The proceedings in this volume follow from the 15^th Center for Developmental Biology meeting on “Advances in Cyclostome Research” that we organized. The meeting was held at the CDB RIKEN Kobe Institute on 24 and 25 January 2008 with support from the CDB.

Jawless vertebrates have been of interest to embryologists and comparative morphologists for more than a century. While the comparative morphology among lampreys, hagfishes, and gnathostomes has long been recognized in contributing to understanding the origin of jaws and other gnathostome traits, the availability of modern molecular methods has rekindled interest in these topics, and evolutionary developmental biology coupled with paleontology has opened new avenues into the study of gnathostome origins. Within the last decade, because of renewed interest in evolutionary developmental biology, the importance of lampreys and hagfishes to our understanding of vertebrate evolution has undergone resurgence in interest, as evidenced by the sea lamprey genome project currently underway at the National Human Genome Research Institute. As new molecular and imaging techniques become available, both paleontological and neontological questions are being readdressed and are providing new insights and speculation into vertebrate evolution. Thus, we determined the timing was optimal to bring together many of the researchers currently contributing to our understanding of the biology of agnathans.

The diversity of speakers at the meeting included evolutionary developmental biologists, phylogenetics and genomics investigators, paleontologists, and endocrinology researchers, because as we move into the 21^st century, integration among these disciplines has encouraged synergistic activities to develop. The goal of this meeting was to highlight in a single setting the most recent advances in this important basal group of vertebrates to facilitate interactions among the cyclostome community. Secondarily, we also hope that this gathering will enhance the visibility of jawless vertebrates as important models in the vertebrate “evo-devo” community.

Several topics for further discussion emerged at this symposium, including: genomic data that have spurred renewed interest in gene duplications and their contribution to our understanding of cyclostome phylogeny and vertebrate evolution; the use of paleontology coupled with modern imaging techniques to clarify vertebrate phylogeny; and the evolution of the neuroendocrine and adaptive immune systems. These were among the topics that led to fruitful discussion. Here we summarize key research topics from the symposium that continue to advance as we move forward in the 21^st century.

The ParaHox genes comprise three Hox-related homeobox gene families, found throughout the animals. They were first discovered in the invertebrate chordate amphioxus, where they are tightly clustered. In this paper we carry out a comparative review of ParaHox gene cluster organization among the deuterostomes, and discuss how the recently published hagfish ParaHox clusters fit into current theories about the evolution of this group of genes.

Interest in understanding the transition from prevertebrates to vertebrates at the molecular level has resulted in accumulating genomic and transcriptomic sequence data for the earliest groups of extant vertebrates, namely, hagfishes (Myxiniformes) and lampreys (Petromyzontiformes). Molecular phylogenetic studies on species phylogeny have revealed the monophyly of cyclostomes and the deep divergence between hagfishes and lampreys (more than 400 million years). In parallel, recent molecular phylogenetic studies have shed light on the complex evolution of the cyclostome genome. This consists of whole genome duplications, shared at least partly with gnathostomes (jawed vertebrates), and cyclostome lineage-specific secondary modifications of the genome, such as gene gains and losses. Therefore, the analysis of cyclostome genomes requires caution in distinguishing between orthology and paralogy in gene molecular phylogeny at the gene family scale, as well as between apomorphic and plesiomorphic genomic traits in larger-scale analyses. In this review, we propose possible ways of improving the resolvability of these evolutionary events, and discuss probable scenarios for cyclostome genome evolution, with special emphasis on the hypothesis that two-round (2R) genome duplication events occurred before the divergence between cyclostomes and gnathostomes, and therefore that a post-2R state is a genomic synapomorphy for all extant vertebrates.

Extant jawless vertebrates, represented by lampreys and hagfishes, have innate immune receptors with variable domains structurally resembling those of T/B-cell receptors. However, they appear to lack cardinal elements of adaptive immunity shared by all jawed vertebrates: major histocompatibility complex molecules and T/B-cell receptors. Thus, it was widely believed that adaptive immunity is unique to jawed vertebrates. Recently, this belief was overturned by the discovery of agnathan antigen receptors named variable lymphocyte receptors. These receptors generate diversity in their antigen-binding sites through assembling highly diverse leucine-rich repeat modules. The crystal structures of hagfish variable lymphocyte receptor monomers indicate that they adopt a horseshoe-shaped structure and likely bind antigens through the hypervariable concave surface. Secreted variable lymphocyte receptors form pentamers or tetramers of dimers and bind antigens with high specificity and avidity. The fact that variable lymphocyte receptors are structurally unrelated to T/B-cell receptors indicates that jawed and jawless vertebrates have developed antigen receptors independently.

