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Homeobox-containing sequences were isolated from a genomic library of zebrafish (Brachydanio rerio). A λ clone containing two homeobox cross-hybridizing regions was characterized. DNA sequencing of one of these regions (ZF-21) revealed that it contains a homeobox closely related to the Antennapedia class of Drosophila homeobox sequences. Moreover, the deduced amino acid sequence of the C-terminal end (81 residues including the homeobox) is identical to the corresponding part of the murine Hox-2.1 protein. Similar to Hox-2.1, a ZF-21 derived transcript of 2.3 kb is present in embryos at the somite forming stages.

The zebrafish genome was found to contain two sequences which cross-hybridize strongly with the engrailed gene of Drosophila. Several independent clones containing one of these cross-hybridizing sequences were isolated from a zebrafish genomic library. Characterization of this region (ZF-EN) by DNA sequencing showed that it shares about 70% sequence identity with the engrailed homeobox. More extensive homeobox homology (greater than 90%) was found relative to the murine En genes. The closest relationship exists between ZF-EN and En-2 where the C-terminal domains (104 amino acids) encoded by these genes are almost identical. We also observed that ZF-EN and En-2 are very similar with respect to their transcript sizes and temporal expression patterns.