Report from the Workshop on Genomic and Genetic Tools for
May 10-11, 1999
|Igor Dawid (Chair)
Preface: The NIH Planning ProcessAdvocate for Zebrafish
Workshop Participants' Report
- Introduction: The Zebrafish in the Context of the Human
- Summary of Current Genomic Resources and Activities
- Genomic Infrastructure Needs
- Mapping tools, ESTs and cDNAs
- Physical map construction
- Sequencing the zebrafish genome
- Budget estimates
- Technologies to Advance the Analysis of Gene Function
- Genetic screens
- Mapping of existing and anticipated mutants
- Technologies for genome manipulation
- Gene expression
- Stock center-related issues
- Budget estimates
- Informatics Issues
- Anatomical Atlases
- Budget estimates
Special Applications of the Zebrafish Model
- Studies of Musculoskeletal and Skin Developmental
- Zebrafish and Cancer Research
- Eye Development and Disease
- Kidney Development and Disease
- Cardiovascular and Other Organogenesis in Zebrafish
- Research on Aging
- Child Health and Human Development
- Research on Ear Morphogenesis and Hearing Disorders
- Developmental Toxicology in Zebrafish
- Genome Research
- Neurogenesis and Neurological Disorders
- Early Developmental Programs in the Zebrafish
- Research on Allergies and Infectious Diseases
- Mutants that Affect Mental Behavior
- Craniofacial and Dental Research
Up to Top
The NIH PLANNING PROCESS for Zebrafish Research
The following report is from the Workshop, "Genomic and Genetic Tools
for the Zebrafish", held May 10-11 at Lister Hill Auditorium on the NIH
campus. The meeting was under the sponsorship of the Trans-NIH Zebrafish Coordinating
Committee (ZFCC), which is comprised of representatives from eighteen of the
NIHs Institutes and Centers. The morning of the first day was devoted
to state of the art talks by leading zebrafish investigators on
genetic and genomic issues followed in the afternoon by three break-out meetings
to discuss genomic infrastructure needs, technologies related to the functional
analysis of gene expression, and related informatics issues. The second day
was devoted to a plenary session to hear and debate the break-out session recommendations.
The report presented here is a summary of these deliberations, prepared by the
Workshop Planning Committee and Break-out Group Chairs.
This was the third in a series of NIH-sponsored activities to address issues
surrounding the zebrafish as a model for development and disease research, particularly
the issue of genomics. It was structured to build upon the previous planning
activities. The first of these meetings "Current Advances in Defining the
Zebrafish Genome", was held in Boston in February 1997. The report of that
workshop contained a set of recommendations that provided the basis for a Request
for Applications resulting in the funding of the five current zebrafish genomics
projects. These projects were initiated in 1998 with cooperative funding from
thirteen institutes, and are scheduled to run three years. They are overseen
by the ZFCC, with the advice of an external oversight committee. Also in response
to the 1997 report, a Program Announcement was published which highlighted the
goals and interests of eighteen participating Institutes and Centers in the
use of the zebrafish model.
In February 1999, Dr. Varmus convened the Workshop on Non-Mammalian Models,
the second meeting at which planning for the zebrafish was addressed. The zebrafish
was one of the model organisms considered in depth at that meeting. A panel
of zebrafish investigators, chaired by Leonard Zon and John Postlethwaite formulated
a report outlining the needs for genetic and genomic tools for the model. This
report was used as a basis for planning the May meeting. The recommendations
in the present report are quite similar to those developed in February, although
they amplify and build upon the recommendations of the earlier panel in important
Also included in the present report are a series of brief essays that outline
the power of this model organism to address important questions
in specific areas of biology and medicine. Zebrafish investigators
prepared these essays after the May meeting through the organizational
efforts of Leonard Zon. The ZFCC believes these excellent, short
reports may be useful to the NIH Institutes in clarifying the utility
of the zebrafish model for their own individual goals.
Up to Top
WORKSHOP PARTICIPANTS REPORT
I. Introduction: Zebrafish in the Context of the Human Genome Project
Vertebrate model organisms complement the human genome project in important
ways. As the sequencing of the human genome nears completion, assigning
functions to large numbers of sequences has taken on increased importance. Linkage
analysis has proved relatively ineffectual in analyzing complex diseases with
small contributions from individual genes. Association studies have the power
to find even small contributions of a gene, but require candidate genes. Large-scale
screens in model organisms provide the perfect next step to identify candidate
genes for human diseases and will help focus the Human Gene Project (HGP).
Phenotype-based genetic screens begin with function. Large-scale phenotypic
screens can define genes as critical to developmental, physiological, or pathological
processes because they identify genes in the living organism. If facile enough
and cost-effective enough, they can be driven to saturation, isolating all genes
informative with regard to any given process. They are limited in such circumstances
only by the ingenuity of investigators. Although yeast, flies, and worm genetics
meet these criteria, they do not speak to many important developmental decisions
that characterize the vertebrate, including man. For example, there is no logical
framework, and only few genes known, for patterns of organ assembly, function,
and disease, and it is difficult to approach genetics of complex behavior.
Zebrafish are vertebrates, and tractable and inexpensive enough for screens
in many labs. Although many important processes may be extrapolated from
invertebrate organisms, including cell fate assignment, many others, such as
organ form and function, are new to vertebrates, and the genes involved not
known. Within vertebrates, however, these processes have been well conserved.
Therefore, the zebrafish is an appropriate and relevant organism for screens
applied to these vertebrate-specific questions. Efficient, saturation phenotypic
screens are possible in zebrafish because the embryo does not develop hidden
inside the mother, as in mammalian embryos, and because the embryos themselves
are translucent allowing the direct visualization of individual developing cells.
Mutations are currently available in nearly 1000 genes essential for embryonic
development, and directed screens are adding more. Methods to store
sperm facilitate the storage of mutant strains, and methods to generate haploid
and homozygous diploid embryos expedite phenotypic screens. American and European
stock centers for the distribution of mutants and a centralized database for
genetic and other types of information have been established.
The zebrafish system, therefore, will open new fields of biology and medicine
Beyond the obvious assets in exploring vertebrate early developmental processes,
genetic screening in zebrafish provides access to these genes for other vertebrate-specific
- Organ system (integrative) biology and physiology
- Complex diseases
- Vertebrate evolution
Rapid gene discovery depends upon rapid cloning of mutations.
For gene discovery to be relevant to other organisms, and to permit biochemical
characterization of pathways involved, these mutant genes must be cloned. If
the zebrafish really is to provide an important biological complement to the
HGP, this must be accomplished in a rapid, efficient, and cost effective way.
Zebrafish mutations can be cloned. Existing maps and BAC and YAC libraries
have sufficed for cloning more than two dozen mutations from chemical mutagenesis
screens, identifying homologs of known genes in addition to novel genes. The
recent successes of viral insertion and gene trap methods show promise in making
rapid cloning of tagged sites feasible, an advance that will make this strategy
a highly attractive approach to genetic screening. In addition, it may prove
feasible to produce a library that could be screened for insertions into any
given gene. Thus, two complementary routes exist to link genes and functions.
One begins with phenotype, most effectively induced by chemical mutagenesis,
and uses positional cloning methods to identify the gene. The second, still
under evaluation for frequency of mutagenesis, begins with a defined gene of
interest, identified as tagged with an insertion, and seeks the mutant phenotype.
The first set of recommendations from this workshop focuses upon infrastructure
needs to go from phenotype to gene -- i.e., to expedite positional
or candidate gene cloning. These are detailed in the chapter entitled
"Genomic Infrastructure Needs." The second group of recommendations
focuses on the need for further targeted strategies to identify
specific genes and understand their function, and are described
in the chapter entitled, "Technologies to Advance the Analysis
of Gene Function." Lastly, recommendations on "Informatics
Needs" are described.
Up to Top
II. Summary of Current Genomic Resources and Activities
Mapping resources: Extensive mapping efforts by individual laboratories
have produced a genetic map, currently anchored with over 2000 independent simple
sequence length polymorphisms (SSLP) and 600 random amplified fragment length
polymorphisms (RAPDs). Over 400 genes have been placed on the map using restriction
fragment length polymorphisms or single strand conformational polymorphisms.
Through the recently funded NIH initiative, over the next two years we
anticipate that enough micro-satellite markers will be placed on the map to
yield a one cM average interval (being done by Mark Fishmans laboratory).
Additional mutant mapping can be accomplished by establishing linkage with micro-satellite
markers. An important advantage of this organism is the ability to perform half
tetrad analysis, which makes use of the ability to produce gynogenetic diploid
fish. When the high-density map becomes available, this will
provide a quick method for establishing mutant gene localization.
Radiation hybrid panels: Two independent zebrafish-mammalian radiation
hybrid panels have been developed for mapping of genes or EST sequences. One
panel was made by Peter Goodfellow and another by Marc Ekker. These two panels
have been typed with 1000 to 2000 SSLP markers, and both have good retention
of zebrafish DNA and high resolution. RH panel mapping under the current initiative
will be performed in the laboratories of Leonard Zon and of Steve Johnson. The
panels are extremely useful for positional cloning approaches and can be utilized
to establish the location of thousands of genes or ESTs. These reagents are
particularly useful for establishing whether a candidate gene or EST is represented
by a zebrafish mutant. In addition to the radiation hybrid panel mapping, there
is another zebrafish genome initiative project that will position 3000 independent
genes on the meiotic map (Wil Talbots lab). Some of these genes also will
be also placed on the radiation hybrid panel, anchoring the RH map to the genetic
map and establishing syntenic relationships between zebrafish and humans.
Genomic DNA libraries: With regard to genomic DNA libraries, there are
two YAC libraries for the zebrafish (Mark Fishmans and Leonard Zons
laboratories). These are useful for positional cloning or chromosomal walking.
A PAC and a BAC library are available from commercial resources. Currently,
Cclone inserts averages are currently 100kb; in the future, it would be helpful
to have a larger insert genomic PAC or BAC libraries. This will be critical
for the generation of a physical map of the genome. The NIH has funded an EST
sequencing project for 50,000 independent ESTs (Steve Johnsons lab). These
ESTs are derived from oligo-fingerprinted libraries available in the community
and should produce an excellent resource for candidate genes.
Other technologies: Another funded project is to develop deletion mutants
representing the entire genome (Marnie Halperns laboratory). This will
be useful to establish the null phenotype for mutant genes and will also evaluate
gene function. Apart from those projects supported by the zebrafish genomics
initiative, other important resources and technologies are being developed.
Transgenic technology for the zebrafish has been mainly developed by Shuo Lin
at the Medical College of Georgia The laboratory of Shuo Lin at the Medical
College of Georgia was the first to show consistent developmental regulation
of transgenesis in zebrafish and continues to develop enhancements such as the
technology to express green fluorescent protein (GFP) in various tissues. Insertional
mutagenesis has been developed by Nancy Hopkins laboratory, and recent
exciting findings regarding gene trap events suggest they will yield an excellent
resource for genomics and genetics of the zebrafish.
In summary, over the past several years, the zebrafish community has amassed
a significant set of reagents and resources to enhance the genetics
and genomics of the system. The community has created many of the
tools necessary for positional and candidate cloning of mutant genes,
thus establishing the basic infrastructure necessary to exploit
the genetic power of this model organism.
Up to Top
III. Genomic Infrastructure Needs
- A. Mapping tools, ESTs and cDNAS
SNPs: Single nucleotide polymorphism maps are needed as the next and
more efficient mapping tool, as they have been in human genetics. New technologies
based on SNP markers offer the potential for rapid, automated genotyping.
