Axis formation in zebrafish

Wolfgang Driever

Massachusetts General Hospital and Harvard Medical School, Boston, USA

Recent advances in our understanding of axis formation and patterning in zebrafish relate the developmental mode of this aspiring genetic model

organism to higher vertebrates. The effect of UV irradiation and lithium

treatment, as well as detailed early lineage analyses, have shed some light

on dorsoventral axis formation. However, the molecular mechanism of axis

formation, as well as the identity of a fish Nieuwkoop center, are still open

issues. A Vgl homolog is expressed in zebrafish, and activin as well as the

mouse nodal gene product have been demonstrated to induce mesoderm and

ectopic axes, respectively, in zebrafish. The zebrafish organizer is defined by

the expression domains of goosecoicf, axial, and liml. The cyclops gene is

involved in maintaining goosecoid expression in axial mesoderm of the

head. Large mutagenesis screens provide the basis for a genetic analysis of

axis formation.

Current Opinion in Genetics & Development 1995, 5:61 O-61 8

Introduction

All vertebrate zygotes, after fertilization, undergo a series of cell divisions to generate a mass of cells from which

the embryo will form. The first patterning step is the establishment of the embryonic axis. At a morphological

level, vertebrate embryos display diversity at this stage.

Some zygotes, such as Xenopus, initially possess clear animal/vegetal polarity and develop dorsoventral polarity

during the first cleavage. For other vertebrates, however, the axes are not predictable before the onset of

gastrulation. At the molecular level, though, there are

clear indications for common themes in axis formation. Many different vertebrate embryos respond in a similar

fashion to the application of inducing agents, such as

lithium, fibroblast growth factor (FGF) or transforming growth factor (TGF)p family members. Gene expression

patterns are homologous, and mutations in homologous

genes result in similar phenotypes in zebrafish and mice

[l-3]. Comparative studies among different vertebrates

have been very important in identifying common basic principles of axis formation.

Traditional vertebrate experimental systems provided

either access to genetic manipulation (mice) or em-

bryological manipulation (chicken, frogs, etc.). Over

the past decade, the zebrafish (Danio rerio) has become

popular for developmental studies because its short

generation time and high fecundity make mutational analysis possible, and the small translucent embryos

are excellent subjects for embryological studies and manipulations. In this review, I will focus on the progress

made during the past two years in our understanding of

axis formation and patterning in zebrafish and will relate this work to classic studies on pattern formation in fish.

Early post-fertilization development

Zebrafish develop in a manner typical of teleosts, and morphological aspects of their development and a normal table of developmental stages have been described in detail [4*]. When eggs are laid, yolk and cytoplasm are intermixed and the egg is surrounded by

a transparent chorion (Fig. 1). The animal/vegetal axis is preset during oogenesis and sperm can enter the egg

only at the future animal pole through the micropyle, a specialization in the otherwise sperm-impermeable chorion [5]. After fertilization, cytoplasm streams to

the animal pole as it segregates from the yolk. About

30 minutes after fertilization, the cytoplasm forms the

blastodisc at the animal pole and surrounds the vegetal

yolk mass as a thin yolk cytoplasmic layer.

At 40 minutes post-fertilization, the first meroblastic

cleavage occurs. Four more cleavages occur in stereo-

typic orientation at 15-minute intervals, followed by five

synchronous, but not oriented, cleavages. During these cleavages, the marginal vegetal blastomeres maintain

large cytoplasmic bridges with the yolk cell. These 10

cleavages generate a mound of blastomeres on top of

the vegetal yolk cell. Subsequently, during mid-blastula

transition, activation of zygotic transcription coincides

with the generation of the first three separate lineages of the embryo [6,7]. Two of these lineages, the enveloping

layer forming the outer surface of the blastoderm and the yolk syncytial layer deriving from the collapse of

vegetal marginal blastomeres into the yolk cell, constitute extraembryonic lineages. The third lineage, termed the

deep cell layer, will form the embryo proper. At the

end of the mid-blastula transition, the first coordinated cell movements occur in the embryo. The cells of the

610

Abbreviations

CNscentral nervous system; FGF-fibroblast growth factor; TCF-transforming growth factor.

