NATIONAL INSTITUTE FOR BASIC BIOLOGY
DIVISION OF CELLULAR COMMUNICATION (ADJUNCT)
- Yoshiki Hotta
- Associate Professor:
- Hitoshi Okamoto
- Research Associates:
- Mika Takahashi
- Institute Research Fellow:
- Akira Chiba
- Graduate Students:
- Keita Koizumi
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.
- 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
- 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
- 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
- We are currently examining how the specification and axonal
pathfindings of primary motoneurons are affected by the ectopic
expression of ZISH genes.
- 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
- 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
- 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
- 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
- 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.
- 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.