The zebrafish
is a powerful model system for the genetic analysis of vertebrate embryogenesis,
organ development, and disease. Its unique power is its tractable, phenotype
driven mutation screens and readily accessible transparent embryos. Because
of its facile forward genetics, zebrafish accelerates gene discovery;
because of its accessible embryos, it promotes deep understanding of gene
function; because of its phylogenetic position, it informs mechanisms
of genome conservation. More than 2000 mutants are currently available
in more than 600 genes, and new phenotypic screens are uncovering more
mutations in many laboratories.
The zebrafish
system relies on the ability to clone and characterize mutant genes rapidly.
Positional cloning and insertional mutagenesis have been successfully
utilized to identify the molecular nature of mutations, but candidate
gene approaches have been the most successful to-date. The infrastructure
of the community and the recent Zebrafish Genome Initiative have added
value to the system by shortening the time to gene isolation, and with
the addition of a few more reagents and services, the cloning of mutations
could become routine. New investigators are becoming interested in zebrafish
and a mission of the current community is to make the system more easily
accessible and tractable. The current RFA and implementation of the suggestions
listed below for additional support will have significant impact on investigator-driven
research in the field.
A. Current RFA
In 1998, the NIH issued a
Request for Applications to improve genomic resources for zebrafish.
Five projects were funded. These three year projects have begun to vastly
increase the number of expressed sequence tags and the number of mapped
expressed sequences and anonymous markers, and are providing a collection
of chromosomal deletions that cover the entire genome. The funded projects
are:
- 100,000
ESTs 5' and 3' sequences
- 10,000
ESTs mapped to RH panel
- 3,000
ESTs mapped meiotically
- 0.5
cM microsatellite map, 5000 microsatellites
- Deletion
panel to cover the genome
B. More Infrastructure Needed
for Genomics
I. ESTs. A
large collection of ESTs benefits all strategies for the molecular identification
of mutations. For positional cloning, sample sequencing of clones in
the critical interval can reveal sequence identity to ESTs. For insertional
mutagenesis, tags isolated from genomic DNA adjacent to inserts can
reveal overlap with ESTs, thereby reducing cDNA library screening. For
the candidate approach, the greater the number of ESTs to serve as candidates,
the greater the likelihood that a mutated gene is represented by a known
EST. Knowing the genetic location and expression pattern further allows
potential candidate genes to be ruled out rapidly, thereby facilitating
cloning of mutations. Therefore, as high priority, our community needs
more EST sequences, and more ESTs characterized by genetic location
and expression pattern.
- Make new, normalized cDNA
libraries from multiple stages and tissues. ($150,000/year x 3 years).
- Sequence 5' and 3' ends
of 100,000 cDNAs. ($400,000/year x 3 years.)
- Map the ESTs to compare
to mutant map positions. ($160/EST X 5,000/year X 3 years = $2,400,000).
- Determine the expression
pattern of the ESTs for comparison to mutant phenotypes. $150,000/year
X 3 years for 10,000 cDNAs. Make these ready for the Zebrafish Gene
Expression Atlas.
- Develop and make widely
available microarrays of 10,000-20,000 ESTs. ($150,000/year X 3 years.)
II. Stock Center.
The power of zebrafish is the collection of mutations isolated on
the basis of mutant phenotypes. Currently, laboratories are unable to
keep all the mutations they recover for lack of space and funds for
maintenance. Requests for the distribution of mutant stocks burdens
all laboratories that have generated substantial mutant collections.
Most mutations that are not of immediate interest must be maintained
as frozen sperm. This hinders research because it requires at least
three months to obtain homozygous embryos starting from frozen sperm,
and stocks adequate for experimentation can take up to 9 months from
the thawing of sperm. Clearly a stock center for the preservation and
distribution of mutant stocks is key to the rapid analysis of zebrafish
mutants. NIH and State of Oregon funds have been obtained to support
the construction of a stock center in Eugene and to partially fund the
preservation and distribution of zebrafish stocks. More funds are required
to maintain all of the current stocks, not to mention the many new mutations
made by new, innovative and directed mutations screens.
- Stock
Center: Additional personnel needed. $250K/year additionally.
III. Data Base.
Ready access of the entire community to genomic and developmental data
is required to turn data into understanding. The zebrafish database
ZFIN is positioned to become a tool for archiving and retrieving data
on zebrafish. Links must be made between the zebrafish database and
the database of other organisms. A substantial increase in database
support is needed to turn ZFIN into the tool the community requires.
- Data
editors to annotate expression and phenotypic data from old and current
papers. ($500,000/year X 3 years.)
- Data
base: Need bioinformaticists to expand capabilities to include conservation
of syntenies, histological atlas, and Zebrafish Gene Expression Atlas.
($500,000/year X 3 years.)
IV. Mutant mapping.
One of the first steps to the molecular characterization of a zebrafish
mutation is its genetic mapping. This rules out many potential EST candidates,
and "rules in" others, as well as providing an initial stage for positional
cloning. It is important to map quickly mutations in each gene mutated
in the two large screens, 600 mutations, by a mechanism that guarantees
that mutant locations are rapidly made public. Only about 150 of these
have been mapped to date.
