Zebrafish transgenic lines co-expressing a hybrid Gal4 activator and eGFP in tissue-restricted patterns.

We have used a Tol2-derived trapping vector, carrying a hybrid Gal4 gene and a UAS:eGFP reporter cassette, to identify 16 transgenic zebrafish lines expressing the fluorescent marker eGFP in tissue-restricted patterns during development. Most lines show co-expression of eGFP and a hybrid Gal4 transcription activator containing a truncated VP16 domain that facilitate induction of other UAS-transgenes (UAS:RFP). Notably, many of the transgenic lines are expressed in particular areas of the central nervous system, such as the retina. We mapped the genomic positions of most of the activated insertions, and for three retina-specific lines we also demonstrate that eGFP reports the expression of particular endogenous genes. One of the identified zebrafish genes shows expression in ventral retina, and encodes a protein containing a repulsive guidance molecule (RGM) domain, suggesting a role in axonal guidance during optic nerve formation. Among the lines labeling other tissues, three show early co-expression of eGFP and Gal4-VP16 in blood vessels, erythrocytes and other hematopoietic cells. Interestingly, the activated insertion in the erythrocyte line was mapped to a site near the globin cluster on chromosome 3. All the reported lines co-expressing eGFP and the hybrid Gal4 activator may have potential as genetic tools to study developmental processes.

Autoregulatory binding sites in the zebrafish six3a promoter region define a new recognition sequence for Six3 proteins.

The homeodomain (HD) transcription factor Six3, which is a member of the Six/Sine oculis family, is essential for development of the eyes and forebrain in vertebrates. It has recently been claimed that the HDs of Six3 and other members of the Six family have a common recognition sequence, TGATAC. However, a different recognition sequence including the typical TAAT core motif, which has not yet been fully defined, has also been proposed for the Six3 HD in mice. Our study of the zebrafish orthologue six3a, which has an identical HD, shows that it binds in vitro to multiple TAAT-containing sites within its promoter region. Comparison of the different binding affinities for these sequences identifies three high-affinity sites with a common TAATGTC motif. Notably, this new recognition sequence, which is supported by our analysis of the influence of single-nucleotide substitutions on the DNA-binding affinity, is distinct from all of the DNA-binding specificities previously described in surveys of HDs. In addition, our comparison of Six3a HD binding to the novel TAATGTC motif and the common recognition sequence of Six family HDs (TGATAC) shows very similar affinities, suggesting two distinct DNA-binding modes. Transient reporter assays of the six3a promoter in zebrafish embryos also indicate that the three high-affinity sites are involved in autoregulation. In support of this, chromatin immunoprecipitation experiments show enrichment of Six3a binding to a six3a promoter fragment containing two clustered high-affinity sites. These findings provide strong evidence that the TAATGTC motif is an important target sequence for vertebrate Six3 proteins in vivo.

Distinct expression of two foxg1 paralogues in zebrafish.

The forkhead proteins (Fox) act as transcription factors in many biological processes in a wide range of species. One member of this superfamily, Foxg1, has essential roles in the development of eyes, telencephalon, ears and olfactory system. Zebrafish foxg1 has been reported to have similar roles as the mouse orthologue Foxg1. However, no data has been reported about possible zebrafish foxg1 paralogues. In this study we identified one zebrafish foxg1 paralogue by enhancer trapping, which we designate foxg1b. A more diverged paralogue, foxg1c, was identified by homology searches. Sequence comparisons indicate that both foxg1b and foxg1c are less related to mouse than the previously characterized foxg1. We report that foxg1b is expressed in a regionally restricted pattern within the developing eye, mainly in the dorsal-nasal retina, which is similar to the retinal expression of mouse Foxg1. By contrast, foxg1c is only expressed transiently in the eyes and forebrain between 14 and 20h post-fertilization, while expression was detected exclusively in the developing inner ear at later stages. Our results suggest that foxg1b and foxg1c have undergone expression pattern divergence during evolution that has resulted in functional specialization.