Department of Molecular Neurobiology and Pharmacology, Graduate School of Medicine, University of Tokyo, and SORST, Japan Science and Technology Agency, Tokyo 113-0033, Japan
* Author for correspondence (e-mail: mishina{at}m.u-tokyo.ac.jp)
Accepted 21 January 2004
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SUMMARY |
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Key words: Zebrafish, Trimethylpsoralen (TMP) mutagenesis, Representational difference analysis (RDA), Chaperonin containing TCP-1 subunit (CCT
), Retinotectal development
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Introduction |
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To investigate the molecular mechanisms underlying neural network
formation, we developed a highly efficient mutagenesis procedure using
4,5',8-trimethylpsoralen (TMP) (Ando
and Mishina, 1998). In a pilot screen, we isolated and
characterized two mutant lines with abnormalities in the nervous system. The
no tectal neuron (ntn) mutation impaired the development of
tectal neurons and eyes, whereas the edawakare mutation affected the
arborization of the trigeminal ganglion and Rohon-Beard sensory neurons. TMP
is a DNA crosslinking agent that can frequently induce small deletions in
combination with UV irradiation in Escherichia coli and
Caenorhabditis elegans (Sladek et
al., 1989
; Yandell et al.,
1994
; Liu et al.,
1999
). A potential advantage of the method would be that one could
isolate mutagenized genes directly by whole-genome subtraction using
TMP-induced deletions as molecular tags. Representational difference analysis
(RDA) is a powerful subtraction method of the entire genome
(Lisitsyn and Wigler,
1993
).
We have applied RDA to characterize the genomic region of the TMP-induced
ntn mutation (Ando and Mishina,
1998). Successful isolation of tightly linked polymorphic markers
by the whole-genome subtraction method led to the construction of genetic and
physical maps of the zebrafish genomic region responsible for retinotectal
development. RT-PCR analysis of transcripts from the ntn region
identified a 143 bp deletion in the cct3 gene encoding the
subunit of chaperonin containing TCP1 (CCT, also called the TCP1 ring complex
or TriC). Injection of antisense cct3 morpholino oligonucleotides
into zebrafish embryos induced characteristic ntn phenotypes,
including the degeneration of retinal ganglion cells and tectal neurons.
Furthermore, injection of cct3 mRNA successfully rescued ntn
mutant embryos. These results revealed that chaperonin CCT
controls
specifically retinotectal development in zebrafish. Our results open a novel
TMP mutagenesis-RDA cloning strategy of zebrafish forward genetics
characterized by high efficiency and rapid cloning.
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Materials and methods |
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RDA procedure
Genomic DNAs were prepared from pools of 40 mutant embryos and 40 wild-type
siblings and RDA was performed essentially as described
(Lisitsyn et al., 1994;
Lisitsyn and Wigler, 1995
).
Genomic DNAs (1 µg of each) were digested with BglII,
EcoRI, HindIII, SpeI and XbaI, and were
PCR-amplified to generate amplicons. Primer sequences were designed according
to the protocol (Lisitsyn and Wigler,
1995
) with some modifications on their cohesive end sequences
compatible for the restriction endonuclease
(Table 1). The iterative
hybridization-amplification step was repeated three times for BglII,
EcoRI, SpeI or XbaI amplicons and four times for
HindIII amplicons. The resulting RDA products were digested with the
corresponding restriction enzymes, agarose gel-isolated and cloned into
pBluescript II SK(+) (Stratagene). Genomic DNA with respective restriction
enzymes and amplicon (5 µg of each) were electrophoresed in 2% agarose gels
and transferred to Hybond-N+ nylon membranes (Amersham). Membranes
were hybridized with RDA products labeled using a random primed DNA labeling
kit (Roche) as probes.
