Li Junling, Chen Zhiliang, Gao Lian-Yong, Colorni Angelo, Ucko Michal, Fang Shengyun, Du Shao Jun
Institute of Marine and Environmental Technology, Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, USA; Shandong Medicinal and Biotechnology Center, Shandong Academy of Medical Sciences, Jinan 250062, Shandong Province, China.
Institute of Marine and Environmental Technology, Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, USA; Center for Biomedical Engineering and Technology, Department of Physiology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
Mech Dev. 2015 Aug;137:33-44. doi: 10.1016/j.mod.2015.04.001. Epub 2015 Apr 16.
Accumulation of misfolded or unfolded proteins in the endoplasmic reticulum (ER) triggers ER stress that initiates unfolded protein response (UPR). XBP1 is a transcription factor that mediates one of the key signaling pathways of UPR to cope with ER stress through regulating gene expression. Activation of XBP1 involves an unconventional mRNA splicing catalyzed by IRE1 endonuclease that removes an internal 26 nucleotides from xbp1 mRNA transcripts in the cytoplasm. Researchers have taken advantage of this unique activation mechanism to monitor XBP1 activation, thereby UPR, in cell culture and transgenic models. Here we report a Tg(ef1α:xbp1δ-gfp) transgenic zebrafish line to monitor XBP1 activation using GFP as a reporter especially in zebrafish oocytes and developing embryos. The Tg(ef1α:xbp1δ-gfp) transgene was constructed using part of the zebrafish xbp1 cDNA containing the splicing element. ER stress induced splicing results in the cDNA encoding a GFP-tagged partial XBP1 without the transactivation activation domain (XBP1Δ-GFP). The results showed that xbp1 transcripts mainly exist as the spliced active isoform in unfertilized oocytes and zebrafish embryos prior to zygotic gene activation at 3 hours post fertilization. A strong GFP expression was observed in unfertilized oocytes, eyes, brain and skeletal muscle in addition to a weak expression in the hatching gland. Incubation of transgenic zebrafish embryos with (dithiothreitol) DTT significantly induced XBP1Δ-GFP expression. Collectively, these studies unveil the presence of maternal xbp1 splicing in zebrafish oocytes, fertilized eggs and early stage embryos. The Tg(ef1α:xbp1δ-gfp) transgenic zebrafish provides a useful model for in vivo monitoring xbp1 splicing during development and under ER stress conditions.
内质网（ER）中错误折叠或未折叠蛋白质的积累会引发内质网应激，从而启动未折叠蛋白反应（UPR）。XBP1是一种转录因子，它通过调节基因表达介导UPR的关键信号通路之一，以应对内质网应激。XBP1的激活涉及由IRE1核酸内切酶催化的非常规mRNA剪接，该剪接从细胞质中的xbp1 mRNA转录本中去除内部26个核苷酸。研究人员利用这种独特的激活机制在细胞培养和转基因模型中监测XBP1的激活，进而监测UPR。在此，我们报告一种Tg(ef1α:xbp1δ - gfp)转基因斑马鱼品系，以绿色荧光蛋白（GFP）作为报告基因监测XBP1的激活，特别是在斑马鱼卵母细胞和发育中的胚胎中。Tg(ef1α:xbp1δ - gfp)转基因是使用包含剪接元件的斑马鱼xbp1 cDNA的一部分构建的。内质网应激诱导的剪接导致cDNA编码一个没有反式激活结构域的GFP标记的部分XBP1（XBP1Δ - GFP）。结果表明，在受精后3小时合子基因激活之前，xbp1转录本主要以剪接后的活性异构体形式存在于未受精卵母细胞和斑马鱼胚胎中。除了在孵化腺中有微弱表达外，在未受精卵母细胞、眼睛、大脑和骨骼肌中观察到强烈的GFP表达。用二硫苏糖醇（DTT）孵育转基因斑马鱼胚胎可显著诱导XBP1Δ - GFP表达。总的来说，这些研究揭示了斑马鱼卵母细胞、受精卵和早期胚胎中母源xbp1剪接的存在。Tg(ef1α:xbp1δ - gfp)转基因斑马鱼为在发育过程中和内质网应激条件下体内监测xbp1剪接提供了一个有用的模型。
