Michael A. Gates

Zebrafish organizer development and germ-layer formation require nodal-related signals

The vertebrate body plan is established during gastrulation, when cells move inwards to form the mesodermal and endodermal germ layers. Signals from a region of dorsal mesoderm, which is termed the organizer, pattern the body axis by specifying the fates of neighbouring cells,. The organizer is itself induced by earlier signals. Although members of the transforming growth factor-β (TGF-β) and Wnt families have been implicated in the formation of the organizer, no endogenous signalling molecule is known to be required for this process. Here we report that the zebrafish squint (sqt) and cyclops (cyc) genes have essential, although partly redundant, functions in organizer development and also in the formation of mesoderm and endoderm. We show that the sqt gene encodes a member of the TGF-β superfamily that is related to mouse nodal. cyc encodes another nodal-related protein,, which is consistent with our genetic evidence that sqt and cyc have overlapping functions. The sqt gene is expressed in a dorsal region of the blastula that includes the extraembryonic yolk syncytial layer (YSL). The YSL has been implicated as a source of signals that induce organizer development and mesendoderm formation,. Misexpression of sqt RNA within the embryo or specifically in the YSL induces expanded or ectopic dorsal mesoderm. These results establish an essential role for nodal-related signals in organizer development and mesendoderm formation.

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.

Mapping of Mhc class I and class II regions to different linkage groups in the zebrafish, Danio rerio

Abstract The mammalian major histocompatibility complex (Mhc) consists of three closely linked regions, I, II, and III, occupying a single chromosomal segment. The class I loci in region I and the class II loci in region II are related in their structure, function, and evolution. Region III, which is intercalated between regions I and II, contains loci unrelated to the class I and II loci, and to one another. There are indications that a similar Mhc organization exists in birds and amphibians. Here, we demonstrate that in the zebrafish (Danio rerio), a representative of the teleost fishes, the class II loci are divided between two linkage groups which are distinct from the linkage group containing the class I loci. The β2-microglobulin-encoding gene is loosely linked to one of the class II loci. The gene coding for complement factor B, which is one of the region III genes in mammals, is linked neither to the class I nor to the class II loci in the zebrafish. These results, combined with preliminary data suggesting that the class I and class II regions in another order of teleost fish are also in different linkage groups, indicate that close linkage of the two regions is not necessary either for regulation of expression or for co-evolution of the class I and class II loci. They also raise the question of whether linkage of the class I and class II loci in tetrapods is a primitive or derived character.

fast1 is required for the development of dorsal axial structures in zebrafish

Nodal-related signals comprise a subclass of the transforming growth factor-beta (TGF-beta) superfamily and regulate key events in vertebrate embryogenesis, including mesoderm formation, establishment of left-right asymmetry and neural patterning [1-8]. Nodal ligands are thought to act with EGF-CFC protein co-factors to activate activin type I and II or related receptors, which phosphorylate Smad2 and trigger nuclear translocation of a Smad2/4 complex [8-12]. The winged-helix transcription factor forkhead activin signal transducer-1 (Fast-1) acts as a co-factor for Smad2 [12-20]. Xenopus Fast-1 is thought to function as a transcriptional effector of Nodal signals during mesoderm formation [17], but no mutations in the Fast-1 gene have been identified. We report the identification of the zebrafish fast1 gene and show that it is disrupted in schmalspur (sur) mutants, which have defects in the development of dorsal midline cell types and establishment of left-right asymmetry [21-25]. We find that prechordal plate and notochord are strongly reduced in maternal-zygotic sur mutants, whereas other mesendodermal structures are present - a less severe phenotype than that caused by complete loss of Nodal signaling. These results show that fast1 is required for development of dorsal axial structures and left-right asymmetry, and suggest that Nodal signals act through Fast1-dependent and independent pathways.

Cloning of two loci for synapse protein Snap25 in zebrafish: Comparison of paralogous linkage groups suggests loss of one locus in the mammalian lineage

Synaptosome‐associated protein of 25 kDa (Snap25) is an intracellular protein that is defined as a target receptor for synapse vesicles prior to neurotransmitter release. Snap25 is highly conserved, with 61% identity between human and Drosophila melanogaster. Whereas mammals and chicken have a single locus for Snap25, the tetraploid goldfish has at least three loci. We report that the zebrafish has two loci with 91% amino acid identity to each other. The alternative splicing of exon 5 arose before the gene duplication. The expression patterns of the two loci are virtually identical in adult zebrafish. The two zebrafish snap25 loci are located in paralogous linkage groups that seem to correspond to human chromosome 20, which harbors the SNAP locus, and human chromosome 14. Because no additional Snap25 homologue has been reported for any mammal or chicken, snap25.2 may have been lost in the amniote or even tetrapod lineage. J. Neurosci. Res. 54:563–573, 1998.

Using Random Amplified Polymorphic DNAs in Zebrafish Genomic Analysis

Publisher Summary This chapter explores random amplified polymorphic DNAs (RAPDs) and its help in zebrafish genomic analysis. The chapter discusses its identification, various types of RAPDs and their advantages and disadvantages with respect to other genetic markers and, finally, the use of RAPDs in constructing maps and mapping mutations. RAPDs have been used extensively as a method to generate a genetic map for a species in a short period of time. RAPDs are only one of the many types of genetic markers that are suitable for building genetic maps. There are several ways to detect DNA polymorphisms that have been used with success in zebrafish research. These can be divided into two basic strategies: the first requires availability of a locus as a clone, which is either directly used as a probe, or which is sequenced to provide information for the design of polymerase chain reaction (PCR) primers. This category includes classical restriction fragment length polymorphisms (RFLPs), microsatellite markers, and single strand conformation polymorphisms (SSCPs). Another category of DNA polymorphisms requires no previous knowledge of the loci identified; these include RAPDs and arbitrary fragment length polymorphisms (AFLPs). Each type has its own peculiar advantages, and different methods are more useful under different situations.