Generation of a Triple-Transgenic Zebrafish Line for Assessment of Developmental Neurotoxicity during Neuronal Differentiation

Koiwa Koiwa,Nakatani Nakatani,Ikejiri Ikejiri,Shiromizu Shiromizu,Tanaka Tanaka,Nishimura Nishimura,Adachi Adachi

doi:10.3390/ph12040145

Koiwa Koiwa, Nakatani Nakatani + Show 5 more

Open Access

https://doi.org/10.3390/ph12040145

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Journal: Pharmaceuticals	Publication Date: Sep 24, 2019
Citations: 6	License type: CC BY 4.0

Affiliation: Mie University, Mie University Hospital

Abstract

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.

Highlights

The developing brain is more vulnerable than the adult brain to most chemicals [1,2,3,4,5,6]
Differentiation of neural stem/progenitor cells into neurons, astrocytes, and oligodendrocytes has been analyzed by immunohistochemical staining with antibodies against cell-specific proteins such as NeuN/RNA-binding Fox3 (Rbfox3) for neurons, glial fibrillary acidic protein (GFAP) for astrocytes, and myelin basic protein (MBP) for oligodendrocytes [26,29]
Distinguishing between multiple cell types requires a multiplexed fluorescent reporter system and, this has been employed in some human stem/progenitor cell models [31], to our knowledge, it has not been used to distinguish between neurons, astrocytes, and oligodendrocytes in the same organism

Summary

Introduction

The developing brain is more vulnerable than the adult brain to most chemicals [1,2,3,4,5,6]. In human stem/progenitor cell models, various endpoints have been used to assess DNT, including viability, apoptosis, proliferation, migration, differentiation, and neuronal network formation [24,26,27]. These endpoints facilitate the identification of adverse outcome pathways and the development of integrated approaches to test and assess chemical-induced DNT [22,28]. To facilitate DNT testing, we have developed a triple-fluorescent Tg zebrafish line in which progenitor cell differentiation into neurons, astrocytes, and oligodendrocytes is accompanied by cell type-specific expression of the fluorescent proteins Cerulean, mCherry, and mCitrine, respectively, thereby enabling the effects of neurotoxicants on neuronal differentiation to be assessed in vivo

Results

Ethics Statement

Compounds

Zebrafish Husbandry

Exposure of Triple-Tg Zebrafish to Chemicals

In Vivo Imaging of Triple-Tg Zebrafish

Statistical Analysis