Objective To study the role and related mechanisms of pax5 gene during the development of the embryos, especially in the early development of B cell and midbrain, and to observe the expression of pax5 gene in wildtype zebrafish embryos. MethodsTotal RNA of Tuebingen wildtype zebrafish embryos was extracted to obtain cDNA of pax5 gene by RT-PCR with specific primers. The cDNA of pax5 gene and pCS2+ vector were double digested with EcoRⅠ and XbaⅠ, and then ligated by T4 DNA ligase. The recombinant vector was verified by double digestion, colony PCR screening and sequencing. The verified recombinant vector was then used to synthesize digoxin labeled anti-sense mRNA probe of pax5 gene using T3 RNA polymerase in vitro transcription system. The generated probes were used to detect pax5 gene expression in zebrafish embryos by whole-mount in situ hybridization. ResultsThe pCS2+-pax5 recombinant plasmid and the probe of digoxin-labeled anti-sense mRNA of pax5 gene was successfully constructed. Whole-mount in situ hybridization showed that pax5 gene was expressed in the cerebellum and midbrain hindbrain boundary from 18-72 h post-fertilization(hpf). In the cochlea pax5 gene was expressed from 24-72 hpf and the expression was increased as time went by. From 18-48 hpf pax5 gene is found in the notochord. ConclusionPax5 gene is highly expressed in the brain and notochord of zebrafish embryos. Pax5 gene expression is found in the cochlea of the Tuebingen wildtype zebrafish’s embryos for the first time. It is suggested that pax5 may play an important role in the development of the nervous system in the early zebrafish embryos.