Development and assessment of new risk assessment methods

Title of Research:
13_PT01-01

Development of in vivo fluorescent imaging of neuronaldifferentiaon in zebrafish for developmental neuotoxicity testing
Principal Investigator:
Yuhei Nishimura (Department of Pharmacogenomics, Mie University Graduate School ofMedicine)Collaborators:
Toshio Tanaka and Reiko Kawase (Department of Pharmacogenomics, Mie University Graduate
School of Medicine)
Summary of Research:
The developing brain is extremely sensitive to many chemicals. Exposure to neurotoxicantsduring development has been implicated in various neuropsychiatric and neurological disorders,
including autism spectrum disorder and attention deficit hyperactive disorder. Although rodents
have been widely used for developmental neurotoxicity testing, experiments using large numbersof rodents are time-consuming, expensive, and raise ethical concerns. Using alternative
non-mammalian animal models may relieve some of these pressures by allowing testing of large
numbers of subjects while reducing expenses and minimizing the use of mammalian subjects.
In this study, we have tried to make the three-color zebrafish in which cerulean, a fluorescentprotein with maximum excitation (Ex) /emission (Em) at 433/475 nm, would express in neurons,mCitrine, a fluorescent protein with Ex/Em at 516/529 nm, would express in oligodendrocytes, andmCherry, a fluorescent protein with Ex/Em at 587/610 nm, would express in astrocytes. We wereable to make one-color zebrafish expressing cerulean in neurons, mCitrine in oligodendrocytes, ormCherry in astrocytes. We were also able to confirm that the zebrafish expressing cerulean inneurons and the zebrafish expressing mCitrine in oligodendrocytes could transmit the phenotype
to their offspring. We will cross these two zebrafish to make two-color zebrafish. We will also
examine whether zebrafish expressing mCherry in astrocytes could transmit the phenotype to theiroffspring. Finally, we will cross the two-color zebrafish and the mono-color zebrafish to makethree-color zebrafish expression cerulean in neurons, mCitrine in oligodendrocytes, and mCherry
in astrocytes.
It has been demonstrated that various developmental neurotoxicants can affect thedifferentiation of neuron, oligodendrocyte, and astrocytes and that the dysregulated differentiation
may cause various neuropsychiatric disorders. The tricolor zebrafish we will develop would makeit possible to examine the developmental neurotoxicity of many chemicals and provide a soundbasis for human risk assessments.
Timeline:Nov 2013 ~ Feb 2015
Topics:Publications:Zebrafish as a systems toxicology model for developmental neurotoxicity testing.
Nishimura Y, Murakami S, Ashikawa Y, Sasagawa S, Umemoto N, Shimada Y, Tanaka T.
Congenital Anomalies 55(1):1-16 (2015)
In vivo fluorescent imaging of blood-brain barrier disruption in zebrafish using a novel dye.
Nishimura Y, Murakami S, Ashikawa Y, Sasagawa S, Umemoto N, Shimada Y, Tanaka T.
DNT4, Philadelphia, May 2014
Evaluation of developmental neurotoxicity of nicotine using gene knockout in zebrafish
Nishimura Y, Murakami S, Ashikawa Y, Sasagawa S, Kawabata M, Umemoto N, Ariyoshi M, Zhang
B, Shimada Y, Tanaka T. The 41st Annual Meeting of Japanese Society of Toxicology, Kobe, July
2014