0 Current Biology Ltd ISSN 0959-437X

Axis formation in zebrafish Driever 611

blastoderm spread vegetal-ward over the yolk cell during epiboly [8,9*,10]. No dorsoventral asymmetry has been detected during these stages.

Castrulation

Shortly before the onset of gastrulation movements, the future dorsoventral axis becomes obvious through an asymmetry in the thickness of the blastoderm [ll]. The formation of the hypoblast (the mesendodermal germ layer) is initiated on the dorsal side of the embryo and soon continues all around the margin. Formation of the hypoblast leads to appearance of the germ-ring, a thickened marginal region all around the blastoderm rim. On the dorsal side forms a pronounced thickening, the embryonic shield [12]. Gastrulation in zebrafish is characterized by movement of single cells rather than coherent cell layers. In clear distinction to the involution of layers of cells in amphibia, ingression might be a more precise term to describe the movements of cells during hypoblast formation [10,13*,14,15*].

Soon, molecular differences between epiblast and hy- poblast can be detected. For instance, ~0~2 transcripts, present throughout the blastula, become restricted to the epiblast during gastrulation [ 16,171. As gastrulation continues, the hypoblast extends toward the animal pole, while epiboly expands both layers towards the vegetal pole. At the same time, both hypoblast and epiblast cells converge from ventral and lateral positions toward the dorsal side. Mediolateral intercalation of cells converging to the dorsal side leads to elongation of the embryonic shield along the anteroposterior axis of the embryo [12,18]. At the end of epiboly, the embryo extends along the dorsal side of the yolk sphere, with the head positioned at the former animal pole and the tailbud developing at the former vegetal pole of the egg.

Axis formation, fate maps and determination

At the onset of gastrulation, analysis of cellular fates reveals that the endoderm will derive from the vegetal- most marginal blastomeres (Fig. 1). Mesoderm forms from the vegetal one-third of the blastoderm, whereas ectoderm originates from the animal half of the blastoderm. Neuroectoderm in particular derives from the dorsal section of the animal half [19]. Notochord derives from the dorsal side, where the shield forms, whereas somitic mesoderm, blood and heart develop fi-om lateral and ventral positions, respectively (see also [20]). The organization of the zebrafish fate map is similar to that of Xenoplrs [19].

Attempts have been made to establish a fate map based on the first cleavages, which are of stereotype orientation and, at the eight-cell stage, produce an asymmetric array of two x four cells on top of the yolk cell [21]. Individual blastomeres at the eight-cell stage were labeled with different fluorescent lineage tracers. A correlation between the second cleavage plane and the dorsoventral axis was suggested, but three independent

studies were later able to demonstrate that no correlation exists between any of the early cleavage planes and the dorsoventral axis [22”,23”,24], confirming earlier studies [25]. These earlier studies scored the position of dorsal tissue at the shield stage (rather than at the end of somitogenesis) [21], which afforded a less ambiguous assignment of dorsal position. A mechanism by which localized (dorsal?) determinants are distributed in a stereotype fashion among the early blastomeres in correlation to the cleavage planes can be excluded from being involved in axis formation in zebrafish.

Portions of the zebra&h embryo retain their relative position in the early embryo from blastula to gastrula and, consequently, rudiments of a fate map can be observed prior to gastrula stages [23”,26,27]. For instance, the descendants of central blastomeres (located at the center of the blastoderm when viewed from the animal pole) at the 64-cell stage frequently end up in the animal-most portion of the early gastrula and give rise to neural structures, as well as ectoderm and mesoderm. The fate of central blastomeres is obscured, as their descendants are subject to extensive cell mixing during epiboly. In contrast, marginal blastomeres are subject to less extensive mixing [28-l. Thus, it has been possible to demonstrate that cardiac progenitors are located at the margin of the blastula-stage embryo [20,29].

