Evolution of cis elements in the differential expression of two Hoxa2 coparalogous genes in pufferfish ( Takifugu rubripes )

Abstract

Sequence divergence in cis-regulatory elements is an important mechanism contributing to functional diversity of genes during evolution. Gene duplication and divergence provide an opportunity for selectively preserving initial functions and evolving new activities. Many vertebrates have 39 Hox genes organized into four clusters (Hoxa-Hoxd); however, some ray-finned fishes have extra Hox clusters. There is a single Hoxa2 gene in most vertebrates, whereas fugu (Takifugu rubripes) and medaka (Oryzias latipes) have two coparalogous genes [Hoxa2(a) and Hoxa2(b)]. In the hindbrain, both genes are expressed in rhombomere (r) 2, but only Hoxa2(b) is expressed in r3, r4, and r5. Multiple regulatory modules directing segmental expression of chicken and mouse Hoxa2 genes have been identified, and each module is composed of a series of discrete elements. We used these modules to investigate the basis of differential expression of duplicated Hoxa2 genes, as a model for understanding the divergence of cis-regulatory elements. Therefore, we cloned putative regulatory regions of the fugu and medaka Hoxa2(a) and -(b) genes and assayed their activity. We found that these modules direct reporter expression in a chicken assay, in a manner corresponding to their endogenous expression pattern in fugu. Although sequence comparisons reveal many differences between the two coparalogous genes, specific subtle changes in seven cis elements of the Hoxa2(a) gene restore segmental regulatory activity. Therefore, drift in subsets of the elements in the regulatory modules is responsible for the differential expression of the two coparalogous genes, thus providing insight into the evolution of cis elements.Hox gene regulation ͉ hindbrain ͉ vertebrate development ͉ fugu

Elephant shark ( Callorhinchus milii ) provides insights into the evolution of Hox gene clusters in gnathostomes

“…Another possibility is that the loss of transcription factor binding sites in one location might be compensated by de novo evolution of binding sites in another location, as has been demonstrated for the stripe 2 enhancer of even-skipped gene in Drosophila (44). Notwithstanding these possibilities, several studies have shown that changes in cis-regulatory sequences, ranging from subtle differences to extensive restructuring, can account for the altered expression pattern of Hox genes (6,7,(45)(46)(47). For example, the duplicate fugu HoxA2a and HoxA2b genes show distinct expression patterns in the hindbrain.…”

“…In vertebrates, Hox cluster genes also exhibit temporal collinearity; that is, genes located in the 3Ј-end of the cluster are expressed early during development, whereas genes in the 5Ј-end are expressed later (2). Recent comparative studies of Hox clusters in model genomes have shown that Hox clusters have experienced repeated molecular changes, including fragmentation, cluster duplication, gene loss, coding-sequence divergence, and cisregulatory element evolution (3)(4)(5)(6)(7). Because of their critical role in defining the identities of body segments, Hox genes are believed to have played a key role in driving the morphologic diversification of animals (8)(9)(10) and thus are of particular interest in understanding the genetic basis of morphologic diversity of metazoans.…”

A regulatory module embedded in the coding region of Hoxa2 controls expression in rhombomere 2

“…Segmental expression of Hoxa2 in the vertebrate hindbrain involves the combined activities of multiple cis-regulatory modules. It has been shown that the intergenic region between Hoxa2 and Hoxa3 contains conserved enhancers implicated in directing expression in r3 and r5 (r3/5) (11)(12)(13)(14) and cranial neural crest cells (ncc) (15). A conserved regulatory module located in the intron of Hoxa2 mediates expression in r4 (16).…”

“…1B). The 3Ј deletion series (constructs [12][13][14] revealed that the short region (region d) when deleted in construct 13 also, results in a complete loss of reporter activity (Fig. 1B).…”

“…Progressive 5Ј and 3Ј deletion of the MluI-PstI fragment (construct 6) produced a series of nested fragments (constructs [7][8][9][10][11][12][13][14] that were tested for r2 activity ( Fig. 1 B and E).…”