Zebrafish Embryo as a Developmental System

Sharon L Amacher, University of California - Berkeley, Berkeley, California, USA

Zebrafish are simple, rapidly-developing animals that are amenable to detailed developmental and genetic analyses. Despite their relatively simple morphology, they share many developmental features with other vertebrates. By combining developmental studies in zebrafish with those in other animals, we will begin to understand the complex events that occur as an embryo develops from a one-celled zygote to a complex, multicellular organism.

Introductory article
Article Contents
. Introduction . Why Study the Zebrafish? . Genetic Analysis of Zebrafish Development . Summary

Introduction
In the last twenty years, the tropical freshwater zebrash, Danio rerio, has emerged as an excellent model of vertebrate development. Much of the pioneering work done to advance zebrash as a model system was initiated in the laboratory of Dr George Streisinger, but now laboratories all over the world use zebrash to study developmental processes. This article will describe the features of zebrash that make them particularly tractable to embryological and genetic analyses.

Zebrafish embryonic development

Zygote period (0 3 4 h) The newly fertilized egg, or zygote, is about 0.7 mm in diameter at the time of fertilization. The embryo develops within a transparent eggshell, called the chorion, which can be easily removed without harming the embryo. During this period, cytoplasmic streaming separates the zygote into two visibly dierent parts: a clear blastodisc at the animal pole and a yolky cytoplasm at the vegetal pole. The blastodisc is the part of the zygote that undergoes cleavage and gives rise to blastomeres, the cells that give rise to the embryo.

Why Study the Zebrafish?

Zebrash are simple, rapidly developing animals that are amenable to detailed developmental analyses. Despite their relatively simple morphology, they share many developmental features with other commonly-studied vertebrates, such as frogs, chickens, and mice. Thus, studies of zebrash will teach us about the development of other organisms, including humans. Several features make zebrash an excellent animal to use in studies of genetics and development. First, zebrash adults reach sexual maturity quickly and are very fecund. Thus, large numbers of embryos can be collected for study. Second, zebrash embryos develop externally. Eggs that are extruded into the surrounding water by adult females are immediately fertilized by sperm from adult males. Embryos are thus easily accessible for observation and experimentation. Third, zebrash embryos are optically clear. Under a microscope, one can visualize tissues that lie deep within the embryo, follow individual cells during development, and recognize developmental mutants. Fourth, zebrash embryos develop rapidly, as illustrated in Figure 1. To emphasize the rapid embryonic development and to review important developmental milestones, the seven periods of embryonic development are described below.
1 Cleavage period (3 4 24 h)

This period includes the rst six regular, rapid, and synchronous divisions of the embryo. At the end of the cleavage period, the developing embryo sits as a mound on top of the large yolk.

1 Blastula period (21 4 54 h, Figure 1a,b)

Many important processes occur during the blastula period. Prior to this period, the embryo has utilized proteins and RNA that the mother deposited in the egg. During midblastula stages, the embryo begins to transcribe its own genes. Not only does transcription begin, but embryonic genes are regionally expressed, reecting the fact that blastomeres are now becoming dierent from one another. When embryonic transcription begins, cell division times gradually lengthen, so that blastomeres no longer divide together. Another major event that begins during the blastula period is epiboly, which describes the thinning and spreading of the cellular blastodisc over the yolk. Near the end of this period, the blastomeres are referred to collectively as the blastoderm, a sheet-like array of cells that sits like a cap on top of the large yolk.
1

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Zebrafish Embryo as a Developmental System

Figure 1 Zebrafish developmental series. Hours post-fertilization (h) are shown in the lower right of each panel. (a) Early blastula. (b) Midblastula. (c) Early gastrula. (d) Midgastrula. The arrows in (c) and (d) mark the position of the blastoderm margin and indicate the process of epiboly; epiboly is the movement of the blastoderm as it crawls over the yolk toward the vegetal pole. (e) Early segmentation stage embryo. Arrowheads in (e) mark developing somites. (f) Midsegmentation stage embryo. (g) Pharyngula. (h) Hatching embryo. The dark cells scattered over the head, body and yolk and in the eyes are pigment cells.