The EST projects will provide a large pool of sequences that are already paid
for and can be used to identify SNPs. Although the frequency of SNPs will
be lower within ORFs than in non-coding regions, it will carry the added benefit
of targeting known and/or candidate genes.
ESTs and cDNA libraries: ESTs provide candidate genes, useful in positional
cloning (during walks and for recognizing ORFs) and for ORF recognition in
cloning of insertion sites. New cDNA libraries are needed as EST sources,
and for BAC hybridization during positional cloning. These libraries should
be from different representative stages and organs, normalized and gridded,
and constructed in appropriate vectors for sequencing and, of course, made
From these libraries, an additional 100,000 ESTs should be sequenced
(divided between 5'- and 3'-end reads). These could include the
sequencing of insertion sites from retroviruses.
RH panel mapping of ESTs is critical to correlate candidate genes
with mutations at the highest rate, and to examine conserved synteny
with other model organisms and with the human. An additional 30,000
ESTs should be mapped on RH panels, in addition to the 10,000
Up to Top
B. Physical map construction
A physical map is critical crucial to expedite positional cloning, and may
provide a critical step towards sequencing the genome. Having such a map will
expedite walks, and thus reduce reducing the costs and increase increasing
the speed with which individual laboratories can clone their mutations of
interest. There are several ways to assemble all of the DNA fragments into
contigs. The current standard is fingerprinting by enzymatic digestion. There
will be gaps left after fingerprinting. These are best closed (as will be
needed to sequence the genome) by end-sequencing of BACs, themselves anchored
to the genetic map.
Currently, because of the YAC cloning techniques utilized, telomeres are
under-represented, and telomeric YAC libraries need to be constructed. Similarly,
current BAC coverage is insufficient, so new BAC libraries need to be constructed,
with 200 kb inserts.
The key needs here are:
- High quality BAC library.
- BAC fingerprinting
- BAC anchoring by EST to genetic maps.
- Half YAC (telomere) library construction.
Up to Top
C. Sequencing the zebrafish genome
Because the zebrafish is the only vertebrate now amenable to large-scale
saturation genetic screens, it can provide the single strongest functional
underpinning to the Human Genome Project. Relating the zebrafish and human
genomes will facilitate gene discovery and reveal connections to human biology,
but this requires that zebrafish mutations be cloned rapidly. This is the
most important reason to sequence the genome. The cost saving to the many
small laboratories involved would more than outweigh the expense of the project.
Just reducing the cost of sequencing BACs for the several hundred genes that
will be cloned by walking over the next few years will go along way to offset
the cost of the project. Obviously, the accomplishment of genome sequencing
depends upon, but dwarfs, the other infrastructure projects, and is more properly
compared only with efforts now underway to sequence the human and mouse genomes.
The value of both of those projects will be greatly enhanced by the international
zebrafish program for functional gene discovery.
The recommendation of this workshop is that immediate steps should be taken
to sequence enough of the genome to establish the quality of the libraries
and maps, and to acquire insight into gene structure and repeats, both of
which would aid current mutation cloning efforts. These goals could be achieved
by sequencing approximately 5% of the zebrafish genome. There are two options
for such a project: either focus on sequences scattered through the genome
(designated by investigator interest) or attempt to identify one long contiguous
piece, preferably an entire chromosome. The recommendation of the breakout
group on genomics at this workshop was for the latter option, but it was recognized
that this issue should be the topic of further discussion. It was also recommended
that the quality for current purposes need not be "fully polished".
Recommendations for sequencing:
- BAC end sequencing to close contig gaps.
- Sequence 5% of the genome.
Up to Top
D. Budget estimates
Workshop participants developed the following direct cost estimates:
ESTs: map 30,000 additional on RH panels, over 3 years
[$6,000,000 total (= 30,000 ESTs x $200/EST)]
Single nucleotide polymorphisms (SNPs): map 10,000 SNPs over 3 years [$240,000
total (= [10,000 reads x 4 strains x $5/read] + [$1/pcr x 4 strains x 10,000
2. Expressed Sequence Tags and Libraries
ESTs (3 years)
[$1,000,000 (= 100,000 clones x 2 reads/clone x $5 per read)]
Construction of new cDNA libraries (normalized, gridded, many stages and
organs, in the best vectors, publicly available) (3 years)
[$450,000 (= $150,000/y x 3 y)]
3. Physical map
High quality BAC libraries of 200 kb inserts (1 year)
[$250,000 (2 libraries@10xcoverage x $125,000/library)]
Fingerprinting BACs. (1 year)
[$1,000,000 (=200,000 clones x $5/clone)]
Anchoring BACs to the genetic map. (2 years)
[$1,000,000 (= 10,000 markers x $100/marker)]
Half-YAC (telomere) library (1 year)
4. Genome sequence
BAC end sequencing (3 years)
Full shotgun sequence 5% of genome (3 years)
[$12,000,000 (=$0.15/base x 80,000,000 bases]
TOTALS ESTIMATES OF DIRECT COSTS
Year 01= $4,480,000
Total (3 years) = $10,090,000
Sequencing 5% of genome
Year 01 = $5,000,000
Total (3 years) = $15,000,000
Up to Top
IV. Technologies to Advance the Analysis of Gene Function in Zebrafish
Genetic tools. This portion of this report deals with the continued
potential of mutagenesis screens and with new technologies that will greatly
accelerate the analysis of gene function in normal and mutant phenotypes, and
the identification of genes affected in mutants. Expenditure of relatively small
amounts of money on the tools described here has the potential to advance the
field significantly by facilitating more sophisticated analysis of gene function,
sometimes at reduced cost and effort, and to make the field accessible to more
Up to Top
A. Genetic screens
The need for continued emphasis on mutagenesis screening and phenotype.
The most powerful and unique feature of the zebrafish is that it is a vertebrate
model organism in which large-scale forward mutagenesis screens can be performed
with relative ease. Participants in this workshop were unanimous in expressing
the conviction that work to date had only begun to exploit the power of mutagenesis
screening to detect developmental genes and pathways of interest. The ultimate
goal should be saturation mutagenesis of the zebrafish genome. The community
felt that such screens are best performed in individual laboratories, and that
centralized mutagenesis centers offered few advantages.
To date, screens have focused exclusively on phenotypes involved in embryonic
development. The potential of phenotypic screens that focused on the adult was
emphasized by many participants, with potential possible applications envisaged
in a variety of disease areas as well as in response to environmental toxins
It was also emphasized that despite decades of research that have proven over
and over again the phenomenal power of the genetic screen for opening new and
unexpected avenues of research, the approach is often looked upon unfavorably
by study sections. Genetic screens, even when carefully focused on defined developmental
pathways, may be seen as fishing expeditions relative to more tightly
hypothesis-driven science. For this reason, although genetic screens are the
raison d'etre of the fish field, and receive much favorable lip service, they
are difficult to fund via the R01 mechanism. Thus, there was considerable support
at the workshop for special mechanisms for that would increasing increase funding
for this type of research. Particularly hard to fund is the acquisition of fish
tank systems for mutagenesis screens. It is interesting to note that no large-scale
genetic screen in the zebrafish has ever been supported entirely or even primarily
by NIH funds. With the advent of promising insertional mutagenesis strategies,
it is more pressing than ever to develop a strong support base for creative
and rigorous mutagenesis screens.
High priority is given to the following:
- Support of additional genetic screens in individual laboratories focusing
on identifying mutations that affect the structure and function of specific
- Development of phenotypic tools and new genetic screens to analyze the genetic
basis of adult phenotypes including behavior, aging, heart disease, cancer,
response to environmental toxins, etc.
- Genetic screens utilizing altered gene expression patterns, as a tool to
identify components of genetic pathways.
- Support of screens using new strategies for insertional mutagenesis.
- Support for expanding institutional fish facilities and purchasing fish
Up to Top
B. Mapping of existing and anticipated mutants
Previously and newly isolated mutations need to be linked to
candidate genes. If all chemically induced mutations were placed
on the zebrafish genetic map, then as an ever-increasing number
of gene sequences become mapped, the probability of cloning mutants
by matching them to candidate genes will continue to increase. It
was felt that obtaining rough map positions for a large number of
mutants was valuable for this reason. This will be an ongoing endeavor,
as mutant screens will continue to be a central activity of the
Up to Top
C. Technologies for genome manipulation
To In order to exploit the power of the zebrafish to clarify gene function,
we need to rapidly expand the application of several novel techniques that have
been developed over the past five years. As we have learned in other model organisms,
a handful of molecular genetic tricks can determine the ease, speed, and cost-effectiveness
of genetic studies in each organism. DNA-mediated or transposable element transgenesis,
insertional mutagenesis, knockouts, embryonic stem cells, the availability of
cell lines, methods for freezing and storing organisms, have all transformed
fields in the case of specific model organisms. The zebrafish still lags in
this regard.developing and taking advantage of these technologies.
Technologies and methods that could greatly accelerate the pace of analyzing
gene function in the fish include the following:
- Generation of transgenic fish expressing GFP, in specific lineages and on
each chromosome, in order to i) facilitate better and more rapid analysis
of mutant phenotypes; and ii) facilitate carrying mutant lines of fish; .
crossing Crossing mutations into the appropriate line bearing a (linked) GFP-marked
chromosome, would permit rapid identification of progeny carrying the mutation.
- Development of other types of transgenics including the UAS-gal4 system,
flp, cre, etc.
- Full exploitation of existing, or additional, libraries of fish in which
gene trap viruses have integrated into large numbers of genes, creating screenable
collections of knock-out libraries.
Up to Top
D. Gene expression
i. Array technology: Array methods for systematic assessment of patterns of
gene expression were felt by many participants to be important tools for the
- Making new cDNA libraries from specific tissues and at multiple developmental
- Getting more expressed sequence tags (ESTs) from these libraries.
- Making these reagents available in a suitable form to use in microarrays
of 10,000-20,000 ESTs to begin with. Ultimately the goal is that expression
of all cDNAs can be systematically assessed.
ii. Gene expression patterns (Also, see section on Informatics Issues): There
was substantial support for the development of systematic information about
spatial patterns of gene expression. Taking advantage of the transparency of
the zebrafish embryo, high-resolution in situ hybridization can be performed
on whole mount embryos, using probes for known genes and for ESTs.
The goal of this project would be:
- Providing a large collection of cell and/or tissue specific molecular markers
for analysis of wild-type development (establishment of a "molecular anatomy"
of the embryo) as well as for the phenotypic description of mutants obtained
from genetic screens.
- Providing starting points for functional analysis (using overexpression
studies as well as the development of numerous Gal4-UAS lines).
- Providing a powerful way (candidate gene approach) to clone genes disrupted
in mutant embryos based on the correlation between mutant phenotypes, expression
pattern and data concerning sequence and map position of these genes.
All these expression data should be released to the public via the Zebrafish
Information Network [ZFIN] in an integrated database (see anatomical
atlases, below) in a standardized format allowing the search by
names, sequences, genetic map positions, and anatomical structures.
The database will include keyword descriptions of all spatially
restricted expression patterns as well as the corresponding pictures
for all developmental stages analyzed.
Up to Top
E. Stock Center and Training -- Related Issues
The U. S. Stock Center is a major concern of the zebrafish research community.
There are a large number of mutant lines currently spread among many laboratories.