Very detailed fate maps have been generated for the shield region [13*] and for the neuroectoderm at the onset of gastrulation [30*]. For the epiblast of the shield region, some degree of radial organization has been observed: the outermost cell layer frequently gives rise to endoderm, the second to notochord, and the third to neuroectoderm. In contrast to amphibians, cell lineages present in the epiblast of the shield region (neuroectodemal, notochordal, endodermal and, to a lesser extent, somitic) are spatially less well segregated and represent intermingled populations of cells. Whether these findings relate to different patterning mechanisms is unknown. Within the neuroectoderm, a predictable order in the precursors of presumptive major brain sub- divisions indicates that cell mixing in the neuroectoderm is less pronounced [30*].

When do cells become irreversibly committed to a spe- cific fate? Heterotopic and heterochronic transplantation experiments [31] reveal that cells are not committed to a certain fate at late-blastula stages (5 hours post-fertil- ization), when tissue-restricted lineages arise. However, when hypoblast cells are transplanted heterotopically to the epiblast at mid-gastrula (8 hours post-fertilization), they will predominantly give rise to hypoblast fates. Thus, cells become committed to a specific germ layer at mid-gastrula stages. Earlier tissue-specific lineage restrictions do not necessarily reflect the state of commitment of the cell.

Dorsoventral patterning

Establishment of the dorsoventral axis is best understood in Xenopus (reviewed in [32,33]). Cortical rotation

612 Differentiation and gene regulation

Micropy’- _ Chorlon

Cleavage planes

’ Cytoplasm

Zygote (0 hrs) 2-Cell(0.7 hrs) a-Cell (1.2 hrs)

AN

VE

1000 Cell (3.0 hrs) 30% Epiboly (4.7 hrs) Fate map at late blastula (50% Epiboly

Shield (6.0 hrs) 90% Epiboly (9 hrs)

Anterior Head region

He 1

1 Somite (10.3 hrs)

0 Endoderm

q Mesendoderm , overlap enddmesoderm on tatemap

n Mesoderm

n Overlap area mesoderm C neuroectodermkctoderm on fatemap

n Ectoderm , neuroectoderm

Axis formation in zebrafish Driever 613

during the first cell cycle is microtubule dependent, W sensitive, and results in the formation of the Nieuwkoop center in dorsal vegetal blastomeres. The Nieuwkoop center appears to induce the Spemann organizer in the dorsal blastopore lip, which again is the source of signals patterning the dorsoventral and anteroposterior axes. Are there similarities to zebrafish development?

W irradiation of zebrafish eggs between 10 and 25 minutes after fertilization depletes dorsal structures and results in radially symmetric embryos [8]. Within the zebrafish yolk cytoplasmic layer at that stage, there is a prominent parallel array of microtubules oriented in animal/vegetal direction [8]. However, the target of W action is unknown-so far, no evidence exists that cortical rotation takes place in zebrafish, and the target of W action could be maternal RNAs as well as the microtubules.

Similarities to amphibia are evident in the sensitivity to lithium chloride exposure during cleavage stages, which results in dorsalized zebrafish embryos [34]. Upon lithium treatment, dorsal genes such as postmid are expressed all around the margin of the gastrula, whereas the expression of ventral and posterior genes such as eve1 are suppressed [35]. In lithium-treated radialized embryos, which appear to have no dorsoventral axis, the anteroposterior order of gene expression is maintained between the animal pole and the vegetal margin at late-gastrulation stages [36-l. The lithium-sensitive period is between the 16-cell and 1024-cell stage [34], before mid-blastula transition. Therefore, the activities of maternal factors appear to be responsible for the initiation of dorsoventral axis formation.

Experiments performed in other teleosts indicate a possible involvement of the yolk syncytial cell in the specification of dorsal fates. Transplantation of younger rainbow trout blastoderms onto gastrula-stage yolk syncytial cells, from which the blastoderm had been removed, led to development of dorsal structures by those cells located atop the dorsal side of the host yolk cell [37]. Further, the capacity of the blastoderm to differentiate autonomously after separation from the yolk has been tested for several teleosts. Fundulur blastoderms from embryos older than 32-cell stage [38] and salmon or loach blastoderms Gem embryos after mid-blastula

stage [39,40] differentiate into embryonic structures, often with well defined axes. In cgntrast, younger blastoderms were reported to form morphologically less differentiated cell aggregates, sometimes with columnar epithelia, which might indicate ectoderm. In a few cases, incompletely differentiated nervous tissue was reported. Oppenheimer [38] suggested that some substances are required to be passed on from the yolk cell to the blastomeres to induce differentiation. Whether these postulated substances are Nieuwkoop center derived dorsal signals and/or general mesoderm inducers remains to be demonstrated.