Gastrula period (51 4 10 h, Figure 1c,d) Epiboly continues during the gastrula period. During this period, cell movements dramatically reorganize the embryo, so that by the end of this period, three embryonic axes are clearly visible. That is, the anterior head of the embryo is clearly distinguished from the posterior tail bud, dorsal tissues are distinct from ventral ones, and medial tissues are easily discernible from lateral ones. Many embryonic genes are expressed in region-specic patterns. Cell movements during gastrulation also produce the germ layers (ectoderm, mesoderm and endoderm) of the embryo. Finally, near the end of the gastrula period, the neural plate forms; this is the rst morphological sign of central nervous system development. Segmentation period (10 24 h, Figure 1e g) The embryo elongates considerably during this period. A complete complement of about 30 bilateral somite pairs
2

forms in an anterior to posterior sequence, giving the segmentation period its name. The rudiments of primary organs, such as notochord, kidney, and blood, become visible. The central nervous system undergoes dramatic changes; the neural plate forms a solid neural keel, which then hollows out to form a neural tube. Additionally, the brain becomes regionalized, with forebrain, midbrain and hindbrain subdivisions becoming distinct from one another. Finally, muscular contractions occur, and the embryo moves within the chorion. To contrast, a mouse embryo at one day after fertilization has only just completed the rst cell division to form two cells. Pharyngula period (24 48 h, Figure 1g) This period is named for the seven pharyngeal arches that rapidly form during this period. Pharyngeal arch development is common to all vertebrates, and in zebrash, pharyngeal arches later form the jaw and gills. Also during

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Zebrafish Embryo as a Developmental System

the second day, the head straightens, ns form, pigment cells dierentiate, the circulatory system forms and the heart beats, and coordinated swimming movements begin. Embryos become responsive to touch, revealing the development of sensory-motor reexive circuits. Hatching period (48 72 h, Figure 1h) Embryos hatch from their chorions during the third day. By this time, many of the organ rudiments have formed; however, the gut and its associated organs are still developing. In addition, this period is marked by rapid development of pectoral ns, jaws and gills. During the two days after hatching (72120 h), the zebrash juvenile starts to swim and feed. The embryonic yolk, which has sustained the embryo until this time, is almost depleted. The zebrash embryo has rapidly become a small version of the adult.

Experimental methods for studying zebrafish development

This section will review powerful experimental methods available to study zebrash, such as cell lineage analyses and cell transplantation, that elucidate when and where important developmental events occur. When these experiments are coupled with genetic analyses (as will be reviewed later), we learn what genes are important for developmental processes, as well as when and where gene function is required for normal development. Cell lineage analysis and the zebrafish fate map The rapid development of the transparent zebrash embryo allows researchers to do many kinds of experiments quickly and easily. For example, zebrash cells can be injected with lineage tracer dyes, which are uorescent dyes that permanently identify the injected cell and its descendants. Since zebrash embryos are transparent, the

fates of injected cells can be followed in living embryos by looking for uorescent cells at later times. We can use timelapse analysis to watch the movements, divisions and dierentiation of uorescently-labelled cells. In other animals that are not optically clear, one would have to serially section the embryo in order to nd labelled cells. Lineage tracing experiments have taught us a great deal about zebrash embryogenesis. When a zebrash blastomere is injected with lineage tracer dye during cleavage and blastula stages, the labelled cell and its progeny contribute later to many dierent tissues. The labelled blastomeres begin to be fate-restricted during gastrula stages. Fate restriction means that a labelled cell and its progeny will contribute to only one tissue type, demonstrating that, at the time of labelling, the cell was specied to a particular fate. By injecting single cells at the gastrula stage and following the fates of their progeny, a zebrash fate map has been constructed. This fate map is shown in Figure 2 and tells us to what tissue a cell in a particular position of the gastrula is likely to contribute. Cells located near the blastoderm margin will make mesodermal derivatives. A marginal blastomere on the dorsal side will normally make notochord, and a marginal blastomere on the ventral side will normally make blood. Cells located farther from the blastoderm margin and closer to the animal pole will contribute to epidermis and neural tissue (brain and spinal cord). When we look at the live gastrula embryo in Figure 2a, it looks deceptively simple, much like a knitted cap pulled over a large yolk cell. The fate map shows us that even though the embryo may look simple, complicated cell specication events are occurring, and many cells are already dierent from their neighbours. In fact, as shown in Figure 2c, cells in one fate map domain often express dierent genes from cells in a neighbouring fate map domain, revealing that dierential gene expression correlates well with cell fate choice.

