We report evidence for a mechanism for the maintenance of long-range conserved synteny across vertebrate genomes. We found the largest mammal-teleost conserved chromosomal segments to be spanned by highly conserved noncoding elements (HCNEs), their developmental regulatory target genes, and phylogenetically and functionally unrelated "bystander" genes. Bystander genes are not specifically under the control of the regulatory elements that drive the target genes and are expressed in patterns that are different from those of the target genes. Reporter insertions distal to zebrafish developmental regulatory genes pax6.1/2, rx3, id1, and fgf8 and miRNA genes mirn9-1 and mirn9-5 recapitulate the expression patterns of these genes even if located inside or beyond bystander genes, suggesting that the regulatory domain of a developmental regulatory gene can extend into and beyond adjacent transcriptional units. We termed these chromosomal segments genomic regulatory blocks (GRBs). After whole genome duplication in teleosts, GRBs, including HCNEs and target genes, were often maintained in both copies, while bystander genes were typically lost from one GRB, strongly suggesting that evolutionary pressure acts to keep the single-copy GRBs of higher vertebrates intact. We show that loss of bystander genes and other mutational events suffered by duplicated GRBs in teleost genomes permits target gene identification and HCNE/target gene assignment. These findings explain the absence of evolutionary breakpoints from large vertebrate chromosomal segments and will aid in the recognition of position effect mutations within human GRBs.

Murine retroviral vectors carrying an enhancer detection cassette were used to generate 95 transgenic lines of fish in which reporter expression is observed in distinct patterns during embryonic development. We mapped 65 insertion sites to the as yet unfinished zebrafish genome sequence. Many integrations map close to previously known developmental genes, including transcription factors of the Pax, Hox, Sox, Pou, Otx, Emx, zinc-finger and bHLH gene families. In most cases, the activated provirus is located in, or within a 15 kb interval around, the corresponding transcriptional unit. The exceptions include four insertions into a gene desert on chromosome 20 upstream of sox11b, and an insertion upstream of otx1. In these cases, the activated insertions are found at a distance of between 32 kb and 132 kb from the coding region. These as well as seven other insertions described here identify genes that have recently been associated with ultra conserved non-coding elements found in all vertebrate genomes.

The zebrafish (Danio rerio) is a useful model for genetic studies of vertebrate development. Its embryos are transparent and develop rapidly outside the mother, making it feasible to visualize and manipulate specific cell types in the living animal. Zebrafish is well suited for transgenic manipulation since it is relatively easy to collect large numbers of embryos from adult fish. Several approaches have been developed for introducing transgenes into the zebrafish germline, from the injection of naked DNA to transposon-mediated integration. In particular, the Tol2 transposable element has been shown to create insertions in the zebrafish genome very efficiently. By using Tol2, gene trap and enhancer trap vectors containing the GFP reporter gene or yeast transcription activator Gal4 gene have been developed. Here we outline methodology for creating transgenic zebrafish using Tol2 vectors, and their applications to visualization and manipulation of specific tissues or cells in vivo and for functional studies of vertebrate neural circuits.

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Anteroposterior patterning of the vertebrate forebrain during gastrulation involves graded Wnt signaling, which segregates anterior fields (telencephalon and eye) from the diencephalon. How the telencephalic and retinal primordia are subsequently subdivided remains largely unknown. We demonstrate that at late gastrulation the Paired-like homeodomain transcription factor Rx3 biases cell specification choices towards the retinal fate within a population of bipotential precursors of the anterior forebrain: direct cell tracing demonstrates that retinal precursors acquire a telencephalic fate in embryos homozygous for the rx3-null allele ckh ne2611 , characterized by an enlarged telencephalon and a lack of eyes. Chimera analyses further indicate that this function of Rx3 is cell autonomous. Transfating of the eye field in the absence of Rx3 function correlates with a substantial posterior expansion of expression of the Wnt antagonist Tlc and the winged-helix transcription factor Foxg1. These results suggest that the process segregating the telencephalic and eye fields is isolated from diencephalic patterning, and is mediated by Rx3.