Mechanism Research Of Multi-mode Gjb2 Gene Knockout Inducing Hearing Impairment In Mice

PART IMechanism of time-dependent Gjb2 knockout model inducing hearing impairment in miceObject:To study the effects of knocking out connexin26 at different stages of cochlear development on mouse hearing and the development of cochlear supporting cells.Methods:The experimental animals were divided into early knockout group（P0 KD）,late knockout group（P8 KD）and corresponding early control group（P0 Control）,and late control group（P8 Control）.Cx26^flox/flox mice were used to hybridize with Rosa26-CreER mice to construct Cx26^flox/flox;Rosa26-CreER mice that knocked out the Gjb2 gene induced by TMX.Tamoxifen（TMX）was given subcutaneously to Cx26^flox/flox;Rosa26-CreER mice at P0-P1 and P8-P9.Cx26^flox/flox;Rosa26-CreER mice were chosen as knockout group,littermate Cx26^flox/flox mice as control group.The flattened cochlear preparations with immunofluorescence staining and Western Blot to detect of Cx26 knockout;ABR detected hearing threshold 18 days after TMX injection;resin section to detect morphology of ogan of Corti;scanning electron microscopy to detect organ of Corti’s space structure;transmission electron microscopy to detect organ of Corti’s ultrastructure;the cytoskeleton of the organ of Corti was detected by immunofluorescence staining.Results:In the early knockout group and the late knockout group,Cx26 expression was successfully down-regulated,and there was no significant difference in knockout rate.ABR examination showed significant full-frequency severe hearing loss in the early knockout mice 18 days after TMX injection.Resin sectioning and flattened cochlear preparations showed that the organ of Corti with obvious developmental disorder only in the early knockout group,which the height of organ of Corti was decreased,and the distance between the column cell nuclei was shortened.Scanning electron microscopy showed that the Deiter cells in the early knockout group had abnormal development of the phalangeal processes,and the pillar cells maintained a naive status.At the same time,the horizontal sections were observed by transmission electron microscopy,the Neul’s space between the outer hair cells was completely full with abnormal phalangeal process of Deiters’cells.Transmission electron microscopy showed that the number of microtubules in the pillar cells was observed to be significantly reduced only in the early knockout group.Immunofluorescence staining showed that the acetylatedα-tubulin in the pillar cells of the early knockout group was significantly decreased,and the microtubule cytoskeleton development disorder was observed.Conclusion:Connexin26 is involved in the early development of cochlear supporting cells,and knockout of inner ear Connxin26 at early stage affects the formation of supporting cells’microtubules,which leading to supporting cell developmental disorders and developmental malformation of the organ of Corti.Knocking out the inner ear Connexin26after the organ of Corti developed does not lead to obvious supporting cell developmental disorders and organ of Corti malformations,which suggesting that the well-developed organ of Corti’s morphology in the mature cochlea does not rely on the expression of Connexin26.PART IIMechanism of dose-dependent Gjb2 knockout model inducing hearing impairment in miceObject: To investigate the effect of different degrees of Connexin26 knockdown on the mouse hearing and the development of cochlear supporting cells.Methods: The experimental animals were divided into a low dose knockout group（Low KD）,a middle dose knockout group（Middle KD）,a high dose knockout group（High KD）,and corresponding control groups（Control）.Cx26^flox/flox mice were used to hybridize with Rosa26-CreER mice to construct Cx26^flox/flox;Rosa26-CreER mice that knocked out the Gjb2 gene induced by TMX.Tamoxifen（TMX）was injected subcutaneously to mice on postnatal day0-1（P0-P1）with a dose of 0.6 mg/10 g body weight（Low KD）,1.1 mg/10 g body weight（Middle KD）and 1.6 mg/10 g body weight（High KD）,Cx26^flox/flox;Rosa26-CreER as a knockout group,and littermates Cx26^flox/flox mice as corresponding control groups.Frozen sections immunofluorescence staining and Western Blot were used to detect the knockout of Cx26 in each group;flattened cochlear preparations immunofluorescence staining was used to detect the knockout pattern of Cx26 in the cochlea;ABR were used to detect the hearing threshold of each group at P20 and P60;resin sections to detect morphological changes and spiral ganglion density of cochlea;ultrastructure of pillar cells were detected by transmission electron microscopy;hair cell damage of mice in different groups was determined by cell counting;CtBP2 immunostaining,DPOAE and transmission electron microscopy were used to evaluate hair cells function.Results: In each knockout group,Cx26 expression was successfully down-regulated,and Cx26 expression was negatively correlated with TMX injection dose.Cx26-negative supporting cells counts revealed that Cx26 was almost completely knocked out in the high-dose knockout group;Cx26 signaling was still observed in a few supporting cells in the medium-dose knockout group,whereas in the