Analyses Of Two Tob Family Genes In Zebrafish

A tissue-specific gene Tob1a was isolated by screening a zebrafish embryonic cDNA library with labeled mix of total RNA and by in situ hybridization screening. BLAST Search indicated the presence of a duplicate/homolog of tob1a in the genome of zebrafish. Subsequently, this deduced gene, tob1b, was cloned by PCR from a cDNA library. Either tob1a or tob1b encodes a peptide of 322 residues, which shares an identity of over 80% with mammalian Tob protein and thus belongs to new members of TOB/BTG family. The overall homology between Tob1a and Tob1b is 82%. Analyses of expression pattern reveal that transcripts of tob1a and tob1b are present in one-cell embryos throughout blastulation, suggesting a maternal origin. During subsequent stages, tob1a is expressed in the germ ring, the notochord, the hatching gland and the tail bud. The zygotic expression of tob1b occures in the middle of segmentation and persists throughout 48 h. The major tissues expressing tob1b include the notochord, somites, eyes and fin. Subcelluar localization analysis using Tob1a-GFP fusion gene indicates that Tob1a is mainly localized in the nuclei of mammalian cells, suggesing a function on gene transcription. Overexpression of the C-terminal domain of Tob1a, which lacks N-terminal BoxA and BoxB, leads to dorsalization of the injected embryos, slight increase of the embryonic shield marker goosecoid, and decrease of the ventral marker eve1. It suggests that Tob1a plays a role in regulating dorsal-ventral patterning of zebrafish embryos. However, the specific functions of Tob proteins and the involved mechanism need to be further studied.