Why study zebrafish?
An understanding of the roles of genes in development requires that we identify as many of the genes involved in development as is possible. Vertebrates are not as amenable to genetic analysis as invertebrates such as Drosophila and Caenorhabditis. The classical vertebrate for genetic analysis has been the mouse. However, the traits that traditionally have been the targets of genetic analysis have been those that are expressed postnatally, not those that lead to embryonic lethality. Consequently, discovery of mutations that interfere with the earliest steps in development has been rare. Gene knockout technology has improved our understanding of the roles of specific genes in mouse development. However, mouse developmental genetics is impeded by the high cost of maintenance of animals and by the intrauterine mode of development (Driever et al., 1996).
George Streisinger (1981) of The University of Oregon recognized that the zebrafish, a small tropical teleost, has many of the advantages of C. elegans and Drosophila; namely:
He also developed a technique for producing homozygous diploid fish, which made it possible to detect rare recessive mutations and to produce large clones of genetically identical fish. Streisinger's colleagues in Oregon continued to exploit zebrafish for developmental genetics, establishing it as one of the most tractable developmental systems. Normal development has been described in considerable detail, and a small number of mutations that affect embryogenesis were described and analyzed to determine their effects on the developmental process. In 1994, 1995 and 1996, Christiane Nüsslein-Volhard and her colleagues and Wolfgang Driever and his colleagues conducted large scale screens for mutations that affect embryonic development, which led to publication of the "Zebrafish" issue of Development, which constitutes volume 123 of the journal.
As mentioned, one of the advantages of the zebrafish embryo is its translucency, which enables the investigator to examine its development readily with the dissecting microscope. Figure 1A gives an overview of development during the first 24 hours (Haffter et al., 1996), and development from 1 to 5 days is shown in Figure 1B.
Figure 1. Living zebrafish embryos. (A) The first 24 hours of development. (B) Embryos at 29 hours, 48 hours and 5 days of development. (From Haffter et al., 1996. Reproduced with permission from The Company of Biologists.)
The following brief description of zebrafish development is derived predominantly from Kimmel et al. (1995).
The teleost egg is telolecithal; i.e., a mound of cytoplasm (the blastodisc) sits on the large mass of yolk and undergoes incomplete (meroblastic) cleavage. The embryo proper is derived from the blastodisc, and the remainder of the zygote becomes the yolk sac, which is later digested.
After cleavage, the embryo enters the blastula stage as the blastodisc forms a sphere of cells sitting atop the yolk. There is no defined blastocoel; rather, small irregular extracellular spaces are formed between the deep cells of the blastodisc. The blastomeres at the margin of the blastoderm have a unique fate. They lie against the yolk and remain cytoplasmically connected to it throughout cleavage. During the blastula stage, these cells release their cytoplasm and nuclei together into the immediately adjoining cytoplasm of the yolk cell, producing the yolk syncytial layer (YSL). Begining in the late blastula stage, the YSL and the blastodisc spread over the yolk cell, much like pulling a knitted ski cap over your head, in a process known as epiboly. Eventually, at the end of gastrulation, the yolk cell is completely surrounded by the spreading YSL and blastodisc. The blastodisc becomes considerably thinner in the process.
The onset of gastrulation occurs at 50% epiboly. At this time, a thickened marginal region termed the germ ring appears around the blastoderm rim. (Note mistake in the figure legend: it should read dorsal on the right.) The germ ring is formed by a folding of the blastoderm back upon itself (involution). Hence, within the germ ring there are two germ layers. The upper layer (the epiblast) continues to feed cells into the lower (the hypoblast) throughout gastrulation. The cells remaining in the epiblast when gastrulation ends correspond to the ectoderm and will give rise to such tissues as epidermis, the central nervous system, neural crest, and sensory placodes. The hypoblast gives rise to both the mesoderm and endoderm, although it is unclear how this layer subdivides into endoderm and mesoderm. A fate map of the gastrula has been produced by injecting single cells with lineage-tracer dye and following their fates. This is, of course, facilitated by the transparency of the embryo.
A marked streaming of cells toward the presumptive dorsal side of the germ ring in both the epiblast and the hypoblast produces the embryonic shield. A narrowing and elongation of the primary embryonic axis occurs as the shield extends toward the animal pole. The dorsal epiblast begins to thicken rather abruptly anteriorly and at the midline near the end of gastrulation, producing the first indication of development of the rudiment of the central nervous system: the neural plate. Below this is the axial hypoblast, flanked by the paraxial hypoblast. In the trunk region, these will form the notochord and somites, respectively.
After epiboly, the somites and neural tube develop, the rudiments of the primary organs become visible, the tail bud becomes more prominent and the embryo elongates. The first somites form anteriorly (see also somite morphogenesis), and the posterior ones form last. Soon, the first cells differentiate morphologically, and the first body movements appear. Formation of the neural tube occurs by a process known as "secondary neurulation". The lumen of the neural tube, the neurocoele, forms by a process of cavitation, rather than by an uplifting and fusion of neural folds as in the amphibia, for example.
When searching for mutations that affect development, investigators examine embryos for alterations in the normal developmental pattern. Some of the landmarks in the embryo that may be affected by mutation in later stages of development are labeled in Figure 2 (Haffter et al., 1996).
Figure 2. Drawings of zebrafish embryos at 24 hours (A), 48 hours (B,D) and 5 days (C,E) of development. For clarity, the pigmentation pattern is omitted from B and C. It is shown in D and E. Most of the structures that can be seen in a living embryo with a compound microscope are labeled. (From Haffter et al., 1996. Reproduced with permission from The Company of Biologists.)