Genomic insights of body plan transitions from bilateral to pentameral symmetry in Echinoderms.

Echinoderms are an exceptional group of bilaterians that develop pentameral adult symmetry from a bilaterally symmetric larva. However, the genetic basis in evolution and development of this unique transformation remains to be clarified. Here we report newly sequenced genomes, developmental transcriptomes, and proteomes of diverse echinoderms including the green sea urchin (L. variegatus), a sea cucumber (A. japonicus), and with particular emphasis on a sister group of the earliest-diverged echinoderms, the feather star (A. japonica). We learned that the last common ancestor of echinoderms retained a well-organized Hox cluster reminiscent of the hemichordate, and had gene sets involved in endoskeleton development. Further, unlike in other animal groups, the most conserved developmental stages were not at the body plan establishing phase, and genes normally involved in bilaterality appear to function in pentameric axis development. These results enhance our understanding of the divergence of protostomes and deuterostomes almost 500 Mya.

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

BACKGROUND: In the previous paper published in 2017, we described the structure of Hox gene cluster of the ascidian, Halocynthia roretzi, and discussed the scenario for the disintegration of Hox gene clusters during evolution of ascidians. The description about the Hox gene cluster structure still represents the latest information, hence it has been left unchanged. In contrast, some points in Discussion, the description on the phylogenetic relationships among tunicates and the theoretical scenario for the disintegration of Hox gene cluster during evolution of ascidians, should be changed because the phylogenetic relationships among tunicates have recently been updated. The above mentioned points were made in accordance with the phylogenetic tree for tunicates based on the mitochondrial DNA sequences, which was the latest at the time of publication. In 2018, however, Kocot et al. and Delsuc et al. proposed new phylogenetic trees for tunicates based on a large number of nuclear gene sequences. The trees obtained by the two groups are essentially the same and different from the previous one in the phylogenetic positions of Appendicularia and Thaliacea, which leads to a change in the order of the emergence of ascidians and the Hox gene cluster disintegration during evolution of ascidians or tunicates. RESULTS: We add here a note to update the previous description on the phylogenetic relationships among tunicates and the theoretical scenario, including one Figure, so as to coincide with the new phylogenetic relationships among tunicates based on the nuclear gene sequences. CONCLUSION: The previous summarized conclusion remains unchanged: we suggest that the Hox gene cluster of the ancestral ascidian experienced extensive genome shuffling during the course of evolution to Hr and Ci. Nevertheless, some features are shared in Hox gene components and gene organization on the chromosomes, suggesting that Hox gene cluster disintegration in ascidians involved early events common to all ascidians and later lineage-specific events.

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

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.

Transcriptional regulation of a horizontally transferred gene from bacterium to chordate.

The horizontal transfer of genes between distantly related organisms is undoubtedly a major factor in the evolution of novel traits. Because genes are functionless without expression, horizontally transferred genes must acquire appropriate transcriptional regulations in their recipient organisms, although the evolutionary mechanism is not known well. The defining characteristic of tunicates is the presence of a cellulose containing tunic covering the adult and larval body surface. Cellulose synthase was acquired by horizontal gene transfer from Actinobacteria. We found that acquisition of the binding site of AP-2 transcription factor was essential for tunicate cellulose synthase to gain epidermal-specific expression. Actinobacteria have very GC-rich genomes, regions of which are capable of inducing specific expression in the tunicate epidermis as the AP-2 binds to a GC-rich region. Therefore, the actinobacterial cellulose synthase could have been potentiated to evolve its new function in the ancestor of tunicates with a higher probability than the evolution depending solely on a spontaneous event.

Polarization of PI3K Activity Initiated by Ooplasmic Segregation Guides Nuclear Migration in the Mesendoderm.