Because the jaw is thought to have evolved as a dorsal–ventral articulation of the anterior pharyngeal arch, knowledge of developmental patterning in the pharyngeal arch is critical to understanding the origin and evolution of the jaw in gnathostomes. It is particularly important to determine whether Agnatha already possessed developmental polarity along the dorsal–ventral axis of the pharyngeal arch. We used the Weigert staining method to examine the development of cartilage in whole-mount lamprey specimens. We found that although the transverse rods showed symmetrical patterning along the dorsal–ventral axis, the hypobranchial bar and subchordal rod showed distinct developmental patterning. Thus, our observations suggest that pharyngeal cartilage also differentiates along the dorsal–ventral axis. In addition, the parachordal rods were shifted dorsally compared to the subchordal rods. Although the development of cartilage occurred earlier in the anterior arches, the fusion of the subchordal rods occurred earlier in the posterior arches. We also noted a unique morphology of cartilage in the ninth pharyngeal arch. Our descriptions and the methods used here facilitate the observation of pharyngeal cartilage in whole-mount specimens, and will aid in the study of developmental patterning in the lamprey pharyngeal arch.

Vertebrates are defined by the presence of the neural crest. These cells contribute to the increased complexity of vertebrates relative to non-vertebrate chordates. It is widely accepted that an increase in vertebrate complexity is also related to gene duplications that occurred at the base of vertebrates and may be related to the origin of the neural crest. Study of the development of one neural crest derivative, pharyngeal cartilage, in the lamprey and comparison to chondrogenesis in other chordates may provide clues regarding acquisition of a chondrogenic fate by the neural crest. The transcription factor Sox9 is thr product of a SoxE gene (Col2a1) that regulates expression of Type II collagenin development of vertebrate cartilage. Duplication of the ancestral SoxE transcription factor into the three SoxE genes present in vertebrates might have been important in partitioning the chondrogenic neural crest role of Sox9. In this review, I discuss evidence that duplicated SoxE genes might have been key in the evolution of chondrogenic neural crest. The recent identification of duplicated SoxE genes, and identification of two Type II collagen genes in lamprey, coupled with their expression in branchial arches suggested ancient regulation of chondrogenesis by a SoxE gene. Examination of SoxE and Type II collagen expression in the developing lamprey branchial arches shows SoxE genes are expressed in developing branchial arch cartilage and Col2a1 genes are expressed in surrounding mesenchymal cells. Lack of cellular co-expression of SoxE genes with Col2a1 suggests additional steps might have been required for direct regulation of Col2a1 by Sox9 in jawed vertebrates.

Here I consider ways to test two hypotheses of the origin of jawed vertebrates: my neoclassical hypothesis, which derived from comparative morphology; and the heterotopic hypothesis, which derives from modern developmental findings. The heterotopic hypothesis, unlike the neoclassical hypothesis, says that major developmental changes had to occur before the upper jaws could evolve: a caudal shift in the expression domains of genes that pattern oral structures; and the loss of ancestral, lamprey-like upper lips. To test whether these did occur, I propose studies on the development of chondrichthyans (sharks and chimaeroid ratfishes), an understudied group that is likely to retain primitive features of the jaw region. The heterotopic hypothesis says no gnathostome retains the upper lip that is so prominent in larval lampreys, yet the neoclassical hypothesis identified such lips in sharks and ratfishes, and it predicts that their lip-skeleton likewise develops from premandibular neural crest. The development and innervation of upper-lip muscles in lampreys and chimaeroids can also be compared. The proposed studies can determine if the upper lips of chondrichthyans and lampreys are homologous (which would support the neoclassical hypothesis), or homoplasious (which would support the heterotopic hypothesis).

Also, I argue that the evolution of the upper jaws (as lateral structures) was not linked to a nasohypophyseal complex (which is a median structure), although such a link is claimed by the heterotopic hypothesis. Finally, I update the neoclassical hypothesis to address recent evidence that the trabeculae of the lamprey skull are not homologous to the trabeculae cranii of gnathostomes.

The apparently primitive features of hagfishes are recognized as a crucial problem in the study of vertebrate evolution, although the monophyletic relationship between these animals and lampreys has been confirmed by large amounts of molecular data, including genome and EST sequences. To solve this problem requires knowledge of the developmental biology of hagfishes. We attempted to obtain embryos from the Japanese inshore hagfish (Eptatretus burgeri) and succeeded in preparing several nicely fixed embryos. Based on detailed histological observations and comparison of gene expression patterns with those of conventional vertebrates, we examined the developmental processes involved in some important morphological traits, including the neural crest, placode, pharyngeal arches, and others. Our data revealed that some apparently primitive morphological traits can be regarded as artifacts deriving mainly from fixation conditions. In addition, our long-term observations of live embryos revealed a slow developmental rate in this animal. In this review, we summarize recent developmental data from these hagfish embryos and discuss a plausible evolutionary scenario for vertebrate development, making comparisons with some old descriptions.

