Genetic screens for mutations affecting adult traits and parental-effect genes.

Forward genetics remains an important approach for the unbiased identification of factors involved in biological pathways. Forward genetic analysis in the zebrafish has until now largely been restricted to the developmental period from zygotic genome activation through the end of embryogenesis. However, the use of the zebrafish as a model system for the analysis of late larval, juvenile and adult traits, including fertility and maternal and paternal effects, continues to gain momentum. Here, we describe two approaches, based on an F3-extended family and gynogenetic methods, that allow genetic screening for, and recovery of mutations affecting post-embryonic stages, including adult traits, fertility, and parental effects. For each approach, we also describe strategies to maintain, map, and molecularly clone the identified mutations.

Zebrafish vasa RNA but not its protein is a component of the germ plasm and segregates asymmetrically before germline specification.

Work in different organisms revealed that the vasa gene product is essential for germline specification. Here, we describe the asymmetric segregation of zebrafish vasa RNA, which distinguishes germ cell precursors from somatic cells in cleavage stage embryos. At the late blastula (sphere) stage, vasa mRNA segregation changes from asymmetric to symmetric, a process that precedes primordial germ cell proliferation and perinuclear localization of Vasa protein. Analysis of hybrid fish between Danio rerio and Danio feegradei demonstrates that zygotic vasa transcription is initiated shortly after the loss of unequal vasa mRNA segregation. Blocking DNA replication indicates that the change in vasa RNA segregation is dependent on a maternal program. Asymmetric segregation is impaired in embryos mutant for the maternal effect gene nebel. Furthermore, ultrastructural analysis of vasa RNA particles reveals that vasa RNA, but not Vasa protein, localizes to a subcellular structure that resembles nuage, a germ plasm organelle. The structure is initially associated with the actin cortex, and subsequent aggregation is inhibited by actin depolymerization. Later, the structure is found in close proximity of microtubules. We previously showed that its translocation to the distal furrows is microtubule dependent. We propose that vasa RNA but not Vasa protein is a component of the zebrafish germ plasm. Triggered by maternal signals, the pattern of germ plasm segregation changes, which results in the expression of primordial germ cell-specific genes such as vasa and, consequently, in germline fate commitment.

A mutation in the zebrafish maternal-effect gene nebel affects furrow formation and vasa RNA localization.

BACKGROUND: In many animals, embryonic patterning depends on a careful interplay between cell division and the segregation of localized cellular components. Both of these processes in turn rely on cytoskeletal elements and motor proteins. A type of localized cellular component found in most animals is the germ plasm, a specialized region of cytoplasm that specifies the germ-cell fate. The gene vasa has been shown in Drosophila to encode an essential component of the germ plasm and is thought to have a similar function in other organisms. In the zebrafish embryo, the vasa RNA is localized to the furrows of the early cellular divisions. RESULTS: We identified the gene nebel in a pilot screen for zebrafish maternal-effect mutations. Embryos from females homozygous for a mutation in nebel exhibit defects in cell adhesion. Our analysis provides genetic evidence for a function of the microtubule array that normally develops at the furrow in the deposition of adhesive membrane at the cleavage plane. In addition, nebel mutant embryos show defects in the early localization of vasa RNA. The vasa RNA localization phenotype could be mimicked with microtubule-inhibiting drugs, and confocal microscopy suggests an interaction between microtubules and vasa-RNA-containing aggregates. CONCLUSIONS: Our data support two functions for the microtubule reorganization at the furrow, one for the exocytosis of adhesive membrane, and another for the translocation of vasa RNA along the forming furrow.

A radiation hybrid map of the zebrafish genome.

Recent large-scale mutagenesis screens have made the zebrafish the first vertebrate organism to allow a forward genetic approach to the discovery of developmental control genes. Mutations can be cloned positionally, or placed on a simple sequence length polymorphism (SSLP) map to match them with mapped candidate genes and expressed sequence tags (ESTs). To facilitate the mapping of candidate genes and to increase the density of markers available for positional cloning, we have created a radiation hybrid (RH) map of the zebrafish genome. This technique is based on somatic cell hybrid lines produced by fusion of lethally irradiated cells of the species of interest with a rodent cell line. Random fragments of the donor chromosomes are integrated into recipient chromosomes or retained as separate minichromosomes. The radiation-induced breakpoints can be used for mapping in a manner analogous to genetic mapping, but at higher resolution and without a need for polymorphism. Genome-wide maps exist for the human, based on three RH panels of different resolutions, as well as for the dog, rat and mouse. For our map of the zebrafish genome, we used an existing RH panel and 1,451 sequence tagged site (STS) markers, including SSLPs, cloned candidate genes and ESTs. Of these, 1,275 (87.9%) have significant linkage to at least one other marker. The fraction of ESTs with significant linkage, which can be used as an estimate of map coverage, is 81.9%. We found the average marker retention frequency to be 18.4%. One cR3000 is equivalent to 61 kb, resulting in a potential resolution of approximately 350 kb.

Function of zebrafish beta-catenin and TCF-3 in dorsoventral patterning.

We report the molecular cloning and expression of the zebrafish tcf-3 homologue and study its function and that of zebrafish betacat in dorsoventral patterning. Overexpression of mutant zTcf-3 products and Cadherin leads to a reduction in the expression of the dorsal-specific genes goosecoid and chording at the blastula stages, indicating a conserved role for betacat and tcf-3 in zebrafish dorsal axis induction. Later during gastrulation, overexpression of these same products leads to the ectopic expression of dorsal-specific genes in the marginal zone and the induction of ectopic axes, suggesting an additional role for betacat and Tcf-3 at these later stages in the repression of dorsal fates.

dino and mercedes, two genes regulating dorsal development in the zebrafish embryo.