- Mapping
mutations. ($2000/Mutation x 500 mutations = $1,000,000 over 3 years.)
V. Physical Map of
Genome. DNA pooling methods and the huge number of anonymous
polymorphisms available by microsatellites, RAPDs, and AFLPs permit
the identification of DNA polymorphisms very closely linked to zebrafish
mutations. Chromosome walks from those linked markers requires large
insert DNA libraries, and is facilitated by a physical map of the genome.
A physical map that includes telomeres furthermore provides reagents
for genome sequencing.
- BAC
libraries, 200 kb clones: ($250,000 for 10-genome coverage.)
- BACs
contiged and anchored to the genetic map. ($1,000,000/year x 3 years.)
- YAC
telomere library. Knowing the end of the chromosomes will aid positional
cloning of genes in these regions. ($150,000)
VI. Genome Sequencing.
Complete knowledge of the sequence of the zebrafish genome would provide
a tool to revolutionize our efforts to understand zebrafish biology.
- Phase
1, start sequencing of the genome. $3M first year. Initially science
driven as to regions to start sequencing, focussing on regions of
highest interest and regions orthologous to those already sequenced
in other important model systems such as human and pufferfish. This
will define repeat structure of the genome and will provide information
on the structure of intron-exon boundaries.
- Phase
2. Ramping up to sequence the entire genome beginning in 2000 with
targeted completion 2008.
VII. Technology development.
Although zebrafish has tremendous advantages as a system for forward
genetics, the development of several additional methodologies would
further improve the system. Key among these needs is gene knockout technology
and the construction of more transgenic zebrafish. $1.5m/y
- ES cells
- transgenesis to provide
more GFP lines
- Gal4 UAS system to investigate
gene interactions
- RNAi to eliminate gene
functions
C. More Infrastructure Needed
for Cell Biology
I. Imaging.
The beauty of zebrafish lies in its optically clear embryos and their
accessibility to experimental intervention. Some additional descriptive
studies exploiting these attributes would significantly enhance our
ability to understand mutant phenotypes. Chief among these would be
a four dimensional fish, that is, a time-lapse reconstruction of development
in real time. Substantial computer assisted imaging must accompany this
so that different organ systems, tissues, and cell types can be highlighted
and so that the images can be viewed from any orientation desired by
the viewer. Furthermore, the gene expression patterns of the ESTs generated
in item B1 above and other isolated genes must be visible on this 4D
fish. Furthermore, the images must be retrievable from a database in
any of a number of ways determined by the user (for example, the expression
patterns of all loci that begin expression within 1 hour of the appearance
of sonic hedgehog transcript in the floorplate).
- 4D Fish. X,Y,Z images
in real time, including gene expression patterns
- More fate maps for later
stages. This would help in the interpretation of gene expression patterns.
II. Novel and Improved
Phenotyping. Members of the community perceive that more effort
needs to go into developing novel means for phenotyping zebrafish, for
example by electrophysiological methods or to assay behaviors, or to
detect changes in ions such as calcium. These goals are probably best
achieved by RO1's.
III. Protein Expression
and Biochemistry. Although the community feels at this point
that infrastructure for genomics should take priority to infrastructure
for proteomics, the construction of monoclonal antibody panels from
various stages and tissues, and the establishment of tissue-specific
cell lines would be useful.
- Monoclonal antibodies
for various stages
- Cell lines from various
tissues
D. Budget
Here is listed the budget
for the genomics items in part B above. The cell biology items are also
needed, but we were less able to put dollar figures on them. To accomplish
the aims in part B would cost about $20 million spent over a three year
period.
|
Items
|
Per
Year
|
Years
|
Total
|
|
|
($
in thousands)
|
|
($
in thousands)
|
ESTs
|
|
|
|
|
|
cDNA libraries
|
150
|
3
|
450
|
|
End sequences
|
400
|
3
|
1,200
|
|
Map ESTs
|
800
|
3
|
2,400
|
|
Expression
|
150
|
3
|
450
|
|
Microarrays
|
150
|
3
|
450
|
Stock Center
|
|
250
|
3
|
750
|
Data Base
|
|
|
|
|
|
Data editors
|
500
|
3
|
1,500
|
|
Bioinformaticists
|
500
|
3
|
1,500
|
Mutant Mapping
|
|
333
|
3
|
1000
|
Physical
Map
|
|
|
|
|
|
BAC libraries
|
250
|
1
|
250
|
|
Contigs
|
1,000
|
3
|
3,000
|
|
Telomeric YACs
|
150
|
1
|
150
|
Genome Sequence
|
|
|
|
|
|
Phase 1
|
3,000
|
1
|
3,000
|
|
Phase 2
|
1,500
|
3
|
4,500
|
Total
|
|
9,133
|
|
20,600
|
|
|