|
Genomic sequences flanking the restriction fragment length polymorphism
(RFLP) sites of RDAjt5B430, RDAjt5B460 and RDAjt5E340 were cloned by
vectorette-PCR using a GenomeWalker kit (Clontech), and primers were designed
to identify cleaved amplified polymorphic sequences (CAPS): RDAjt5B430 RFLP
site, 5'-TGGCGGTTCATTCTGCTGTGCGAC-3' and
5'-CGACAAGACTTTTGTCAGGTAG-3'; RDAjt5B460 RFLP site,
5'-CAATACCGGCAACTTTCAAC-3' and
5'-CAAGGACAAGAAATCATGCC-3'; and RDAjt5E340 RFLP site,
5'-GTCAAAATGCTCACTATACTAACTGCTGTC-3' and
5'-AGTTTCGGCTTGGTTACGGAATCTC-3'. Mapping of YD1 and YH2 loci was
carried out by PCRs using primers flanking the deletions; YD1 locus,
5'-GACAGTGGAAATGCGGCTAT-3' and
5'-TACCCATGTCTTCTGCGTAG-3'; YH2 locus,
5'-GGCCAGAGTTTACATAGGGGT-3' and
5'-GGTTTTTGCTGTGTCTGCCTG-3'. Radiation hybrid (RH) mapping was
performed on the Goodfellow zebrafish T51 RH panel (Research Genetics)
(Geisler et al., 1999) using
the primers for the RDAjt5E340 RFLP site and YD1 locus.
Genomic DNA library screening and cDNA cloning
YAC clones D04128 and H0145 were obtained by PCR screening of a zebrafish
YAC library (Resource Center/Primary Databank, Germany) with the primers for
RDAjt5E340 RFLP site and their terminal sequences were determined as described
(Zhong et al., 1998). PAC
10J03 and BAC 18M9 clones were isolated from zebrafish BAC and PAC libraries
(Genome Systems) using 32P-labeled RDAjt5B430 and RDAjt5E340 as
probes, respectively. The inserts of these clones were sized by pulsed-field
gel electrophoresis using CHEF-Mapper (BioRad). For YAC clonality analysis,
the blot was probed with the 32P-labeled YAC arm pRML plasmid.
Zebrafish genomic DNA in YAC D04128 clone purified by pulsed-field gel
electrophoresis was biotinylated using a random primed DNA labeling kit
(Roche). A random primed zebrafish cDNA library was synthesized using RNA from
zebrafish embryos at 36 hpf as templates (SuperScript Choice System,
Invitrogen) and PCR-amplified after ligation to adapters
5'-TAGTCCGAATTCAAGCAAGAGCAGA-3' and
5'-CTCTTGCTTGAATCGGACTA-3'. After preincubation with 2 µg
sonicated zebrafish genomic DNA and 1 µg HaeIII-digested yeast
genomic DNA, 1 µg preamplified cDNA was hybridized with 100 ng biotinylated
zebrafish genomic DNA in YAC D04128 clone as described
(Del Mastro and Lovett, 1996).
The sequences of the 5' and 3' regions of the cct3 cDNA
were obtained by 5'- and 3'-rapid amplification of cDNA ends
(RACE) using a SMART cDNA kit (Clontech), respectively. The entire coding
sequence of cct3 cDNA was cloned into pCRII vector (Invitrogen) by
RT-PCR with primers, 5'-TGTCCGGTACCGGTGATCTAAC-3' and
5'-AAATGGATTTCTGATGAGAACGTTGT-3' to yield pCRII-CCT3. The deletion
mutation in the cct3 mRNA was identified by sequencing RT-PCR
products of mRNAs from
200 ntn homozygous embryos and
650
wild-type siblings at 50 hpf according to the protocol of the SuperScript
Choice System (Invitrogen). The mutation in the cct3 gene was
confirmed by PCR on 100 ng genomic DNAs from
300 wild-type siblings and
100 ntn embryos with primers flanking the 143 bp deletion,
5'-GCCATGCAAGTGTGTCGTAATG-3' and
5'-CTCAGAGAAGTGAGCACACGAATG-3'. Genotyping of embryos was
performed using the same primer set.
In situ hybridization
The entire coding sequence of the zebrafish brn3b/pou4f1 was
obtained by RT-PCR using primers 5'-CGGTCGCAAATATGATGATG-3' and
5'-ATGATTCCACATCCCCTTTG-3', and was cloned into pCRII vector to
yield pCRII-BRN3B (GenBank Accession Number AB122025). We carried out
whole-mount in situ hybridization with antisense RNA probes prepared with a
DIG RNA labeling kit (Roche), paraffin-embedding and sectioning of whole
embryos as described previously (Mori et
al., 1994; Jowett,
1999
). Deparaffinized sections were counterstained with 0.5%
Methyl Green for 10 minutes. Probes for ath5/lakritz, dlx2, hlx1, krox20,
myod, ntl, pax2a/noi (previously pax2.1), shh and
zash1 mRNAs were as described previously
(Masai et al., 2000
;
Akimenko et al., 1994
;
Seo et al., 1999
;
Oxtoby and Jowett, 1993
;
Weinberg et al., 1996
;
Schulte-Merker et al., 1994
;
Krauss et al., 1991
;
Krauss et al., 1993
;
Allende and Weinberg,
1994
).
