Fig. 1

Fig. 1

Identification of the transcriptional regulatory region that drives hes6 expression in the PSM and tailbud of zebrafish embryos. (A) Schematic representation of the genomic region that encompasses hes6 in zebrafish. Exons in each gene are shown by dark brown boxes and the arrows indicate the direction of transcription. The numbers above the genomic region represents the distance from the translation start site in hes6. The genomic regions encompassed by the four BAC clones used in this study are shown below the genomic region. The insert length of each BAC clone is shown above the bar. The green boxes represent the EGFP reporter inserted in the first exon of hes6 in each BAC clone. At this scale, the hes6 gene is represented by a single box; however, hes6 actually comprises four exons. (B) Injection of the CHORI73-20O6 BAC EGFP reporter at a concentration of 10 ng/µl induced EGFP signals in the PSM and tailbud regions at the 6-somite stage; lateral view. (C) Dissection of the flanking genomic region of hes6 from the CHORI73-20O6 EGFP BAC reporter via BAC homologous recombination. The genomic regions encompassed by the deleted BAC clones are indicated at the right of each bar. Each modified BAC clone was injected into embryos at a concentration of 10 ng/µl and the presence of the EGFP signal in the PSM and tailbud was examined at the 10–15-somite stage. (D) Deletion analyses of the intergenic region between hes6 and per2. Each genomic DNA was isolated and inserted upstream of the hsp70l-EGFP reporter. The constructed plasmids were injected into embryos at a concentration of 10 ng/µl. Enhancer activities in the PSM and tailbud were examined at the 10–15-somite stage. (E) The 1.7-kb DNA fragment, located 2.8-kb downstream of the hes6 translation initiation site, was inserted upstream of the hsp70l-EGFP reporter. Injection of this EGFP reporter gene at a concentration of 10 ng/µl produced specific EGFP signals in the PSM and tailbud at the 16-somite stage. Scale bars in B and E are 200 µm.