering the important role of supporting cells in hair cell regeneration,we will further study the function of Aim1a in this processIn this study,the 4 bases deletion genotype of Aim1a-/-zebrafish strain was successfully constructed by using CRISPR-Cas9 gene editing technology,which resulted in the termination of Aimla expression from isoleucine at 390th position,and the final expression of Aim1a was truncated.The growth and development of Aim1a-/-zebrafish are basically normal,and the phenotype can be inherited stably.To explore the function of Aimla gene in embryonic zebrafish lateral line,neuoromasts were recognized by staining with alkaline phosphatase and 4-DI-2-ASP,the results showed that there was no significant difference about the number of neuromasts between WT and Aim1a-/-zebrafish at the early stage of embryonic lateral line development.Further detection of lateral line hair cells found that the average number of hair cells in Aimla-/-zebrafish has no difference compared with WT zebrafish.Therefore,knockout of Aim1a did not affect the early development in zebrafish lateral line.In order to further reveal the function of Aimla in zebrafish hair cells regeneration,we use neomycin to construct the regeneration model of zebrafish lateral line hair cells.The effect of Aimla knockout on the number of new hair cells in zebrafish was detected by labeling hair cells with myosin VI antibody.Compared with wild-type zebrafish,the average number of hair cells in Aimla-/-zebrafish was significantly increased at 48 h,72 h and 96 h after neomycin injury.After the treatment of neomycin and recovery for 12 hours,we observed that there were more proliferating cells in the neuromast of Aim1a-/-zebrafish compared to wild-type zebrafish by BrdU stainingIn conclusion,Aimla was specifically expressed in supporting cells of zebrafish.We have successfully established an Aimla knockout zebrafish strain by CRISPR-Cas9 technology.There are no obviously defects in the morphological development of this strain,and the development of lateral line system in larval zebrafish is hardly affected.What's more,knockout of Aimla gene can increase the cell proliferation in zebrafish neuromast and promote the neomycin-induced hair cell regeneration after neomycin,which may provide a new idea for further study on the mechanism of hair cell regeneration. |
PDF Full Text Request