Zebrafish have become a popular organism for the study of vertebrate gene function. The virtually transparent embryos of this species, and the ability to accelerate genetic studies by gene knockdown or overexpression, have led to the widespread use of zebrafish in the detailed investigation of vertebrate gene function and increasingly, the study of human genetic disease. However, for effective modelling of human genetic disease it is important to understand the extent to which zebrafish genes and gene structures are related to orthologous human genes. To examine this, we generated a high-quality sequence assembly of the zebrafish genome, made up of an overlapping set of completely sequenced large-insert clones that were ordered and oriented using a high-resolution high-density meiotic map. Detailed automatic and manual annotation provides evidence of more than 26,000 protein-coding genes, the largest gene set of any vertebrate so far sequenced. Comparison to the human reference genome shows that approximately 70% of human genes have at least one obvious zebrafish orthologue. In addition, the high quality of this genome assembly provides a clearer understanding of key genomic features such as a unique repeat content, a scarcity of pseudogenes, an enrichment of zebrafish-specific genes on chromosome 4 and chromosomal regions that influence sex determination.

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The zebrafish reference genome sequence and its relationship to the human genome.

Targeted gene disruption in the mouse shows that the Sonic hedgehog (Shh) gene plays a critical role in patterning of vertebrate embryonic tissues, including the brain and spinal cord, the axial skeleton and the limbs. Early defects are observed in the establishment or maintenance of midline structures, such as the notochord and the floorplate, and later defects include absence of distal limb structures, cyclopia, absence of ventral cell types within the neural tube, and absence of the spinal column and most of the ribs. Defects in all tissues extend beyond the normal sites of Shh transcription, confirming the proposed role of Shh proteins as an extracellular signal required for the tissue-organizing properties of several vertebrate patterning centres.

Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function.

In a large-scale screen, we isolated mutants displaying a specific visible phenotype in embryos or early larvae of the zebrafish, Danio rerio. Males were mutagenized with ethylnitrosourea (ENU) and F2 families of single pair matings between sibling F1 fish, heterozygous for a mutagenized genome, were raised. Egg lays were obtained from several crosses between F2 siblings, resulting in scoring of 3857 mutagenized genomes. F3 progeny were scored at the second, third and sixth day of development, using a stereomicroscope. In a subsequent screen, fixed embryos were analyzed for correct retinotectal projection. A total of 4264 mutants were identified. Two thirds of the mutants displaying rather general abnormalities were eventually discarded. We kept and characterized 1163 mutants. In complementation crosses performed between mutants with similar phenotypes, 894 mutants have been assigned to 372 genes. The average allele frequency is 2.4. We identified genes involved in early development, notochord, brain, spinal cord, somites, muscles, heart, circulation, blood, skin, fin, eye, otic vesicle, jaw and branchial arches, pigment pattern, pigment formation, gut, liver, motility and touch response. Our collection contains alleles of almost all previously described zebrafish mutants. From the allele frequencies and other considerations we estimate that the 372 genes defined by the mutants probably represent more than half of all genes that could have been discovered using the criteria of our screen. Here we give an overview of the spectrum of mutant phenotypes obtained, and discuss the limits and the potentials of a genetic saturation screen in the zebrafish.

The identification of genes with unique and essential functions in the development of the zebrafish, Danio rerio

Systematic genome-wide mutagenesis screens for embryonic phenotypes have been instrumental in the understanding of invertebrate and plant development. Here, we report the results from the first application of such a large-scale genetic screening to vertebrate development. Male zebrafish were mutagenized with N-ethyl N-nitrosourea to induce mutations in spermatogonial cells at an average specific locus rate of one in 651 mutagenized genomes. Mutations were transmitted to the F1 generation, and 2205 F2 families were raised. F3 embryos from sibling crosses within the F2 families were screened for developmental abnormalities. A total of 2337 mutagenized genomes were analyzed, and 2383 mutations resulting in abnormal embryonic and early larval phenotypes were identified. The phenotypes of 695 mutants indicated involvement of the identified loci in specific aspects of embryogenesis. These mutations were maintained for further characterization and were classified into categories according to their phenotypes. The analyses and genetic complementation of mutations from several categories are reported in separate manuscripts. Mutations affecting pigmentation, motility, muscle and body shape have not been extensively analyzed and are listed here. A total of 331 mutations were tested for allelism within their respective categories. This defined 220 genetic loci with on average 1.5 alleles per locus. For about two-thirds of all loci only one allele was isolated. Therefore it is not possible to give a reliable estimate on the degree of saturation reached in our screen; however, the number of genes that can mutate to visible embryonic and early larval phenotypes in zebrafish is expected to be several-fold larger than the one for which we have observed mutant alleles during the screen. This screen demonstrates that mutations affecting a variety of developmental processes can be efficiently recovered from zebrafish.