Asymmetric localization of RNA is a widely observed mechanism of cell polarization. Using embryos of the ascidian, Halocynthia roretzi, we previously showed that mesoderm and endoderm fates are separated by localization of mRNA encoding a transcription factor, Not, to the future mesoderm-side cytoplasm of the mesendoderm cell through asymmetric positioning of the nucleus. Here, we investigated the mechanism that defines the direction of the nuclear migration. We show that localization of PtdIns(3,4,5)P3 to the future mesoderm region determines the direction of nuclear migration. Localization of PtdIns(3,4,5)P3 was dependent on the localization of PI3Kα to the future mesoderm region. PI3Kα was first localized at the 1-cell stage by the ooplasmic movement. Activity of localized PI3Kα at the 4-cell stage was required for the localization of PI3Kα up to the nuclear migration. Our results provide the scaffold for understanding the chain of causality leading to the separation of germ layer fates.

Hox10-regulated endodermal cell migration is essential for development of the ascidian intestine.

Hox cluster genes play crucial roles in development of the metazoan antero-posterior axis. Functions of Hox genes in patterning the central nervous system and limb buds are well known. They are also expressed in chordate endodermal tissues, where their roles in endodermal development are still poorly understood. In the invertebrate chordate, Ciona intestinalis, endodermal tissues are in a premature state during the larval stage, and they differentiate into the digestive tract during metamorphosis. In this study, we showed that disruption of a Hox gene, Ci-Hox10, prevented intestinal formation. Ci-Hox10-knock-down larvae displayed defective migration of endodermal strand cells. Formation of a protrusion, which is important for cell migration, was disrupted in these cells. The collagen type IX gene is a downstream target of Ci-Hox10, and is negatively regulated by Ci-Hox10 and a matrix metalloproteinase ortholog, prior to endodermal cell migration. Inhibition of this regulation prevented cellular migration. These results suggest that Ci-Hox10 regulates endodermal strand cell migration by forming a protrusion and by reconstructing the extracellular matrix.

Analysis of the Transcription Regulatory Mechanism of Otx During the Development of the Sensory Vesicle in Ciona intestinalis.

Establishment of the anterior-posterior axis is an important event in the development of bilateral animals. A homeodomain transcription factor, Otx, is important for the formation of the anterior part of the embryo, and its mRNA is expressed in a continuous manner in a wide range of animals. This pattern of expression is thought to be important for the formation of anterior neural structures, but the mechanism that regulates Otx expression remains largely unknown. Towards understanding how the transcription of Otx is maintained in the cells of anterior neural structure, the sensory vesicle, during embryogenesis, we examined transcription regulatory mechanisms of Otx, using embryos of the ascidian, Ciona intestinalis, from the gastrula to tailbud stages, which have not been studied previously. We identified two genomic regions capable of mimicking the Otx expression pattern from the gastrula to tailbud stages. Putative transcription factor binding sites required for this activity were identified. Notably, distinct sets of transcription factor binding sites were required at different developmental stages for the expression of Otx, suggesting that the continuity of Otx is supported by distinct transcriptional mechanisms in the gastrula and neurula stages. Along with previous studies using Halocynthia roretzi, the present results provide insight into the evolution of transcriptional regulatory mechanism of Otx.

Continuous expression of Otx in the anterior neural lineage is supported by different transcriptional regulatory mechanisms during the development of Halocynthia roretzi.

The process of establishing the anterior-posterior axis is an important event in the development of bilateral animals. Otx, which encodes a homeodomain transcription factor, is continuously expressed in the anterior part of the embryo in a wide range of animals. This pattern of expression is thought to be important for the formation of anterior neural structures, but the regulatory mechanism that sustains the expression is not known. Here, using embryos of the ascidian, Halocynthia roretzi, we investigated how the transcription of Otx is maintained in the cells of the anterior neural lineage during embryogenesis. We identified an enhancer region sufficient to mimic the Otx expression pattern from the gastrula to tailbud stages. Several putative transcription factor binding sites that are required for generating the Otx expression pattern were also identified. Distinct sets of sites were required at different developmental stages, suggesting that distinct transcriptional mechanisms regulate Otx transcription in each of the gastrula, neurula and tailbud stages. Along with previous studies on the transcriptional regulatory mechanism of Otx during the pre-gastrula stages, the present results provide the first overview of the mechanism that sustains Otx expression in the anterior neural lineage during ascidian embryogenesis and demonstrate the complexity of a developmental mechanism that maintains Otx transcription.