Stainings
Whole-mount immunostaining of zebrafish embryos with anti-acetylated
tubulin antibody (Sigma) were carried out as previously described
(Hammerschmidt et al., 1996)
except that Alexa 488 anti-mouse IgG antibody was used as secondary antibody.
Immunostaining of cryosectioned embryos using a monoclonal antibody zn5
(Oregon Monoclonal Bank) was performed as described
(Masai et al., 2003
). Terminal
deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) was
carried out in cryosections according to the manufacturer's protocol (ApopTag
Apoptosis Detection Kit; Serologicals Corporation).
Dechorionated embryos were soaked in ERS containing 5 µg/ml Acridine Orange (Sigma) or 100 µM Bodipy-ceramide (Fl C5, Molecular Probes) and 2% DMSO for 30 minutes in the dark. The embryos were washed, anesthetized by 0.02% 3-aminobenzoic acid ethyl ester (tricaine, Sigma) and embedded in low-melting temperature agarose gels during microscopic observation. The eyes and tectum of Bodipy-ceramide-stained embryos were scanned by confocal microscopy.
Phenocopy by antisense morpholino oligonucleotides and phenotypic rescue by RNA injections
The fluorescein-tagged morpholino oligonucleotide complementary to the
nucleotide residues 16 to +9 of the zebrafish cct3 mRNA
(nucleotide residues are numbered from the putative translational initiation
codon, GenBank Accession Number AF506209)
(Golling et al., 2002) was
obtained from GeneTools. The antisense or control oligonucleotide at a
concentration of 4 µg/µl in 1x Danieau buffer
(Nasevicius and Ekker, 2000
)
was injected into the yolk of one- to four-cell stage wild-type or transgenic
embryos carrying PAR-EGFP using a microinjector (IM-300, Narishige).
Distribution of oligonucleotides in embryos was monitored under fluorescent
microscopy. Fluorescence of EGFP was much stronger and easily distinguishable
from that of fluorescein. Fluorescence of EGFP-labeled retinal ganglion cell
(RGC) axons was observed as described
(Tokuoka et al., 2002
).
The 1.8 kb EcoRV-SpeI fragment from pCRII-CCT3 was cloned
into the StuI and XbaI sites of pCS2+ vector
(Turner and Weintraub, 1994)
to yield pCS-CCT3. Capped cct3 mRNA was prepared from 1 µg
pCS-CCT3 linearized with NotI using an mMessage mMachine kit
(Ambion). We injected 110-270 pg mRNA into the cytoplasm of embryos produced
by crossing heterozygous fish.
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Results |
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We further determined the genotypes of 432 ntn mutant embryos for the B430 and E340 loci using CAPS markers. There was one recombination event between the ntn and B430 loci, the calculated genetic distance being 0.12 cM (95% confidence interval, 0.00-0.64 cM). However, we found no recombination events between the ntn and E340 loci. Thus, among isolated polymorphic RDA markers, E340 was the marker closest to the ntn locus.
We next screened zebrafish genomic libraries with E340 as a probe and obtained two YAC clones and one PAC clone (Fig. 1E). YAC H0145 contained a terminal sequence of BAC clone 18M9 obtained by screening with B430 as a probe. By comparing the 2.4 kb terminal sequences of YAC D04128 and the 2.9 kb terminal sequences of YAC H0145 with the corresponding genomic sequences of wild-type and ntn mutant embryos, we found 28 bp and 107 bp deletions in the ntn genome, designated as YD1 and YH2, respectively (Fig. 1E). Genotyping of 432 ntn mutant embryos by PCR using primers flanking the deletions in respective YAC ends identified one recombination event out of 864 meioses between the ntn and YH2 loci and one between the ntn and YD1 loci. We detected ten recombinations between the ntn and B460 loci by genotyping 432 ntn mutants using B460 CAPS marker. Thus, the relative order of markers around the ntn locus on the chromosome was B460-YH2-ntn/E340-YD1-B430 (Fig. 1E). These analyses localized the ntn locus within a 0.24 cM region between YH2 and YD1 markers.