Figure 2 Zebrafish gastrula fate map and regional gene expression. (a) Live early gastrula. (b) Schematic drawing of the fate map at a similar stage. The embryonic blastoderm is shown in red. Mesodermal fates (notochord, muscle and blood) arise near the blastoderm margin, whereas neural tissue and epidermis arise farther from the blastoderm margin. Only a subset of tissue fates is shown. AP, animal pole; VP, vegetal pole; D, dorsal; V, ventral. (Redrawn from Kimmel CB et al. (1990) Development 108: 581 594.) (c) Early gastrula embryo fixed and processed to detect expression of the floating head (flh) gene. The dorsal cells at the blastoderm margin that express flh (stained blue) are found within the notochord domain of the fate map.

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Zebrafish Embryo as a Developmental System

Cell transplantation analyses The information from the fate map allows us to ask more sophisticated questions. We know from the gastrula fate map that cells in certain positions are fated to give rise to specic tissues. In addition, region-specic gene expression patterns are also apparent. Thus, during early gastrulation, cell fate specication is occurring; that is cells are becoming distinct from their neighbours and begin to express characteristics appropriate for their future fates. However, the fate map does not reveal whether those cells are committed to a particular fate or to express particular genes. One can only learn about cell commitment, the irreversible decision to adopt a particular fate, if a cell is challenged by moving it to a new environment. If a cell is committed, it will adopt the fate consistent with its original position, even though it is surrounded by neighbouring cells that adopt dierent fates. If a cell is not committed, it will develop like its new neighbours and adopt the fate appropriate for its new environment. In zebrash, single cells can be aspirated into a micropipette and transplanted from their original positions to ectopic positions. When a cell is transplanted during early gastrulation from one region of the fate map to another, it adopts the fate appropriate for its new location. This result shows that early gastrula cells are not committed to a particular fate. When single cells are transplanted at midgastrulation, they adopt fates appropriate for their original position. Taken together, these results show that cell fate specication begins at early gastrulation, but cell commitment (the irreversible decision to adopt a particular fate) does not begin until midgastrulation, two hours later. Below, we discuss how the powerful techniques of cell lineage analysis and cell transplantation are used to characterize the defects in developmental mutants.

Using cell lineage and transplantation to analyse developmental mutants

One of the rst zebrash developmental mutations described was the recessive lethal spadetail mutation. Figure 3a shows how spadetail (spt) mutants got their name; at the end of the rst day of development, a distinctive ball of cells has accumulated at the end of the spt mutant tail (arrow). The other prominent phenotype of spt mutants is seen in Figure 3b. In wild-type embryos, bilateral pairs of somites (which later give rise to muscle) form next to the notochord. In contrast, spt mutants lack organized trunk somites, and later in development trunk muscle is severely depleted. When spt mutant cells at the trunk muscle position of the gastrula fate map are labelled with lineage tracer, they adopt tail fates instead of trunk fates. These cell lineage experiments suggest the defective migration of somitic precursors to the spt mutant trunk may explain the abnormal accumulation of cells in the spt mutant tail. Lineage analyses in spt mutant embryos do not address whether the behaviour of mutant cells is intrinsic or extrinsic to the mutant cells. To explain these terms, consider the following analogy. Suppose a car is travelling erratically down a road o in the distance. Just by watching, we cannot tell whether the car is moving erratically because of extrinsic factors (e.g. because the road is in poor condition) or because of intrinsic factors (e.g. because the driver is blindfolded). How might we discriminate between these two possibilities? We could observe the car travel on a road we knew to be in good condition. If the car still drove erratically, we would deduce that the original road was in good condition and that the driver was impaired. If the car now drove awlessly, we would deduce the opposite, that the driver was perfectly competent, but the original road was in need of repair. The same sort of experiment can be done with spt mutant cells by creating a genetic mosaic embryo that contains both wild-type and mutant cells. When a few spt mutant gastrula cells are transplanted among trunk somitic precursors of a wild-type host gastrula, they do not make somitic tissue, but instead they migrate abnormally during gastrulation and end up at the tip of the wild-type hosts tail. Thus, because spt mutant cells behave abnormally even when they are surrounded by wild-type cells, we say that spt acts intrinsically, or cell-autonomously, in trunk somitic precursors. Even if the molecular identity of the spt gene is not known, these transplantation experiments suggest the type of molecule spt might encode. Examples of intrinsic (cell-autonomous) factors are transcription factors, intracellular structural or signalling molecules, or cell surface receptors. These types of molecules, if expressed at the expected time and place, would be good candidates for the spt gene. Examples of extrinsic (non-cell-autonomous) factors are secreted signalling molecules or extracellular matrix proteins.

