Background

Zinc is one of the vital micronutrients required through various developmental stages in animals. Zinc transporter-1 (ZnT1; Slc30a1) is essential in vertebrates for nutritional zinc uptake and cellular zinc extrusion. Knockout of ZnT1 is lethal in vertebrates and there are therefore few functional studies of this protein in vivo.

Methods

In the present study we characterised the embryonic development in a zebrafish Znt1a mutant (Znt1a^sa17) which is lacking the last 40 amino acids of Znt1a as generated by TILLING. In parallel experiments, we compared the development of a zebrafish embryo Znt1a morphant (Znt1a^MO) which was generated by knockdown of Znt1a using morpholino-modified oligonucliotides.

Results

The homozygous Znt1a^sa17 embryo is viable, but displays a subtle phenotype informing on the biological roles of Znt1a. The Znt1a^sa17 fish have delayed development, including attenuated epiboly. They further show a decrease in phosphorylated extracellular signal-regulated kinases 1 and 2 (pERK1/2), retarded yolk resorption, and impaired clearance of free Zn²⁺ from the vitelline fluid and its storage in hatching gland cells. All these aberrations are milder versions of those observed upon knockdown of Znt1a by morpholinos. Interestingly, the phenotype could be rescued by addition of the cell-permeable zinc chelator, N,N,N′,N′-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN) to the incubation medium and was aggravated by addition of zinc(II). Thus, the Znt1a^sa17 mutant has a reduced ability to handle zinc and can be characterised as a hypomorph.

Conclusion

This study is the first to show that the last 40 amino acids of Znt1a are of importance for its role in zinc homeostasis and ability to activate the MAPK/ERK pathway contrary to what was previously thought.

中文翻译：

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斑马鱼Znt1a ^sa17突变体揭示锌转运蛋白1a在胚胎发育中的作用

背景

锌是动物各个发育阶段所需的重要微量营养素之一。锌转运蛋白-1（ZnT1; Slc30a1）在脊椎动物中对于营养性锌吸收和细胞锌挤出至关重要。敲除ZnT1在脊椎动物中是致命的，因此在体内对该蛋白的功能研究很少。

方法

在本研究中，我们表征了斑马鱼Znt1a突变体（Znt1a ^sa17）的胚胎发育，该突变体缺少TILLING生成的Znt1a的最后40个氨基酸。在平行实验中，我们比较了斑马鱼胚胎Znt1a morphant（Znt1a ^MO）的发育，该突变体是通过使用吗啉代修饰的寡核苷酸敲除Znt1a而产生的。

结果

纯合的Znt1a ^sa17胚胎是可行的，但显示出微妙的表型，说明Znt1a的生物学作用。Znt1a ^sa17鱼的发育延迟，包括^外延衰减。他们进一步显示磷酸化的细胞外信号调节激酶1和2（pERK1 / 2）减少，卵黄吸收受阻，卵黄液中游离Zn ^2+的清除及其在孵化腺细胞中的贮存受损。所有这些像差是通过吗啉代敲除Znt1a时观察到的温和版本。有趣的是，可以通过向培养液中添加可渗透细胞的锌螯合剂N，N，N'，N'-四（2-吡啶基甲基）-1,2-乙二胺（TPEN）挽救表型，并通过加重添加锌（II）。因此，Znt1a^sa17突变体的锌处理能力降低，可被表征为亚同型。

结论

这项研究首次表明Znt1a的最后40个氨基酸对于其在锌稳态中的作用以及激活MAPK / ERK途径的能力具有重要意义，这与以前的想法相反。