Transcripts from the ntn region
Using YAC D04128 as a probe, we isolated 300 cDNA clones by screening
a cDNA library prepared from zebrafish embryos at 36 hpf. Sequence analysis
suggested that these clones encoded at least three genes. The first group of
26 cDNA clones and zebrafish ESTs (EST269466, fb13f04, fc26d03, fc72f03 and
fe18d09) from the database of the Washington University zebrafish EST project
encoded the
subunit of zebrafish chaperonin CCT, which shared 87%,
86%, 85%, 70% and 58% amino acid sequence identities with the Xenopus
laevis, mouse, human, Drosophila melanogaster and yeast
counterparts, respectively (Chen et al.,
1994
; Kubota et al.,
1994
; Dunn and Mercola,
1996
; Walkley et al.,
1996a
; Walkley et al.,
1996b
; Walkley and Malik,
1996
). The second group of five cDNA clones encoded a putative
protein that had 69% amino acid sequence identity with human C18orf1, a
transmembrane protein with a LDL receptor type A domain
(Yoshikawa et al., 1998
). A
putative protein encoded by the third group of seven cDNAs showed 32% amino
acid sequence identity with mouse semaphorin 6C precursor
(Kikuchi et al., 1999
). One
cDNA contained a single open reading frame, but there found no proteins
homologous to the putative protein. The rest of the cDNA clones contained
(CA)n dinucleotide repetitive sequences of various length and were not
characterized further.
By RT-PCR analysis of mRNAs, we found a 143 bp deletion in the CCT
subunit gene (cct3) transcript from the mutant embryos
(Fig. 2A,B). The deletion was
present in the cct3 gene of mutant embryos, but not in the wild-type
gene (Fig. 2C). The
subunit of zebrafish CCT consisted of 543 amino acids and had five regions
common to the CCT subunit proteins (Kim et
al., 1994
), the N- and C-terminal equatorial ATPase domains, two
intermediate domains and the apical substrate-binding domain
(Fig. 2A). The cct3
transcript in the mutant embryos completely lacked a putative ATPase motif and
the reading frame was shifted by the deletion. There were no mutations within
the coding sequences of three other candidate gene transcripts.
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Identification of the ntn mutant gene
The characteristic phenotypes of ntn mutant embryos were selective
impairment of development of the eyes and tectum at 40 hpf
(Ando and Mishina, 1998
). At
later stages (4 dpf), ntn mutants can be macroscopically
distinguished from their wild-type siblings as having small eyes and turbidity
in the developing tectum (Fig.
3A). There appeared no other abnormalities even at this stage
except for small pectoral fins and some underdeveloped jaw skeletons in
ntn mutant embryos.
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To examine whether the mutation in the cct3 gene was responsible
for the ntn phenotypes, we injected an antisense morpholino
oligonucleotide complementary to the cct3 mRNA sequence encompassing
the translation start codon into the wild-type embryos. The injected embryos
showed small eyes at 4 dpf (Fig.
3F) and Acridine Orange staining in the tectum at 36 hpf
(Fig. 3G). Bodipy-ceramide
staining revealed loss of tectal cells
(Fig. 3H). Anti-acetylated
tubulin antibody immunostaining demonstrated that the formation of tectal
neuropil and optic nerve was impaired in the embryos injected with an
antisense morpholino oligonucleotide (Fig.
3I,J). Injection of a control morpholino oligonucleotide with the
inverted antisense sequence exerted little effect on the development of the
tectal and retinal neurons (Fig.
3F-J). These results suggest that suppression of CCT
expression by the antisense morpholino oligonucleotide induces the
characteristic phenotypes of ntn mutant embryos.
We next injected wild-type cct3 mRNA into one-cell stage embryos
derived from crosses of heterozygous (+/) fish to examine whether the
cct3 gene could rescue ntn mutant embryos. The injected
embryos were stained with Acridine Orange for testing the ntn mutant
phenotypes (Fig. 3K). Among 77
embryos injected with cct3 mRNA, only four embryos (5%) showed strong
Acridine Orange staining in the tectum at 36 hpf and 73 embryos (95%) appeared
normal, which deviated significantly from recessive inheritance
(2 test, P<0.001). The genotyping of embryos with
wild-type phenotypes revealed that 11 of 73 embryos were
cct3-/- at the ntn locus. The genotype at the
ntn locus of 20 embryos was cct3+/+ and that of
remaining 42 embryos was cct3+/. Among 137
mock-injected embryos, 37 embryos (27%) showed the ntn mutant
phenotypes (Fig. 3K) and 100
embryos (73%) exhibited the wild-type phenotypes, which was consistent with a
recessive mode of inheritance (
2 test, P=0.70). In
100 mock-injected embryos showing wild-type phenotypes, there were no embryos
with cct3-/- genotype at the ntn locus. All the
embryos with wild-type phenotypes had genotypes of either
cct3+/+ or cct3+/ at the
ntn locus. The development of the eyes at 3 dpf was normal in the
embryos that did not show an increase in Acridine Orange staining in the
tectum. These results suggest that injection of cct3 mRNA rescued
ntn mutant embryos.
