Abstract
pax6 is a canonic master gene for eye formation. Knockout of pax6 affects the development of craniofacial skeleton and eye in mice. Whether pax6 affects the development of spinal bone has not been reported yet. In the present study, we used CRISPR/Cas9 system to generate Olpax6.1 mutant in Japanese medaka. Phenotype analysis showed that ocular mutation caused by the Olpax6.1 mutation occurred in the homozygous mutant. The phenotype of heterozygotes is not significantly different from that of wild-type. In addition, knockout Olpax6.1 resulted in severe curvature of the spine in the homozygous F2 generation. Comparative transcriptome analysis and qRT-PCR revealed that the defective Olpax6.1 protein caused a decrease in the expression level of sp7, col10a1a, and bglap, while the expression level of xylt2 did not change significantly. The functional enrichment of differentially expressed genes (DEGs) using the Kyoto Encyclopedia of Genes and Genomes database showed that the DEGs between Olpax6.1 mutation and wild-type were enriched in p53 signaling pathway, extracellular matrix (ECM) -receptor interaction, et al. Our results indicated that the defective Olpax6.1 protein results in the reduction of sp7 expression level and the activation of p53 signaling pathway, which leads to a decrease in the expression of genes encoding ECM protein, such as collagen protein family and bone gamma-carboxyglutamate protein, which further inhibits bone development. Based on the phenotype and molecular mechanism of ocular mutation and spinal curvature induced by Olpax6.1 knockout, we believe that the Olpax6.1-/- mutant could be a potential model for the study of spondylo-ocular syndrome.
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This study was supported by the National Natural Science Foundation of China (31771648 & 32273127 & 31672653), the Scientific Research Foundation of Jimei University (ZQ2020003), and the National Key Basic Research Program of China (2013CB967700).
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Qihua Pan designed and performed the experiment, analyzed experimental data, and wrote the original draft; Ke Lu, Junzhi Luo, Yuewen Jiang, Bilin Xia, Lei Chen, Mengyang Wang, and Ronggui Dai participated in part of the experiment and edited the manuscript; Tiansheng Chen designed and supervised the experiment, analyzed experimental data, and wrote and edited the manuscript. All authors have read and agreed to the published version of the manuscript. The authors have full access to all the data in the study and take responsibility for the integrity and security of the data.
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ESM 1:
Figure S1 Screening Olpax6.1△26 and Olpax6.1△677 mutation types by PCR and T7EI. Figure S2 validation the predicted off-target by PCR and sanger sequencing. Figure S3 The clustering heatmap of all DEGs. Table S1 the primers used in this study. Table S2 the predicted offtarget site of Olpax6.1 gE5. Table S3 the predicted offtarget site of Olpax6.1 gE6
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Pan, Q., Lu, K., Luo, J. et al. Japanese medaka Olpax6.1 mutant as a potential model for spondylo-ocular syndrome. Funct Integr Genomics 23, 168 (2023). https://doi.org/10.1007/s10142-023-01090-4
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DOI: https://doi.org/10.1007/s10142-023-01090-4