Role of the Connexin C-terminus in skin pattern formation of Zebrafish
Role of the Connexin C-terminus in skin pattern formation of Zebrafish
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DOI：
10.1016/j.bbadva.2021.100006
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发表时间：
2021-01-01
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影响因子：
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通讯作者：
Watanabe，Masakatsu
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作者：
Usui，Yuu;Watanabe，Masakatsu
斑马鱼身上的皮肤呈条纹状；两种连接蛋白，即Connexin39.4 （Cx39.4）和Connexin41.8 （Cx41.8）参与条纹模式的形成。在此，我们研究了Cx39.4和cx41.8的c端（CT）结构域在体内和体外的作用。方法为了研究CT结构域在体内的作用，我们在斑马鱼色素细胞中建立了表达CT结构域修饰连接蛋白系列的转基因斑马鱼系，并观察了斑马鱼的皮肤形态。为了研究CT结构域在体外的作用，我们在神经-2a （N2a）细胞中表达了CT结构域修饰的连接蛋白系列，并计算了斑块的形成频率。结果在x39.4−/−突变体和incx39.4和cx41.8双敲除突变体斑马鱼中，缺乏CT结构域的Cx39.4过表达产生的皮肤图案与全长Cx39.4相似。荧光蛋白融合的ct结构域修饰的Cx39.4在N2a细胞之间形成间隙连接斑块。过表达ct截短的Cx41.8在x41.8−/−突变体中挽救了突变体表型，但在双敲除斑马鱼中不起作用。荧光蛋白融合的ct截断的Cx41.8在不含Cx39.4的N2a细胞之间几乎不形成斑块，但与Cx39.4共表达时形成间隙连接斑块。结论Cx39.4的CT结构域与斑马鱼色素细胞的蛋白功能无关。然而，需要Cx41.8的CT域取决于Cx39.4的表达。这些结果为斑马鱼色素细胞中Cx39.4和Cx41.8之间的相互作用提供了证据，并表明至少有一个连接蛋白具有CT结构域。
BackgroundZebrafish display a striped skin pattern on their body; two types of connexins, namely, Connexin39.4 (Cx39.4) and Connexin41.8 (Cx41.8), are involved in stripe pattern formation. Herein, we investigated the role of the C-terminal (CT) domains of Cx39.4 and Cx41.8in vivoandin vitro.MethodsTo investigate the role of CT domainsin vivo, we established transgenic zebrafish lines expressing the CT-domain-modified connexin series in pigmented cells and observed skin patterns in fish. To investigate the role of the CT domainsin vitro, we expressed the CT-domain modified connexin series in Neuro-2a (N2a) cells and calculated the plaque formation frequency.ResultsThe overexpression of Cx39.4 lacking a CT domain produced skin patterns similar to that produced by full-length Cx39.4 in thecx39.4−/−mutant and incx39.4andcx41.8double-knockout mutant zebrafish. Fluorescence-protein-fused CT-domain-modified Cx39.4 formed gap junction plaques between N2a cells. The overexpression of CT-truncated Cx41.8 rescued the mutant phenotype in thecx41.8−/−mutant but did not function in the double knockout zebrafish. Fluorescence-protein-fused CT-truncated Cx41.8 hardly formed plaques between N2a cells without Cx39.4 but formed gap junction plaques when co-expressed with Cx39.4.ConclusionsThe CT domain of Cx39.4 is not required for protein function, at least in the pigment cells of zebrafish. However, the need for the CT domain of Cx41.8 depends on Cx39.4 expression.General significanceThese results provide evidence for the interactions between Cx39.4 and Cx41.8 in pigment cells of zebrafish and suggest that at least one connexin must have a CT domain.