Functional study of retinoic acidactivated regulatory sequences and their role in the expression of hox genes in zebrafish

Abstract

This master’s thesis examines the functional role of retinoic acid-responsive regulatory sequences and their impact on the expression of hox genes in zebrafish (Danio rerio). Hox genes are crucial for vertebrate embryonic development, particularly in body segmentation and the formation of structures along the anterior-posterior axis. Retinoic acid, a metabolite of vitamin A, plays a key role in regulating the expression of these genes. In this study, we focused our investigations on the role of four "RARE" regions, R1 to R4, located far downstream of the hoxbb complex (between 10 and 60 kb) and within the neighbouring skap1 gene. Since the skap1 gene is not regulated by retinoic acid, these R1-R4 regions were suspected to regulate hoxbb genes over a long distance. We tested this hypothesis by analysing the expression profile of four hox genes—hoxb1b, hoxb5b, hoxb6b, and hoxb8b—at various developmental stages in mutants where the R1 to R4 regions were removed. The results revealed that deletion of sequences containing the R2, R3, and R4 elements significantly decreased the expression of the four tested hoxbb genes. The expression levels of hox genes were more significantly reduced at the late stage (48 hpf) than at early stages (15 hpf). However, we observed that at the 15 hpf stage, the expression profile of the hoxb6b gene was altered, showing a shift in expression towards the posterior part of the embryo. Surprisingly, the deletion of the R2 region alone did not significantly alter the expression of the hoxbb genes, although this region is the most conserved among the four RARE sequences. This suggests potential functional redundancy or compensation, particularly between the R2 and R4 regions. Experiments using transgenic lines, where the R2 and R3 sequences were inserted upstream of the cfos promoter linked to GFP-coding sequences, demonstrated that the R3 region is not sufficient to confer retinoic acid regulation, in contrast to the R2-cfos line. DEAB treatments on the R2 line showed that this region was more active during somitogenesis and remained active between 24 and 48 hpf. These results were consistent with observations from the RARE element deletion experiments. Although further studies are necessary for a more comprehensive understanding of the actions of these regions, this research has significantly deepened our knowledge of the molecular mechanisms governing the regulation of hoxbb genes by retinoic acid in zebrafish.