The gene encoding nAChRß3 is an early differentiation marker of RGCs
(Matter-Sadzinski et al.,
2001). To examine the differentiation of RGCs, we crossed the
heterozygous ntn fish with a transgenic zebrafish carrying the
nAChRß3 gene promoter-directed EGFP expression vector
(Tokuoka et al., 2002
).
Crossing of doubly transgenic fish with heterozygous ntn fish yielded
homozygous mutant embryos with EGFP-labeled RGCs. Expression of EGFP signals
in ntn embryos injected with cct3 mRNA indicated the
restoration of development of RGCs (Fig.
3L). In control mock-injected embryos, no EGFP signals appeared in
the retina. Genotypes of ntn embryos injected with cct3 mRNA
showing wild-type phenotypes were confirmed to be cct3-/-
at the ntn locus by PCR using the primers flanking the deletion in
the cct3 gene (Fig.
3M).
Impairment of retinotectal development in ntn mutant zebrafish
Chaperones play an important role in folding of many proteins and CCT is a
member of two major chaperone systems implicated in cytoplasmic protein
folding in eukaryotes (Bukau and Horwich,
1998; Hartl and Hayer-Hartl,
2002
). By 12 hpf, the strong expression of the cct3 mRNA
started in entire zebrafish embryos and continued thereafter
(Fig. 2D). In the segmentation
period (10-24 hpf), a variety of morphogenetic movements occur: the somites
develop, the rudiments of the primary organs become visible and the overall
body length of the embryo increases very rapidly
(Kimmel et al., 1995
). Thus,
we investigated whether there were any changes in body patterning or
neurogenesis in ntn mutant embryos at 30 hpf. Two zebrafish homologs
of the neurogenic gene achaete-scute, zash1a (asha
Zebrafish Information Network) and zash1b, were strongly expressed in
the entire neural retina (Fig.
4A), and in the midbrain and hindbrain
(Fig. 4B) of both wild-type and
ntn embryos at 30 hpf, respectively. Between wild-type and
ntn embryos, there were no differences in the expression patterns of
the homeobox gene hlx1 (dbx1a Zebrafish Information
Network) in the midbrain (Fig.
4C), dlx2 in the telencephalon and diencephalon
(Fig. 4D), krox20
(egr2b Zebrafish Information Network) in rhombomeres 3 and 5
(Fig. 4E), and pax2a
in the midbrain-hindbrain boundary (Fig.
4G). The expression patterns of dlx2 in the pharyngeal
arches (Fig. 4D), shh
in midline structures (Fig.
4F), pax2a in the optic stalk, otic vesicle and
pronephric duct (Fig. 4G),
ntl in the notochord (Fig.
4H) and myod in the myotomes
(Fig. 4I) were also comparable
between wild-type and mutant embryos. Thus, the development of zebrafish
embryos appeared to have proceeded normally until 30 hpf without the
CCT
subunit.
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We also analyzed the development of tectal neurons by staining with
Bodipy-ceremide (Fig. 6A).
Zebrafish tectal cells proliferated over the whole extent of the tectal plate
at 24 hpf and many tectal precursor cells turned into postmitotic cells
forming the central differentiated zone by 48 hpf, while cells in the
peripheral marginal zone still remained proliferative
(Wullimann and Knipp, 2000).
Large and round cells representing mitotically active cells
(Cooper et al., 1999
) were
found along the edge of the tectum in wild-type embryos at 28 hpf and 36 hpf.
