Linghui Cao 1 , Lingyu Li 1 , Yongqing Li 1 , Jian Zhuang 2 , Yu Chen 1 , Yuequn Wang 1 , Yan Shi 1 , Jimei Chen 2 , Xiaolan Zhu 2 , Yongqi Wan 1 , Fang Li 1 , Wuzhou Yuan 1 , Xiaoyang Mo 1 , Xiangli Ye 1 , Zuoqiong Zhou 1,2 , Guo Dai 1 , Zhigang Jiang 1 , Ping Zhu 2 , Xiushan Wu 1 , Xiongwei Fan 1 Show Affiliations »
Abstract
BACKGROUND: Transforming growth factor-β (TGF-β)/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. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.
BACKGROUND: Transforming growth factor-β (TGF-β)/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 . Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.
Entities: Chemical
Disease
Gene
Species
Keywords:
CRISPR/Cas9; chondrogenesis; crest cells; in situ; nomo; zebrafish.
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Year: 2018
PMID： 30539698 DOI： 10.2174/1566524019666181212095307
Source DB: PubMed Journal: Curr Mol Med ISSN： 1566-5240 Impact factor: 2.222