Generation of a Triple-Transgenic Zebrafish Line for Assessment of Developmental Neurotoxicity during Neuronal Differentiation
Generation of a Triple-Transgenic Zebrafish Line for Assessment of Developmental Neurotoxicity during Neuronal Differentiation
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DOI：
10.3390/ph12040145
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发表时间：
2019-12-01
期刊：
影响因子：
4.6
通讯作者：
Nishimura, Yuhei
中科院分区：
文献类型：
--
作者：
Koiwa, Junko;Shiromizu, Takashi;Nishimura, Yuhei
发育中的大脑对许多化学物质非常敏感。在发育过程中暴露于神经毒物与各种神经精神和神经系统疾病有关，包括自闭症谱系障碍和精神分裂症。各种筛选方法已被用于评估化学品的发育神经毒性（DNT），大多数试验集中在细胞活力，凋亡，增殖，迁移，神经元分化和神经元网络的形成。然而，在祖细胞分化为神经元、星形胶质细胞和少突胶质细胞期间的毒性评估通常需要免疫组织化学，这是一种可靠但劳动密集且耗时的测定。在这里，我们报告了一个三转基因斑马鱼线，表达不同的荧光蛋白在神经元（Cerulean），星形胶质细胞（mCherry），少突胶质细胞（mCitrine），可用于检测DNT在神经元分化的发展。利用活体荧光显微镜，我们可以检测到6的10种神经毒物暴露于斑马鱼受精后12小时至5天的DNT。此外，基于荧光模式，化学物质可以被聚类为三个主要的DNT组：（i）抑制神经元和少突胶质细胞分化以及刺激星形胶质细胞分化;（ii）抑制神经元和少突胶质细胞分化;以及（iii）抑制神经元和星形胶质细胞分化，这表明报告基因表达反映了化学物质的毒理动力学。因此，在这里开发的三转基因斑马鱼线可能是一个有用的工具，以评估DNT在神经元分化。
The developing brain is extremely sensitive to many chemicals. Exposure to neurotoxicants during development has been implicated in various neuropsychiatric and neurological disorders, including autism spectrum disorders and schizophrenia. Various screening methods have been used to assess the developmental neurotoxicity (DNT) of chemicals, with most assays focusing on cell viability, apoptosis, proliferation, migration, neuronal differentiation, and neuronal network formation. However, assessment of toxicity during progenitor cell differentiation into neurons, astrocytes, and oligodendrocytes often requires immunohistochemistry, which is a reliable but labor-intensive and time-consuming assay. Here, we report the development of a triple-transgenic zebrafish line that expresses distinct fluorescent proteins in neurons (Cerulean), astrocytes (mCherry), and oligodendrocytes (mCitrine), which can be used to detect DNT during neuronal differentiation. Using in vivo fluorescence microscopy, we could detect DNT by 6 of the 10 neurotoxicants tested after exposure to zebrafish from 12 h to 5 days' post-fertilization. Moreover, the chemicals could be clustered into three main DNT groups based on the fluorescence pattern: (i) inhibition of neuron and oligodendrocyte differentiation and stimulation of astrocyte differentiation; (ii) inhibition of neuron and oligodendrocyte differentiation; and (iii) inhibition of neuron and astrocyte differentiation, which suggests that reporter expression reflects the toxicodynamics of the chemicals. Thus, the triple-transgenic zebrafish line developed here may be a useful tool to assess DNT during neuronal differentiation.