Molecular basis for functional diversification of duplicated hoxb5 genes in zebrafish

Abstract

The Duplication-Degeneration-Complementation (DDC) model predicts that division of the original function between duplicate genes (subfunctionalization) is a common mechanism for their preservation. The Hox genes constitute a good system to test this hypothesis as they underwent significant expansion during evolution. Mammals have four Hox complexes whereas teleosts have additional complexes resulting from duplication event(s) that took place after the separation of the ray-finned and lobe-finned lineages. Thus, zebrafish and Takifugu have two hoxb complexes, each containing a hoxb5 gene. The zebrafish hoxb5a and hoxb5b genes are expressed in overlapping, yet distinct, domains during development with their combined expression patterns similar to that of the single mouse Hoxb5 gene. To determine if changes in cis-acting regulatory elements account for hoxb5a and hoxb5b subfunctionalization, we compared the sequences of the Hoxb5 loci of human, mouse, zebrafish and Takifugu and identified four conserved non-coding elements (CNEs). One of the CNEs, named J3, is only retained in the hoxb5a locus while three others are present in both teleost hoxb5 loci. We examined individual and collective regulatory activity of the CNEs in transgenic assays. When tested individually, the enhancer activity of individual cognate CNEs from the hoxb5a and hoxb5b genes extensively overlapped. In contrast, large fragments encompassing multiple CNEs were able to target reporter gene expression to unique domains of hoxb5a and hoxb5b expression. The deletion of J3 from the hoxb5a locus and its insertion in the hoxb5b locus drastically altered the regulatory activity of the original loci. Our results suggest that patterns of regulatory evolution of hoxb5 duplicates in teleosts may be more complex than ones taken in consideration by the DDC model.