Moreover, many laboratories are generating new mutant and transgenic lines at
an increasing pace. The community is concerned about whether the this Stock
Center can adequately house all these lines safely. The current plan for the
Stock Center calls for space to maintain a maximum of about 5,000 lines in tanks;
the remainder will be kept as frozen sperm. The mutant collection in Germany
will not be expanded, meaning 400 or fewer lines will be maintained there. To
In order to ensure that all lines are available, it is suggested that the Stock
Center concentrate on providing lines quickly and efficiently from frozen sperm.
This will require additional personnel.
For many laboratories, in vitro fertilization and recovery of mutant lines
is difficult. The Stock Center should provide these services. Additional staff
will also be required to meet these goals because both services are more labor
intensive than simply maintaining lines.
The community felt that putting all mutants in one Stock Center, without
some backup was not a good idea. Funding should be provided for banking sperm
at a second site that would serve as a safety backup. This would require money
for liquid nitrogen freezers and at least a part time salary.
Concern was expressed that the number of individuals with expertise in fish
pathology is very limited. Expanded training in this field was a recommendation
of this workshop.
- Expanded support for existing stock center for providing mutant fish from
frozen sperm and in providing training opportunities in techniques needed
to recover mutant lines.
- Support for an additional back-up center in the U.S. for sperm preservation
to prevent catastrophic loss of mutants.
- Expanded training of individuals with expertise in fish pathology.
Up to Top
F. Budget estimates
Workshop participants developed the following direct cost estimates:
1. Genetic screens (including adult phenotypes) $3.0 M/yr
2. Technologies for genome manipulation Total/yr: $3.5 Million
- Methods improvement and novel developments: $1.5 M/yr
- GFP-marked chromosomes: $1 M/yr
- KO-viral insert approach: $1 M/yr
3. Stock centers $ 0.35 M/yr
4. Array technology $1 M/yr
5. Mapping mutants $0.5 M/yr
It is anticipated that each of the above initiatives will be funded for
TOTAL ESTIMATE OF DIRECT COSTS
Year 01 = $8,350,000
Total (3 years) = $25,050,000
Up to Top
V. Informatics Issues
With the increasing complexity of biological, sequence and mapping data being
generated, the need to develop and maintain a community-based database becomes
paramount. The recommendations in this chapter clearly impact on the research
activities described in the preceding two chapters since the integration of
information at a common site will provide a valuable resource to the zebrafish
community. This important role is to be fulfilled by ZFIN, currently funded
through a grant to the University of Oregon. However, the cost of maintaining
such a sophisticated database is high and one that appropriately should be shared
by Institutes and organizations supporting research using zebrafish. It is a
complicated issue and the recommendations cited below look at both long-term
needs of development and maintenance of the system as well as the more short-term
need for developing an anatomical atlas and dictionary.
The utility of genomic, mapping and biological information to the community
of investigators interested in the zebrafish is heavily dependent upon the existence
of mechanisms that facilitate access to this information. The sharing of materials
and data in a timely manner is an essential element if rapid progress is to
be made in the construction and use of genetic maps. The personnel who develop
and maintain ZFIN are crucial to its success in implementing the goals of the
zebrafish community. The data editors who annotate expression and phenotypic
data and the informaticists who are needed to expand the capabilities of the
website are essential to ensure that ZFIN is able to fulfill its mission of
providing the entire community with ready access to genomic and developmental
One of the important needs is the development of standard terminology and staging
criteria as well as standardized formats that will allow for the rapid transfer
and submission of sequence, mapping, and biological data to ZFIN. This will
also be important for the integration of the zebrafish database with those of
other model organisms. Community standards for data acquisition as well as rules
regarding intellectual properties and full disclosure of potential restrictions
on the use of data need to be developed. Many of these activities will be covered
in the costs associated with the maintenance of the informatics infrastructure
indicated below. Recommendations include:
- Data editors to annotate expression and phenotypic data
- Bioinformaticists to expand capabilities of database
- Develop "data model" for minimal criteria for submission and
acceptance of data to ZFIN
- Establish community standards for access and use of database
- Interface ZFIN with stock center database so that requests for orders
can be input through ZFIN
- Define terminology and staging criteria for the establishment of a controlled
vocabulary and anatomical dictionary (see below)
- Integrate ZFIN with the databases of other organisms
- Copy ZFIN so that private contractors cant resell database information
Up to Top
A. Anatomical atlases
Anatomical atlases will be important for the rapid development of the fish
field. Because the fish has only recently become a popular model organism, anatomical
descriptions of the development of structures from embryo to adulthood are incomplete.
For this reason, the zebrafish community needs, with some urgency, to build
an anatomical atlas and establish an anatomical dictionary. Development of uniform
terminology is a critical first step in the larger problem of providing systematic
annotated information about gene expression.
The specific goals of this project are to:
- Establishment of a common language so that the same key words are used
by the whole zebrafish community. Thus, we need to build a zebrafish anatomical
- Development of a set of images that illustrate these terms throughout
zebrafish development, and make these available through ZFIN.
- Use of this dictionary to assess and annotate in a systematic fashion
the gene expression patterns coming from large-scale in situ hybridization
screens and phenotypic data coming from mutagenesis screens, including that
available in the literature.
- Making annotated high quality anatomic images of gene expression available
in ZFIN. The long-term goal would be a four-dimensional space and
time - description of the pattern of expression of key genes.
- Making compatible links between the zebrafish anatomic database information
and other databases.
Up to Top
B. Budget Estimates
1. Informatics infrastructure $1.0
Data editors $0.5
Bioinformaticists $0.5 M/yr
2. Gene expression atlas $1.5
Total Estimate of Direct Costs
Year 01 $2,500,000
Total (3 Years) $7,500,000
Up to Top
SPECIAL APPLICATIONS OF THE ZEBRAFISH
Studies of Musculoskeletal and Skin Developmental Diseases
Washington University School of Medicine
University of Oregon
The zebrafish offers a number of advantages for studying the
biology of the skin and skeletal/muscle systems. Because embryonic
development is external to the mother and the embryos are transparent,
development of skin, pigment cells, bones and muscles are easily seen in
the living embryo.
One major area of research in zebrafish is development of the
melanocyte pigment pattern in zebrafish. Abnormal melanocyte development
or survival in humans leads to a variety of syndromes or diseases,
including piebaldism, vitiligo, moles, and skin cancer. Studies of
development of the adult zebrafish melanocyte stripes reveals that these
cells are recruited from a population of undifferentiated precursors or
stem cells that persist throughout the life of the fish. Mutations have
been identified that affect how the precursor cells are established or
later recruited to proliferate and differentiate. Other mutations affect
migration of melanocytes, or the distance between stripes. More than one
hundred mutations have been identified that affect the development of
The control of bone growth and regeneration has been investigated
in zebrafish. Dozens of mutations affecting jaw cartilage or bone formation
have been identified. Mutations affecting the regeneration of the adult
fin may help us better understand the genetic control of bone remodeling
and healing. For example, when adult fins are amputated, normally
post-mitotic differentiated bone cells at the amputation plane are
recruited to divide and form the regeneration blastema. Screens for
mutations that affect fin regeneration have identified seven
temperature-sensitive mutations that affect aspects of the regeneration or
morphogenesis of the regenerating bone in the fin. Other mutants have been
identified that affect the rate of growth of the fin ray, either causing
abnormal morphology of the bone, or bones that grow too slow or too fast.
Molecular characterization of these mutations may reveal previously unknown
players in these processes shared among all vertebrates.
Important recent work in zebrafish has advanced our understanding
of vertebrate muscle development and function. The isolation of a large
collection of mutants affecting the development and function of muscles
provides an important resource for investigation. These mutants affect a
variety of processes including: somite formation (5 genes), horizontal
myoseptum formation (11 genes), myoblast differentiation (3 genes),
myofiber development and/or organization (8 genes), muscle tissue
maintenance (4 genes), and locomotion (>40 genes). Although preliminary
characterization has been done on these mutants, further analysis and
molecular characterization is important. For example, double mutant
combinations must be made to understand gene interactions and to order the
genes identified by mutation into a regulatory hierarchy. Furthermore, we
must discover the molecular nature of the encoded gene products.
Significant advances have been made in understanding the
organization of the segmental plate and the origin of somites in zebrafish.
For example, slow and fast muscle precursors become specified very early
in development (long before overt muscle differentiation) and then undergo
dramatic morphogenetic movements to assume their final positions in the
embryo. Indeed, some zebrafish mutations, particularly those that affect
horizontal myoseptum formation, may identify components involved in
specifying these two muscle cell types.
A wide variety of molecular markers that identify different
regions of the developing somites and segmental plate have been
characterized in zebrafish. One such gene is her-1, a homolog of the
Drosophila pair-rule gene hairy. As in Drosophila, this gene marks the
developing segments in zebrafish in a pair-rule fashion. In fact, her-1
"prefigures" somite development by several hours, making it the earliest
known marker of specified somites. Mutations have been isolated that
disrupt the expression pattern of her-1 in zebrafish embryos, and thus
identify genes important for the initial specification of somites.
The molecular characterization of genes responsible for mutant
phenotypes may identify genes involved in human disease. For example,
zebrafish mutants that display muscle-specific degeneration may identify
components involved in the destructive degeneration-regeneration process
that occurs in human muscle dystrophies. Besides identifying genes
involved in these diseases, these mutant lines would then become useful
model systems to test potential therapies. Other mutants, such as the
large number of genes affecting motility and locomotion, may help us
identify genes involved in proper neuromuscular function and in human
Up to Top
Zebrafish and Cancer Research
Keith C. Cheng
Penn State University College of Medicine
June 2, 1999
The fundamental phenotypes of the human cancer cell include
abnormal growth control, abnormal cell differentiation, invasion,
metastasis, and genomic instability. The identification of the genes
controlling these processes among the 50,000-100,000 genes in the human
genome will be one of the major charges of NIH-funded research in the next
several decades. We propose that the zebrafish can play a key role in
defining those genes, through the generation and study of mutants.
A variety of screens can yield information about cancer. Among
the hundreds of genes identified in the large scale developmental mutant
screens, many may be expected to affect growth control, cell communication,
differentiation and and cell migration. Some of the temperature-sensitive
fin regeneration mutations generated at the University of Oregon may lead
to discovery of basic cell growth control mechanisms. Among the
hematopoiesis mutants are some whose phenotype is reminiscent of
myelodysplasia or pre-leukemia.
Two Penn State screens may yield tumor suppressor genes. One,
currently funded by the National Science Foundation, has yielded mutants
that show elevated frequencies of somatic loss of heterozygosity. These
genomic instability mutations are expected to have defects in chromosome
segregation, recombination, and gene regulation. Preliminary data suggest
potential tumor susceptibility, consistent with the hypothesis that some of
these mutations will function as tumor suppressor genes. The second screen
is based upon the high sensitivity of light microscopy to detect defects in
cell differentiation, as is done routinely in human pathology. A
particularly interesting mutant affects multiple organs and shows
cytological abnormalities that are common in cancer, including
pleomorphism, nuclear atypia, and hyperchromatic nuclei. This work shows
that a histology-based screen can produce potentially important mutants.
Large scale zebrafish screens have required funding by companies
or private research institutes. The corresponding difficulty of individual
labs to gain funding for small-scale screens suggests that "small labs"
need a mechanism for funding of large facilities for collaborative research
endeavors. The building of new zebrafish facilities around new areas of
investigation not covered in developmental biology screens is one potential
approach. To facilitate the exploration of cancer-related research, it
will be best to maintain a cross-Institute effort in the support of
Up to Top
Eye Development and Disease
Harvard Biological Labs
The zebrafish visual system, especially the eye, is particularly
amenable for genetic analysis. Development of the eye is rapid; within 24
hours post-fertilization (pf) a well-formed eye is present.
