Shigehiro Kuraku

The African coelacanth genome provides insights into tetrapod evolution.

The discovery of a living coelacanth specimen in 1938 was remarkable, as this lineage of lobe-finned fish was thought to have become extinct 70 million years ago. The modern coelacanth looks remarkably similar to many of its ancient relatives, and its evolutionary proximity to our own fish ancestors provides a glimpse of the fish that first walked on land. Here we report the genome sequence of the African coelacanth, Latimeria chalumnae. Through a phylogenomic analysis, we conclude that the lungfish, and not the coelacanth, is the closest living relative of tetrapods. Coelacanth protein-coding genes are significantly more slowly evolving than those of tetrapods, unlike other genomic features. Analyses of changes in genes and regulatory elements during the vertebrate adaptation to land highlight genes involved in immunity, nitrogen excretion and the development of fins, tail, ear, eye, brain and olfaction. Functional assays of enhancers involved in the fin-to-limb transition and in the emergence of extra-embryonic tissues show the importance of the coelacanth genome as a blueprint for understanding tetrapod evolution.The discovery of a living coelacanth specimen in 1938 was remarkable, as this lineage of lobe-finned fish was thought to have become extinct 70 million years ago. The modern coelacanth looks remarkably similar to many of its ancient relatives, and its evolutionary proximity to our own fish ancestors provides a glimpse of the fish that first walked on land. Here we report the genome sequence of the African coelacanth, Latimeria chalumnae. Through a phylogenomic analysis, we conclude that the lungfish, and not the coelacanth, is the closest living relative of tetrapods. Coelacanth protein-coding genes are significantly more slowly evolving than those of tetrapods, unlike other genomic features. Analyses of changes in genes and regulatory elements during the vertebrate adaptation to land highlight genes involved in immunity, nitrogen excretion and the development of fins, tail, ear, eye, brain and olfaction. Functional assays of enhancers involved in the fin-to-limb transition and in the emergence of extra-embryonic tissues show the importance of the coelacanth genome as a blueprint for understanding tetrapod evolution.

Hagfish embryology with reference to the evolution of the neural crest

Hagfish, which lack both jaws and vertebrae, have long been the subject of intense interest owing to their position at a crucial point in the evolutionary transition to a truly vertebrate body plan. However, unlike the comparatively well characterized vertebrate agnathan lamprey, little is known about hagfish development. The inability to analyse hagfish at early embryonic stages has frustrated attempts to resolve questions with important phylogenetic implications, including fundamental ones relating to the emergence of the neural crest. Here we report the obtainment of multiple pharyngula-stage embryos of the hagfish species Eptatretus burgeri and our preliminary analyses of their early development. We present histological evidence of putative neural crest cells, which appear as delaminated cells that migrate along pathways corresponding to neural crest cells in fish and amphibians. Molecular cloning studies further revealed the expression of several regulatory genes, including cognates of Pax6, Pax3/7, SoxEa and Sox9, suggesting that the hagfish neural crest is specified by molecular mechanisms that are general to vertebrates. We propose that the neural crest emerged as a population of de-epithelialized migratory cells in a common vertebrate ancestor, and suggest that the possibility of classical and molecular embryology in hagfish opens up new approaches to clarifying the evolutionary history of vertebrates.

Time Scale for Cyclostome Evolution Inferred with a Phylogenetic Diagnosis of Hagfish and Lamprey cDNA Sequences

Abstract The Cyclostomata consists of the two orders Myxiniformes (hagfishes) and Petromyzoniformes (lampreys), and its monophyly has been unequivocally supported by recent molecular phylogenetic studies. Under this updated vertebrate phylogeny, we performed in silico evolutionary analyses using currently available cDNA sequences of cyclostomes. We first calculated the GC-content at four-fold degenerate sites (GC4), which revealed that an extremely high GC-content is shared by all the lamprey species we surveyed, whereas no striking pattern in GC-content was observed in any of the hagfish species surveyed. We then estimated the timing of diversification in cyclostome evolution using nucleotide and amino acid sequences. We obtained divergence times of 470–390 million years ago (Mya) in the Ordovician–Silurian–Devonian Periods for the interordinal split between Myxiniformes and Petromyzoniformes; 90–60 Mya in the Cretaceous–Tertiary Periods for the split between the two hagfish subfamilies, Myxininae and Eptatretinae; 280–220 Mya in the Permian–Triassic Periods for the split between the two lamprey subfamilies, Geotriinae and Petromyzoninae; and 30–10 Mya in the Tertiary Period for the split between the two lamprey genera, Petromyzon and Lethenteron. This evolutionary configuration indicates that Myxiniformes and Petromyzoniformes diverged shortly after the common ancestor of cyclostomes split from the future gnathostome lineage. Our results also suggest that intra-subfamilial diversification in hagfish and lamprey lineages (especially those distributed in the northern hemisphere) occurred in the Cretaceous or Tertiary Periods.