Formation of the digestive tract in Ciona intestinalis includes two distinct morphogenic processes between its anterior and posterior parts.

BACKGROUND: In the ascidian Ciona intestinalis, the digestive tract, an essential system for animals, develops during metamorphosis from the two primordial tissues, the endoderm and endodermal strand, located in the larval trunk and tail, respectively. However, it has been largely unknown how the digestive tract develops from these primordial tissues. We examined the metamorphosing larvae for the tubular formation of the digestive tract, focusing on the epithelial organization of the endoderm, by combined confocal microscopy and computational rendering. RESULTS: The tubular structure of the esophagus to the stomach was formed through the folding and closure of the endodermal epithelia in the central-to-right posterior trunk. By contrast, the intestine was formed in the left posterior trunk through the accumulation and rearrangement of the cells originated from the endodermal strand. This was confirmed by the cell-tracing experiment using Kaede expression construct driven in the endodermal strand. Thus, the tubular formation of the digestive tract in C. intestinalis includes distinct morphogenetic processes and cell lineages between its anterior and posterior parts. CONCLUSION: This study provides the first detailed description of the digestive tract morphogenesis in C. intestinalis and serves as an important basis toward thorough understanding of its digestive tract development.

Identification of an intact ParaHox cluster with temporal colinearity but altered spatial colinearity in the hemichordate Ptychodera flava.

BACKGROUND: ParaHox and Hox genes are thought to have evolved from a common ancestral ProtoHox cluster or from tandem duplication prior to the divergence of cnidarians and bilaterians. Similar to Hox clusters, chordate ParaHox genes including Gsx, Xlox, and Cdx, are clustered and their expression exhibits temporal and spatial colinearity. In non-chordate animals, however, studies on the genomic organization of ParaHox genes are limited to only a few animal taxa. Hemichordates, such as the Enteropneust acorn worms, have been used to gain insights into the origins of chordate characters. In this study, we investigated the genomic organization and expression of ParaHox genes in the indirect developing hemichordate acorn worm Ptychodera flava. RESULTS: We found that P. flava contains an intact ParaHox cluster with a similar arrangement to that of chordates. The temporal expression order of the P. flava ParaHox genes is the same as that of the chordate ParaHox genes. During embryogenesis, the spatial expression pattern of PfCdx in the posterior endoderm represents a conserved feature similar to the expression of its orthologs in other animals. On the other hand, PfXlox and PfGsx show a novel expression pattern in the blastopore. Nevertheless, during metamorphosis, PfXlox and PfCdx are expressed in the endoderm in a spatially staggered pattern similar to the situation in chordates. CONCLUSIONS: Our study shows that P. flava ParaHox genes, despite forming an intact cluster, exhibit temporal colinearity but lose spatial colinearity during embryogenesis. During metamorphosis, partial spatial colinearity is retained in the transforming larva. These results strongly suggest that intact ParaHox gene clustering was retained in the deuterostome ancestor and is correlated with temporal colinearity.

Identical genomic organization of two hemichordate hox clusters.