Differentiation of the neural retina occurs between 1 and 3 days pf; so
that by three days pf the retina appears functional. Visual responses can
first be elicited at this time, and by 5-6 days pf robust optokinetic
reflex responses can be obtained from 98% of normal animals. Initially the
retina is cone dominated; abundant rods are not evident until the second
week of life. In adult fish, the retina contains four morphologically
distinct cone types. Short single cones are ultraviolet-sensitive whereas
long single cones are blue-sensitive. The principal and accessory members
of the double cones are red- and green-sensitive respectively. In
addition, the cones are arranged in a precise mosaic pattern across the
retina, aiding in their identification.
In genetic studies carried out so far, numerous mutations affecting
retinal development have been observed (Malicki et al., 1996; Heisenberg et
al., 1996; Fadool et al., 1997). Most of these mutants were detected
because of a small eye phenotype. Many show both retinal and brain defects
histologically, but some show only retinal deficits. Mutants with abnormal
retinal lamination have been observed, as well as mutants showing selective
retinal cell loss. In some cases cells fail to form; in other cases,
specific cell types rapidly degenerate after differentiation. Visual
behavioral studies at 5-7 days (pf) (Brockerhoff et al., 1995; Brockerhoff
et al., 1997) have revealed functional defects in morphologically normal
fish. One mutant, for example, appears to have a defect in synaptic
transmission between photoreceptor and second-order cells. Another mutant
loses all its red-cones between 3 and 5 days pf, resulting in an animal
deficient in red-sensitive vision. This latter mutation does not involve
the opsin gene, suggesting that this mutation represents a new form of
cone-specific color blindness. Finally, a dominant mutation causing slow
photoreceptor cell degeneration has been detected behaviorally in adult
fish (>4 months pf) (Lei and Dowling, 1997). When homozygous, this
mutation causes an early (~2 day pf) massive retinal and tectal cell
degeneration and death of the animal by 5 days of age, suggesting that the
gene involved is not photoreceptor cell-specific. Again, this mutation
appears to represent a new type of inherited retinal degeneration. Another
promising approach is the study of retinal-tectal projections. In
Friedrich Bonhoeffer has been able to detect over 100 mutants which have
defects of the ganglion cell axons finding their way to the tectum.
Future studies will include isolation of visual mutations using
more subtle and sophisticated behavioral tests, as well as the further
characterization of both behavioral and morphological mutants. So far the
focus has been on retinal mutations, but mutations affecting higher visual
processing and eye movement mechanism are likely to be found. Molecular
genetic studies to isolate the mutated genes represent an important next
step in the enterprise.
Brockerhoff, S. E., Hurley, J. B., Janssen-Bienhold, U., Neuhauss,
S. C., Driever, W. and Dowling, J. E. A behavioral screen for isolating
zebrafish mutants. Proc. Natl. Acad. Sci., 92:10545-10549, 1995.
Brockerhoff, S. E., Hurley, J. B., Niemi, G. A. and Dowling, J. E.
A new form of inherited red-blindness in zebrafish, J. Neurosci.
Fadool, J. M., Brockerhoff, S. E., Hyatt, G. A. and Dowling, J. E.
Mutations affecting eye morphology in the developing zebrafish, Danio
rerio. Developmental Genetics. 20:288-295, 1997.
Heisenberg, C.-P., Brand, M., Jiang, Y.-J., Warga, R. M., Beuchle,
D., van Eeden, F. J. M., Furutani-Seiki, M., Granato, M., Haffter, P.,
Hammerschmidt, M., Kane, D. A., Kelsh, R. N., Mullins, M. C., Odenthal, J.
and Nüsslein-Volhard, C. Genes involved in forebrain development in the
zebrafish, Danio rerio. Development, 123:179-190.
Li, L., and Dowling, J. E. A dominant form of inherited retinal
degeneration caused by anon-photoreceptor cell-specific mutation, Proc.
Natl. Acad. Sci., 94:11645-11650, 1997.
Malicki, J., Neuhauss, S. C. F., Schier, A. F., Solnica-Krezel, L.,
Stemple, D. L., Stainier, D. Y. R., Abdelilah, S., Zwartkruis, F., Rangini,
Z. and Driever, W. Mutations affecting development of the zebrafish ear.
Development, 123:263-273, 1996.
Up to Top
Kidney Development and Diseases
Mass. General Hospital - East, Boston
The zebrafish, as a model system for vertebrate development, offers
distinct experimental advantages for studies of kidney development. The
zebrafish pronephros is a remarkably simple kidney, consisting of only two
nephrons with glomeruli fused at the midline, pronephric tubules connecting
directly to the glomeruli via a neck segment, and paired bilateral
pronephric ducts which convey the altered blood filtrate outside the animal
(1). Large scale mutant screens taking advantage of the optical
transparency of zebrafish embryos have isolated many mutants with obvious
visible kidney defects (1,2,3). In addition, a recent mutagenesis screen in
the laboratory of Dr. Mark Fishman employing wt1 as an in situ probe for
glomerular development has suceeded in isolating new mutants with more
subtle kidney defects. The combined yield of all screens has provided
mutants affecting each of the principle stages of nephrogenesis including
1) specification of the nephrogenic mesoderm, 2) nephron patterning, 3) the
development of epithelial polarity, and 4) vascularization of the
glomerulus. In addition, fifteen loci have been isolated which result in
cystic maldevelopment in the pronephros.
Formation of the nephrogenic mesoderm.
Dorsalized mutants swirl and somitobun lack signals necessary for ventral
mesodermal development and show a reduction or elimination of
pax2.1-positive, presumptive kidney intermediate mesoderm (4). Conversely,
the ventralized mutant chordino lacks a dorsal determinant and shows an
expansion of the pax2.1 expression domain (5). The dorsalized mutants swirl
and somitobun carry mutations in zebrafish BMP2b and SMAD5 respectively
(6-8), while the ventralized mutant chordino carries mutations in zebrafish
chordin, an inhibitor of BMP activity (9). The data from these mutants
suggests that BMP signaling, most likely in concert with additional
signals, is required to specify kidney mesoderm, as well as other ventral
Zebrafish pronephric podocytes and tubules differentiate from a single
primordium, suggesting that nephron patterning events must occur to
restrict podocyte and tubule cell differentiation to their appropriate cell
fates and positions in the nephron (1). In the zebrafish pax2.1 mutant no
isthmus (noi) we have observed a failure to develop pronephric tubules and
an expanded expression of podocyte markers wt1 and vegf in cells normally
destined to differentiate into the pronephric duct. These results shed new
light on the role of pax2.1 in restricting podocyte development and suggest
that pronephric tubule and podocyte cell differentiation may be regulated
by antagonistic signals.
The development of epithelial polarity
Several mutants affecting epithelial polarity and the terminal
differentiation of pronephric duct epithelial cells have been found (1).
The function of the duct cells in recovering ions and metabolites from the
glomerular filtrate is dependent upon the establishment of apical-basal
membrane polarity and the asymmetric distribution of membrane transporters.
The driving force for many transport processes is the sodium gradient
established by the Na+/K+ ATPase, a membrane bound ion pump normally
located basolaterally in kidney tubule and collecting system epithelia
(10). In several mutants including double bubble, fleer and dizzy, antibody
staining for the Na+/K+ ATPase is strongly apical and diminished on
basolateral membranes (1), suggesting a failure to properly target or
retain the Na+/K+ ATPase on the basolateral cell surface. Studies of these
mutants are likely to contribute to our understanding of mechanisms
underlying the generation of cell polarity and terminal epithelial cell
Vascularization of the glomerulus and podocyte morphological differentiation
In zebrafish the nephron primordia rest directly on the ventral surface of
the dorsal aorta where the glomerulus will form. Between 40 and 48 hours
post-fertilization (hpf) glomerular formation occurs starting with the
extension of podocyte foot processes and the interdigitation of the
glomerular basement membrane (GBM) with endothelial cells (1). This
infolding of the GBM correlates with capillary loop formation and the onset
of glomerular filtration. The zebrafish mutant cloche survives into the
larval period desptie the fact that it is nearly completely lacking in
endothelial cells (11). Zebrafish cloche embryos thus provide a unique
opportunity to investigate the role of endothelial cell derived signals in
the differentiation of pronephric podocytes. We find that the
differentiation of podocytes in cloche proceeds normally despite the
complete absence of glomerular endothelial cells (12). cloche podocytes
express wt1 and vegf and form extensive foot processes arranged as pedicels
along a basement membrane (12). These findings indicate that once
initiated, podocyte differentiation can proceed independently of
endothelial cells or endothelial cell derived signals. The idea that
podocytes may initiate glomerulogenesis by recruiting endothelial cells is
substantiated in studies of the mutant floating head. floating head embryos
lack a notochord and as a consequence also lack a dorsal aorta (13), the
normal blood supply for the pronephros. We have found that in the absence
of the dorsal aorta, clusters of podocytes develop at ectopic lateral
locations and appear to recruit flk-1-positive endothelial cells from
alternate sources. Remarkably, these clusters of podocytes proceed to form
two separate vascularized nephrons, fully capable of blood fitration. The
availablity of these mutants has allowed us to demonstrate that podocytes
play a major role in glomerular morphogenesis, independently of associated
cells and tissues.
Pronephric mutants as models of cystic maldevelopment.
Fifteen different zebrafish genetic loci have been identified which when
mutated result in the development of pronephric cysts followed by general
edema (1). The total number of mutants having a kidney cyst phenotype is
now thirty-eight and includes mutants from the original screens in Tübingen
and Boston as well as a recently completed in situ hybridization screen in
Mark Fishman's laboratory at MGH. This large number of mutants and our
results that additional mutants are falling into the original fifteen
complementation groups suggests that a genetic pathway responsible for
proper epithelial development may be close to saturated with mutations. The
mutant double bubble, develops glomerular cysts at the stage when blood
filtration is getting established and later, defects in the glomerular
basement membrane are evident (1). As noted above, double bubble mutants
also show defects in Na+/K+ ATPase targeting in the pronephric ducts which
may result in altered ion gradients across pronephric epithelia. Despite
the cloning of several genes associated with human polycystic kidney
disease, the mechanism of cyst formation remains unknown. In human disease,
developmental or cellular defects in epithelia caused by cystic mutations
are often obscured by compensatory tissue responses, making it difficult to
define the primary cause of cyst formation. The mechanism of cyst
development in zebrafish cyst mutants is also unknown but could be due to
either 1) an inability of the forming glomerulus to withstand the onset of
filtration pressure or 2) to altered osmotic forces in the mutant
pronephroi. To distinguish among these possibilities we have created a
double mutant homozygous for a the dbb cystic mutation and the myocardial
function mutation silent heart. These double mutants have no blood pressure
to drive filtration and would be not be expeccted to develop cysts if
glomerular filtratration were the primary force driving cyst formation but
presumably would develop cysts if osmotic forces within the pronephros were
the cause of glomerular and tubule distension. These types of experiments
underscore the utility of zebrafish as a genetic system to perform types of
analysis that cannot reasonably be performed in any other model organism.