We describe two genes, dino and mercedes, which are required for the organization of the zebrafish body plan. In dino mutant embryos, the tail is enlarged at the expense of the head and the anterior region of the trunk. The altered expression patterns of various marker genes reveal that, with the exception of the dorsal most marginal zone, all regions of the early dino mutant embryo acquire more ventral fates. These alterations are already apparent before the onset of gastrulation. mercedes mutant embryos show a similar but weaker phenotype, suggesting a role in the same patterning processes. The phenotypes suggests that dino and mercedes are required for the establishment of dorsal fates in both the marginal and the animal zone of the early gastrula embryo. Their function in the patterning of the ventrolateral mesoderm and the induction of the neuroectoderm is similar to the function of the Spemann organizer in the amphibian embryo.

Mutations affecting development of the midline and general body shape during zebrafish embryogenesis.

Tissues of the dorsal midline of vertebrate embryos, such as notochord and floor plate, have been implicated in inductive interactions that pattern the neural tube and somites. In our screen for embryonic visible mutations in the zebrafish we found 113 mutations in more than 27 genes with altered body shape, often with additional defects in CNS development. We concentrated on a subgroup of mutations in ten genes (the midline-group) that cause defective development of the floor plate. By using floor plate markers, such as the signaling molecule sonic hedgehog, we show that the schmalspur (sur) gene is needed for early floor plate development, similar to one-eyed-pinhead (oep) and the previously described cyclops (cyc) gene. In contrast to oep and cyc, sur embryos show deletions of ventral CNS tissue restricted to the mid- and hindbrain, whereas the forebrain appears largely unaffected. In the underlying mesendodermal tissue of the head, sur is needed only for development of the posterior prechordal plate, whereas oep and cyc are required for both anterior and posterior prechordal plate development. Our analysis of sur mutants suggests that defects within the posterior prechordal plate may cause aberrant development of ventral CNS structures in the mid- and hindbrain. Later development of the floor plate is affected in mutant chameleon, you-too, sonic-you, iguana, detour, schmalhans and monorail embryos; these mutants often show additional defects in tissues that are known to depend on signals from notochord and floor plate. For example, sur, con and yot mutants show reduction of motor neurons; median deletions of brain tissue are seen in sur, con and yot embryos; and cyc, con, yot, igu and dtr mutants often show no or abnormal formation of the optic chiasm. We also find fusions of the ventral neurocranium for all midline mutants tested, which may reveal a hitherto unrecognized function of the midline in influencing differentiation of neural crest cells at their destination. As a working hypothesis, we propose that midline-group genes may act to maintain proper structure and inductive function of zebrafish midline tissues.

Mutations affecting morphogenesis during gastrulation and tail formation in the zebrafish, Danio rerio.

We have identified several genes that are required for various morphogenetic processes during gastrulation and tail formation. Two genes are required in the anterior region of the body axis: one eyed pinhead (oep) and dirty nose (dns).oep mutant embryos are defective in prechordal plate formation and the specification of anterior and ventral structures of the central nervous system. In dns mutants, cells of the prechordal plate, such as the prospective hatching gland cells, fail to specify. Two genes are required for convergence and extension movements. In mutant trilobite embryos, extension movements on the dorsal side of the embryo are affected, whereas in the formerly described spadetail mutants, for which two new alleles have been isolated, convergent movements of ventrolateral cells to the dorsal side are blocked. Two genes are required for the development of the posterior end of the body axis. In pipetail mutants, the tailbud fails to move ventrally on the yolk sac after germ ring closure, and the tip of the tail fails to detach from the yolk tube. Mutants in kugelig (kgg) do not form the yolk tube at the posterior side of the yolk sac.

A role of polycomb group genes in the regulation of gap gene expression in Drosophila.

Anteroposterior polarity of the Drosophila embryo is initiated by the localized activities of the maternal genes, bicoid and nanos, which establish a gradient of the hunchback (hb) morphogen. nanos determines the distribution of the maternal Hb protein by regulating its translation. To identify further components of this pathway we isolated suppressors of nanos. In the absence of nanos high levels of Hb protein repress the abdomen-specific genes knirps and giant. In suppressor-of-nanos mutants, knirps and giant are expressed in spite of high Hb levels. The suppressors are alleles of Enhancer of zeste (E(z)) a member of the Polycomb group (Pc-G) of genes. We show that E(z), and likely other Pc-G genes, are required for maintaining the expression domains of knirps and giant initiated by the maternal Hb protein gradient. We have identified a small region of the knirps promoter that mediates the regulation by E(z) and hb. Because Pc-G genes are thought to control gene expression by regulating chromatin, we propose that imprinting at the chromatin level underlies the determination of anteroposterior polarity in the early embryo.

Developmental regulation of embryonic genes in plants.

Somatic embryogenesis from cultured carrot cells progresses through successive morphogenetic stages termed globular, heart, and torpedo. To understand the molecular mechanisms underlying plant embryogenesis, we isolated two genes differentially expressed during embryo development. The expression of these two genes is associated with heart-stage embryogenesis. By altering the culture conditions and examining their expressions in a developmental variant cell line, we found that these genes were controlled by the developmental program of embryogenesis and were not directly regulated by 2,4-dichlorophenoxyacetic acid, the growth regulator that promotes unorganized growth of cultured cells and suppresses embryo morphogenesis. These genes are also expressed in carrot zygotic embryos but not in seedlings or mature plants.