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A genetic screen for mutations affecting embryogenesis in zebrafish

Cell signaling plays a key role in the development of all multicellular organisms. Numerous studies have established the importance of Hedgehog signaling in a wide variety of regulatory functions during the development of vertebrate and invertebrate organisms. Several reviews have discussed the signaling components in this pathway, their various interactions, and some of the general principles that govern Hedgehog signaling mechanisms. This review focuses on the developing systems themselves, providing a comprehensive survey of the role of Hedgehog signaling in each of these. We also discuss the increasing significance of Hedgehog signaling in the clinical setting.

Developmental roles and clinical significance of hedgehog signaling.

The notochord is a midline mesodermal structure with an essential patterning function in all vertebrate embryos. Zebrafish floating head (flh) mutants lack a notochord, but develop with prechordal plate and other mesodermal derivatives, indicating that flh functions specifically in notochord development. We show that floating head is the zebrafish homologue of Xnot, a homeobox gene expressed in the amphibian organizer and notochord. We propose that flh regulates notochord precursor cell fate.

A homeobox gene essential for zebrafish notochord development

Using fate mapping techniques, we have analyzed development of cells of the dorsal marginal region in wild-type and mutant zebrafish. We define a domain in the early gastrula that is located just at the margin and centered on the dorsal midline, in which most cells generate clones that develop exclusively as notochord. The borders of the notochord domain are sharp at the level of single cells, and coincide almost exactly with the border of the expression domain of the homeobox gene floating head (flh; zebrafish homologue of Xnot), a gene essential for notochord development. In flh mutants, cells in the notochord domain generate clones of muscle cells. In contrast, notochord domain cells form mesenchyme in embryos mutant for no tail (ntl; zebrafish homologue of Brachyury). A minority of cells in the notochord domain in wild-type embryos develop as unrestricted mesoderm, invariably located in the tail, suggesting that early gastrula expression of flh does not restrict cellular potential to the notochord fate. The unrestricted tail mesodermal fate is also expressed by the forerunner cells, a cluster of cells located outside the blastoderm, adjacent to the notochord domain. We show that cells can leave the dorsal blastoderm to join the forerunners, suggesting that relocation between fate map domains might respecify notochord domain cells to the tail mesodermal fate. An intermediate fate of the forerunners is to form the epithelial lining of Kupffer9s vesicle, a transient structure of the teleost tailbud. The forerunners appear to generate the entire structure of Kupffer9s vesicle, which also develops in most flh mutants. Although forerunner cells are present in ntl mutants, Kupffer9s vesicle never appears, which is correlated with the later severe disruption of tail development.

Specification of cell fates at the dorsal margin of the zebrafish gastrula

The zebrafish homeobox gene floating head (flh) is essential for notochord development and is one of the earliest genes to be expressed in notochord precursors. To understand how flh is regulated during notochord development, we compared the wild-type flh expression pattern to that in embryos mutant for flh and no tail (ntl), the zebrafish homologue of Brachyury. In the early gastrula, the pattern of flh expression is not affected in either flh or ntl mutants, implying that the initial establishment of a gastrula notochord domain is independent of the function of these genes. However, flh RNA is expressed at lower levels in flh mutants, suggesting that flh positively regulates its own expression. During gastrulation, flh mutants show an abrupt loss of flh expression in cells which have involuted and entered the hypoblast, while the rest of the expression pattern appears normal, thus flh+ function is specifically required to maintain flh expression in hypoblast cells. The anterior-most part of the notochord rudiment differentially maintains flh expression in both wild types and flh mutant embryos, suggesting that there is unique regulation of flh in this region of the developing notochord. In ntl mutants, the spatial pattern of flh expression is altered as early as the late gastrula stage, becoming broad and diffuse. We hypothesize that ntl+ is required for the proper convergence movements of flh-expressing cells. Dev. Dyn. 209: 156-165, 1997. © 1997 Wiley-Liss, Inc.

Anna E. Melby

Papers

A homeobox gene essential for zebrafish notochord development

Specification of cell fates at the dorsal margin of the zebrafish gastrula

Spatial regulation of floating head expression in the developing notochord