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.

Retinal ganglion cell genesis requires lakritz, a Zebrafish atonal Homolog.

Mutation of the zebrafish lakritz (lak) locus completely eliminates the earliest-born retinal cells, the ganglion cells (RGCs). Instead, excess amacrine, bipolar, and Müller glial cells are generated in the mutant. The extra amacrines are found at ectopic locations in the ganglion cell layer. Cone photoreceptors appear unaffected by the mutation. Molecular analysis reveals that lak encodes Ath5, the zebrafish eye-specific ortholog of the Drosophila basic helix-loop-helix transcription factor Atonal. A combined birth-dating and cell marker analysis demonstrates that lak/ath5 is essential for RGC determination during the first wave of neurogenesis in the retina. Our results suggest that this wave is skipped in the mutant, leading to an accumulation of progenitors for inner nuclear layer cells.

CMIX, a paired-type homeobox gene expressed before and during formation of the avian primitive streak.

We cloned a chicken homeobox gene closely related to the Xenopus Mix. 1 gene. CMIX is expressed early in embryogenis in a sickle-shaped area in the posterior zone of the blastoderm. With the beginning of gastrulation, CMIX transcripts are found in the primitive streak primordium, then in the young and medium-sized streak, however not in the mesoderm after its emergence. In the fully-extended streak, CMIX is restricted to its middle, i.e. the prospective ventral mesoderm. CMIX RNA is undetectable by whole-mount in-situ analysis in later stages. We compare CMIX expression to the early pattern of the brachyury gene.

Nuclear beta-catenin and the development of bilateral symmetry in normal and LiCl-exposed chick embryos.

Studies in Xenopus laevis and zebrafish suggest a key role for beta-catenin in the specification of the axis of bilateral symmetry. In these organisms, nuclear beta-catenin demarcates the dorsalizing centers. We have asked whether beta-catenin plays a comparable role in the chick embryo and how it is adapted to the particular developmental constraints of chick development. The first nuclear localization of beta-catenin is observed in late intrauterine stages of development in the periphery of the blastoderm, the developing area opaca and marginal zone. Obviously, this early, radially symmetric domain does not predict the future organizing center of the embryo. During further development, cells containing nuclear beta-catenin spread under the epiblast and form the secondary hypoblast. The onset of hypoblast formation thus demarcates the first bilateral symmetry in nuclear beta-catenin distribution. Lithium chloride exposure also causes ectopic nuclear localization of beta-catenin in cells of the epiblast in the area pellucida. Embryos treated before primitive streak formation become completely radialized, as shown by the expression of molecular markers, CMIX and GSC. Lithium treatments performed during early or medium streak stages cause excessive development of the anterior primitive streak, node and notochord, and lead to a degeneration of prospective ventral and posterior structures, as shown by the expression of the molecular markers GSC, CNOT1, BMP2 and Ch-Tbx6L. In summary, we found that in spite of remarkable spatiotemporal differences, beta-catenin acts in the chick in a manner similar to that in fish and amphibia.

Segregating expression domains of two goosecoid genes during the transition from gastrulation to neurulation in chick embryos.

We report the isolation and characterization of a chicken gene, GSX, containing a homeobox similar to that of the goosecoid gene. The structure of the GSX gene and the deduced GSX protein are highly related to the previously described goosecoid gene. The two homeodomains are 74% identical. In the first few hours of chick embryogenesis, the expression pattern of GSX is similar to GSC, in the posterior margin of the embryo and the young primitive streak. Later during gastrulation, expression of the two genes segregate. GSC is expressed in the anterior part of the primitive streak, then in the node, and finally in the pre-chordal plate. GSX is expressed in the primitive streak excluding the node, and then demarcating the early neural plate around the anterior streak and overlying the pre-chordal plate. We demonstrate that the GSX-positive part of the primitive streak induces gastrulation, while the GSC-expressing part induces neurulation. After full extension of the streak, the fate of cells now characterized by GSX is to undergo neurulation, while those expressing GSC undergo gastrulation. We discuss the effect of a duplicated basic goosecoid identity for the generation of a chordate nervous system in ontogeny and phylogeny.