low-dose knockout group more expression was observed than the medium dose knockout group.ABR test showed that the high-dose knockout group showed severe full-frequency hearing loss at P20,the middle-dose knockout group had mild hearing loss,while the low-dose knockout group had no obvious hearing loss;at P60,the hearing loss was aggravated in the medium-and high-dose knockout group,and the upper threshold was also observed in the low dose knockout group,but there was no statistical difference.The morphology of organ of Corti was found to be abnormal in the high-dose knockout group.The organ of Corti in the medium-dose knockout group was generally normal,but the height was reduced,while the low-dose knockout group showed no significant change.Spiral ganglion counts showed no significant change in SGN of each group of mice at P20.Ultrastructural observation of pillar cells showed that the microtubules in the high-dose knockout group decreased significantly,but the microtubule density did not change significantly in the medium-dose knockout group,but the length of the pillar cells and the distance between the nucleus of the pillar cells were shortened.The hair cells count showed that there was obvious hair cell death in the high-dose knockout group at P20,and the hair cell death was mainly in the middle turn.At P60,the outer hair cells death was also observed in the basal turn of the low-and medium-dose knockout groups.More hair cells death in the medium-dose knockout group than low-dose knockout groups.Evaluation of hair cell function revealed no significant changes.Conclusion: The above results suggest that the degree of hearing loss is positively correlated with the degree of Connexin26 deletion in the Connexin26 knockout mouse model.The lack of Connexin26 expression in a small amount of supporting cells does not affect the development of the supporting cells and hearing.In the medium-dose knockout group,the supporting cells were mildly dysplastic,and the hair cells showed no degeneration and functional changes,when hearing loss appeared,suggesting that supporting cell developmental disorders may be the cause of deafness related to GJB2 deletion mutation.PART IIIMechanism of spatially specific Gjb2 knockout model inducing hearing impairment in miceObject: To study the effect of spatial specific connexin26 knockout on mouse hearing and the development of cochlear supporting cells.Methods: By hybridizing Lgr5-CreER mice and Atoh1-Cre mice to Cx26^flox/flox mice and tdTomato reporter mice,respectively.Cx26^flox/flox;Lgr5-CreER mice with partial supporting cells specific knockout of Cx26 and Cx26^flox/flox;Atoh1-Cre mice,which specifically knock out Cx26 in hair cells,were constructed.Transgenic tdTomato reporter mice were used to examine Cre expression and activation regions.The hearing threshold of each spatial specific Cx26 knockout mouse was detected by ABR.Transmission electron microscopy and scanning electron microscopy were used to detect the development and ultrastructure of organ of Corti in each spatial specific Cx26 knockout mouse.Hair cell counts were used to detect hair cell damage patterns in each spatial specific Cx26 knockout mouse.Results: By use different transgene mice with Cre expression and activation in different regions,and hybridizing them with Cx26^flox/flox mice,we successfully constructed two Cx26 knockout mouse models with different spatial knockout patterns.Cx26^flox/flox;Lgr5-CreER mice specifically knocked out Cx26 in the inner phalangeal cells,inner pillar cells and the third row of Deiter cells,while Cx26^flox/flox;Atoh1-Cre specifically knocked out Cx26 in the hair cell region.In the Lgr5-CreER-mediated partial supporting cells knockout of Cx26 mice,no obvious hearing changes were detected for a period of time.Morphological examination showed that the Corti tunnel was still open,but the third row of Deiter cells was found by scanning electron microscopy,the phalangeal processes showed a certain thickening and deformity and the surface of the cell body was irregular.There was no obvious abnormality in the transmission electron microscope,and a few outer hair cells in the third row of the basal turn were degenerated.In the hair cell-specific knockout group,there was no significant change in hearing,organ of Corti morphology and hair cells.Conclusion: Knocking out the inner phalangeal cells,inner pillar cells and the third row of Deiter cells alone did not significantly affect the development of the corresponding supporting cells and the morphology of the organ of Corti,suggesting that the development of the supporting cells depends not only on their own Cx26 expression.After knockout Cx26 in the third row of Deiter cells,a few third outer hair cell death of the basal turn was observed,suggesting that Cx26 of the supporting cell may play a role in maintaining the survival of the corresponding hair cells.However,no significant cochlear morphological changes and hearing loss were observed in hair cells-specific knockout Cx26 mice models,suggesting that even in the previous study,Cx26 mRNA expression in hair cells was detected,but it may not be expressed as protein in hair cells or cx26 may not play an important role hair cells.