It is interesting to note that both glomerulocystic kidneys and apical
mislocalization of the Na+/K+ ATPase have been associated with mutations in
the PKD1 gene which accounts for the majority of cases of autosomal
dominant polycystic kidney disease (ADPKD) in humans (14,15). In a
candidate gene approach to cloning the zebrafish cystic genes, we have
isolated zebrafish homologs of the alpha1 subunit of the Na/K ATPase which
is mislocalized in dbb, as well as homologs of polycystin (PKD1) and the
mouse cystic gene tg737. Two zebrafish cyst mutants, fleer and elipsa,
display a combined renal-retinal dysplasia (1,16), a disease in humans
referred to as Senior-Loken syndrome (17). No human loci or candidate genes
have yet been identified for Senior-Loken syndrome. Complementation tests
of fleer and elipsa as well as additional cyst mutants isolated in the
Tübingen screen have shown that fleer and elipsa are distinct loci with one
allele for fleer and two alleles for elipsa. Current efforts to map and
positionally clone these zebrafish genes will potentially speed the
isolation of human genes responsible for similar heritable defects.
1. Drummond IA, Majumdar A, Hentschel H, Elger M, Solnica-Krezel L, Schier
AF, Neuhauss SC, Stemple DL, Zwartkruis F, Rangini Z, Driever W, and
Fishman MC (1998) Early development of the zebrafish pronephros and
analysis of mutations affecting pronephric function. Development
2. Haffter P, Granato M, Brand M, Mullins MC, Hammerschmidt M, Kane DA,
Odenthal J, van Eeden FJ, Jiang YJ, Heisenberg CP, Kelsh RN, Furutani-Seiki
M, Vogelsang E, Beuchle D, Schach U, Fabian C, and Nusslein-Volhard C
(1996) The identification of genes with unique and essential functions in
the development of the zebrafish, Danio rerio. Development 123:1-36.
3. Driever W, Solnica-Krezel L, Schier AF, Neuhauss SC, Malicki J, Stemple
DL, Stainier DY, Zwartkruis F, Abdelilah S, Rangini Z, Belak J, and Boggs C
(1996) A genetic screen for mutations affecting embryogenesis in zebrafish.
4. Mullins MC, Hammerschmidt M, Kane DA, Odenthal J, Brand M, van Eeden FJ,
Furutani-Seiki M, Granato M, Haffter P, Heisenberg CP, Jiang YJ, Kelsh RN,
and Nusslein-Volhard C (1996) Genes establishing dorsoventral pattern
formation in the zebrafish embryo: the ventral specifying genes.
5. Hammerschmidt M, Pelegri F, Mullins MC, Kane DA, van Eeden FJ, Granato
M, Brand M, Furutani-Seiki M, Haffter P, Heisenberg CP, Jiang YJ, Kelsh RN,
Odenthal J, Warga RM, and Nusslein-Volhard C (1996) dino and mercedes, two
genes regulating dorsal development in the zebrafish embryo. Development
6. Nguyen VH, Schmid B, Trout J, Connors SA, Ekker M, and Mullins MC (1998)
Ventral and lateral regions of the zebrafish gastrula, including the neural
crest progenitors, are established by a bmp2b/swirl pathway of genes. Dev
7. Kishimoto Y, Lee KH, Zon L, Hammerschmidt M, and Schulte-Merker S (1997)
The molecular nature of zebrafish swirl: BMP2 function is essential during
early dorsoventral patterning. Development 124:4457-66.
8. Hild M, Dick A, Rauch GJ, Meier A, Bouwmeester T, Haffter P, and
Hammerschmidt M (1999) The smad5 mutation somitabun blocks Bmp2b signaling
during early dorsoventral patterning of the zebrafish embryo. Development
9. Schulte-Merker S, Lee KJ, McMahon AP, and Hammerschmidt M (1997) The
zebrafish organizer requires chordino [letter]. Nature 387:862-3.
10. Drubin DG, and Nelson WJ (1996) Origins of cell polarity. Cell 84:335-344.
11. Stainier DY, Weinstein BM, Detrich HW, 3rd, Zon LI, and Fishman MC
(1995) Cloche, an early acting zebrafish gene, is required by both the
endothelial and hematopoietic lineages. Development 121:3141-50.
12. Majumdar A, and Drummond IA (1999) Podocyte differentiation in the
absence of endothelial cells as revealed in the zebrafish avascular mutant,
cloche [In Process Citation]. Dev Genet 24:220-9.
13. Fouquet B, Weinstein BM, Serluca FC, and Fishman MC (1997) Vessel
patterning in the embryo of the zebrafish: guidance by notochord. Dev Biol
14. Torra R, Badenas C, Darnell A, Bru C, Escorsell A, and Estivill X
(1997) Autosomal dominant polycystic kidney disease with anticipation and
Caroli's disease associated with a PKD1 mutation. Rapid communication.
Kidney Int 52:33-8.
15. Wilson PD, Sherwood AC, Palla K, Du J, Watson R, and Norman JT (1991)
Reversed polarity of Na(+) -K(+) -ATPase: mislocation to apical plasma
membranes in polycystic kidney disease epithelia. Am J Physiol 260:F420-30.
16. Malicki J, Neuhauss SC, Schier AF, Solnica-Krezel L, Stemple DL,
Stainier DY, Abdelilah S, Zwartkruis F, Rangini Z, and Driever W (1996)
Mutations affecting development of the zebrafish retina. Development
17. Gusmano R, Ghiggeri GM, and Caridi G (1998) Nephronophthisis-medullary
cystic disease: clinical and genetic aspects. J Nephrol 11:224-8.
Up to Top
Cardiovascular and Other Organogenesis in Zebrafish
Mark C. Fishman
Mass General Hospital and Harvard Medical School
Zebrafish mutations discovered in the two original, and several subsequent,
large screens have already revealed an organizing logic to organ
development, physiology, disease and evolution. Each of the genes
discovered is an entrance point to pathways not previously imagined.
Screens for organ mutants are of biologic and medical importance
1. Organs can be dissected as genetic "modules". Single gene mutations in
the zebrafish can remove or disrupt components of organs with relative
selectivity, leaving the remainder of development to proceed with impunity.
In this way, they reveal separable units, as did screens in the fly for
units of body form. These include genes for formation of the ventricle of
the heart, for heart valves, or for the glomerulus of the kidney.
2. Mutations reveal candidates for disease. Single gene heritable
arrhythmias and heart failure mutants speak to common adult disorders.
Single gene-related disorders of early organ form or laterality are models
of common congenital abnormalities. Complex diseases have not proved
readily decipherable by whole genome linkage studies. However, the power
of association studies, using candidate genes, is such that it can reveal
the role of genes contributing even minimal components of risk. Screens in
zebrafish provide such candidates.
3. The mutations perturb "new" vertebrate evolutionary additions. The
global form of heart, kidney, pancreas, etc. are new on the evolutionary
scene, not present in primitive chordates. Interestingly, the modules
perturbed by mutations are precisely those for these new evolutionary
additions, and therefore speak to how large-scale innovations may have
occurred by single gene mutations during evolution.
The zebrafish is the most tractable and relevant genetic system for organ screens
Unlike Drosophila or C. elegans, the organs of zebrafish are
similar to those of man. Whereas Drosophila lacks vessels and the heart has
no ventricle, endocardium, valves, or conduction system, the zebrafish
heart is essentially identical to that of the three week gestation human.
The mouse embryo is not as good for organ screens because of its relative
opacity. More importantly, and in contrast to the zebrafish, the mouse
embryo depends upon ongoing delivery of nutrients and removal of waste
products, leading to rapid deterioration of mutants which affect these
Some examples of zebrafish organ mutants which have proved important
1. Models of disease.
The gridlock mutation lacks circulation to the tail, because of failure to
assemble one branch point, embryologically identical to the region affected
in congenital coarctation of the aorta (Weinstein et al., 1995). The
asymmetry mutants, in which the right-left axis of the heart is reversed,
affect the process believed perturbed in ventricular inversion (Chen et
al., 1997). The mutation with a diminutive ventricle (Stainier et al.,
1996) affects processes relevant to the hypoplastic heart syndromes.
Many common disorders are impossible to model in animals, and are
approachable in humans only by total genome scanning. For example, heart
failure is a common and polygenic disorder, frequently "idiopathic" in
nature. Several mutants evidence the same dilated poorly contractile
physiology as do these patients. Other individual mutants model each
common rhythm disorder, including heart block, fibrillation, and
bradycardia (Baker et al., 1997). Mutants which cause disarray of
epithelial patterning in the gut (Pack et al., 1996) may be relevant to
cancer, as are the mutants which affect angiogenesis. Those which perturb
one pancreatic lineage (Pack et al., 1996) speak to issues important in
tissue replacement for cystic fibrosis and diabetes.
2. New paradigms in biology
These mutants have revealed steps of organ assembly, steps we could not
have predicted to exist. In terms of generation of heart form, for
example, there are genes needed specifically to generate one chamber but
not the other, to assemble a single heart tube rather than two, to elicit
valve formation or to generate the endocardium (Fishman and Olson, 1997).
Mutants separate lineages, such as those for pancreatic exocrine and
endocrine cells. In each case, the mutation is discrete and other aspects
of development are relatively intact. These mutants provide an entrance to
pathways to understand development of the ventricle, or valves, or
endocardium, etc. In addition, the mutants provide evidence that discrete
genetic pathways regulate important physiological parameters, such as heart
size (Stainier et al., 1996).
Biological and molecular work-up of current mutants
In vitro assay systems have been useful. Patch clamping of isolated
cardiocytes, for example, has identified the channel defect in pacemaker
mutants. Cell transplantation between mutant and wild-type fish has
revealed the existence of unexpected signals, for example, how tissues
regulate assembly of neighboring vessels. The movements of cells in the
heart field, and essential control over regulation in the field to
perturbants, can be analyzed at a single cell level by caged fluorescent
lineage markers (Serbedzija et al., 1998). Positional cloning is underway.
Using the microsatellite map , there is linkage for about 20 organ mutants,
and positional cloning has delimited the region, for several, to a BAC.
There are opportunities for new types of screens with regard to organs.
Screens based on molecular probes can identify mutations in organ systems
which are difficult to visualize directly, such as the kidney, or in the
precursor populations which generate the organs. Insertional screens,
taking advantage of sequenced sites around insertions to reveal
tissue-related ESTs, could provide mutants in defined genes. Dominant
mutations, in adults, will allow correlation with physiology and discovery
of modifier mutations.
Candidate genes for the human genome
It goes without saying that, once cloned, the genes for mutants with
medically relevant phenotypes become candidates for disease genes. The
phenotypes suggest that these might be especially useful in the study of
complex and common diseases, for which uniform human populations may be
elusive, such as heart failure, sudden death, and diabetes.
Baker, K., Warren, K. S., Yellen, G., and Fishman, M. C. (1997). Defective
"pacemaker" current (Ih) in a zebrafish mutant with a slow heart rate.
Proc. Natl. Acad. Sci. USA 94, 4554-4559.
Chen, J.-N., van Eeden, F. J. M., Warren, K. S., Chin, A. J.,
Nusslein-Volhard, C., Haffter, P., and Fishman, M. C. (1997). Left-right
pattern of cardiac BMP4 may drive asymmetry of the heart in zebrafish.
Development 124, 4373-4382.
Fishman, M. C., and Olson, E. N. (1997). Parsing the heart: genetic modules
for organ assembly. Cell 91, 153-156.
Pack, M., Solnica-Krezel, L., Malicki, J., Neuhauss, S. C. F., Schier, A.
F., Stemple, D. L., Driever, W., and Fishman, M. (1996). Mutations
affecting development of zebrafish digestive organs. Development 123,
Serbedzija, G. N., Chen, J.-N., and Fishman, M. C. (1998). Regulation in
the heart field of zebrafish. Development 125, 1095-1101.