Genetic Analysis of Zebrafish Development

Genetic analysis oers an important advantage to any developmental system. By isolating mutations that disrupt normal development, we let the embryo tell us what genes are important and when gene function is required. If a gene required for normal development is destroyed by mutation, the developmental consequences reveal the role of the gene during normal development. This section reviews how zebrash mutants are analysed, explains how zebrash mutants are isolated, and describes some of the interesting zebrash genes that have been identied by mutational analyses.

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Zebrafish Embryo as a Developmental System

Figure 3 Wild-type and spadetail (spt) mutant zebrafish embryos. (a) Wild-type and spt mutant embryos at 24 h. Arrow marks the abnormal accumulation of cells in the spt tail bud. (b) High-magnification dorsal views of wild-type and spt mutant embryos at 14 h; spt mutants have a notochord, but clearly lack somites.

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Zebrafish Embryo as a Developmental System

It should be clear from this discussion of the spt gene that studying the defects in genetic mutants can teach us an enormous amount about normal development. Studies of spt mutant embryos demonstrate that, during gastrulation, a gene called spadetail is required intrinsically in trunk somitic precursors to ensure that they migrate to the correct position and adopt the correct fate. The rest of the article discusses in more detail how developmental mutations are found and highlights some of the interesting classes of mutations that have recently been discovered.

Zebrafish genetic screens

How are developmental mutations isolated? Two largescale zebrash screens (hunts for mutants) have been performed, and both screens used a traditional twogeneration breeding scheme, followed by screening of embryos in the third generation. Figure 4a illustrates the traditional screening strategy. Male parental (P) sh are
(a) Traditional screening strategy

treated with a chemical mutagen to induce mutations in their germ line. Each mutagenized male is crossed to a wildtype female to generate F1 lines; individual sh in each F1 line carry dierent mutations inherited from their father. For simplicity, only a single mutation carried by a female F1 sh is shown to be segregating. If these mutations are recessive, no mutant phenotype will be observed since the wild-type allele will mask the eect of the mutant recessive allele. Sibling matings between F1 sh generate F2 families; in the F2 generation, about half of all sh in each family carry the same mutation. Now, crosses of F2 siblings will reveal the mutant phenotype when both the female and male parents carry the same mutant allele. If the mutation is recessive, then 25% of the F3 progeny of such a cross will display the mutant phenotype and 75% will be phenotypically normal. A variation of this traditional approach utilizes genetic trickery to generate gynogenetic haploids during the screening step. As the name implies, gynogenetic haploids
(b) Haploid screening strategy

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Figure 4 Zebrafish mutagenesis screening strategies. (a) Traditional screening strategy. (b) Haploid screening strategy. +, Wild-type allele; m, mutant allele. Red embryos represent mutant diploids (m/m) or haploids (m).

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Zebrafish Embryo as a Developmental System

are embryos that have half the normal number of chromosomes, all of which are derived from the mother. The haploid screening strategy, illustrated in Figure 4b, reduces the time and space required for screening. How does haploid screening work? As in the traditional strategy, male parental (P) sh are treated with a chemical mutagen to induce mutations. Mutagenized males are crossed to females to generate a mutagenized F1 line. Now the procedure diverges from the traditional approach. Instead of performing sibling crosses to generate F2 families, the haploid progeny of F1 females are examined directly for mutant phenotypes. Eggs are gently expressed from F1 females and mixed with sperm that has been treated with ultraviolet irradiation to destroy its genetic material. The irradiated sperm triggers development of the egg, but does not contribute any genetic material to the developing haploid embryo. If the F1 female carries a recessive mutation that aects embryonic development, the mutant phenotype will be observed in about 50% of her haploid F2 progeny. Many embryonic structures, especially during early embryogenesis, can be observed in haploids. However, haploid embryos live for only about three days, and therefore haploid screens for mutations that aect late embryogenesis are not feasible. Additionally, development of haploid embryos is not completely normal. The haploid body is short, the brain is

abnormally sculptured, and blood circulation is abnormal. Despite these potential drawbacks, many interesting developmental mutants have been discovered using a haploid screening strategy.