Other observations are consistent with an involvement of the yolk syncytial cell in axis formation. Blastomeres appear to have equal developmental potentials prior to onset of gastrulation [41], and transplantation of blastomeres prior to gastrulation never results in the induction of a second axis in hosts. The developmental modes of some other teleosts make it difficult to consider dorsalizing determinants in blastomeres. Teleosts with annual lifestyles often go through a phase of dispersion of blastomeres at the end of epiboly, and blastomere move- ment is amoeboid [42,43]. Reaggregation at one site on the yolk syncytial cell precedes embryo formation. Reaggregation could be guided by specialization of a region of the yolk cell, or by sorting out of blastomeres that would have differentiated into separate lineages before the migratory phase, similar to chick mesoderm formation [44].

So far, no zebrafish mutations have been described in maternal-effect genes involved in the establishment of the dorsoventral axis. The maternal-effect mutation jantrs can give rise to axis duplications in the embryo [22**]. Mutant blastoderms tend to split at the 4-16-cell stage into two half-blastoderms, which remain next to each other and continue cleavage and epiboly independently. Only in the fraction of embryos with dorsal structures induced at the place where both blastoderms touch each other are two embryonic shields formed adjacent to each other, resulting in axis duplications. It is important to note that only one dorsal center is induced per embryo, not one per blastoderm. It is not yet known from where the dorsal-inducing signal (the zebrafish homolog of the Nieuwkoop center?) derives.

Fig. 1. Early development of zebrafish. The development of zebrafish embryos from fertilization to the end of gastrulation is represented by schematic drawings of mid-saggital sections of the embryos. Developmental times are in hours post-fertilization at 28’C. Only for the zygote and the two-cell embryo is the chorion shown. Fertilization occurs at a structurally specific site, the micropyle. In the zygote, yolk (grey) and cytoplasm (light blue) are mixed, but separate during the first two hours of development by cytoplasmic streaming to the animal pole (blue arrows). The stereotypical cleavage planes are indicated above the eight-cell embryo. The lOOO-cell embryo represents the mid-blastula. The different embryonic and extraembryonic lineages can be clearly distinguished: DL, deep layer (embryo proper); EVL, enveloping layer; YSL, yolk syncytial layer; YSN, yolk syncytial nuclei; YCL, yolk cytoplasmic layer. Fate map at 50% epiboly, just before the onset of gastrulation: V, ventral; D, dorsal; AN, animal pole; VE, vegetal pole. During fate-mapping experiments, significant regions of overlap have been found. Orange represents areas with cells that give rise to both endodermal and mesodermal fates. Purple represents areas where cells with both mesodermal and ectodermal or neuroectodermal fate were found. At the shield stage, gastrulation movements have created the epiblast. For the first time, the dorsoventral axis can be identified by a morphological criterion: the thickening of the embryonic shield on the dorsal side. The red arrows represent hypothetical mesoderm-inducing signals, the green arrow a dorsal-inducing signal. At 90% epiboly during late gastrula, the hypoblast has almost reached the animal pole. Convergence of cells to the dorsal side results in a thickening of the embryonic axis. On the ventral side of the yolk, only very thin layers remain. At the one-somite stage, gastrulation is complete and the major regions along the anteroposterior axis (head, trunk, and tail) can be distinguished. The organization of germlayers in the tailbud (green) is not known for zebrafish.

614 Differentiation and acne regulation

If the zebrafish Nieuwkoop center were a structure

autonomously generated during blastula stages in the

blastoderm itself, one would expect to find two spatially

separated dorsal centers in some of thejanus blastoderms,

which is never the case. However, a Nieuwkoop center

could form during the first cleavages and segregate

among prospective dorsal marginal blastomeres or with

the yolk syncytial cell. Whether maternal gene products

are localized in the zebrafish yolk syncytial cell, and

whether there is any contribution to patterning by

transcription from the yolk syncytial nuclei, needs to be

determined.