Stainier, D. Y. R., Fouquet, B., Chen, J. N., Warren, K., Weinstein, B.,
Meiler, S., Mohideen, M. A. P. K., Neuhauss, S. C. F., Solnica-Krezel, L.,
Schier, A. F., Zwartkruis, F., Stemple, D. L., Malicki, J., Driever, W.,
and Fishman, M. C. (1996). Mutations affecting the formation and function
of the cardiovascular system in zebrafish embryos. Development 123, 285-292.
Weinstein, B. M., Stemple, D. L., Driever, W., and Fishman, M. C. (1995).
gridlock, a localized heritable vascular patterning defect in the
zebrafish. Nature Med. 1, 1143-1147.
Up to Top
Research on Aging
University of Utah
The most outstanding feature of the recent studies with the
zebrafish is the broad range of developmental and physiological
characteristics that can be probed by mutational analysis in this organism.
In addition to the well-known isolation of mutants with embryonic
developmental phenotypes, ongoing studies that are yielding palpable
results include the recovery of mutations that affect cardiovascular
physiology and the rate at which somatic mutations arise spontaneously.
Hence it is evident that zebrafish research can be applied toward the study
of genetic, cell biological, and developmental processes that influence or
correlate with aging. We foresee four areas of research that may
contribute significantly to our understanding of aging:
1) research concerning the accumulation of somatic mutations, including the
study of genetic variants with altered somatic mutation rates
2) research concerning the biological effects of alterations in DNA
metabolism, including the study of genetic variants that affect DNA
replication, repair, or recombination
3) research concerning the biological effects of altered metabolic rates,
including the study of genetic variants that display alterations in the
accumulation of specific metabolite species, such as free radicals
4) research concerning factors that influence the rate of development,
including the study of the potential correlation between the timing with
which developmental landmarks are attained and other markers of aging or
The areas of research highlighted here reflect the priorities of
ongoing research in the zebrafish field. We anticipate that as the field
matures and expands, a broader range of developmental and physiological
characteristics will come under study, and some of these additional areas
of research will be of significance to aging processes.
Up to Top
Child Health and Human Development
University of Oregon
Fundamental developmental genetic mechanisms have been conserved in all
vertebrates. Because of this fact and because zebrafish have marked
advantages for both genetic and developmental studies, the zebrafish
provides a useful model for understanding many aspects of human
At the genetic level, conservation is apparent from mutant phenotypes. For
example, our earlier work revealed that the zebrafish no tail gene is the
homologue of the Brachyury gene in the mouse, and the zebrafish valentino
gene is the homologue of the kreisler gene in the mouse. Both genes were
initially identified in both species by their mutant phenotypes.
Brachyury/no tail mutants lack tail and notochord development in both
zebrafish and mouse. kreisler/valentino mutants lack development of
hindbrain segments r5 and r6 in both zebrafish and mouse. These were the
first two cases in which two vertebrate species have been connected by
functional (mutational) analyses of orthologous genes. In both of these
cases the phenotypic analysis available in zebrafish has pointed to new
understanding of the defects in the mouse. These initial two cases
demonstrate that zebrafish is a useful system for understanding how genes
encode early development in all vertebrates, including humans.
Since these studies, thousands of mutations have become available through
continued and expanded genetic screens. My lab has collaborated with
others in the study about 10 of these mutations, and during the past two
years we have identified 5 of them moleularly. They include genes of the
PBX family that control hindbrain segmentation, a T-box that specifies
muscle development in dorsal mesoderm, and transcription and cell signaling
factors that control craniofacial skeletal patterning. All of these areas
are crucial ones to understand with respect to our eventual abilities to
deal with human disorders and birth defects
Our excellent progress has depended on advances in zebrafish genomics.
All of the initial identification of the genes relied on our new mapping
abilities, including making use of the radiation hybrid panels that have
become recently available. Cloning the genes depended on new libraries,
and one of them depended on the very newly available EST databases. This
progress is only a taste of what we will witness in the future, because we
have only begun to scratch the surface in terms of both mutational analyses
and utilizing genomics. We will most certainly continue to learn that many
of the genes now known only by their mutant phenotypes in zebrafish have
direct counterparts in humans. This will help us understand what is likely
happening when these human genes function incorrectly or not at all, as in
many types of congenital diseases.
Up to Top
Leonard I. Zon
Children's Hospital, Boston
The zebrafish is an excellent system to study hematopoiesis. Blood
is formed within 24 hours and the optical clarity of the embryo allows
visualization of mutants that affect the number and differentiation of
circulatory blood cells. Normal hematopoiesis occurs in a region above the
yolk sac called the intermediate cell mass. This region yields primitive
embryonic cells initially and then subsequently c-myb positive cells in the
ventral wall of the dorsal aorta, similar to the aorta-gonad-mesonephros
(AGM) region of higher vertebrates. The posterior region of the ICM
contains cells that are poorly differentiated and have a blast-like
morphology. These cells are likely to be stem cells or progenitors.
Zebrafish globins have been characterized and embryonic to adult hemoglobin
switching occurs after day 21 of development. Over 80 genes have been
cloned that are homologs of human or mouse factors that participate in
hematopoiesis. cDNA homologs have been isolated for every gene that has
been knocked-out in murine embryonic stem cells which have a defect in
hematopoiesis. This includes SCL, LMO2, GATA-1, GATA-2, TTG2, PU.1, myb,
flk1, flt4, and fli1. Thus, the hematopoietic program has been largely
conserved throughout vertebrate evolution.
We have collected over 26 complementation groups of hematopoietic mutants
and have recently derived 8 complementation groups of mutants affecting the
T cell program. Mutants have defects in either dorsal-ventral patterning,
hemagioblast formation, hematopoietic stem call formation, proliferation or
differentiation. A number of the mutants have difficulty in making
hemoglobin. Recently, the gene for two of the mutants with defects in
hemoglobin production were isolated. These encoded the ALAS-E gene and
UROD gene, two enzymes involved in heme biosynthesis. Thus, the fish is an
excellent model for studying hemoglobin production. In addition, the fish
model has a relevance to human disease in that missense mutations in these
genes cause congenital sideroblastic anemia or phorpheryia, respectively.
The sauternes gene was isolated by positional cloning approaches, thereby
demonstrating the success of this technique. Isolation of many of the
mutant genes are in progress and should provide interesting novel genes for
regulation of hematopoiesis as well as disease models for understanding the
pathophysiology of the disorder.
Genetic epistasis is possible in the zebrafish. cloche is a defect in
which there is no blood or blood vessel formation. Recent evidence has
demonstrated that the cloche mutant can be rescued by the overexpression of
the helix-loop-helix protein SCL. Overexpression of SCL leads to rescue of
hematopoiesis and vasculogenesis. Thus, overexpression of downstream genes
can rescue mutant phenotypes i.e. SCL acts downstream of cloche.
In the future, it should be possible to do focus screens for mutants that
affect other hematopoietic tissues or to envision studying earlier the
process of stem cell formation in vivo in the embryo. With this respect,
transgenic lines have been made in which green fluorescent protein has been
driven by either GATA-1 or GATA-2 promoters. This allows an early
definition of the hematopoietic program in vivo and should allow for
isolation of interesting cell populations and characterization of the
mutants. Finally, dominant suppressor screens may be possible in the
zebrafish, thus developing genetic pathways surrounding mutant phenotypes.
Up to Top
Research on Ear Morphogenesis and Hearing Disorders
Eric S. Weinberg
University Of Pennsylvania
Mass Eye and Ear, Boston
Animal models are extremely valuable for learning the genetic basis
of inner ear disorders. Because of obvious similarities in the pathology
of humans and mice with inner ear defects, the murine model is particularly
important model for genetic deafness. It is now evident from recent work
with the zebrafish that a second powerful animal model is now available for
studying the genetic basis of inner ear development and function. In
particular, the ability to trace the fate and lineage of early embryonic
cells, the feasibility of large scale screens of mutants with early
embryonic phenotypes, the ease of assaying the developmental effects of
gene products produced ectopically from injected RNAs, and the possibility
of constructing transgenic and chimeric animals all make the zebrafish a
potentially valuable animal model.
Recent mutant screens have yielded over 90 mutations (affecting
approximately 40 loci) that primarily affect development of the inner ear.
Mutations in at least nine genes affect morphology and patterning of the
inner ear epithelium, including semicircular canal development and
development of maculae and cristae. Other mutants affect ear size, otolith
formation, or initial specification of the otic placode. Many of the ear
mutants also exhibit abnormal behaviors such as swimming in circles or
upside down. Work on these mutants is only just beginning, and
undoubtedly, further analysis will increase our understanding of inner ear
development. One particularly well studied case is the valentino mutation,
which is a homolog of the mouse kreisler gene. In both zebrafish and mouse,
a malformation of the inner-ear is accompanied in mutant individuals by
abnormalities in the hindbrain, the site at which the gene is active,
suggesting conserved signaling mechanisms in inner-ear formation.
The vestibular part of the ear of teleosts and mammals is
remarkably conserved. Inner ears of both taxa have semicircular canals, a
utriculus, a sacculus, and types of otolith structures; the main auditory
organ in mammals (and birds), however, the cochlea, is absent in the
teleost ear. Nevertheless, for auditory functions, teleosts appear to use
the sensory epithelium of the sacculus and other structures that are
primarily vestbular in mammals. Since the sensory epithelia of both
vestibular and auditory structures of mammals and other vertebrates share a
common basic tissue architecture of hair cells and supporting cells,
mutants defective in such structures in fish ARE relevant to studies of
human sensory epithelial defects. At least two mutants with deffective
hair cell cillia have been recently identified in zebrafish: sputnik (spu)
and mariner (mar). Related to this previous point is the finding that some
types of mammalian deafness are accompanied by impairment of vestibular
function; such syndromes (e.g., Usher syndrome type 1B) are caused by
defects in components of the sensory epithelium common to the vestibular
and auditory regions of the ear. Moreover, different alleles of the same
gene can cause loss of vestibular function without impairment of hearing,
or loss of hearing without loss of vestibular function. Therefore,
identification of mutants with purely vestibular defects (easily
identifiable in zebrafish mutant screens) may ultimately be useful for
identifying genes that have both vestibular and auditory functions in
Even at first glance, many of the recently identified zebrafish
phenotypes resemble human inherited auditory disorders. For example,
branchio-oto-renal syndrome (BOR), mandibulofacial dystosis, craniofacial
dystosis, Alpert syndrome and some others are associated with craniofacial
abnormalities. Similar combination of phenotypes is obvious in the
zebrafish mutations quadro and little richard which affect both otic
vesicle and branchial arches. Likewise, selected genetic defects of both
human and zebrafish auditory system are associated with pigmentation
defects. In humans, wardenburg syndrome, piebaldness, vitiligo and
universal dyschromatosis involve depigmentation. Among fish mutants golas,
piegus, mizerny, punktata and others affect both auditory system and
pigmentation. The current collection of genetically defective zebrafish
strains, although already impressive, is likely to expand even more. New,
creative screening methods offer opportunities to search for mutations
affecting more and more specific developmental processes. In the area of
ear research, particularly promising are screens for defects of hair cell
development. Zebrafish lateral line hair cells can be visualized by
immersing fish larvae in solution of a fluorescent dye for a few minutes
allowing for efficient detection of many aberrations of this important cell
type. As hair cells are essential for auditory perception of both fish and
humans, this type of screening may have substantial medical importance.
