Pathogenic Mechanism Of NOTCH2 In Zebrafish Model Of Congenital Hypothyroidism

Objective:Congenital hypothyroidism（CH）is a common preventable endocrine disorder disease causing mental retardation in children.It is caused by 85%hypothyroidism（TD）and15%hormone dyssynthesis（TDH）.If the children are not clearly diagnosed and treated in time,irreversible growth retardation and mental retardation will occur in childhood.The molecular genetic mechanism of most TD cases is not clear,and there are new gene mutations that have not been discovered.The research team used whole exome resequencing association analysis to find NOTCH2 as a new candidate causative gene in 587 cases of TD.Taking zebrafish as model animal,the effects of notch2 on the development of zebrafish thyroid was observed by constructing gene knockdown model and gene knockout model,and the pathogenic mechanism of notch2 was preliminarily discussed.Methods:（Part 1）The notch2 gene knock-down model was constructed by injecting morpholino（MO）rings into zebrafish embryos,and the whole embryonic protein was extracted and the effect of MO knock-down was detected by Western blotting technique.Subsequently,embryos at different stages were collected for follow-up experiments.At the same time,the zebrafish notch2 gene knockout model was constructed by CRISPR/cas9technology to make the stop codon appear in advance so as to silence the expression of the gene.The mutants were obtained through the passage of zebrafish and the screening fish with T7 digestion.（Part 2）Then,further experiments were carried out on the successfully constructed model,using fluorescence in vivo imaging technology to observe the development of the thyroid gland of tg transgenic zebrafish after gene knockdown.The effect of q PCR on m RNA expression of thyroid related genes by extracting whole embryonic RNA at different periods.The effect of the spatial expression of thyroid transcription factor pax2a was observed by in situ hybridization of zebrafish whole embryo.Zebrafish with gene knockout heterozygotic mutant notch2^+/-1 month old were selected for RNA-seq,and the information of differentially expressed genes was obtained for bioinformatics analysis,including GO and KEGG enrichment analysis.Results:（Part 1）Western blotting results showed that the expression level of notch2 protein in embryos injected with Atg-targeted MO（atg-MO）decreased significantly,indicating that the injection of atg-MO successfully knocked down the expression of notch2 protein in zebrafish embryos,and the gene knockdown model was successfully constructed.The sequencing results of zebrafish mutant notch2^+/-showed that the frameshift mutation of the mutant notch2 resulted in the advanced appearance of the terminator,and all subsequent sequences were silenced,achieving the purpose of gene knockout.（Part 2）The results of fluorescence in vivo imaging showed thyroid dysplasia and ectopic phenomena in zebrafish embryos injected with atg-MO.Through analysis of fluorescence intensity,it was found that there were significant differences in the expression of tg in zebrafish embryos at 100hpf,5dpf and 6dpf stages.The results of q PCR showed that after notch2 knockdown,the expression of pax2a was significantly up-regulated,while tg and tshba were significantly down-regulated.At 36hpf,pax2a,tg,tshba and thrb were significantly down-regulated.At48hpf,hhex was significantly up-regulated,while nis was down-regulated.At 72hpf,tg and thra showed a downward trend.In 96hpf,nkx2.1a and pax2a all showed an upward trend.In situ hybridization results showed that notch2 knockdown affected the temporal and spatial expression of pax2a in 24hpf and 36hpf zebrafish embryos,thus affecting thyroid development.Transcriptome sequencing of the knockout heterozygous mutant notch2^+/-showed that 232 differentially expressed genes were detected,of which 130 genes were up-regulated and 102 genes were down-regulated,including duox,a homologous gene of the CH pathogenic gene DUOX2.notch signaling pathway related genes her1,her11,bbs1 and dtx2,differential genes are mainly enriched in apoptosis,cell aging and phagosome related signaling pathways,protein function is mainly related to MHC and antigen processing and presentation..Conclusions:（1）notch2 deficiency could cause thyroid dysplasia in zebrafish.（2）notch2may regulate thyroid development and thyroid hormone metabolism,and play different roles in different periods.（3）The knockout of notch2 may affect the expression of the CH pathogenic gene duox,thus leading to the occurrence of CH.