Mesoderm induction

In Xenopus, activin, Vg-1, wnt-1 1, bone morphogenetic

protein 4, and FGF have been suggested to be major

components of the mesoderm-induction pathway (for review, see [45]). In contrast to the vegetal localization

of Vgl (DVR-1) mRNA in Xenopus, the maternally expressed zebrafish zDVR-1 mRNA is equally dis-

tributed among all blastomeres, but appears to be absent from the vegetal yolk cell [46]. Processing of the inactive Vgl precursor is highly controlled, and

the active form can induce dorsal mesoderm [47].

As zDVR-1 is processed in Xenupus and can induce mesoderm, it is likely to be a true Vgl homolog

(C Dohrmann, DS Kessler, DA Melton, personal communication). The reason for the difference in

localization between zebrafish and Xenopus is unknown. Helde and Grunwald [46] suggest that the indeterminate

state of the zebrafish blastomeres and the more extensive cell mixing, when compared to Xenopus, require that all blastomeres harbor equivalent maternal information for

developmental potential. Therefore, it could be possible

that zDVR-1 is processed to the active form in only a subset of the zebrafish blastomeres.

The mouse nodal gene product, another member of the

TGFB family, when injected into zebrafish embryos,

causes the formation of ectopic shields and, subsequently, axes including notochord and somites, but no head

duplications [48*]. The mouse nodal gene is disrupted

in a mouse mutant deficient in mesoderm formation

[49]. The axis-determining function of nodal might

be mediated by activating early-response genes such as

~~yoose& and lirlr 1 [48’].

The role of zebrafish activin has been studied in embryos

of a similar teleost, medaka [50-l. Activin PB is expressed during oogenesis and from the late-blastula stage on.

To distinguish between maternal and zygotic activin,

distinct mutant forms were generated. Embryos injected with RNAs encoding dominant-negative processing-

defective activins, which act upon cotranslation on the

cellular form by dimer formation, do not generate a mesoderm-deficient phenotype in fish. In contrast, dominant-negative activins, which appear to interfere with the activin receptors, do result in mesoderm-defi-

cient phenotypes. The authors conclude that maternally derived activin receptor activating activity might be

sufficient for mesoderm formation. However, as the

dominant-negative activin might interfere with TGFP

family receptors other than activin receptors, a specific

role for the maternal activins has yet to be demonstrated.

Activin has the ability to induce in zebrafish animal

cap explants mesodermal markers, such as no tail [l]

(the zebrafish Bruchyury homolog), snail1 [51], and the

hepatocyte nuclear factor B (HNF3p) homolog axial [52]. Zygotic expression of both snail1 [51,53] and no tail is initially established within the whole germ-ring,

prospective mesendodermal cells. Animal cap cells transplanted to the germ-ring start to express no tail [l]. Thus activins, and/or other related mesoderm-inducing

factors, are active in the germ-ring. The role of FGFs

has not yet been investigated, but FGF receptors are

expressed in the early embryo [54].

The ‘fish organizer’

Classic transplantation experiments demonstrate that the zebrafish shield has organizer activities similar to

the dorsal blastopore lip of amphibia. In the teleosts F&tr/us [55] and zebrafish [56], shield mesoderm, when ectopically transplanted to the ventral side of a host embryo, induces secondary axis formation. The

molecular nature of the signals from the organizer appears to be conserved, as zebrafish shield can induce

axis duplications in amphibia [57].

The earliest gene known to be expressed specifically in the organizer region is the zebrafish goosecoid

homolog [34,58,59]. Transcripts are detected as early

as late-blastula stages in one-half of the blastula, in a graded distribution with a high point at the future dorsal side. Cells expressing goosecoid are among the

first to enter the hypoblast, and expression continues in the anterior hypoblast until onset of somitogenesis.

