Hox gene cluster of the ascidian, Halocynthia roretzi, reveals multiple ancient steps of cluster disintegration during ascidian evolution

Abstract

Background: Hox gene clusters with at least 13 paralog group (PG) members are common in vertebrate genomes and in that of amphioxus. Ascidians, which belong to the subphylum Tunicata (Urochordata), are phylogenetically positioned between vertebrates and amphioxus, and traditionally divided into two groups: the Pleurogona and the Enterogona. An enterogonan ascidian, Ciona intestinalis (Ci), possesses nine Hox genes localized on two chromosomes; thus, the Hox gene cluster is disintegrated. We investigated the Hox gene cluster of a pleurogonan ascidian, Halocynthia roretzi (Hr) to investigate whether Hox gene cluster disintegration is common among ascidians, and if so, how such disintegration occurred during ascidian or tunicate evolution.

Results: Our phylogenetic analysis reveals that the Hr Hox gene complement comprises nine members, including one with a relatively divergent Hox homeodomain sequence. Eight of nine Hr Hox genes were orthologous to Ci-Hox1, 2, 3, 4, 5, 10, 12 and 13. Following the phylogenetic classification into 13 PGs, we designated Hr Hox genes as Hox1, 2, 3, 4, 5, 10, 11/12/13.a, 11/12/13.b and HoxX. To address the chromosomal arrangement of the nine Hox genes, we performed two-color chromosomal fluorescent in situ hybridization, which revealed that the nine Hox genes are localized on a single chromosome in Hr, distinct from their arrangement in Ci. We further examined the order of the nine Hox genes on the chromosome by chromosome/scaffold walking. This analysis suggested a gene order of Hox1, 11/12/13.b, 11/12/13.a, 10, 5, X, followed by either Hox4, 3, 2 or Hox2, 3, 4 on the chromosome. Based on the present results and those previously reported in Ci, we discuss the establishment of the Hox gene complement and disintegration of Hox gene clusters during the course of ascidian or tunicate evolution.

Conclusions: The Hox gene cluster and the genome must have experienced extensive reorganization during the course of evolution from the ancestral tunicate to Hr and Ci. Nevertheless, some features are shared in Hox gene components and gene arrangement on the chromosomes, suggesting that Hox gene cluster disintegration in ascidians involved early events common to tunicates as well as later ascidian lineage-specific events.

Keywords: Ascidian; Halocynthia roretzi; Hox gene cluster; Tunicate (urochordate) evolution.

Fig. 1

Phylogenetic analysis of Hox gene candidates of Halocynthia roretzi. The ML tree was constructed using homeodomain sequences and the adjacent 20 N-terminal and seven C-terminal amino acid residues (Additional file 1: Figure S1) and MEGA5 software package. The percentage of 1000 replicated trees in which clustering of genes was supported is indicated at nodes. Within a clade consisting only of vertebrate Hox genes, the percentage is not indicated. Hr Hox gene candidates and Hox genes of Ciona intestinalis (Ci-Hox) are indicated by larger and smaller colored circles, respectively. Here, Hr Hox gene candidates are tentatively designated according to their Ci counterparts, except for HoxX, which did not show apparent orthology to Ci-Hox genes. The color-code indicates distinct paralog groups (PGs). Taxonomic abbreviations are Mm for Mus musculus, Lm for Latimeria menadoensis, Hf for Heterodontus francisci, Ci for Ciona intestinalis and Hr for Halocynthia roretzi. The bar at the bottom indicates one amino acid substitution per position in the sequence

Fig. 2

Phylogenetic analysis of Hox gene candidates of Halocynthia roretzi, using PGs 5–8 a and PGs 9–13 b genes, and PG1 genes were used as an out group. The ML tree was constructed using homeodomain sequences and the adjacent 20 N-terminal side and seven C-terminal side amino acid residues (see Additional file 1: Figure S1) with MEGA5 software and 1000 replicates. The percentage of replicated trees in which the clustering of genes was supported is indicated at the nodes. Within a clade consisting of only vertebrate Hox genes, the percentage was not indicated at the node. Colored circles to indicate ascidian genes and taxonomic abbreviations are the same as in Fig. 1. The bars at the bottom indicate amino acid substitutions per position in the sequence

