Loss of the Nodal Modulator Nomo Results in Chondrodysplasia in Zebrafish
Loss of the Nodal Modulator Nomo Results in Chondrodysplasia in Zebrafish
复制标题
节点调节器 Nomo 的丢失导致斑马鱼软骨发育不良
DOI：
10.2174/1566524019666181212095307
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发表时间：
2018-01-01
影响因子：
2.5
通讯作者：
Fan, Xiongwei
中科院分区：
文献类型：
--
作者：
Cao, Linghui;Li, Lingyu;Fan, Xiongwei
背景：转化生长因子-β（TGF-β）/nodal信号参与脊椎动物早期胚胎模式的形成。Nodal modulator（Nomo，也称为pM 5）是节点信号的负调节器。目前，nomo基因在脊椎动物软骨发育中的作用尚不清楚。方法：在斑马鱼基因敲除模型中，利用成簇规则间隔短回文重复序列（clustered regularly interspaced short palindromic repeats，CRISPR）/CRISPR-associated protein 9（CRISPR/Cas9）靶向纤连蛋白III型结构域，构建Nomo基因突变体。通过整体原位杂交和qRT-PCR分析软骨形成关键的相关基因的表达。结果：nomo在包括软骨在内的多种组织中均有高表达。我们成功构建了斑马鱼nomo基因敲除模型。NOMO纯合突变体在4和5DPF时表现出不同程度的发育不全和畸形，这与人类的软骨发育不良相似。与野生型相比，软骨和骨骼发育的关键基因sox 9a、sox 9 b、dlx 1a、dlx 2a、osx、col 10a 1和col 11 a2在nomo突变体中表达下调。结论：nomo基因正调控着骨形成和软骨发育的主要调控因子和其他关键发育基因的表达，是软骨发育所必需的。
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.