The zebrafish is rapidly becoming a valuable model system for the
studies of human hearing disorders. In the immediate future, we expect
progress in two areas: identification of additional mutations of zebrafish
hearing and molecular characterization of mutant genes. The current and
future mutagenesis screens will involve increasingly more sophisticated
screening approaches, such as behavioral tests. One of the currently
performed screens, for example, takes advantage of the startle response to
identify hearing deficient fish. The second area of future growth is
molecular characterization of mutant genes. Rapid development of genomic
tools, such as genome maps, large insert libraries and est databases, has
already produced benefits in that several mutant genes have been
characterized via positional cloning and many others through the candidate
approach. These prospects should be a major factor in encouraging research
on the basic development and sensory physiology of the zebrafish inner ear,
and hence, the middle ear of all vertebrates.
Up to Top
Developmental Toxicology In Zebrafish
R.E. Peterson and W. Heideman
University of Wisconsin, Madison, WI
The zebrafish has been extensively utilized for toxicologic
studies, allowing rapid testing of various agents on normal embryogenesis.
The zebrafish system has the potential to genetically dissect pathways that
regulate response to toxins.
The polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans
(PCDFs), and biphenyls (PCBs) are global environmental contaminants and
potent developmental toxicants. Human exposure occurs during pregnancy and
breast feeding when these chemicals are transferred transplacentally to the
embryo and fetus and lactationally to the neonate posing a human health
risk. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the prototype compound
used to study this type of toxicity. In all vertebrates, the developing
embryo/fetus is significantly more sensitive to TCDD than the adult,
suggesting that TCDD has the ability to perturb critical developmental
events. Therefore, a thorough mechanistic understanding of TCDD action
during early development is necessary to assess the risk that such exposure
poses to children. Toxic effects of TCDD occur by TCDD binding with high
affinity to the aryl hydrocarbon receptor (AhR) followed by changes in gene
expression. After TCDD binding, the activated AhR translocates to the
nucleus and dimerizes with its partner ARNT. AhR-ARNT heterodimers
recognize and bind dioxin response elements found in promoters of
responsive genes to alter gene expression. AhR and ARNT are members of the
basic helix-loop-helix/PAS family of transcription factors that include
drosophila PER and SIM, murine ARNT-2, and human hypoxia inducible factor
1a. Members of this family are master developmental regulators, and it is
intriguing to speculate similar roles for AhR and ARNT.
The rationale for studying AhR and ARNT function in zebrafish is that
much of our current understanding of TCDD action involves characterizing
endpoints of TCDD exposure, and very little is know about the actual
mechanism of toxicity. It is largely accepted that TCDD developmental
toxicity in fish like mammals is AhR-mediated, but the gene targets of
activated AhR-ARNT that are causally related to the expression of toxicity
are poorly understood. Furthermore, the understanding of the physiological
role of AhR and ARNT in normal development is meager at best. The
zebrafish are being used to investigate the developmental toxicity of TCDD
and the physiological function of the AhR signaling pathway. Zebrafish
embryos exposed to graded doses of TCDD for 1 hour, from 2-5 hours post
fertilization, are responsive to TCDD (Toxicol. Appl. Pharmacol. 142:
56-68, 1997). The developmental toxicity is characterized by pericardial
and yolk sac edema, craniofacial malformations, and mortality.
Importantly, severe hemodynamic changes, manifested as slowed blood flow in
vascular beds of the trunk, head, and gills and slowed heart rate, occur prior to or
coincident with the onset of lesions. Visual inspection of the transparent
embryos shows that TCDD does not inhibit initial development of vasculature
or prevent initiation of blood flow to the above structures. Rather it
appears to interfere with the maintenance of peripheral vascular beds after
they are formed.
Future research will be directed at determining whether the
hemodynamic changes are due to a direct effect of TCDD on maintenance of
the vasculature and/or are secondary to cardiac insufficiency. Mutagenesis
screening will be used to identify key genes involved in the developmental
cardiovascular toxicity of TCDD and for this purpose a zebrafish genetic
map would be helpful. The human health significance of this research is
that development and maintenance of a vascular supply is a fundamental
requirement for organogenesis in the embryo. TCDD exposure does not
appear to affect vasculogenesis. Rather the subsequent process of
angiogenesis which includes sprouting, growth, migration, and remodeling of
endothelial cells seems altered. Therefore, our future research will focus
on the hypothesis that the developmental cardiovascular toxicity of TCDD is
caused by an interference with angiogenesis. Angiogenesis is also
implicated in the pathogenesis of a variety of human diseases such as
proliferative retinopathies, age-related macular degeneration, tumors,
rheumatoid arthritis, and psoriasis. Findings in zebrafish may ultimately
demonstrate whether or not the AhR signaling pathway is an important
regulator of angiogenesis with the degree of AhR activity in vascular
endothelial cells correlating with physiological and/or pathological
regulation of blood vessel growth.
Up to Top
University of Oregon
Knowing the sequence of the human genome will revolutionize our
understanding of human biology, human development, and human health. But
how can we go quickly from human genome sequence to human gene function?
The thesis of the paragraphs below is that research on zebrafish is the
most efficient way to understand the functions of genes necessary for the
development and physiology of all vertebrates, and thus zebrafish research
can efficiently illuminate the function of human genes known only by
sequence and map position.
The schedule for a complete, polished human genome sequence has
recently been revised to the end of 2003, two years ahead of earlier
projections. And a "working draft" will be available already by the end of
2001, barely two years away. We are faced with a surfeit of information and
a deficit of understanding. A major challenge is the interpretation of the
immense amount of sequence data -- among many questions, one reigns
supreme: What does each gene do?
An efficient way to find out what genes do is to isolate mutations
that disrupt the gene's abilities to direct the construction and operation
of the body's organs and organ systems. Screens for such mutations must be
driven by phenotype, because it is the phenotype that one wants to
understand: How is the phenotype constructed? What makes the phenotype
function? Such screens are difficult and expensive to conduct in mammals,
where embryos are opaque and hidden inside the mother. Zebrafish embryos,
however, develop outside the mother and embryos are optically translucent
so that each individual cell is visible until late in development.
Therefore, one can conduct efficient, cost effective, phenotype driven
screens at near saturation levels for mutations essential for zebrafish
development and physiology. In addition to providing an understanding of
the molecular genetic mechanisms of development and physiology, such
mutations become models for disease in humans and other animals.
Mutations are currently available in nearly 1000 genes essential
for embryonic development in zebrafish, and more are being added by
directed screens. For example, by staining for the expression of specific
genes, researchers have found mutations that block a single segment of the
hindbrain, or a specific valve of the heart. Because the early development
of human embryos, including the development of the heart, the brain, the
body axis, the ear, the liver, and so on, are highly similar in zebrafish
and human embryos, the genes that actually effect zebrafish development
will have close relatives that will direct similar processes in human
development and organ function.
An important addition to our understanding of human biology will
come through the identification of human gene functions suggested by the
phenotypes of zebrafish mutations. The ability to move from zebrafish
mutation to human gene, however, depends on the ability to move from
zebrafish genome to human genome. Fortunately, recent results have shown
that zebrafish and humans share large chromosome segments which have been
conserved intact during the 420 million years of evolution since the
divergence of the two phylogenetic lineages. For example, the apparent
orthologues of 14 genes localized along the full stretch of the long arm of
human chromosome 2 (about 5% of the human genome) have been mapped in
zebrafish. Eleven of these genes reside on one zebrafish chromosome, and
the remaining three on one other chromosome. This same group of genes is
also on two chromosomes in mouse. Analysis of the full set of mapped
zebrafish genes suggests that the number of chromosome segments shared
between zebrafish and human genomes may be just slightly more than the
number of segments shared between the genomes of mouse and humans.
The implication of the extensive conservation of chromosome
segments between human and zebrafish genomes is that comparative mapping
should be almost as effective between zebrafish and humans as between mouse
and humans. The pressing need now is to place enough orthologous markers on
the zebrafish genetic map (which is currently at about 1 cM (about 600kb)
average resolution due to microsatellite markers) to clearly define the
borders of conserved segments. This could be achieved by an expanded EST
project, using normalized libraries from a variety of tissues and
developmental stages, and the funding of projects to map the ESTs in the
genome, and to systematically catalogue their expression patterns in
embryos, larvae, and adult fish. Furthermore, we need to establish a
physical map of the zebrafish genome, both to improve the ability of
workers to molecularly characterize the sequences disrupted by the
mutations they study, but also to provide a framework for subsequent
sequencing of the complete zebrafish genome.
Because of the similarity of development and physiology in
zebrafish and humans, because of the ability to conduct efficient, near
saturation, phenotype driven screens in zebrafish, because of the ease of
single cell investigation in zebrafish embryos, and because of the
similarity of genomes in zebrafish and humans, the zebrafish should be the
next organism on the list of those to be sequenced in their entirety after
mouse. This would enormously facilitate the cloning of mutations, and hence
speed suggestions for functions of human genes.
Furthermore, the phylogenetic distance of zebrafish from humans is
sufficient that sequences that are not highly constrained by natural
selection will have been randomized when corresponding parts of the two
genomes are compared. This will greatly facilitate the identification of
regulatory elements and other functional features of the genome. Because of
the ability to work with zebrafish embryos, the functions of these
sequences can be directly tested in transgenic animals.
The unique feature that zebrafish brings to the analysis of
vertebrate genomes is the ability to perform an efficient mutational -- and
hence a functional -- analysis of a vertebrate genome. If, for example, a
zebrafish mutation maps to a chromosome segment known to be conserved
between zebrafish and humans, then the human genome can suggest candidate
genes for the zebrafish mutation. Molecular genetic experiments can then
test whether the candidate gene is in fact disrupted by the mutation.
Reciprocally, and importantly, the phenotype of the zebrafish mutation can
suggest functions for the human genes, which might otherwise be known only
by sequence and position. In this way, a greater knowledge of the zebrafish
genome could help move the human genome project from descriptive genomics
to functional genomics.
Up to Top
Neurogenesis and Neurological Disorders
Alexander F. Schier
Skirball Instutute, NYU School of Medicine
The study of neural development and function in the zebrafish is
facilitated by the translucence and accessibility of the embryo, the
relative simplicity of its nervous system, and the availability of a large
number of mutations disrupting genes that are essential for normal
development and physiology. Despite its simplicity, many of the features of
the zebrafish nervous system are conserved in higher vertebrates and
humans, including the patterning of the neural tube, the differentiation of
neurons, the routes of axon tracts, neuronal signaling, and many aspects of
behavior. Large-scale mutant screens in zebrafish have led to the
identification of more than 50 genes affecting various aspects of neural
development and function. The identified genes are involved in neural
induction and patterning, axon pathfinding, neuronal differentiation, and
behavior. Another 100 genes were found to be required for neural survival.
To name a few examples,
- cyclops and one-eyed pinhead mutants lack the ventral regions of the
neural tube, resulting in cyclopia.
- mind bomb mutants display supernumerary neurons.
- unplugged mutants lead to the abnormal axon pathfinding of motorneurons.
- who-cares mutants affect the topographic mapping of retinal axons on the
tectum target area.
- accordion mutants display abnormal locomotion.
- nic mutants are immotile and affect acetylcholin receptor function.
- space cowboy mutants display degeneration of the central nervous system.