During development from 28 hpf to 36 hpf, the total volume of the tectum
remained relatively constant, whereas each tectal neuron precursor became
smaller in wild-type embryos. The ventricle between the tectum and the
posterior tectal membrane became less prominent in wild-type embryos from 36
hpf to 40 hpf. There were no detectable abnormalities in the organization of
the tectal neuroepithelium, alignment and mitotic cell images of tectal
precursor neurons in ntn embryos at 28 hpf and 36 hpf. At 40 hpf,
however, staining revealed cell-free spaces in the central zone of the tectum
in mutant embryos. Consistently, TUNEL staining signals appeared in the tectum
of mutant embryos at 40 hpf, but not at 30 hpf and 36 hpf
(Fig. 6B). Immunostaining with
anti-acetylated tubulin visualized the axons of trigeminal ganglion cells
extending along the epidermis over the tectum and there were no significant
differences in the immunostaining patterns of the tectum between wild-type and
mutant embryos at 30 hpf and 36 hpf (Fig.
6C). At 40 hpf, immunostaining showed the formation of tectal
neuropil by vigorous neurite extension of tectal neurons in wild-type embryos,
but there was little staining in mutant embryos. However, the expression
pattern of brn3b in tectal cells was comparable between wild-type and
mutant embryos at 48 hpf (Fig.
6D), indicating the presence of tectal neurons. These results
suggest that the ntn mutation of the cct3 gene exerted
little effect on the production of tectal cells but suppressed their
differentiation to form tectal neuropil.
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Discussion |
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Our results demonstrate that the combination of TMP mutagenesis and
genetically directed RDA provides a highly efficient and rapid cloning
strategy for zebrafish forward genetics. Using RDA products from a TMP-induced
zebrafish mutant, vibrato, with defects in the spontaneous
contraction and touch response, we also successfully constructed a
high-resolution physical map of a genomic region of 720 kb containing the
mutant locus (Sato and Mishina,
2003). The whole-genome subtraction method will be also applicable
to zebrafish mutants induced by ENU. In addition to the 143 bp deletion in the
cct3 gene, there were two small deletions in
5 kb ntn
genomic region, indicating successful TMP deletion mutagenesis in zebrafish.
We also found larger deletions in the genome of
edawakarejt10 mutant zebrafish obtained by TMP mutagenesis
(T. Morita, unpublished). Thus, direct selection by RDA of the mutated genes
from TMP-induced mutant fish would be feasible depending on the sizes of
deletions induced. In C. elegans, deletion sizes were dependent on
TMP concentrations (Gengyo-Ando and
Mitani, 2000
).
Chaperonin CCT is essential for retinotectal development
In the present investigation, we identified the subunit of
chaperonin CCT as an essential regulator of retinotectal development in
zebrafish by whole-genome subtraction cloning from TMP-induced ntn
mutants. Induction of ntn phenotypes by injection of cct3
antisense morpholino oligonucleotide into wild-type embryos and rescue of
ntn mutants by injection of wild-type cct3 mRNA clearly
showed that the impaired retinotectal development in the ntn mutant
fish was caused by the mutation in the chaperonin cct3 gene. The
ntnjt5 mutation appears to be null because the deletion
causes frameshift of translation. Available information suggests the presence
of a single gene for the CCT
subunit as well as the
,
,
,
,
and
subunits in the zebrafish genome
(http://www.ensembl.org/Danio_rerio/).
A cct3 zebrafish mutant was on the list of retroviral insertion
mutants but no characterization was reported
(Golling et al., 2002
).
Molecular chaperones play an important role in folding of many proteins and
CCT is a member of two major chaperone systems implied in cytoplasmic protein
folding in eukaryotes (Bukau and Horwich,
1998; Hartl and Hayer-Hartl,
2002
). In mammalian cells,
15-20% of newly synthesized
proteins transiently bind to Hsp70 and
9-15% of them interact with CCT
(Thulasiraman et al., 1999
).