Evolutionary biology: Lamprey Hox genes and the evolution of jaws

Arising from: M. J. Cohn 416, 386–387 (2002).In vertebrates with jaws (gnathostomes), the jaws are formed from the first pharyngeal arch (PA1), which does not express homeobox (Hox) genes. Cohn describes expression of the HoxL6 gene in the PA1 of the lamprey Lampetra fluviatilis, a jawless (agnathan) vertebrate, and postulates that a retreat of Hox expression from PA1 might have favoured the evolution of jaws in the gnathostome lineage after the split from agnathans. Here we examine the distribution of Hox genes in another lamprey species, Lethenteron japonicum, and find that none are expressed in the PA1. We conclude that Cohns finding is not a general feature within the lamprey group and is therefore unlikely to be related to jawlessness.

aLeaves facilitates on-demand exploration of metazoan gene family trees on MAFFT sequence alignment server with enhanced interactivity

We report a new web server, aLeaves (http://aleaves.cdb.riken.jp/), for homologue collection from diverse animal genomes. In molecular comparative studies involving multiple species, orthology identification is the basis on which most subsequent biological analyses rely. It can be achieved most accurately by explicit phylogenetic inference. More and more species are subjected to large-scale sequencing, but the resultant resources are scattered in independent project-based, and multi-species, but separate, web sites. This complicates data access and is becoming a serious barrier to the comprehensiveness of molecular phylogenetic analysis. aLeaves, launched to overcome this difficulty, collects sequences similar to an input query sequence from various data sources. The collected sequences can be passed on to the MAFFT sequence alignment server (http://mafft.cbrc.jp/alignment/server/), which has been significantly improved in interactivity. This update enables to switch between (i) sequence selection using the Archaeopteryx tree viewer, (ii) multiple sequence alignment and (iii) tree inference. This can be performed as a loop until one reaches a sensible data set, which minimizes redundancy for better visibility and handling in phylogenetic inference while covering relevant taxa. The work flow achieved by the seamless link between aLeaves and MAFFT provides a convenient online platform to address various questions in zoology and evolutionary biology.

Comprehensive survey of carapacial ridge-specific genes in turtle implies co-option of some regulatory genes in carapace evolution.

Summary The turtle shell is an evolutionary novelty in which the developmental pattern of the ribs is radically modified. In contrast to those of other amniotes, turtle ribs grow laterally into the dorsal dermis to form a carapace. The lateral margin of carapacial primordium is called the carapacial ridge (CR), and is thought to play an essential role in carapace patterning. To reveal the developmental mechanisms underlying this structure, we systematically screened for genes expressed specifically in the CR of the Chinese soft‐shelled turtle, Pelodiscus sinensis, using microbead‐based differential cDNA analysis and real‐time reverse transcription‐polymerase chain reaction. We identified orthologs of Sp5, cellular retinoic acid‐binding protein‐I (CRABP‐I), adenomatous polyposis coli down‐regulated 1 (APCDD1), and lymphoid enhancer‐binding factor‐1 (LEF‐1). Although these genes are conserved throughout the major vertebrate lineages, comparison of their expression patterns with those in chicken and mouse indicated that these genes have acquired de novo expression in the CR in the turtle lineage. In association with the expression of LEF‐1, the nuclear localization of β‐catenin protein was detected in the CR ectoderm, suggesting that the canonical Wnt signaling triggers carapace development. These findings indicate that the acquisition of the turtle shell did not involve the creation of novel genes, but was based on the co‐option of pre‐existing genes.

