Advantage of sea urchin embryos for the analysis of GRN Koji Akasaka Misaki Marine Biological Station, Graduate School of Science, The University of Tokyo Sea urchin embryos have made great contribution to the study of gene regulatory network (GNR) in metazoans. The availability of huge numbers of synchronized embryos, sufficient amounts of nuclei extract to use for purification of transcription factors, and the use of an in vivo transcription system using developing embryonic cells have all shown that sea urchin embryos are extraordinary animals for research. Furthermore, morpholino antisense oligonucleotides have accelerated the study of GRN in sea urchin embryos. Taking advantage of superior properties of sea urchin embryos, we analyzed mechanism of regulation of gene expression of arylsulfatase (Ars), which serves as a model gene for study of spatial and temporal gene expression. The major enhancer element of Ars composed of the Otx target sequence and CAAT sequences is located in the 1st intron. Otx binding sites alone have little effect on the activity of an Ars promoter, but when both Otx binding sites and CAAT sequences are present in the enhancer region of Ars, the DNA fragment shows a high enhancer activity. A gel mobility shift assay reveals that Otx and CAAT box binding proteins bind to the enhancer. The activation domain of Otx resides in the C terminal region. The N-terminal region is responsible for the enhancement of transactivation of the Ars promoter, although that region itself does not function as an activation domain. These findings suggest that Otx regulates Ars by interacting with different co-factors in sea urchin development. Screening of target genes using morpholino antisense oligonucleotides is a powerful method for analysis of GRN; although, these findings suggest that black box still remains poised between the regulatory gene and the target gene. In order to uncover the black box, analysis of coordination with different transcription factors and interactions of these transcription factors with the cis-regulatory elements is required. Experimental system using sea urchin embryos should contribute to clarification of the molecular mechanisms of regulation of gene expression and contribute to the network diagram of the GNR.

Advantage of sea urchin embryos for the analysis of GRN

Koji Akasaka

Misaki Marine Biological Station, Graduate School of Science, The University of Tokyo

Sea urchin embryos have made great contribution to the study of gene regulatory network (GNR) in metazoans. The availability of huge numbers of synchronized embryos, sufficient amounts of nuclei extract to use for purification of transcription factors, and the use of an in vivo transcription system using developing embryonic cells have all shown that sea urchin embryos are extraordinary animals for research. Furthermore, morpholino antisense oligonucleotides have accelerated the study of GRN in sea urchin embryos. Taking advantage of superior properties of sea urchin embryos, we analyzed mechanism of regulation of gene expression of arylsulfatase (Ars), which serves as a model gene for study of spatial and temporal gene expression. The major enhancer element of Ars composed of the Otx target sequence and CAAT sequences is located in the 1st intron. Otx binding sites alone have little effect on the activity of an Ars promoter, but when both Otx binding sites and CAAT sequences are present in the enhancer region of Ars, the DNA fragment shows a high enhancer activity. A gel mobility shift assay reveals that Otx and CAAT box binding proteins bind to the enhancer. The activation domain of Otx resides in the C terminal region. The N-terminal region is responsible for the enhancement of transactivation of the Ars promoter, although that region itself does not function as an activation domain. These findings suggest that Otx regulates Ars by interacting with different co-factors in sea urchin development. Screening of target genes using morpholino antisense oligonucleotides is a powerful method for analysis of GRN; although, these findings suggest that black box still remains poised between the regulatory gene and the target gene. In order to uncover the black box, analysis of coordination with different transcription factors and interactions of these transcription factors with the cis-regulatory elements is required. Experimental system using sea urchin embryos should contribute to clarification of the molecular mechanisms of regulation of gene expression and contribute to the network diagram of the GNR.