4-6 Zebrafish

The zebrafish is a vertebrate with a short life cycle and rapid embryonic
development

The zebrafish (Danio rerio) is a small tropical freshwater fish that is easy to raise and breed
in the laboratory. Like man, it is a vertebrate, but its embryonic development is spectacu-
larly fast: it generates a typical vertebrate body plan—with a neural tube, muscles, a vascu-
lar system and a beating heart—within 24 hours of fertilization at 28 °C. Its life cycle is
illustrated in Figure 4-6.1. Adults breed year-round, and natural matings generate large and
synchronous batches of embryos; the eggs are fertilized externally, and an average clutch size
is about 200.
30 min chorion
fertilized blastodisc
Soon after laying, cytoplasmic streaming movements in the egg generate an area of yolk-free
egg cytoplasm—the blastodisc—at the top (animal) pole of the embryo. Early cleavages occur
yolk
every fifteen minutes. They divide the blastodisc, but not the yolk, generating a mound of cells
on top of, and in cytoplasmic contact with, the ball of yolky cytoplasm below. The embryo is
now termed a blastula. In the tenth cell cycle, the embryo undergoes a characteristic mid-blas-
cleavage tula transition: the cell cycle slows and becomes asynchronous, cells become motile and tran-
scription from the zygotic genome begins. In the late blastula, the blastodisc thins and forms
2 hours
a blastoderm that spreads over the yolk, eventually completely engulfing it. This spreading
8-cell
movement, termed epiboly, provides a useful marker of the stage of development, which can
stage be measured by the percentage of yolk covered, or “percent epiboly”. At 50% epiboly, the lead-
ing edge of the blastoderm margin thickens (generating the germ ring), and migrating cells
begin to converge on the prospective dorsal side of the embryo, generating the embryonic
shield. These thickenings are the first sign of the gastrulation movements that now begin to
build the body plan; this is evident by the 18 somite stage, a mere 18 hours after fertilization.
Muscular contractions now begin, and the rudiments of several organs (brain, eye, ear) become
4 hours blastodisc visible. In the next few hours, the tail extends, the heart begins to beat and the embryo takes
sphere on a more fish-like appearance. By day 4, the larva is able to swim and feed. Sexual maturity
stage yolk is reached within three months.

The external development and optical transparency of the zebrafish

epiboly embryo facilitate phenotypic analysis

germ ring
Because cleavages do not divide the yolk cell, cells in the embryo proper do not contain yolk;
6 hours initially, they are also unpigmented. This means that the early embryo is completely optically
shield shield clear, which allows for detailed observations of internal structures, even under the dissecting
stage microscope. Developing organ systems can be observed directly in the live embryo, making
section animal pole view screening for morphological defects relatively easy (Figure 4-6.2). Many individual neurons
can be identified and followed as they develop. The optical transparency of the embryo also
gastrulation facilitates the visualization of gene expression patterns in stained and fixed tissue, or of fluo-
and epiboly rescently labeled cells in live specimens.

8 hours The external fertilization and large clutch size of the zebrafish mean that its embryo is well
75%
suited to microinjection. With a diameter of 0.7 mm, the embryo is also a convenient size;
epiboly section large numbers can be manipulated under the dissecting microscope. RNA or DNA can be
injected for misexpression studies, the rescue of mutant phenotypes or the generation of
transgenic lines. Gene activity can be inhibited by the injection of antisense morpholino
segmentation and oligonucleotides, which block mRNA splicing or translation in a sequence-specific manner,
organogenesis

18 hours Figure 4-6.1 Life cycle of the zebrafish Male and female fish are distinguished by the rounder belly of
the silvery female (gravid with eggs) and the slimmer, pinker male (top panel). Each egg is surrounded
18 somite by a chorion (shown only for the fertilized egg; second panel). Cleavage of the blastodisc creates a
stage
mound of cells sitting on top of the yolk (third panel). These proceed to envelop the yolk, beginning at
the sphere stage (fourth panel). Gastrulation begins at 50% epiboly. Involution movements around the
margin produce the thickened germ ring, while involution and convergence movements produce a more
prominent thickening, the shield, on the future dorsal side of the embryo (fifth panel). Epiboly and
organogenesis gastrulation progress until the entire yolk ball is covered with cells, most of them on the dorsal side
and hatching (sixth panel). Between 10 and 24 h after fertilization, segmentation generates pairs of somites on either
5 days
larva side of the midline, useful as a staging tool. By 18 hpf, 18 somite pairs have formed, the tail begins to
extend and the rudiments of the brain and sensory organs (eye and ear) are readily apparent (seventh
panel). The embryo hatches from its chorion between the second and third days. By day 5, it has an
inflated swimbladder and actively pursues live prey.