Zebrafish developmental mutants

What types of developmental mutations are discovered in morphological screens? Hundreds of mutations have been identied that aect virtually every aspect of early development. The phenotype caused by a mutation in the spadetail gene is shown in Figure 3. Figure 5 illustrates other examples of mutations that have been discovered in morphological screens. Embryos homozygous for a mutation in the no tail gene have no tail (as the name implies) and also lack the notochord, the embryonic backbone. The gene disrupted by the no tail mutation is a transcription factor that is conserved across animal phyla, and it turns out that mice homozygous for a mutation in a homologous gene (called Brachyury) have a very similar phenotype. Socalled cyclops mutant embryos are cyclopic (having one centrally located eye) and also lack the oor plate, a distinct population of ventral spinal cord cells located just above the notochord. Like cyclops mutant embryos, embryos homozgous for a mutation in the one-eyed pinhead gene also are cyclopic and lack a oor plate. The

Figure 5 Zebrafish mutants at one day of development. The notochord (arrowhead) and an eye (open arrowhead) are indicated on the wild-type embryo. The other eye in the wild-type embryo is in another plane of focus. See text for descriptions of mutants. The boxed inset in the lower right shows high-magnification views of wild-type (wt) and no isthmus (noi) mutant embryo heads, showing the lack of the midbrain hindbrain boundary (arrow) in no isthmus mutants.

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Zebrafish Embryo as a Developmental System

one-eyed pinhead gene has recently been shown to encode a novel protein, illustrating that genetic screens in zebrash will uncover new developmental genes. Another example of a mutation aecting brain development is the no isthmus mutation. Embryos lacking a functional no isthmus gene lack the isthmus, a structure that demarcates the division between the midbrain and the hindbrain and later becomes the cerebellum (see Figure 5 inset). Embryos with the no isthmus mutation are more subtle than the other mutants shown in Figure 5. How might one screen easily for even more subtle defects? One way to do this is to screen directly for disruptions in gene expression patterns. Figure 6 shows wild-type and mutant embryos that were xed and processed to detect the transcripts of three genes that have nonoverlapping, region-specic expression patterns. Two of these genes are expressed in discrete regions of the midbrain and hindbrain; engrailed3 (eng3) is expressed in the midbrainhindbrain boundary, and krox20 is expressed in two hindbrain segments (3 and 5). Comparing a wild-type embryo with an embryo carrying a mutation in the valentino gene shows that valentino mutant embryos have normal krox20 expression in hindbrain segment 3, but virtually no krox20 expression in segment 5. This strongly suggests that the valentino mutation is not in the krox20 gene itself, but instead is a mutation in a gene that regulates krox20 expression specically in hindbrain segment 5. Thus, one important feature of the expression screening approach is that it allows isolation of regulatory genes that control expression of downstream genes. Another important feature is that it allows identication of genes that control subtle patterning of the embryo. Live valentino mutant embryos do have a morphologically identiable defect, but it is a subtle defect in hindbrain segmentation that can be picked out only by an experienced eye. On the other hand, even an untrained eye can sort out valentino mutant embryos from their wild-type siblings using gene expression patterns! We have discussed only a few of the hundreds of mutations that have been described that disrupt zebrash embryonic development. It is beyond the scope of this

article to review them all, and instead we briey mention the classes of mutations that aect virtually all aspects of embryonic development. These classes include mutations that disrupt gastrulation movements, establishment of the body axes, tissue and organ formation (including the notochord, blood, heart, neural crest derivatives, placodes, and central nervous system), formation of neural connections, and behaviour. Genetic screens using novel approaches will identify additional developmental genes, since it is clear that there are still many new genes to be identied and studied. By characterizing mutant phenotypes using cell lineage and transplantation analyses and by examining the genetic interactions among dierent mutations, we can begin to understand regulatory pathways that govern embryonic development.