Genomic comparisons of chordates, hemichordates, and echinoderms can inform hypotheses for the evolution of these strikingly different phyla from the last common deuterostome ancestor. Because hox genes play pivotal developmental roles in bilaterian animals, we analyzed the Hox complexes of two hemichordate genomes. We find that Saccoglossus kowalevskii and Ptychodera flava both possess 12-gene clusters, with mir10 between hox4 and hox5, in 550 kb and 452 kb intervals, respectively. Genes hox1-hox9/10 of the clusters are in the same genomic order and transcriptional orientation as their orthologs in chordates, with hox1 at the 3' end of the cluster. At the 5' end, each cluster contains three posterior genes specific to Ambulacraria (the hemichordate-echinoderm clade), two forming an inverted terminal pair. In contrast, the echinoderm Strongylocentrotus purpuratus contains a 588 kb cluster of 11 orthologs of the hemichordate genes, ordered differently, plausibly reflecting rearrangements of an ancestral hemichordate-like ambulacrarian cluster. Hox clusters of vertebrates and the basal chordate amphioxus have similar organization to the hemichordate cluster, but with different posterior genes. These results provide genomic evidence for a well-ordered complex in the deuterostome ancestor for the hox1-hox9/10 region, with the number and kind of posterior genes still to be elucidated.

Transcription regulatory mechanism of Pitx in the papilla-forming region in the ascidian, Halocynthia roretzi, implies conserved involvement of Otx as the upstream gene in the adhesive organ development of chordates.

Pitx genes play important roles in a variety of developmental processes in vertebrates. In an ascidian species, Halocynthia roretzi, Hr-Pitx, the only Pitx gene of this species, has been reported to be expressed in the left epidermis at the tailbud stage. In the present study, first, we have shown that Hr-Pitx is also expressed in the papilla-forming region at the neurula to tailbud stages, and then we addressed transcription regulatory mechanisms for the expression of Hr-Pitx in the papilla-forming region. We have identified the genomic region ranging from 850 to 1211 bp upstream from the translation start site of the Hr-Pitx gene as an enhancer region that drives the transcription of Hr-Pitx in the papilla-forming region. Within the enhancer region, putative transcriptional factor binding sites for Otx as well as Fox were shown to be required for its activity. Finally, we carried out knocking down experiments of Hr-Otx function using an antisense morpholino oligonucleotide, in which the knocking down of Hr-Otx function resulted in reduction of the enhancer activity and loss of the expression of Hr-Pitx in the papilla-forming region. In Xenopus laevis, it has been reported that Pitx genes are expressed downstream of Otx function during development of the cement gland, an adhesive organ of its larva. Taken together, it is suggested that the expression regulatory mechanism of Pitx, involving Otx as the upstream gene, in the developing adhesive organ is conserved between ascidians and vertebrates.

Repression of Rx gene on the left side of the sensory vesicle by Nodal signaling is crucial for right-sided formation of the ocellus photoreceptor in the development of Ciona intestinalis.

Nodal signaling plays an essential role in the establishment of left-right asymmetry in various animals. However, it is largely unknown how Nodal signaling is involved in the establishment of the left-right asymmetric morphology. In this study, the role of Nodal signaling in the left-right asymmetric ocellus formation in the ascidian, Ciona intestinalis was dealt with. During the development of C. intestinalis, the ocellus pigment cell forms on the midline and moves to the right side of the midline. Then, the photoreceptor cells form on the right side of the sensory vesicle (SV). Ci-Nodal is expressed on the left side of the SV in the developing tail bud embryo. When Nodal signaling is inhibited, the ocellus pigment cell form but remain on the midline, and expression of marker genes of the ocellus photoreceptor cells is ectopically detected on the left side as well as on the right side of the SV in the larva. Furthermore, Ci-Rx, which is essential for the ocellus differentiation, turns out to be negatively regulated by the Nodal signaling on the left side of the SV, even though it is required for the right-sided photoreceptor formation. These results indicate that Nodal signaling controls the left-right asymmetric ocellus formation in the development of C. intestinalis.

Segregation of germ layer fates by nuclear migration-dependent localization of Not mRNA.