1 Chapter 4 Model Organisms ©2004 New Science Press Ltd

 Zebrafish 4-6
and provide a reliable reverse genetic approach. Chimeric embryos can be generated by a sim-
ple cell transplantation technique: in general, a donor embryo is labeled during the first
cleavages with a marker (for example a fluorescent dye), and a few cells are transplanted at
the blastula stage into a host embryo of differing genotype. The fate of the labeled trans-
planted cells can then be followed as the host embryo develops. Such experiments provide an
exquisite test of the cell autonomy of protein function in the embryo, unparalleled in any
other vertebrate model system.
Figure 4-6.2 Optical transparency of the
Several methods to expedite forward genetic analysis have been embryo makes it easy to screen for
developed in the zebrafish morphological defects Lateral views of a 6 day
old colourless mutant (upper panel) and its
Mutations in zebrafish can be generated using a variety of techniques, including chemical phenotypically normal sibling. Black pigment is
mutagenesis, γ-irradiation or viral insertion. Forward genetic screens have generated an abun- evident in the wild-type sibling, but virtually all
pigment (apart from in the retina) is missing in
dance of mutant phenotypes, many of which correspond to human disorders, ranging from the mutant. Defects are also present in the ear
cardiovascular, haematopoietic and kidney disease to deafness (Figure 4-6.2). Some of these are (arrow) and the peripheral nervous system (not
illustrated in detail in Chapter 10. It is an important advantage of the zebrafish that, although shown). The swimbladder is also not inflated, a
it is diploid, animals displaying a mutant phenotype can be generated from a single heterozy- defect found in many embryonic lethal lines.
Mutations in colourless disrupt the sox10 gene;
gous parent by manipulation of the ploidy of the fertilized egg. This is usually done by UV in humans, haploinsufficiency at the SOX10
irradiation of sperm prior to fertilization. This treatment inactivates the paternal genome; fer- locus results in Waardenburg-Shah syndrome,
tilization with such irradiated sperm results in gynogenetic haploid embryos, which carry only characterized by pigmentation defects of the
a single maternal copy of each gene. It is also possible to generate gynogenetic diploid embryos, skin, hair and iris, a loss of enteric ganglia
(Hirschsprung’s disease) and deafness.
which carry two copies of each gene, both of maternal origin. These techniques are especially
useful where breeding through several generations is impractical (for example in a screen for
maternal-effect mutations), and are also used for genetic mapping, complementation analysis
and in the production of inbred lines.

The zebrafish genome contains duplicate genes, but shares a high degree Related organisms
Puffer fish (Takifugu rubripes, Tetraodon
of sequence and syntenic conservation with the human genome nigroviridis). Fish with exceptionally small,
gene-rich genomes (350–400 Mb, c.f. 1700 Mb
Despite its recent introduction to the laboratory as a model vertebrate system, the ease of in the zebrafish), which are used extensively for
genetic analysis in the zebrafish and its promise as a model for human disease has led to a huge comparative genomic studies, and are expected
effort to establish genomic tools for this organism within the last ten years. These include a to share a high level of conserved synteny and
dense genetic map, genomic libraries and the full genome sequence, all of which aid in the sequence with the zebrafish genome. Neither
species of puffer fish, however, is easy to breed
cloning of genes identified through mutagenesis screens. in captivity, and so they cannot be used for
developmental or genetic analyses. Websites:
The genomes of ray-finned fish in general, of which the zebrafish is one, show a wide range http://fugu.hgmp.mrc.ac.uk/PFW/
in size and chromosome number, reflecting the huge diversity of morphology and physiol- http://www.genoscope.cns.fr/externe/tetraodon/
ogy for this group, which, in terms of species number, accounts for half of all vertebrates. Medaka (Oryzias latipes). A small, hardy fish,
native to the rice fields of East Asia, with a long
At 1.7x109 bp, the zebrafish genome is about half the size of the human genome. Although history of genetic research. The medaka has
it is separated from it by about 420 million years, there are nevertheless large stretches of similar advantages as a genetic model to the
conserved synteny between the two. There are also significant differences: an analysis of zebrafish. In addition, stable embryonic stem
Hox gene organisation in the zebrafish and other fish suggests that the ray-finned group cell lines are available, which are being
developed to establish targeted gene disruption
may have undergone a third whole genome duplication in addition to the two genome in this species, a technology currently
duplications that are proposed to have accompanied the evolution of vertebrate character- unavailable in the zebrafish. Website:
istics. Alternatively, duplication may have occurred many times at the level of individual http://biol1.bio.nagoya-u.ac.jp:8000/
genes. Whatever their origin, the zebrafish frequently has two copies of genes that are Xiphophorus spp. Interspecies hybrids form
genetically controlled models for tumor
unique in mammals, and the functions of the ancestral gene may now be subdivided formation. Website: www.xiphophorus.org
between the zebrafish duplicates.

Definitions
gynogenetic: containing genetic material derived from
the female parent only. Gynogenetic haploid embryos
contain a haploid chromosome complement; each rep-
resents a single product of a meiotic division (one quar-
ter of a tetrad). Haploid embryos do not develop entire-
ly normally, and are only viable for about 4 days, but this
is long enough to identify many phenotypes of interest.
Gynogenetic diploid embryos contain a diploid chro-
mosome complement, also derived from the mother
only, and are obtained by treatments to prevent either
the second meiotic division in an egg fertilized with UV- Patton, E.E. and Zon, L.I.: The art and design of genetic
irradiated sperm (generating a half-tetrad), or the first screens: zebrafish. Nature Reviews Genetics 2001,
mitotic cleavage division in a gynogenetic haploid 2:956–966.
embryo. The latter form of gynogenetic diploid, which
are homozygous for all loci, have poor viability, but half- Streisinger, G. et al.: Production of clones of homozy-
tetrad embryos can be raised to generate fertile adults. gous diploid zebra fish (Brachydanio rerio). Nature
1981, 291:293–296.
References
Fishbase:
Nüsslein-Volhard, C. and Dahm, R. (eds): Zebrafish: A www.fishbase.org
Practical Approach (Oxford University Press, Oxford,
2002). Zebrafish Information Network:
http://zfin.org/ZFIN/

©2004 New Science Press Ltd Model Organisms Chapter 4 2