In addition to their importance in the analysis of basic
neurobiological processes, the identified genes might also have relevance
to the study of disease states: defects in cyclopic mutants resemble the
human congenital disorder holoprosencephaly, degenerative mutants might
serve as models for neural degeneration conditions in humans, and retinal
axon pathfinding mutants might serve as models for human defects such as
The recent molecular cloning and phenotypic characterization of
zebrafish mutations has already led to some exciting new insights into
neural development and function. The isolation of cyclops as a
nodal-related gene and of one-eyed pinhead as an EGF-CFC protein has
identified a previously unexpected role for TGF-beta signaling in neural
patterning. The cloning of sonic you as sonic hedgehog and you-too as gli2
has shown that the hedgehog pathway plays an important role in forebrain
development and axon pathfinding. In vivo imaging techniques have allowed
monitoring the neuronal activity during locomotion and escape behavior with
single-cell resolution. The on-going molecular isolation of the disrupted
genes and their phenotypic characterization using modern imaging approaches
promises unprecedented insights into the molecular mechanisms underlying
neural function and development in vertebrates.
Up to Top
Early Developmental Programs in the Zebrafish
William S. Talbot
Stanford University School of Medicine
Max-Planck-Institut fur Entwicklungsbiologie
The zebrafish has emerged as an important model organism for the
study of early development in vertebrates. Zebrafish and other vertebrates
share many fundamental similarities in the organization of their body plans
and developmental pathways. The experimental advantages of the zebrafish
make it uniquely suited for the investigation of dynamic processes such as
axis formation and gastrulation. For example, the optical clarity and
external development of the embryo allow one to trace the movements of
individual cells in the living embryo as the animal develops. Moreover,
large-scale genetic screens have identified more than two thousand
mutations that collectively define the functions of hundreds of genes
essential for many aspects of early development. For example, dino and
swirl disrupt neural induction and the dorsal-ventral axis, bozozok and
squint eliminate the Spemann organizer, trilobite alters the morphogenetic
movements that reshape the embryo during gastrulation, and spadetail, no
tail, floating head and numerous others perturb the development of specific
cell types and organs that are established during embryogenesis. The
elegant phenotypic analysis of these mutants that is possible in zebrafish
will provide an unparalleled understanding of the functions of the
disrupted genes. For example single-cell labeling experiments demonstrated
that the floating head mutation alters the fates of a specific population
of cells in the Spemann organizer, such that they form muscle instead of
notochord. This and many other examples show that cellular analysis in
zebrafish provides an understanding of gene function that cannot be matched
in other vertebrates.
Over the last two years, advances in the molecular analysis of
zebrafish mutations have demonstrated that zebrafish genetics can discover
new genes and new gene functions. One striking example is the one-eyed
pinhead gene, first described in 1996 as a mutation disrupting endoderm,
Spemann organizer, and floor plate development. In 1998, positional
cloning identified the oep gene as a novel membrane-associated protein of
unknown function. Homologs were known in mammals and frog, but not
Drosophila or C. elegans. Thus oep provided the first evidence for an
essential function of a new gene family that is apparently specific to
vertebrates. Recent evidence shows that the Oep protein is a co-factor for
signaling by nodal-related proteins, a sub-class of the TGF-beta
superfamily of secreted growth factors. The analysis of oep illustrates
that mutational analysis in zebrafish can reveal the functions of novel,
There are many other recent examples of new gene functions
identified by analysis of zebrafish mutations. In one case, a previously
unsuspected role for the nodal-related signals in patterning of the neural
tube was established from the study of the cyclops mutation.
Characterization of the extensive collection of other mutations that
perturb neural patterning will allow a detailed understanding of the
pathways that establish the floor plate and other specialized cell types in
the ventral neural tube. Two additional genes essential for neural
patterning have been shown to encode components of the hedgehog signaling
pathway. Isolation of the remaining genes will likely identify novel
components of the hedgehog and nodal signaling pathways and elucidate the
interactions between these pathways in the development of the neural tube
and other organs.
Many of the genes defined by zebrafish mutations have human
homologs implicated hereditary diseases and cancer. For example, loss of
hedgehog signaling can result in Grieg cephalopolysyndactyly or
holoprosencephaly whereas ectopic hedgehog signaling results in basal cell
carcinoma. In addition to the hedgehog and TGF-beta signals, zebrafish
mutations define components of other signaling pathways with important
roles in human disease, including members of the Wnt and FGF families of
secreted growth factors. Thus zebrafish mutations provide powerful tools
for understanding and modulating the cell signaling events that underlie
human disease and vertebrate biology.
In summary, recent advances have established the value of the
zebrafish as a system for the discovery of new genes and new gene
functions. The molecular analysis of mutations will identify factors
regulating fundamental processes conserved in vertebrates. New screens
will discover more mutants with developmental and behavioral phenotypes
that have not been scored previously, and genetic modifier screens will
define genes that interact with key players in established pathways.
Further development of transgenic technology and refinement of imaging
techniques will take functional analysis of these genes to a new level,
allowing the in vivo detection of gene expression.
Up to Top
Research on Allergies and Infectious Diseases
Children's Hospital, Boston
Similar to higher vertebrates, the teleost zebrafish has T and B
lymphocytes and is thus an excellent system to study lymphopoiesis. Using
degenerate oligonucleotide polymerase chain reaction (PCR) and low
stringency hybridization, zebrafish homologs of early hematopoietic
transcription factors, such as c-myb, PU.1 and tal-1/scl, as well as the
immunoglobulin heavy chain gene (cm), the lymphocyte specific marker
ikaros, the T-cell tyrosine kinase lck, and rag-1 and rag-2 were obtained.
Expression of the above genes in wild-type zebrafish assayed by in situ
hybridization shows presence of T cells in the bilateral thymi as early as
day 3 of life. The analysis of lymphocyte development is currently being
extended to include 22 complementation groups of zebrafish with defects in
blood production. These mutants potentially represent genes required for
lymphopoiesis and are of particular interest as they are expected to have
defects in stem cell differentiation at various stages. One of these
mutants, cloche, appears to produce neither erythrocytes nor lymphocytes.
By analogy to erythropoiesis, where at least 22 complementation
groups have been defined, multiple developmental steps are likely to exist
between the uncommitted HSC and the mature lymphocyte. To derive a panel
of mutants with defects in lymphopoiesis, we have carried out chemical and
radiation mutagenesis of the zebrafish genome. We screened these mutated
zebrafish larvae with the erythroid and lymphoid markers, alpha-globin and
rag-1, respectively. Mutants with defects in both markers are likely to
have a defect in hematopoietic stem cell induction, proliferation, or
differentiation. Mutants with defects in rag-1 alone are likely to solely
In this screen 110 clutches of sufficient size and quality derived from ENU
mutagenized F1 offspring were analyzed. We identified 17 clutches with a
defect in Rag-1 expression, nine with a defect in globin expression and
three with a defect in both. F1 females that these clutches were derived
from were outcrossed, and to date we have confirmed the mutant phenotype in
five Rag-1 deficient and one globin deficient mutants by F2 incrossing.
Two of the Rag-1 mutants (CZ-3 and CZ-5) fall into the same complementation
group. Phenotypic analysis of the mutants shows total absence of Rag-1
expression in CZ-3/5 and CZ-26, while CZ-18 and CZ-32 have faint Rag-1
expression in the thymi of mutant offspring. Further analysis demonstrates
normal neural crest cell development and endodermal marker expression in
the mutants. Pharyngeal arch development and thymic development are
abnormal in CZ-3/5. We have used half tetrad analysis to map these mutants.
Investigation of zebrafish with defective lymphopoiesis will further our
understanding of hematopoiesis and will have therapeutic impact for bone
marrow transplantation, stem cell gene therapy, and leukemia.
Up to Top
Mutants That Affect Mental Behavior
University of Oregon
The small size, optical transparency and rapid development of
zebrafish make them good subjects for studying the mechanisms that regulate
development of nervous system function and behavior. Several labs are
currently funded to study retinal physiology including John Dowling,
Harvard University, who is recording from cone photoreceptors and Douglas
McMahon, University of Kentucky, who is investigating mechanisms of retinal
synaptic plasticity by electrical recording. Joe Fetcho, SUNY Stony Brook,
is recording membrane potentials in hindbrain neurons during escape
behaviors using optical imaging techniques. John Schmidt, SUNY Albany, is
studying activity and trophism in synaptic stabilization of retinal arbors.
In principle, the results of these types of studies can provide not only
important information about normal physiology, but also can be used to
design genetic screens for mutations in genes that regulate these
The first screen for behavioral mutants was carried out by Kimmel
and Westerfield, University of Oregon, in the 1980's and was based on a
simple test of the response to touch. A number of mutations affecting
neural crest cells (sensory neurons), motoneurons, and muscles were
discovered. Additional alleles of some of these mutations as well as
several new mutations were obtained in the recent large scale screens which
looked for paralyzed or weak movement phenotypes.
During the past year, at least two labs have begun new genetic screens for
mutations that affect behavior. The James Hurley lab, University of
Washington, is screening for mutations affecting visual behavior and Kate
Whitlock in the Westerfield lab, University of Oregon, is screening for
olfactory mutants on responses to olfactants. The olfactory screen has
already uncovered several new mutations. Further elaboration of behavioral
mutant screens and molecular characterization of the disrupted genes will
help further out understanding of the genetic basis of vertebrate behavior.
Up to Top
Craniofacial and Dental Research
James A. Weston
Institute of Neuroscience
University of Oregon
Considerable progress has been made in identifying and analyzing
mutations that affect craniofacial development in the embryonic zebrafish.
This work will provide noteworthy opportunities for understanding the
etiology of vertebrate craniofacial abnormalities. For example, 33 alleles
of 19 genetic loci that affect the development of the zebrafish jaw and
branchial arches were revealed by the Tübingen screen and an additional 48
alleles of mutations at 34 loci have been found by the Boston screen. Work
in a number of other labs has revealed a variety of mutations that affect
the patterning of the vertebrate head and the development of neural crest
derived structures. There are also a number of pigmentation mutants with
pleiotropic effects on other neural crest derivatives that will undoubtedly
provide important insights into the mechanisms of lineage diversification
within the neural crest, and the regulation of development of various
crest derivatives. Finally, a number of mutants affecting motility that
express pleiotropic effects on neural crest derivatives suggest that the
functions of a number of other genes in crest development will be uncovered
when analysis of these mutations proceeds.
The special attributes of the zebrafish embryo (e.g., its
transparency and rapid development outside of the mother or an eggshell,
the ability to label individual cells with lineage tracers and to follow
them in living embryos by time-lapse, DIC- fluorescence- and confocal
microscopy), combined with the growing library of mutants that affect
craniofacial structures or the neural
crest from which such structures are thought to arise, provide an
outstanding resource for research on the mechanisms of normal development
of specific craniofacial structures and other neural crest derivatives.
Thus, the zebrafish embryo provides a tractable experimental system to
analyze specification of developmentally distinct lineages derived from the
neural crest and the development of jaw and branchial arch structures.
Although the zebrafish lack dentition in the mandibullar and maxillary bones,
they do possess masticatory teeth. These teeth are embedded in cellular
bone and project into the oral cavity at the level of the pharynx.
They oppose the dorsal hyperkeratinized pad. The teeth are composed
of acellular dentin and have anatomy similar to that seen in humans.
The supporting bone is highly cellular, and shows evidence of rapid
turnover. The maturation of these teeth occurs within the first
week of development, and precedes ossification of the major chondroid
bones. A screen has been initiated to identify mutants that affect
the normal formation of these teeth. These mutations will represent
interesting models for identifying precise molecular and cellular
mechanisms of tooth development. The study of these mutants will
be complimentary to work done in higher vertebrates, and will likely
provide novel genes that would not have been identified in those
Up to Top