An important step in early embryonic development is the allocation and segregation of germ layer fates into distinct embryonic regions. However, the mechanism that segregates the mesendoderm into mesoderm and endoderm fates remains largely unknown in most animals. Here, using ascidians, a primitive chordate, we show that these fates are segregated by partitioning of asymmetrically localized Not mRNA from the mesendoderm cell to its mesodermal daughter. Migration of the mesendoderm cell nucleus to the future mesoderm-forming region, release of Not mRNA from the nucleus, Wnt5α-dependent local retention of the mRNA, and subsequent repositioning of the mitotic spindle to the center of the cell are each required for the asymmetric localization and partitioning of Not mRNA. Our results show that nuclear migration plays an unexpected role in asymmetric cell divisions that segregate germ layer fates in chordate embryos.

Limited functions of Hox genes in the larval development of the ascidian Ciona intestinalis.

In animals, region specific morphological characters along the anteroposterior axis are controlled by a number of developmental genes, including Hox genes encoding homeodomain transcription factors. Although Hox genes have been regarded to play a key role in the evolution of morphological diversity, as well as in the establishment of the body plan, little is known about the function of Hox genes in invertebrates, except for in insects and nematodes. The present study addresses the role of Hox genes in body patterning during the larval development of the ascidian Ciona intestinalis conducting knockdown experiments of the seven Hox genes expressed during embryogenesis. Experimental results have demonstrated that Ci-Hox12 plays an important role in tail development through the maintenance of expression of Ci-Fgf8/17/18 and Ci-Wnt5 in the tail tip epidermis. Additionally, it has been shown that Ci-Hox10 is involved in the development of GABAergic neurons in the dorsal visceral ganglion. Surprisingly, knockdown of Ci-Hox1, Ci-Hox2, Ci-Hox3, Ci-Hox4 and Ci-Hox5 did not give rise to any consistent morphological defects in the larvae. Furthermore, expression of neuronal marker genes was not affected in larvae injected with MOs against Ci-Hox1, Ci-Hox3 or Ci-Hox5. In conclusion, we suggest that the contribution of Hox genes to the larval development of the ascidian C. intestinalis might be limited, despite the fact that Ci-Hox10 and Ci-Hox12 play important roles in neuronal and tail development.

Spatial and temporal expression of two transcriptional isoforms of Lhx3, a LIM class homeobox gene, during embryogenesis of two phylogenetically remote ascidians, Halocynthia roretzi and Ciona intestinalis.

Lhx3 genes are members of one of the six subfamilies of the LIM class homeobox genes. In ascidians, Lhx3 is known to play a critical role in endoderm differentiation, while in vertebrates Lhx3 is involved in the development of pituitary and subsets of motor neurons. It has been shown recently, using RT-PCR analysis, that two transcriptional isoforms a and b are differentially expressed during the larval development of Ciona intestinalis (Christiaen et al., 2009). The present study provides an in-depth description of Lhx3 gene expression during the development of the two remote ascidian species, C. intestinalis and Halocynthia roretzi; for this, 5'RACE and whole-mount in situ hybridization (WISH) were employed. In both species, maternal expression of Lhx3a, but not Lhx3b, is evident. In H. roretzi, the maternal Lhx3a transcripts have been detected by WISH in the animal half of early cleavage stage embryos. In both species, transcriptional isoform a is also zygotically expressed in the sensory vesicle and the visceral ganglion lineages from the neurula stage onward. By contrast, Lhx3b transcripts are expressed only zygotically and localized in the endoderm, notochord and mesenchyme lineages during cleavage stage. Lhx3a, but not Lhx3b, transcripts are subjected to trans-splicing. Additionally, in C. intestinalis, other variations in the 5' region have been identified among Lhx3a transcripts. Although some differences are present, over-all developmental expression of Lhx3 is rather well conserved between the two ascidian species, which is quite different from that of vertebrate counterparts.

Ambulacrarian prototypical Hox and ParaHox gene complements of the indirect-developing hemichordate Balanoglossus simodensis.