Fig. 3

Mapping of Halocynthia roretzi Hox genes onto metaphase chromosomes (a-d). Metaphase chromosome spreads were prepared from cleavage stage Hr embryos and hybridized with two or three probes labeled with digoxigenin (red) or biotin (green) for genes indicated at the top of each panel. Chromosomes were stained with DAPI. Red and green arrowheads indicate signals for the gene of the same color code. In the right bottom corner of each panel, enlargement of one of the chromosomes with signals is shown in inset. Within a chromosome, a pale blue stained region corresponds to the centromeric region. The bar in d indicates 5 μm and is applicable to all panels. BAC clones used for probes were 5 J1 (Hox1), 3C14 (Hox2, Hox3 and Hox4), 6B23 (Hox5 and HoxX) and 1 J20 (Hox10, Hox11/12/13.a and Hox11/12/13.b)

Fig. 4

Schematic representation of the Hox gene cluster of Halocynthia roretzi. The Hox gene cluster structure of Hr as estimated by chromosome/scaffold walking is shown at the top. A horizontal line represents a part of chromosome. The telomeric side is to the left and the centromeric side is to the right. Hox genes are represented as thick arrows, which also indicate transcription direction. The color code is the same as that in Fig. 1. Between HoxX and Hox4, there is a region, from which no clones were available out of the BAC library; hence, no scaffolds available out of the ANISEED database. Grey arrays of short vertical bars indicate 100 kbp, starting at the right and left ends of the region where no scaffolds are available. Dark green or blue horizontal bars below the scales indicate BAC clones corresponding to Hox gene clusters. Dark green bars indicate BAC clones, end regions of which were sequenced. Blue bars indicate BAC clones for which the whole insert sequence was determined. Bars with red dots in the middle are the clones used for probes for chromosomal FISH (Fig. 3). Names of clones are also indicated. Green horizontal bars at the bottom indicate scaffolds in the ANISEED database (Halocynthia roretzi MTP2014; https://www.aniseed.cnrs.fr/fgb2/gbrowse/harore_mtp2014/) that correspond to the Hox gene cluster spanning chromosomal region. The length of each scaffold according to ANISEED database is indicated below scaffold names, except for the scaffolds in the two regions adjacent to S26, where multiple small scaffolds are included. Arc lines with arrowheads on both ends placed upper and lower of the subcluster region of Hox2, 3 and 4 indicate that the orientation of the subcluster has not been determined

Fig. 5

A proposed scheme for Hox gene cluster disintegration during ascidian evolution. The last common ancestor for cephalochordates, tunicates, and vertebrates (represented as Chordata) possessed a single Hox gene cluster consisting of three anterior (red, orange, and yellow), five central (green) and three ancestral posterior genes (blue). After the ancestral cephalochordate diverged, the tunicate ancestor (represented as Tunicata), in turn, diverged from the vertebrate lineage. At this stage, the ancestral tunicate must have experienced extensive changes in the genome, and the Hox gene cluster disintegration started, losing one or two central Hox genes. The ancestral tunicate subsequently evolved into two lineages, and in turn, diverged into Stolidobranchia and Appendicularia lineages (right side, upper) and Phlebobranchia, Aplousobranchia and Thaliacea lineages (right side, lower) [27]. The Hox gene complement of the ancestral tunicate with each three of anterior, central and posterior genes must have been established by the divergence of the two evolutionary lineages stated above. At the same time, early Hox gene cluster disintegration events must have occurred. In one of the two resultant evolutionary lineages, the ancestral stolidobranchial ascidian (Hr) emerged, being separated from the larvacean lineage. In the other evolutionary lineage, the ancestral phlebobranchial ascidian (Ci) emerged, being separated from Aplousobranchia and Thaliacea lineages. The Hox gene cluster subsequently disintegrated in different patterns in the two evolutionary lineages. White or gray ovals indicate Hox genes, probably of the central Hox gene group origin (see text). The Hox gene complement of Oikopleura dioica, consisting of two anterior, one central, and six posterior genes, and that of amphioxus, consisting of 15 members, are schematically represented