Yoshiki Hotta

Associate Professor:
Hitoshi Okamoto

Research Associates:
Mika Takahashi
Shinichi Higashijima

Institute Research Fellow:
Akira Chiba
Nobuyoshi Shimoda

Graduate Students:
Keita Koizumi
Yoko Yasuda

Brain can be seen as an integrated circuit where neurons of various identities are interconnected in a highly ordered manner by their axons. We have been interested in how individual neurons acquire their own identities and how their axons find their own pathways and finally recognize their proper targets. Our current interest is mainly focused on the motoneurons because of their accessibility to various cellular manipulations. Using two different animals, zebrafish (Danio rerio) and fly (Drosophila melanogaster), both of which are suitable for genetic analysis and gene manipulation, we are trying to address to these questions both at the molecular and cellular levels.

I. Molecular Developmental Neurobiology of Zebrafish

Embryos of zebrafish stay transparent throughout most period of their development. Most neurons in the central nervous system of early embryos are identifiable. In the embryonic brain, a primitive neuronal network is formed by the early born neurons (primary neurons) and presents the initial scaffold for the later extending axons from the secondary neurons. Our current goal is to identify the molecules which are involved in the final determination of the identities or the axonal pathways by individual primary neurons.

1. zish (zebrafish isl-1 homologues) genes in the specification of primary motoneurons

Zebrafish embryos have three subtypes of primary motoneurons (RoP, MiP and CaP) per hemisegment, each of which extends the axon along the stereotyped pathway and innervates the specific region of the somites. In the last annual report, we reported the molecular cloning of the cDNA for zebrafish Isl-1. Its expression pattern in the ventral region of the spinal cord suggested that Isl-1 may be involved in the specification of primary motoneurons.

In collaboration with Ziyuan Gong and Choy Hew at University of Toronto, we have recently isolated two novel zebrafish cDNA clones which encode the proteins similar to the original Isl-1, using the salmon pituitary cDNA encoding a novel Isl- 1-like protein as a probe. We named them ZISH (Zebrafish Isl- 1 Homologue)-2 and -3, by counting the original zebrafish Isl- I as ZISH-1.

The expression patterns of the original Isl-1 (ZISH-1) mRNA and ZISH-2 mRNA are almost complementary. ZISH- I mRNA expression is at first observed in randomly distributed ventromedial cells and later restricted to RoP (or RoP and MiP) and the secondary motoneurons surrounding RoP. And the number of ZISH- I mRNA-positive ventromedial cells increases in the tail region and in the spaidtail mutant where the somites surrounding the spinal cord are missing or defective. These data suggest that ZISH-1 mRNA expression is downregulated by the influence of the somites. In contrast, ZISH-2 mRNA is expressed only by CaP and VaP from the moment when its expression starts to be detected (around 15 hours after fertilization), just when the nascent CaPs are about to stop expressing ZISH-1 mRNA. ZISH-2 mRNA is not expressed in the caudal end of the spinal cord where the somites are not yet formed. These subtype specific expression patterns suggest that the Isl-1 (ZISH-1) and ZISH-2 genes may either react oppositely to the influence from the somites or regulate the expression of each other, and together be involved in the determination of cellular identities (specification) by motoneurons in embryonic zebrafish.

Although ZISH-3 mRNA is expressed only by Rohon-Beard cells but not by motoneurons in the spinal cord, its expression pattern is nonetheless very specific. In the body trunk, it is expressed intensely only in the ventral region of the axial muscle which is innervated by CaP. And in the brain, it is expressed both in the entire eye (including the retina and the lens) and in the nascent tectum. These data suggest that the ZISH-3 gene may regulate the expression of the molecules that help CaP and the retinal ganglion cells to correctly find their proper targets, the ventral axial muscle and the tectum respectively.

We are currently examining how the specification and axonal pathfindings of primary motoneurons are affected by the ectopic expression of ZISH genes.

