期刊
CURRENT MOLECULAR MEDICINE
卷 18, 期 7, 页码 448-458出版社
BENTHAM SCIENCE PUBL LTD
DOI: 10.2174/1566524019666181212095307
关键词
nomo; CRISPR/Cas9; chondrogenesis; zebrafish; crest cells; in situ
资金
- National Natural Science Foundation of China [81470449, 81670290, 81470377, 31572349, 81670288, 31872315, 81700338, 81570279]
- Cooperative Innovation Center of Engineering and New Products for Developmental Biology of Hunan Province [2013-448-6]
- National Key Research and Development Program of China [2018YFA0108700, 2017YFA0105602]
- NSFC Projects of International Cooperation and Exchanges [81720102004]
- Research Team Project of Natural Science Foundation of Guangdong Province of China [2017A030312007]
- key program of guangzhou science research plan [805212639211]
- Hunan Provincial Natural Science Foundation of China [2015JJ3087, 2018JJ2666]
Background: Transforming growth factor-beta (TGF-beta)/nodal signaling is involved in early embryonic patterning in vertebrates. Nodal modulator (Nomo, also called pM5) is a negative regulator of nodal signaling. Currently, the role of nomo gene in cartilage development in vertebrates remains unknown. Methods: Nomo mutants were generated in a knockout model of zebrafish by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) targeting of the fibronectin type III domain. The expression of related genes, which are critical for chondrogenesis, was analyzed by whole-mount in situ hybridization and qRT-PCR. Whole-mount alcian staining was performed to analyze the cartilage structure. Results: nomo is highly expressed in various tissues including the cartilage. We successfully constructed a zebrafish nomo knockout model. nomo homozygous mutants exhibited varying degrees of hypoplasia and dysmorphism on 4 and 5 dpf, which is similar to chondrodysplasia in humans. The key genes of cartilage and skeletal development, including sox9a, sox9b, dlx1a, dlx2a, osx, col10a1, and col11a2 were all downregulated in nomo mutants compared with the wildtype. Conclusion: The nomo gene positively regulates the expression of the master regulator and other key development genes involved in bone formation and cartilage development and it is essential for cartilage development in zebrafish.
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