21/7/2017 Zebrafish (Molecular Biology)

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Zebrafish (Molecular Biology)

Zebrafish, Danio (Brachydanio) rerio (Hamilton-Buchanan), is a member of the family Cyprinidae, order
Cypriniformes. A native of rivers in Southeastern Asia, this small, tropical freshwater fish is now a popular
inhabitant of home aquaria throughout the world. A number of features facilitating embryological and
genetic manipulations has made zebrafish the most recent model system in which to study mechanisms of
vertebrate embryonic development (1, 2). Zebrafish are also frequently used in toxicologic studies (3).

Zebrafish are elongated and flattened laterally, with dark blue and silver-gold stripes running along the
longitudinal axis of their body. In their two- to three-year life span, zebrafish grow up to 5 cm in length.
Zebrafish achieve sexual maturity at three months of age. They are omnivorous and thus easy to raise using a
variety of live and dry foods. Furthermore, they tolerate a relatively wide range of water quality. In captive
breeding, zebrafish can produce hundreds of eggs on a weekly basis without seasonal variation. Therefore,
large numbers (105) of zebrafish can be raised and maintained at low cost and labor, facilitating large-scale
genetic screens. The mating behavior is photoperiodic; spawning takes place at dawn or, in laboratory
conditions, at the beginning of the light cycle. In the course of mating, the male closely follows the fast-
swimming female, culminating in the release of eggs and sperm into the water. Eggs and sperm can be
harvested easily from anesthetized fishes and fertilized in vitro. Importantly, the sperm can be preserved by
deep freezing, allowing for cost and space-effective maintenance of genotypes (4).

Embryos from a single clutch develop synchronously outside the mother in a range of temperatures,
(between 23C and 33C, with the optimal temperature being 28.5C). The embryo and surrounding chorion
are translucent, allowing for a detailed microscopic inspection of development. Furthermore, the embryos
can survive several days with major developmental defects (eg, without a functional cardiovascular system).
The large size of the embryo (0.7 mm in diameter) facilitates microinjection, dissection, transplantation, and
other embryological manipulations. All of the above features are critical for effective identification and
subsequent analysis of mutant phenotypes.

1. Genomics
The haploid genome of zebrafish contains about 1.7 x 109bp (5), organized into 25 approximately
metacentric chromosomes of similar size (6). A genetic linkage map of the zebrafish genome has been
developed using PCR (polymerase chain reaction)-based polymorphisms: random amplified polymorphic
markers (RAPID) (7) as well as CA dinucleotide repeats and simple sequence length polymorphisms (SSLP)
(8). Furthermore, about 500 DNA sequences have been reported for zebrafish, and positions on the genetic
map have been determined for 120 of these cloned genes. The current estimate of the size of the map is 3,000
centiMorgan (cM) with an average 600 kb cM-1. Taking into account about 1,200 markers for which map
positions are known, the average interval between the available zebrafish markers is approximately 1,500 kb.
The mapping efforts also revealed a surprising conservation of many large chromosome segments in the
genomes of humans and zebrafish (9).

2. Embryonic Development
Embryonic development is initiated by the segregation of nonyolky cytoplasm in the form of a blastodisc
situated atop a large sphere of translucent yolk. A series of synchronous and rapid cleavages of the blastodisc
leads to the formation of a blastula with a mound of blastomeres on a syncytial yolk cell. The establishment
of dorso-ventral asymmetry of the embryo is not strictly correlated with the planes of the first cleavages (10,
11). This process, however, requires the transport of substances from the vegetal hemisphere into marginal

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blastomeres via cortical microtubules of the yolk cell (12). An important step in the establishment of the
dorsal axis is a transient accumulation of b-catenin in the nuclei on the dorsal side of the zebrafish blastula
(13, 14).

The embryonic body is shaped by three main morphogenetic movements of gastrulation (15). During
epiboly, the blastoderm becomes thinner as its surface expands to cover the yolk cell completely 10 hours
after fertilization. Tissue-restricted lineages can be identified in late blastula, when the blastoderm reaches
the equator of the yolk cell. The three classically defined germ layers exhibit an overall organization
reminiscent of the early gastrula fate maps of other vertebrates, with progenitors of ectoderm arising from
near the animal pole, and the mesoderm and endoderm occupying the most marginal positions of the
blastoderm (16). The germ layers form by involution/ingression movements at the blastoderm margin that
bring prospective mesodermal and endodermal cells underneath the future ectoderm. Concurrent convergent
extension movements bring the precursors of various tissues closer to the dorsal midline and are responsible
for narrowing of the embryonic axis in the mediolateral direction and for its anterior-posterior elongation.
These gastrulation movements create the embryonic shield, a thickening at the dorsal blastoderm margin.
The embryonic shield is equivalent to the Spemann gastrula organizer of frog and other vertebrate embryos
(17, 18). The dorso-ventral pattern specification involves highly conserved antagonistic interactions between
dorsalizing signals emanating from the Spemann gastrula organizer and secreted factors like bone
morphogenetic protein-4 (BMP-4) released by the ventral signaling center (19, 20). Gastrulation movements
bring precursors of various organs into their proper positions in the embryo.