The Roosecoid-positive cells at this stage are located in the prechordal plate (axial head mesoderm), which continues to express the hlxl homeodomain protein during

further development [60]. Cells expressing goosecoid in

the late-blastula most likely define the region of the

early organizer and the head organizer, whereas late

goosemid- and h/xl-expressing cells appear to constitute

axial head mesoderm, the prechordal plate, to which

activities in dorsoventral patterning of the head have

been attributed [61-63]. It appears that at late-gastrula

stages, goosecoid expression spreads also to the inner cell

layer of the epiblast. This might reflect some of the interactions between hypoblast and epiblast, known as

vertical induction [64*]. The fate of these inner epiblast

cells is unknown, but is suggested to be neuroectoderm

[64’].

Midline signaling and dorsoventral patterning

Patterning along the dorsoventral midline of the embryo proper includes dorsoventral specification within the CNS (Rohon Beard cells, motoneurons, and floorplate,

Axis formation in zebrafish Driever 615

among others), formation and differentiation of the notochord, and an endodermal layer of cells between notochord and yolk. The prominent role of axial mesoderm in patterning of the trunk of zebrafish has become obvious from the analysis of the no tail mutation [2], the zebrafish homolog of mouse Bruchyury/T [3]. Embryos mutant for no tail do not develop differentiated notochord, although chordamesodermal precursor cells are present in the trunk. It has been suggested that signaling from these precursors is responsible for the fact that no tail mutants have a normal floorplate in the trunk [2]. However, requirements for patterning of paraxial mesoderm appear to also depend on signaling from differentiated notochord, as rzo tail mutants have abnormal somite patterning. The tail phenotype is much more severe, similar to Brachyury in mice. The tail is truncated, axial mesoderm is absent, and floorplate does not form in the remnants of the tail spinal cord. Expression of the ventro-posterior marker eve1 in the tailbud is absent [35].

The phenotype as well as the expression pattern define three aspects of no tail activity [l]. First, it is widely expressed in the tailbud and is involved in morphogenesis of the tail. Second, its expression in chordamesoderm during gastrulation appears to be required for differentiation into notochord. Third, during gastrulation, no tail is also expressed in the entire germ-ring. Cells from paraxial, lateral, and ventral positions of the germ-ring develop largely normally in no tail mutant embryos. It has been reported, however, that expression of snail occurs at reduced levels during gastrulation of no tail mutant embryos [51,53]. Thus, no tail might contribute toward establishment of ventral and lateral mesoderm, even though it is not the limiting regulator.

Zebrafish cyclops mutants are characterized by a deletion of some ventral parts of the brain and the floorplate [65,66*]. The deletion of ventral midline cells is cell autonomous, whereas other missing ventral midbrain and forebrain structures result from non-autonomous interactions. In mutant embryos, the expression of goosecoid in the epiblast is absent and, during late gastrulation, is reduced in the hypoblast. Thus, cyclops is also involved in specification of mesoderm in the zebrafish head [64*].

Wild-type floorplate cells, when transplanted into the cyclops ventral CNS midline, can rescue neighboring mutant cells to form midline structures [65,66-l. Thus, floorplate cells can homogenetically induce cyclops neighbors to form floorplate. The pathway for homogenetic induction might be distinct from the initial vertical floorplate-inducing signals originating in axial mesoderm [65,67].

A simple notion that cyclops might be defective in receiving the floorplate-inducing signals is challenged by the fact that goosecoid expression is reduced in the hypoblast. Furthermore, analysis of a differentiation marker, a(I) collagen type II, detects abnormalities in

cyclops mutant notochord [68], indicating that defects in axial mesoderm might be responsible in part for the phenotype.

The zebrafish HNF3p homolog axial [52] is first expressed just prior to gastrulation on the dorsal side of the embryo and continues to be expressed in dorsal axial mesendoderm (first the organizer region, later prechordal plate, notochord, and endoderm) along the whole axis until the end of gastrulation. Subsequently, axial expression is turned on in ventral neural plate cells (future forebrain and floorplate) adjacent to axial- expressing mesodermal cells. In cyclops mutant embryos, axial is not expressed in the neuroectoderm. Thus, cyclops is a component of the genetic pathway leading to axial activation. The cyclops gene is also involved in activation of sonic hedgehog expression in ventral CNS [67]. The sonic hedgehog gene is initially expressed in the shield hypoblast, next in axial mesoderm, and then in the ventral midline of the CNS. Ectopic expression of sonic hedgehog leads to ectopic activation of axial expression in the midbrain, where it is usually restricted to the floorplate, indicating that sonic hedgehog is one of the signals inducing ventral structures in the CNS. In cyclops mutants, sonic hedgehog is expressed in axial mesoderm, but not in the CNS.

