Laboratory/Faculty

Bioscience and Biotechnology Center
Group of Animal Organ Functions

ProfessorMasahiko Hibi
Development of cerebellum and cerebellar neural circuits
Associate ProfessorTakashi Shimizu
Functions of cerebellar neural circuits
Assistant ProfessorHisashi Hashimoto
Development of neural crest-derived cells
Laboratory HP
Japanese
Masahiko Hibi Professor
Lab members

During vertebrate development, development of organs (organogenesis) is precisely regulated to elicit their functions. We are studying organogenesis and organ functions by using teleost species, such as zebrafish and medaka. We currently have two major projects. First, we study formation and function of neu1qaal circuits that control complex behaviors. During neural development, the fate of neural tissues is determined along the rostro-caudal axis, depending on their location. In each neural tissue, neurons are generated from neural stem cells and/or neuronal progenitors. Neurons migrate and extend axons and dendrites that form neural circuits. As a model of neural development and functions, we currently focus on the cerebellum. We are trying to understand molecular mechanisms underlying the neural circuit formation, and higher order functions of the cerebellum, such as motor learning and cognitive/emotional functions. We also study cell differentiation from neural crest cells. Neural crest cells are generated on the dorsal side of neural tissues and serve as stem cells in early vertebrate development. They give rise a variety of cells, including pigment cells. We are interested in genetic cascades controlling differentiation of pigment cells from the neural crest cells.

Neural development-formation of cerebellum and cerebellar neural circuits

The cerebellum is a neural tissue derived from the dorsal part of the most rostral hindbrain in the vertebrate central nervous system. The structure of the cerebellar neural circuits is generally conserved among the vertebrates. There are two major cerebellar neurons, glutamatergic granule cells and GABAergic Purkinje cells, which are generated in distinct progenitor domains. Granule cells are derived from neuronal progenitors in the upper (cerebellar) rhombic lip that is located in the superficial domain of the cerebellum primordium. Purkinje cells are derived from neuronal progenitors in the ventricular zone that is located on the ventral side of the cerebellum primordium. They migrate, differentiate, and form neural circuits (Fig. 1). Granule cells and Purkinje cells receive afferent inputs, the mossy fibers (MFs) and climbing fibers (CFs), respectively, from outside the cerebellum. As the information of MFs is conveyed to Purkinje cells, the information of the MFs and CFs is integrated in Purkinje cells. Purkinje cells send outputs through the projection neurons (eurydendroid cells in teleost cerebellum) outside the cerebellum. This simple organization of the cerebellar neural circuits provides a good model to understand development of neural circuits in vertebrate CNS. We visualize the neural circuits by establishing transgenic zebrafish expressing marker proteins, such as fluorescent proteins, and study developmental processes of the neural circuit formation. We further analyze molecular mechanisms that control differentiation of the cerebellar neurons and the input neurons (precerebellar neurons, e.g. neurons in the inferior olive nuclei) by generating mutants with genome editing techniques.

Functions of neural circuits-classical fear conditioning

The cerebellum is known to function in some forms of skillful movements and motor learning. Recent studies reveal that the cerebellum is also implicated in higher cognitive and emotional functions. Furthermore, abnormalities in the cerebellar neural circuitry have been shown to be involved in pathogenesis of psychiatric disorders, such as autism. Therefore, elucidation of the functions of the cerebellar neural circuits provide some clue for understanding pathological conditions in related human diseases. We study roles of the cerebellar neural circuits in motor learning and fear conditioning by using zebrafish. We monitor the activity of the cerebellar neural circuits in the learning processes. We further examine their roles in the higher order functions by manipulating their activity using neurotoxins and optogenetic techniques.

Neural crest cells-differentiation of pigment cells

Pigment cells are derived from multipotent neural crest cells and their diversity in teleosts provides a good model for studying mechanisms controlling the specification of distinct cell types. Zebrafish have three types of pigment cells (melanophores, iridophores, and xanthophores), while medaka have four (three shared with zebrafish, plus leucophores). It remains elusive how different types of pigment cells are generated from the neural crest cells. In the late twentieth century, Hideo Tomita of Nagoya University isolated more than 80 naturally occurring medaka mutant strains called the Tomita collections. We have been studying medaka mutants in the Tomita collections which display abnormalities in pigmentation. By establishing medaka and zebrafish mutants for key genes potentially involved in pigment cell differentiation, we study molecule mechanisms that control the pigment cell differentiation from the neural crest cells. By comparative studies with other teleosts, we try to understand evolution of the pigment cell development.

Fig. 1. Cerebellar neural circuits in zebrafish

(A) Early stage larva stained with anti-Vglut1 (granule cell axons, green) and anti-parvalbumin7 (Purkinje cells, magenta) antibodies.

(B) Transgenic zebrafish larva expressing Venus and mCherry in granule cells and Purkinje cells, respectively.

Schematic drawing of the cerebellar neural circuits and transgenic fish expressing GFP in the cerebellar neural circuits.

Genes expressed in Purkinje cells.

Fig. 2. Classical fear conditioning in zebrafish.

(A) Experimental paradigm for delayed classical fear conditioning. The extinguishment of a white LED light was used as the condition stimulus (CS), and an electric shock was used as the unconditioned stimulus (US). Before the conditioning, bradycardia responses were elicited only by the US. After the paired-associated learning, the conditioned bradycardia occurred shortly after the CS onset.

(B) Conditioning-associated neurons. Ca2+ imaging with transgenic fish expressing GCaMP7a identified two types of cerebellar neurons whose activity was elicited by the CS upon the conditioning (early and late responders). They are located in the corpus cerebelli and likely granule cells.

Fig. 3. Differentiation of pigment cells from neural crest cells.

(A) Pigment cell development in medaka embryo. Neural crest cells are stem cells that give rise to chromatoblasts. Chromatoblast cells differentiate to four-types of pigment cells, melanophores, iridophores, xantophores (X), and leucophores (L) in medaka.

(B) Mutant analyses.

(C) Working model.

References

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