2. Search for new cell surface recognition molecules expressed in a subset of CNS axons.

During neuronal development, growth cones are known to have ability to recognize and extend along specific axonal pathways. Several lines of evidence suggest that cell surface glycoproteins play important roles in this process. Immunocytochemical studies have demonstrated that the monoclonal antibody HNK- 1, originally raised against a human lymphoblastoma, recognizes a subset of CNS and PNS axons in many vertebrates including zebrafish by binding to a carbohydrate determinant in several glycoproteins. Thus, we started characterizing glycoproteins recognized by HNK-1, hoping to isolate new cell surface recognition molecules expressed in a subset of CNS and PNS axons during neuronal development.

Our strategy is as follows: Many brains of adult zebrafish are homogenized, and from this extract, molecules which binds to HNK-1 are intensely enriched by using HNK-1 affinity chromatography. The affinity-purified fractions are used to immunize mice to get a series of monoclonal antibodies (MAbs). The MAbs, thus obtained, are screened with immunohistochemistry to zebrafish tissues. Hopefully, those MAbs which recognize peptides and not carbohydrates are used to isolate and characterize new molecules.

Immunogen which represents several protein family on Western blotting has been prepared. Immunization is now in progress.

II. Drosophila neurogenetics

1. Fasciclin III as a synaptic recognition molecule in Drosophila

The larval neuromuscular system of the Drosophila consists of uniquely identified cells, and is a powerful model system for studying selective synapse formation. During synaptogenesis, the cell adhesion molecule fasciclin III appears in both motoneuron RP3 and its targets, muscle 6 and 7. We have tested whether fasciclin III is necessary and/or sufficient for RP3 target selection using intracellular dye injection.

First, we have found that in the existing fasciclin III null mutant RP3 reliably formed synapse with its normal targets. Therefore, fasciclin III is either irrelevant for the process, or playing a positive role but its absence can be compensated for another redundant mechanism ("X").

We now have demonstrated that the latter is the case. We generated transgenic flies which misexpresses fasciclin III ectopically on all skeletal muscles during neuromuscular synaptogenesis. This was accompanied by creating a construct which placed the fasciclin III gene under the control of the myosin heavy chain promoter, and introducing this construct into the fly genome by P-element mediated genomic transformation. In these flies, PR3 often innervated non-target muscle cells while other identified motoneurons innervated targets normally.

Our results provide the single identified-cell level evidence that a cell adhesion molecule functions as a specific synaptic target recognition molecule in vivo .

2. Analysis of A9 gene which affects larval neurogenesis

P-element insertion line A9 is a pupal lethal mutant which has a diminished brain in the homozygotes. Using BrdU incorporation analysis, we found larval neuroblasts of this mutant are morphologically abnormal and showed almost no sign of proliferation.

We tried to clone this A9 gene and identified a 0.6 kb transcript around the P-element insertion site which was reduced in A9 homozygotes. The molecular cloning of the cDNA and the subsequent sequencing determination and in situ hybridization indicate that the putative product of this transcript is a secretory glycoprotein expressed in larval imaginal cells including those in CNS. These data suggest that the defect in the gene encoding this transcript is responsible for the A9 mutant phenotype. We are now trying to examine if A9 mutants can be rescued by P-element mediated transformation with this gene.

Publication List:

Inoue, A., Hatta, K., Hotta, Y. and Okamoto, H. (1994) Developmental regulation of Islet- I mRNA expression during neuronal differentiation in embryonic zebrafish. Devd Dynam. 199, 1-11.

Masai, I., Okazaki, A., Hosoya, T. and Hotta, Y. (1993) Drosophila retinal degeneration A (rdgA) gene encodes an eye-specific diacylglycerol kinase with cysteine-rich zinc finger motif and ankyrin repeats. Proc. Natl. Acad. Sci. USA 90, 11157-11161.

Okamoto, H. (1993) Neuronal differentiation and specialization in embryonic zebrafish. Neurosci. Res. Suppl. 18, p. 9.