The evolutionary history of the vertebrate mouth has long been an intriguing issue in comparative zoology. When the prevertebrate state was considered, the oral structure in adult lancelets (amphioxus) was traditionally referred to because of its general similarity to that of the ammocoete larva of lampreys. The larval mouth in lancelets, however, shows a peculiar developmental mode. Reflecting this, the affinity of the lancelet mouth has long been argued, but is still far from a consensus. The increase in available data from molecular biology, comparative developmental biology, paleontology, and other related fields makes it prudent to discuss morphological homology and homoplasy. Here, we review how the lancelet mouth has been interpreted in the study of evolution of the vertebrate mouth, as well as recent advances in chordate studies. With this background of increased knowledge, our innervation analysis supports the interpretation that the morphological similarity in the oral apparatus between ammocoetes and lancelets is a homoplasy caused by their similar food habits.

Vertebrate brains are highly organized structures that show remarkable diversity throughout animal groups. The agnathans, which diverged from the gnathostomes early in the evolution of the vertebrates, occupy a key phylogenetic position from which to clarify the origin and evolution of the brain. We studied the developing lamprey brain and compared its developmental plan with that of the gnathostomes, in order to reconstruct the evolutionary processes of the vertebrate brain. We found that the lamprey brain has the basic molecular mechanisms necessary to form neuromeric compartments, and that its mesencephalon and diencephalon exhibit conserved morphological and molecular features. Conversely, the telencephalon and metencephalon display lamprey-specific developmental mechanisms. Thus, the molecular program of the nervous system is thought to have improved in the gnathostome lineage. Changes in the expression domains of some regulatory genes might have facilitated the evolution of the vertebrate central nervous system.

The pituitary gland is present in all vertebrates, from agnathans (jawless fishes) to mammals, but not in invertebrates. Hagfishes, which lack both jaws and vertebrae, are considered the most primitive vertebrate known, living or extinct. Accordingly, studies on hagfishes are indispensable for understanding the origin and evolution of the pituitary hormones. Nevertheless, little is known about the hagfish adenohypophysial hormones. Our recent immunohistochemical and lectin histochemical studies have revealed that gonadotropin (GTH), adrenocorticotropin (ACTH), and growth hormone (GH) are present in the hagfish pituitary gland. This review summarizes the latest data regarding the hagfish adenohypophysial hormones from an evolutionary point of view.

Secretion of the pituitary glycoprotein hormones (GpH) follitropin, lutropin, and thyrotropin in vertebrates is the main mechanism by which neuroendocrine signals are propagated at the level of the peripheral glands, gonads and thyroid. Receptors of these hormones (glycoprotein hormone receptors, GpH-R) evolved from a common ancestor through gene duplication and subsequent functional divergence during the split of gnathostomes from their agnathan ancestors. Here we review the properties of two novel receptors closely related to gnathostome GpH-Rs identified in the sea lamprey. Although these are the oldest members of this family of receptors described so far in vertebrates, their overall structural features are remarkably close to their mammalian counterparts. However, they cannot be classified unequivocally as either gonadotropin (FSH-R, LH-R) or as thyrotropin receptors (TSH-R) since they share characteristics with both these groups. This may indicate that lamprey receptors reflect in part properties of the ancestral molecule(s) from which all vertebrate GpH-Rs originated. Molecular phylogenetic relationships among gnathostome GpH-Rs are heavily dependent on the functional domain used in analysis. This suggests large variation in functional constraints acting at the level of different segments of the receptor molecule.

Undoubted fossil lampreys are recorded since the Late Devonian (358 Ma), and probable fossil hagfishes since the Late Carboniferous (300 Ma), but molecular clock data suggest a much earlier divergence times for the two groups. In the early 20^th century, hagfishes and lampreys were generally thought to have diverged much later from unknown ancestral cyclostomes, in turn derived through ‘degeneracy’ from some Paleozoic armored jawless vertebrates, or ‘ostracoderms.’ However, current vertebrate phylogenies suggest that most, if not all, ‘ostracoderms’ are in fact jawless stem gnathostomes, which retain certain features that were once regarded as unique to the cyclostomes, such as gill pouches or lack of horizontal semicircular canal. The dorsal, median, nasohypophysial complex of osteostracans has been regarded as identical and homologous to that of lampreys, but recent investigation (notably on the galeaspid braincase) now suggests that this resemblance is in fact a convergence. The anatomy and physiology of lampreys and hagfishes are so different that it is difficult to reconstruct an ancestral morphotype of the cyclostomes, assuming that they are a clade, and there is no clear evidence of any fossil taxon that is neither a fossil hagfish nor a fossil lamprey, but would be more closely related to the cyclostomes than to the gnathostomes. A possible exception is the Silurian-Devonian euphaneropids (or ‘naked anaspids’).