Unit on Nervous Development Systems

Zebrafish provide an outstanding opportunity to study the neurons of a vertebrate animal at early stages of development.

We are studying how and when various neurons develop from a fertilized egg. The human brain contains 100 billion nerve cells, andthe human brain has become larger during the course of evolution. However, within the vertebrate classes, brain structure has not changed greatly through evolution. The human brain performs a variety of functions: it receives sensory input, control movement, maintain memories, and so on. The brains of lower vertebrates also have these functions to some extent. We are using zebrafish as a model animal to study the development of the vertebrate brain and nervous system. Zebrafish are widely used because of their advantages over mice and rats. Zebrafish can lay up to a hundred eggs weekly.
Due to the reproductive rate and the fact that the zebrafish embryo is transparent, developmental processes can be easily observed. Combined with gene manipulation technology, it is now possible to observe neuronal cells in living embryos. For instance, using fluorescence microscopy, we can observe neuronal cells expressing the green fluorescent protein in living zebrafish embryos (Fig. 1).

Zebrafish mutants provide a way to understand the mechanisms of neurogenesis.

Fig. 1. Neurons visualized with GFP in huC-GFP transgenic embryos. Lateral views of wild-type and mib mutant huC-GFP embryos show an excess of GFP-expressing cells in mib mutants.

The zebrafish is a unique and powerful vertebrate model organism with a proven track record of easily executed, large-scale forward mutagenesis screens. As many zebrafish genes show a high degree of structural and functional similarity to their human homologues, studies of zebrafish genes may provide useful information about these human homologues. In a landmark issue of Development in 1996, the results of two large ENU (Ethylnitrosourea) mutagenesis screens were published, reporting the discovery of hundreds of zebrafish mutants. Among them was mind bomb (mib), a mutant with increased numbers of neuronal cells relative to the wild-type embryo (Fig. 1). We identified the gene responsible for mib and determined its essential role in neuronal cell fate decisions.

Mechanisms regulating neurogenesis by Notch and Wnt signaling.

In recent years, we have studied how neurogenesis is regulated by various signaling pathways. Analysis of the molecular mechanism by which mib regulates neurogenesis revealed the importance of Notch signaling. Mib facilitates the endocytosis of Notch ligand, Delta, and is therefore essential for Notch signaling in zebrafish (Itoh et al., 2003). Through analysis of the zebrafish Nrarp (Notch-regulated ankyrin repeat protein) knockdown phenotype, it turned out that Nrarp independently regulates canonical Wnt and Notch signaling by modulating LEF1 and Notch protein turnover, respectively (Ishitani et al., 2005). This revealed a novel molecular link between Notch and Wnt signaling mediated by the Notch and LEF1 proteins. In addition, using a biochemical screen, Notch was identified as a substrate for Nemo-like kinase (NLK) (Ishitani et al., 2010). NLK-phosphorylated Notch1ICD is impaired in its ability to form a transcriptionally active ternary complex; knockdown of NLK leads to hyperactivation of Notch signaling, and consequently decreases neurogenesis in zebrafish. These results reveal a previously unrecognized mode of regulation in the Notch signaling pathway that is important for neurogenesis.

Lab members