The Hox genes and its evolutionary sister, the ParaHox genes, are widely distributed among animals. Although it has been expected that hemichordates and echinoderms have a single set of Hox genes and most likely a single set of ParaHox genes, it is not known whether the ortholog of Hox8 is absent in hemichordates, and in turn, consensus view about Hox/ParaHox gene complements in hemichordates has not been established. In this study, we isolated either complete or nearly complete coding sequences of 12 Hox genes, including the ortholog of the Hox8 that has not been reported in the previous studies, and three ParaHox genes from the recently discovered indirect-developing acorn worm, Balanoglossus simodensis. Our data suggest that the ancestral hemichordate had intact complements of ambulacrarian prototypical Hox and ParaHox genes, consisting of 12 and three members, respectively.

Evolutionary origins of blastoporal expression and organizer activity of the vertebrate gastrula organizer gene lhx1 and its ancient metazoan paralog lhx3.

Expression of the LIM homeobox gene lhx1 (lim1) is specific to the vertebrate gastrula organizer. Lhx1 functions as a transcriptional regulatory core protein to exert ;organizer' activity in Xenopus embryos. Its ancient paralog, lhx3 (lim3), is expressed around the blastopore in amphioxus and ascidian, but not vertebrate, gastrulae. These two genes are thus implicated in organizer evolution, and we addressed the evolutionary origins of their blastoporal expression and organizer activity. Gene expression analysis of organisms ranging from cnidarians to chordates suggests that blastoporal expression has its evolutionary root in or before the ancestral eumetazoan for lhx1, but possibly in the ancestral chordate for lhx3, and that in the ascidian lineage, blastoporal expression of lhx1 ceased, whereas endodermal expression of lhx3 has persisted. Analysis of organizer activity using Xenopus embryos suggests that a co-factor of LIM homeodomain proteins, Ldb, has a conserved function in eumetazoans to activate Lhx1, but that Lhx1 acquired organizer activity in the bilaterian lineage, Lhx3 acquired organizer activity in the deuterostome lineage and ascidian Lhx3 acquired a specific transactivation domain to confer organizer activity on this molecule. Knockdown analysis using cnidarian embryos suggests that Lhx1 is required for chordin expression in the blastoporal region. These data suggest that Lhx1 has been playing fundamental roles in the blastoporal region since the ancestral eumetazoan arose, that it contributed as an 'original organizer gene' to the evolution of the vertebrate gastrula organizer, and that Lhx3 could be involved in the establishment of organizer gene networks.

Evolution of CUT class homeobox genes: insights from the genome of the amphioxus, Branchiostoma floridae.

CUT class homeobox genes, including CUX/CASP, ONECUT, SATB and COMPASS family genes, are known to exhibit diverse features in the homeodomain and the domain architecture. Furthermore, the intron/exon organization of CUX/CASP is different between vertebrates and protostomes, and SATB genes are only known for vertebrates, whereas COMPASS genes have only been found in protostomes. These observations suggest a complex evolutionary history for the CUT class homeobox genes, but the evolution of CUT class homeobox genes in the lineage to vertebrates remained largely unknown. To obtain clearer insights into this issue, we searched the genome of amphioxus, Branchiostoma floridae, a lower chordate, for CUT class homeobox genes by extensive BLAST survey and phylogenetic analyses. We found that the genome of Branchiostoma floridae encodes each single orthologue of CUX/CASP, ONECUT, and COMPASS, but not the SATB gene, and one atypical CUT gene likely specific to this species. In addition, the genomic structure of the amphioxus CUX/CASP gene turned out to be protostome-type, but not vertebrate-type. Based on these observations, we propose a model in which SATB is suggested to evolve at the expense of COMPASS and this change, together with the structural change in CUX/CASP, is supposed to take place in the lineage to vertebrates after divergence of the amphioxus and vertebrate ancestors. The present study provides an example of dramatic evolution among homeobox gene groups in the vertebrate lineage and highlights the ancient character of amphioxus, retaining genomic features shared by protostomes.