Enhancer detection in zebrafish: Search for developmentally important genes and regulatory sequences

We have performed a large-scale insertional screen aimed at detecting live gene expression patterns in embryonic zebrafish. For this purpose, a murine retroviral genome was engineered to contain a basal promoter from the zebrafish GATA2 gene driving a yellow fluorescent protein reporter cassette. This construct was shown to assume expression patterns according to insertion sites. We found the majority of the insertions was located in, or within 15kb of the corresponding transcriptional unit. To a surprising extent, the genes found close to the active insertions were developmental regulatory genes, in particular known transcription factors, while genes encoding other biological functions, for instance house keeping genes, were exceedingly rare. In some of the rare cases, we have determined that the expression pattern was that of a gene further away, suggesting that enhancer domains can overlap to a certain extent in vertebrate genomes.

To date, we have screened about 10000 insertions, and isolated and kept over 900 transgenic lines of fish, and have mapped 380 of them to the zebrafish genome. We are currently characterizing the cellular expression patterns and have begun to use bioinformatics to map non-coding conserved sequences around the insertion sites to isolate and test candidate enhancer sequences.

This seminar will give an overview of what can be learned about vertebrate early embryonic gene regulation and expression using the zebrafish as a model system.