The Effects Of Waca/b Loss Of Function On Nervous System Development Of Zebrafish

Neurodevelopmental disorders are caused by the developmental impairment of the central nervous system,in which genetic factors play an important role.Due to their extremely high genetic heterogeneity,it is quite challenging to identify the genetic causes of diseases and to study the underlying molecular mechanisms.In recent years,large-scale next-generation sequencing studies have promoted the identification of many novel neurodevelopmental disease genes,including WAC.The association between WAC mutations and several forms of neurodevelopmental diseases has been reported by many independent studies,a series of clinical symptoms related to WAC loss-of-function is called Desanto-Shinawi syndrome.However,it remains unclear how mutations in WAC cause these diseases.Previous studies have shown that WAC is involved in regulating ubiquitination of certain target proteins such as H2B,as well as in regulating autophagy pathways.However,whether these functions are related to the disease pathology remains unclear.In this study,we use zebrafish as a model to study the function of WAC in neurodevelopment,animal behavior,and neuronal circuit function.Since WAC has two homologs in zebrafish,namely waca and wacb,we constructed waca deletion,wacb deletion and waca/b deletion homozygous mutants respectively.These findings may shed light on the molecular pathology underlying the neurodevelopmental disorders caused by WAC mutations.Part Ⅰ:Spatiotemporal expression profile of waca and wacb and construction of homozygous mutantsObjective:We attempted to explore the spatiotemporal expression profile of waca and wacb respectively,and construct single and double mutant models of waca and wacb in zebrafish as the basis for the subsequent studies.Methods:Whole-mount in situ hybridization and RT-q PCR technique were utilized to explore the expression of waca and wacb.After designing the specific sg RNA targeting on waca and wacb,chimeras were made by CRISPR/Cas9 with microinjection.The homozygous mutants were obtained through several generations of crosses and backcross with genetic sanger screen.Results:Waca and wacb are broadly expressed at the onset of zebrafish embryonic development,and are mainly distributed in the head after 3 dpf.The homozygous mutants are obtained and the double mutant species have been screened.Conclusion:Zebrafish genetic models of waca mutantion,wacb mutantion,and waca/b mutantion with normal fertility were constructed successfully.PartⅡ:Behavior studies on waca/b mutant zebrafishObjective:Intellectual disability,anxiety,and social weaknesses were found in patients with WAC mutations.Therefore,we attempted to evaluate zebrafish from these aspects and identify similar behavior.Methods:The repetitive light flashes and the T-maze test were used to evaluate learning-memory ability.Anxiety levels could be reflected by openfield assay and novel tank test.Mirror test and three-tank test were performed to assess social weaknesses behavior.Results:Zebrafish with waca/b mutations showed defects in non-associative learning and associative learning compared to WT group.They spent more time in the bottom area of the novel tank and the edge of the openfield.Besides,they spent less time in the contact area of the mirror test and in the social zone of the three-tank test.Conclusion:The behavior features of waca^-/-;wacb^-/-zebrafish are in concordance with the phenotype of patients with WAC mutation,namely,intellectual disability,higher anxiety,and social weaknesses.PartⅢ:Cellular and molecular mechanism studies on waca/b mutant zebrafish Objective:We attempted to explore the morphology and neurodevelopment changes in waca/b mutant zebrafish to find out and verify the potential molecular mechanism and biochemistry process.Methods:Zebrafish larvae were photographed and statistical measurements were performed.Immunofluorescence was carried out to detect the neurogenesis,neuron number,cell proliferation,and apoptosis.Afterward,transcriptome sequencing was performed to seek for the potential molecular mechanism and biochemistry process,followed by validation in RNA and protein aspects.Results:Developmental delay and downregulation of neurogenesis appeared in waca/b mutant zebrafish,accompanied by neuronal reduction,decreased proliferation,and increased apoptosis.Transcriptome sequencing and enrichment identified the aberrant activation of JNK pathway,which was validated by q PCR and western blot assay.Conclusion:Loss of function of waca/b gene caused developmental dalay,disorder of neurogenesis,cell proliferation and apoptosis.The aberrant activation of JNK pathway might be responsible for the defects.Part Ⅳ:JNK pathway inhibitor SP600125 can rescue waca/b mutant zebrafish Objective:Since the aberrant activation of JNK pathway was found in partⅢ,a specific inhibitor targeting JNK pathway was used on waca/b mutant zebrafish.The trial was aimed to explore whether it could rescue the phenotype of zebrafish and find new potential therapy targets for clinical practice of Desanto-Shinawi syndrome.Methods:Waca/b mutant zebrafish were treated with SP600125,and the rescue effects were evaluated in aspects of molecular,cellular,and overall functions.Results:Upon SP600125 treatment,waca/b mutant zebrafish showed decreased p-c-JUN levels,recovered neurons,and increased overall developmental status and non-associative learning abilities.Conclusion:SP600125 can partially rescue the phenotype of waca/b mutant zebrafish by inhibition of JNK pathway.These data provide new insights for understanding the molecular pathology of Desanto-Shinawi syndrome and highlighted a potential target for the treatment.