Pharmacological inhibition of Hsp90, which cooperates with Hsp70 in folding of
signal-transduction proteins (Young et
al., 2001
), was lethal and affected the development of various
organs in zebrafish embryos (Lele et al.,
1999
). In addition, compromised Hsp90 activity in
Drosophila and Arabidopsis caused a wide array of
morphological variations, suggesting that Hsp90 acts as a capacitor for
evolution (Rutherford and Landquist,
1998
; Queitsh et al., 2002). Thus, it is surprising that the
impairment of CCT
caused defects specifically in coordinate
retinotectal development of zebrafish. The causal relationship between
CCT
defect and the degeneration of retinal and tectal cells implies the
importance of chaperones in neurodegenerative diseases
(Slavotinek and Biesecker,
2001
). CCT is a large cylindrical complex composed of eight
different subunits providing physically defined compartments inside which a
complete protein or a protein domain can fold while being sequestered from the
cytosol (Kubota et al., 1995
;
Llorca et al., 1999
). It is
possible that the defect of the
subunit in ntn mutants can be
compensated by other CCT subunits to form functional CCT complex that assist
folding of many proteins except for those specifically dependent on the
subunit. The finding that the binding of actin to CCT is both subunit
specific and geometry dependent (Vinh and
Drubin, 1994
; Llorca et al.,
1999
) may be consistent with this view. Major substrates of CCT
are tubulin and actin in mammalian and yeast cells
(Stoldt et al., 1996
;
Thulasiraman et al., 1999
). In
fact, CCT is essential for mitosis and growth in budding yeast
Saccharomyces cerevisiae and conditional mutations in individual CCT
subunit genes affect biogenesis of tubulin and/or actin
(Chen et al., 1994
;
Stoldt et al., 1996
). However,
there was no detectable expression of the cct3 mRNA in zebrafish
embryos from one-cell to 90%-epiboly stages when vigorous cell proliferation
and gastrulation took place. The cct3 mRNA was strongly expressed at
12 hpf in the entire embryo and sustained thereafter, but the defects in
development of ntn mutant embryos became detectable only at
30
hpf and specifically in the retinotectal system. Anti-acetylated tubulin
immunostaining in most of neurons other than RGCs and tectal neuropil was
comparable between wild-type and ntn mutant embryos. It is known that
axonogenesis also involves actin biogenesis and polymerization
(Chien et al., 1993
). Thus, it
is unlikely that the ntn mutation directly impaired the actin and/or
tubulin biogenesis. Transducin
requires CCT activity for folding (Farr
et al., 1993). However, unlike ntn mutants, zebrafish transducin
mutants showed morphologically normal retina
(Brockerhoff et al., 2003
).
ntn mutation impaired differentiation of retinal and tectal neurons
One may speculate that the effect of the ntn mutation of the
CCT gene on the retinotectal development is rather nonspecific, as CCT
complex should assist folding of many proteins and zebrafish mutants affecting
both retina and tectum were frequently found in large-scale screens
(Abdelilah et al., 1996
;
Furutani-Seiki et al., 1996
).
However, the specificity of the ntn phenotypes is threefold. First,
there were no detectable abnormalities in body patterning and neurogenesis in
ntn mutant embryos at 30 hpf, despite the fact that the strong
expression of the cct3 mRNA in the entire embryos started by 12 hpf
and that very active developmental changes occurred in the segmentation period
(10-24 hpf), including a variety of morphogentic movements, the development of
somites and primary organ rudiments and rapid increase in overall body length
of the embryo (Kimmel et al.,
1995
). Second, ntn phenotypes appeared specifically in
the retina and tectum at
2 dpf. At later stages (4 dpf), however,
underdevelopment of pectoral fins and some jaw skeletons were noted in
addition to small eyes and turbid tectum. Such abnormalities may be caused
secondarily or may represent nonspecific effects. Third, a specific step in
RGC differentiation is impaired in ntn mutants. The cellular
organization of the retinal neuroepithelium at 27 and 36 hpf suggested that
the formation of eye primordium, the proliferation of retinal cell progenitors
and retinal patterning proceeded normally in ntn mutant embryos. The
expression patterns of transcription factors ath5 and brn3b,
which are essential for the development and maintenance of RGCs
(Erkman et al., 1996
;
Xiang, 1998
;
Brown et al., 2001
;
Kay et al., 2001
;
Matter-Sadzinski et al.,
2001
), were indistinguishable between wild-type and ntn
mutant embryos, but those of early and late differentiation markers of RGCs,
nAChRß3 and zn5, were diminished in mutant embryos. Immunostaining of
acetylated tubulin also revealed the impairment of RGC axon extension and
optic nerve formation. Thus, ntn mutation of the cct3 gene
exerted little effect on the commitment of retinal neuroepithelial cells to
postmitotic retinal neurons but severely impaired the differentiation of
retinal neuroepithelial cells to RGCs. Similarly, the expression of
brn3b was normal in the tectum of ntn mutants, but tectal
neuropil formation was abolished. These results suggest that the
subunit of chaperonin CCT plays an essential role in retinotectal
development.
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ACKNOWLEDGMENTS |
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