The Center for Heart Development, State Key Lab of Development Biology of Freshwater Fish, The National & Local Joint Engineering Laboratory of Animal Peptide Drug Development, Key Lab of MOE for Development Biology and Protein Chemistry, College of Life Sciences, Hunan Normal University, Changsha, Hunan, China.
Department of Cardiac Surgery, Guangdong Cardiovascular Institute, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou, Guangdong 510100, China.
Curr Mol Med. 2018;18(7):448-458. doi: 10.2174/1566524019666181212095307.
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.
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.
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.
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.
转化生长因子-β（TGF-β）/诺达信号通路参与脊椎动物早期胚胎的模式形成。诺达调节剂（Nomo，也称为 pM5）是诺达信号通路的负调节剂。目前，脊椎动物软骨发育过程中 nomo 基因的作用尚不清楚。
通过纤维连接蛋白 III 结构域的成簇规律间隔短回文重复序列（CRISPR）/CRISPR 相关蛋白 9（CRISPR/Cas9）靶向敲除斑马鱼模型，生成了 nomo 突变体。通过全胚胎原位杂交和 qRT-PCR 分析了对软骨发生至关重要的相关基因的表达。进行全胚胎茜素蓝染色以分析软骨结构。
nomo 在包括软骨在内的各种组织中高度表达。我们成功构建了斑马鱼 nomo 敲除模型。nomo 纯合突变体在 4 和 5 dpf 时表现出不同程度的发育不良和畸形，这与人类的软骨发育不良相似。软骨和骨骼发育的关键基因，包括 sox9a、sox9b、dlx1a、dlx2a、osx、col10a1 和 col11a2，在 nomo 突变体中的表达均低于野生型。
nomo 基因正向调节参与骨形成和软骨发育的主调控因子和其他关键发育基因的表达，它对斑马鱼软骨发育是必需的。