The most prominent phenotype of spadetail mutants is the absence of trunk muscles [69]. Detailed fate mapping and the analysis of snail 1 and snail2 expression in spadetail embryos have demonstrated that the mutation is deficient in convergence and, possibly, specification of paraxial and lateral mesoderm in the trunk as welI as the head [53,70].

Further prospects for genetic analysis of pattern

formation

The unique features of zebrafish as a genetic system [71-73,74*] make it possible to identify a wide variety of mutations affecting pattern formation. So far, only a small number of mutations have been published (and discussed in this review). However, large-scale screens for embryonic phenotypes have been completed during the past few years [75*,76’], and results from the screens, including several hundred novel mutant loci, are expected to be published at the end of this year. Although these mutations will not suffice for solving the problem of pattern formation, they will provide a vast set of novel entry points from which to study axis formation, patterning, and organogenesis. In addition to the existing chemically induced mutations, novel techniques are emerging to induce insertional mutations [77*].

Current rapid improvements in genome resources, such as the genetic linkage map of zebrafish [78*] (for which the number of markers has already doubled within only one year after publication), and large insert libraries will make possible a molecular analysis of novel mutations in the near future. This will be important in order not only

616 Differentiation and gene regulation

to understand interactions between zebrafish mutations by gene expression analysis, but also, on a molecular level, to understand how patterning pathways identified in other vertebrate systems, such as Xenopus and mice, relate to zebrafish development.

Conclusions and perspectives

Despite significant advances in our understanding of zebrafish development, many important questions re- main. How is the dorsoventral axis initially specified? Is there a Nieuwkoop center in the embryo, and where is it? What is the source of the mesoderm-inducing signal? Where is Vg-1 processed? Whether the yolk syncytial cell plays a role as a source of localized signals and whether the yolk syncytial nuclei are transcriptionally active and contribute to patterning are interesting hypotheses to challenge.

Can zebrafish be a model for axis formation in mammals? Early blastomeres, similar to the inner cell mass in mice, appear developmentally non-restricted and equivalent. What role do the ‘extraembryonic’ tissues play in zebrafish is an issue as important as the one of activities of extraembryonic tissues at the onset of mammalian gastrulation.

The recently identified mutations should provide tools to answer some of these questions. The two mutations cydop.c and no tail have provided important information on the mechanism of midline signaling, and recent genetic screens have identified >20 additional mutations involved in dorsoventral patterning and midline signaling (C Niisslein-Volhard et al., personal communication; W Driever et al., unpublished data). Detailed genetic pathways should be constructed from these mutants over the next few years.

Zebrafish will be useful in the unraveling of develop- mental mechanisms by comparative studies. This applies not only to phenotypic and experimental analyses, but also to the study of gene regulation. With ~400 million years’ divergence, only sequences of functionally significant regulatory elements are conserved between mammals and zebrafish (or the puffer fish, another novel fish model system for genome analysis) [79-811. The combination of genetic, embryological, molecular, and comparative studies in zebrafish should enable us to dissect the components of maternal and zygotic genetic contributions to axis formation and to elucidate developmental pathways in the early embryo.

Acknowledgements

Thanks for discussion and comments on earlier versions of the

review to I>errk StempIe, Alexander Schier, Eliza Shah, and

Lilianna Solnlca-Krczel. Thanks to Uill Trevarrow for reference

to thr work of Wourrns on annual fishes. The author is sup-

ported by National Institutes of Health grant HI129761 and by

Umtol-Myers Squibb.

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W Ikievrr, Cardiovascular Research Center, Massachusetts

General Hospital Thirteenth Street, Building 149 (Mail Code

1494X1), Charlestown, Massachusetts 02129-2060, USA.