During the subsequent segmentation period, organ rudiments form; notochord becomes visible as a
prominent dorsal structure and blocks of somites appear on both sides of the notochord sequentially in the
trunk and later in the tail. The primordium of the central nervous system becomes visible above the
notochord as a thickened neural plate. By the process of epithelial infolding, the neural plate first transforms
into a solid neural keel that subsequently hollows into the neural tube (21, 22). The regional morphogenetic
processes lead to the formation of distinctive swellings (neuromeres) in the anterior region of the neural
plate, corresponding to brain rudiments characteristic of all vertebrates. The most anterior forebrain rudiment
consists of telencephalon and ventral diencephalon, from which the eyes develop. The medially located
mesencephalon gives rise to the optic tectum dorsally and tegementum ventrally. The most posterior
hindbrain is subdivided into seven rhombomeres. The neural tube posterior to hindbrain gives rise to spinal
cord, which develops characteristic dorso-ventral organization, dependent on secreted proteins of the
Hedgehog family (23).

During the segmentation period, while the embryo continues to narrow in the mediolateral direction and
elongates, the rudiments of kidney, heart, and gut, as well as sensory placodes, also become distinct. The tail,
originally closely apposed to the round yolk cell, becomes a prominent structure when it everts from the
yolk. At 24 hours, the embryo reaches the pharyngula stage of development. At this phylotypic stage, the
zebrafish embryo is most similar to other vertebrate embryos, exhibiting a prominent notochord in the trunk
and tail, surrounded by chevron-shaped somites (Fig. 1). The nervous system positioned dorsal to the
notochord is hollow and expanded anteriorly, with eyes and brain subdivisions visible in the head. The
embryo exhibits circulation and is motile, and rudiments of most organs can be easily identified. The heart
tube is located against the ventral body wall below the head and consists of an inner endocardial tube and an
outer, myocardial tube. The adult fish heart will consist of a single atrium and a single ventricle, an
arrangement characteristic of the early embryonic stages of other vertebrates. Blood cells develop in the
intermediate cell mass located in the trunk between the notochord and endoderm, accumulating in a blood
island at the trunk/tail border (24). Further development involves the formation of gill arches, including jaws
and an outgrowth of pectoral fins. Hatching from the chorion occurs at 2-3 days of development, the swim
bladder becomes inflated, and larvae start feeding on day 4 of development.

3. Genetics of Zebrafish Development

Efficient methods of induction, recovery and screening for mutations are available in zebrafish. N- ethyl- ^-
nitrosourea is a potent mutagen of fish spermatogonia and sperm (25-28). g-ray-irradiation is used to induce
mutations in embryos or in sperm of adult males (29, 30). Furthermore, insertional mutagenesis can be
achieved by injecting a pseudotyped retroviral vector containing a genome based on the Moloney murine
leukemia virus and the envelope glycoprotein of the vesicular stomatitis virus into zebrafish embryos (31,
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32). Mutations induced by various mutagens in the G0 generation are transmitted to the F1 generation and
are revealed by screening haploid progeny of F1 females (obtained by fertilization with genetically
compromised sperm) or gynogenetic diploid progeny of F1 females (obtained by fertilization with
genetically compromised sperm followed by inhibition of the second meiotic division). Alternatively,
mutations are bred to homozygosity in a classic three-generation screen to be manifest in F3 embryos (33).

Figure 1. Zebrafish embryo at day 1 of development (pharyngula stage). Scale bar, 0.5 mm.

Large-scale genetic screens for mutations that alter embryonic morphology, behavior, or gene
expression patterns resulted in the identification of mutations in over 400 genes (34-36). These screens
identified genes involved in practically every aspect of early embryonic and larval development in the
zebrafish: early pattern formation, notochord, brain, spinal cord, somites, muscles, heart, circulation, blood,
skin, fin, eye, otic vesicle, jaw, and branchial arches (37). It is estimated that fewer than 50% of genes that
could be detected by the screening methods employed have been identified so far. Comparison of the
frequency of mutations in known loci with that of embryonic and early larval lethal mutations led to an
estimate that 1,500-5,000 genes are essential for early development in zebrafish.

Genes identified by virtue of retroviral insertions are rapidly cloned using the retroviral tag (38). Genes
identified by other types of mutagens are cloned by two main strategies. In the candidate gene approach,
genes are identified that have been cloned in other systems and exhibit properties predicted for the mutant
locus (39, 40). The positional cloning approach starts with the identification via genetic mapping of a DNA
marker closely linked to a gene to be cloned. Next, this DNA marker is used to isolate chromosomal walking
clones encompassing the gene, which is subsequently identified by gene expression patterns and sequence
analysis, as well as by functional assays (41).

During the 1990s, zebrafish researchers recovered thousands of mutations that identify hundreds of genes
essential for embryonic development. Furthermore, a genetic map of the zebrafish genome has been
constructed, and its density is increasing rapidly. Considering the powerful tools available for the analysis of
normal and mutant embryos, zebrafish will significantly contribute to our understanding of molecular
mechanisms governing early vertebrate development.

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