The zebrafish genetic map

The challenge now is to identify the molecular nature of genes identied in mutational screens. What kind of proteins do these genes encode? Among the many possibilities are transcription factors, secreted signalling molecules, and cell surface receptors. An important tool for identifying the molecular identity of genes known only by mutation is a genetic linkage map. A genetic linkage map is constructed by examining the inheritance patterns of various markers relative to one another. If two markers are unlinked (for example on dierent chromosomes), then they will assort independently of one another, just as Gregor Mendel observed for the various traits he followed in pea plants. On the other hand, if two markers are closely linked, they will almost always be inherited together. Only rare recombination events will separate these two markers, and the frequency of recombination indicates the genetic distance between the markers. Recently, the 25 chromosomes of the zebrash have been ordered into 25 linkage groups. These 25 linkage groups are collectively called the zebrash genetic map. How can one use the genetic map to identify the molecular nature of a gene known only by mutation? First, the mutation is placed on the genetic map using the same recombinational analysis described above. The mutation will be linked only to markers that are on the same chromosome and will be unlinked to markers on other chromosomes. Recombinational frequency will indicate which markers are most closely linked to the mutation. Sometimes, the most closely linked marker will be a gene that is a good candidate for the one that is disrupted by the mutation. More frequently, the mutation will map to a chromosomal region in which no candidate genes have yet been mapped. In this case, the most closely linked marker can be used to walk along the chromosome towards the gene of interest. Once a good candidate gene is identied, either by serendipity or by chromosomal walking, several criteria are used to determine whether

Figure 6 Mutagenesis screening using gene expression patterns. Wildtype and valentino mutant embryos fixed at 22 h and processed to detect expression of three different genes. Comparing head views of wild-type and mutant embryos shows that engrailed3 (eng3) expression at the midbrain hindbrain boundary and krox20 expression in hindbrain segment 3 is normal in valentino mutant embryos, but krox20 expression in hindbrain segment 5 is severely diminished (*). (Photographs courtesy of Dr Cecilia B. Moens.)

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Zebrafish Embryo as a Developmental System

the candidate gene is indeed the gene disrupted by the mutation. First, recombinational analysis should show that the candidate gene and the mutation always segregate together and are never separated by a recombinational event. Second, the gene should be expressed at the right time and place to explain the defects observed in mutant embryos. Third, sequencing of the candidate gene from mutant embryos should reveal a molecular lesion in the gene that disrupts its function. Finally, the mutant phenotype may be rescued by providing mutant embryos with a wild-type copy of the candidate gene. When these criteria are satised, one can say with condence that the candidate gene is indeed the same gene identied by mutation. To date, the zebrash genetic map has been utilized to identify the molecular nature of several genes originally identied by mutation, and the genetic map will continue to be an important tool in the future.

establishment of molecular and genetic tools such as the zebrash genetic map has allowed us to identify the molecular nature of genes known only by mutation. Together, these attributes of the zebrash allow us to dissect embryonic development at cellular, genetic and molecular levels. By combining developmental studies in zebrash with those in other vertebrate model systems, developmental biologists will begin to unravel the extraordinary events that occur as an embryo develops from a one-celled zygote to a complex, multicellular organism.

Further Reading
Development (1996) vol. 123. [Many articles in this issue contain initial phenotypic descriptions of zebrash developmental mutants found in two large-scale screens.] Driever W, Stemple D, Schier A and Solnica-Krezel L (1994) Zebrash: genetic tools for studying vertebrate development. Trends in Genetics 10: 152159. Eisen J S (1996) Zebrash make a big splash. Cell 87: 969977. Kimmel CB, Kane DA and Ho RK (1991) Lineage specication during early embryonic development of the zebrash. In: Gerhart J (ed.) Cell Cell Interactions in Early Development, pp. 203225. New York: Wiley-Liss. Kimmel CB, Ballard WW, Kimmel SR, Ullmann B and Schilling TF (1995) Stages of embryonic development of the zebrash. Developmental Dynamics 203: 253310. Postlethwait JH and Talbot WS (1997) Zebrash genomics: from mutants to genes. Trends in Genetics 13: 183190. Solnica-Krezel L, Stemple D and Driever W (1995) Transparent things}cell fates and cell movements during early embryogenesis of zebrash. BioEssays 17: 931939.

Summary
We have reviewed the features of the zebrash embryo that make it an excellent vertebrate model system. Zebrash embryos are small, virtually transparent creatures that develop rapidly and synchronously outside the mother. Cell lineage and transplantation analyses allow us to follow the behaviour of individual cells over time in both wildtype and mutant embryos. The relative ease of genetic screens in zebrash has allowed the identication of hundreds of genes that regulate embryonic development, only a few of which were described here. Finally, the

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