On the carapacial ridge in turtle embryos: its developmental origin, function and the chelonian body plan

The chelonian carapace is composed of dorsolaterally expanded ribs; an evolutionary change in the rib-patterning program is assumed to be related to this novelty. Turtle embryos exhibit a longitudinal ridge called the carapacial ridge (CR) on the flank, and its histological resemblance to the apical ectodermal ridge of the limb bud implies its inductive activity in the unique patterning of the ribs. We studied the Chinese soft-shelled turtle, Pelodiscus sinensis, and confirmed by labeling with a lipophilic dye, DiI, that the CR contains the somite-derived dermis and that it is a unique structure among amniotes. Using electroporation of a dominant-negative form of LEF-1, the CR-specific gene, we showed that CR-specific genes function in the growth and maintenance of the CR. Microcauterization or implantation of the CR did not change the dorsoventral pattern of the ribs, and only their fan-shaped pattern was arrested by CR removal. We conclude that the CR is a true embryonic novelty among amniotes and, because of the specific expression of regulatory genes, it functions in the marginal growth of the carapacial primordium, thereby inducing the fan-shaped arrangement of the ribs.

Involvement of Hedgehog and FGF signalling in the lamprey telencephalon: evolution of regionalization and dorsoventral patterning of the vertebrate forebrain

Dorsoventral (DV) specification is a crucial step for the development of the vertebrate telencephalon. Clarifying the origin of this mechanism will lead to a better understanding of vertebrate central nervous system (CNS) evolution. Based on the lamprey, a sister group of the gnathostomes (jawed vertebrates), we identified three lamprey Hedgehog (Hh) homologues, which are thought to play central signalling roles in telencephalon patterning. However, unlike in gnathostomes, none of these genes, nor Lhx6/7/8, a marker for the migrating interneuron subtype, was expressed in the ventral telencephalon, consistent with the reported absence of the medial ganglionic eminence (MGE) in this animal. Homologues of Gsh2, Isl1/2 and Sp8, which are involved in the patterning of the lateral ganglionic eminence (LGE) of gnathostomes, were expressed in the lamprey subpallium, as in gnathostomes. Hh signalling is necessary for induction of the subpallium identity in the gnathostome telencephalon. When Hh signalling was inhibited, the ventral identity was disrupted in the lamprey, suggesting that prechordal mesoderm-derived Hh signalling might be involved in the DV patterning of the telencephalon. By blocking fibroblast growth factor (FGF) signalling, the ventral telencephalon was suppressed in the lamprey, as in gnathostomes. We conclude that Hh- and FGF-dependent DV patterning, together with the resultant LGE identity, are likely to have been established in a common ancestor before the divergence of cyclostomes and gnathostomes. Later, gnathostomes would have acquired a novel Hh expression domain corresponding to the MGE, leading to the obtainment of cortical interneurons.

Evidence from cyclostomes for complex regionalization of the ancestral vertebrate brain

The vertebrate brain is highly complex, but its evolutionary origin remains elusive. Because of the absence of certain developmental domains generally marked by the expression of regulatory genes, the embryonic brain of the lamprey, a jawless vertebrate, had been regarded as representing a less complex, ancestral state of the vertebrate brain. Specifically, the absence of a Hedgehog- and Nkx2.1-positive domain in the lamprey subpallium was thought to be similar to mouse mutants in which the suppression of Nkx2-1 leads to a loss of the medial ganglionic eminence. Here we show that the brain of the inshore hagfish (Eptatretus burgeri), another cyclostome group, develops domains equivalent to the medial ganglionic eminence and rhombic lip, resembling the gnathostome brain. Moreover, further investigation of lamprey larvae revealed that these domains are also present, ruling out the possibility of convergent evolution between hagfish and gnathostomes. Thus, brain regionalization as seen in crown gnathostomes is not an evolutionary innovation of this group, but dates back to the latest vertebrate ancestor before the divergence of cyclostomes and gnathostomes more than 500 million years ago.

Impact of asymmetric gene repertoire between cyclostomes and gnathostomes

Extant vertebrates are divided into the two major groups, cyclostomes and gnathostomes (jawed vertebrates). The former includes jawless fishes, hagfishes and lampreys, and the latter includes all extant jawed vertebrates. In many research fields, the phenotypic traits of the cyclostomes have been considered crucial in understanding the evolutionary process from invertebrates to vertebrates. Recent studies have suggested that the common ancestor of the extant vertebrates including hagfishes and lampreys underwent two-round of whole genome duplications, and thus the genome expansion solely does not account for phenotypic differences between cyclostomes and gnathostomes. Emerging evidence from molecular phylogeny of individual gene families indicates that the gene repertoire expanded at the common ancestor of vertebrates were later reshaped asymmetrically between the two lineages, resulting in the retention of differential gene sets. This also confuses interpretation of conserved synteny which often serves as indicator of orthology and the ploidy level. In this review, current controversy and future perspectives of cyclostome genomics are discussed with reference to evolutionary developmental biology.