Masaaki Okuno

Identification, Tissue Expression, and Functional Characterization of Otx3, a Novel Member of the Otx Family

Transcription factors containing a homeodomain play an important role in the organogenesis of vertebrates. We have isolated a novel homeodomain transcription factor, Otx3, which is structurally and functionally related to Otx1 and Otx2, transcription factors that are critical in brain morphogenesis. Mouse Otx3 is a protein composed of 376 amino acids. Otx3 mRNA was expressed in mouse embryos from 10.5 to 13.5 days postcoitum (dpc) and in adult cerebellum as assessed by Northern blotting. Whole-mountin situ hybridization of mouse embryos from 9.5 to 11.5 dpc revealed strong expression of Otx3 mRNA in the diencephalon, mesencephalon, metencephalon, myelencephalon, and developing eye, indicating an expression pattern largely overlapping but distinct from those of Otx1 and Otx2. In addition, Otx3 was shown by electrophoretic mobility shift assay to bind to the TAATCC motif, the consensus binding sequence for Otx1, Otx2, and Crx. Results of a transcription reporter assay suggest that Otx3 functions as a transcription repressor by binding to this motif. These results suggest that Otx3 is a novel member of the Otx family and may be involved in the development of the central nervous system.

Functional analysis of transcriptional repressor Otx3/Dmbx1

Otx3/Dmbx1 is a member of paired class homeodomain transcription factors. In this study, we found that Otx3/Dmbx1 represses the Otx2‐mediated transactivation by forming heterodimer with Otx2 on the P3C (TAATCCGATTA) sequence in vitro. The 156 amino acid region (residues 1–156) of Otx3/Dmbx1 is required for its repressor activity, and interacts directly with Otx2. Co‐localization of Otx3/Dmbx1 and Otx2 in brain sections was confirmed by in situ hybridization. These data suggest that Otx3/Dmbx1 represses Otx2‐mediated transcription in the developing brain. We also identified the consensus binding sequence [TAATCCGATTA and TAATCC(N2‐4)TAATCC] of Otx3/Dmbx1.