Physiology, biomechanics, and biomimetics of hagfish slime.

Hagfishes thwart attacks by fish predators by producing liters of defensive slime. The slime is produced when slime gland exudate is released into the predator's mouth, where it deploys in a fraction of a second and clogs the gills. Slime exudate is composed mainly of secretory products from two cell types, gland mucous cells and gland thread cells, which produce the mucous and fibrous components of the slime, respectively. Here, we review what is known about the composition of the slime, morphology of the slime gland, and physiology of the cells that produce the slime. We also discuss several of the mechanisms involved in the deployment of both mucous and thread cells during the transition from thick glandular exudate to ultradilute material. We review biomechanical aspects of the slime, along with recent efforts to produce biomimetic slime thread analogs, and end with a discussion of how hagfish slime may have evolved.

The hagfish genome and the evolution of vertebrates.

As the only surviving lineages of jawless fishes, hagfishes and lampreys provide a crucial window into early vertebrate evolution1-3. Here we investigate the complex history, timing and functional role of genome-wide duplications4-7 and programmed DNA elimination8,9 in vertebrates in the light of a chromosome-scale genome sequence for the brown hagfish Eptatretus atami. Combining evidence from syntenic and phylogenetic analyses, we establish a comprehensive picture of vertebrate genome evolution, including an auto-tetraploidization (1RV) that predates the early Cambrian cyclostome-gnathostome split, followed by a mid-late Cambrian allo-tetraploidization (2RJV) in gnathostomes and a prolonged Cambrian-Ordovician hexaploidization (2RCY) in cyclostomes. Subsequently, hagfishes underwent extensive genomic changes, with chromosomal fusions accompanied by the loss of genes that are essential for organ systems (for example, genes involved in the development of eyes and in the proliferation of osteoclasts); these changes account, in part, for the simplification of the hagfish body plan1,2. Finally, we characterize programmed DNA elimination in hagfish, identifying protein-coding genes and repetitive elements that are deleted from somatic cell lineages during early development. The elimination of these germline-specific genes provides a mechanism for resolving genetic conflict between soma and germline by repressing germline and pluripotency functions, paralleling findings in lampreys10,11. Reconstruction of the early genomic history of vertebrates provides a framework for further investigations of the evolution of cyclostomes and jawed vertebrates.

The oldest three-dimensionally preserved vertebrate neurocranium.

The neurocranium is an integral part of the vertebrate head, itself a major evolutionary innovation1,2. However, its early history remains poorly understood, with great dissimilarity in form between the two living vertebrate groups: gnathostomes (jawed vertebrates) and cyclostomes (hagfishes and lampreys)2,3. The 100 Myr gap separating the Cambrian appearance of vertebrates4-6 from the earliest three-dimensionally preserved vertebrate neurocrania7 further obscures the origins of modern states. Here we use computed tomography to describe the cranial anatomy of an Ordovician stem-group gnathostome: Eriptychius americanus from the Harding Sandstone of Colorado, USA8. A fossilized head of Eriptychius preserves a symmetrical set of cartilages that we interpret as the preorbital neurocranium, enclosing the fronts of laterally placed orbits, terminally located mouth, olfactory bulbs and pineal organ. This suggests that, in the earliest gnathostomes, the neurocranium filled out the space between the dermal skeleton and brain, like in galeaspids, osteostracans and placoderms and unlike in cyclostomes2. However, these cartilages are not fused into a single neurocranial unit, suggesting that this is a derived gnathostome trait. Eriptychius fills a major temporal and phylogenetic gap in our understanding of the evolution of the gnathostome head, revealing a neurocranium with an anatomy unlike that of any previously described vertebrate.

Inner ear development in cyclostomes and evolution of the vertebrate semicircular canals.

Jawed vertebrates have inner ears with three semicircular canals, the presence of which has been used as a key to understanding evolutionary relationships. Ostracoderms, the jawless stem gnathostomes, had only two canals and lacked the lateral canal1-3. Lampreys, which are modern cyclostomes, are generally thought to possess two semicircular canals whereas the hagfishes-which are also cyclostomes-have only a single canal, which used to be regarded as a more primitive trait1,4. However, recent molecular and developmental analyses have strongly supported the monophyly of cyclostomes5-7, which has left the evolutionary trajectory of the vertebrate inner ear unclear8. Here we show the differentiation of the otic vesicle of the lamprey Lethenteron camtschaticum and inshore hagfish Eptatretus burgeri. This is the first time, to our knowledge, that the development of the hagfish inner ear is reported. We found that canal development in the lamprey starts with two depressions-which is reminiscent of the early developmental pattern of the inner ear in modern gnathostomes. These cyclostome otic vesicles show a pattern of expression of regulatory genes, including OTX genes, that is comparable to that of gnathosomes. Although two depressions appear in the lamprey vesicle, they subsequently fuse to form a single canal that is similar to that of hagfishes. Complete separation of the depressions results in anterior and posterior canals in gnathostomes. The single depression of the vesicle in hagfishes thus appears to be a secondarily derived trait. Furthermore, the lateral canal in crown gnathostomes was acquired secondarily-not by de novo acquisition of an OTX expression domain, but by the evolution of a developmental program downstream of the OTX genes.

Collagen IV of basement membranes: II. Emergence of collagen IVα345 enabled the assembly of a compact GBM as an ultrafilter in mammalian kidneys.

The collagen IVα345 (Col-IVα345) scaffold, the major constituent of the glomerular basement membrane (GBM), is a critical component of the kidney glomerular filtration barrier. In Alport syndrome, affecting millions of people worldwide, over two thousand genetic variants occur in the COL4A3, COL4A4, and COL4A5 genes that encode the Col-IVα345 scaffold. Variants cause loss of scaffold, a suprastructure that tethers macromolecules, from the GBM or assembly of a defective scaffold, causing hematuria in nearly all cases, proteinuria, and often progressive kidney failure. How these variants cause proteinuria remains an enigma. In a companion paper, we found that the evolutionary emergence of the COL4A3, COL4A4, COL4A5, and COL4A6 genes coincided with kidney emergence in hagfish and shark and that the COL4A3 and COL4A4 were lost in amphibians. These findings opened an experimental window to gain insights into functionality of the Col-IVα345 scaffold. Here, using tissue staining, biochemical analysis and TEM, we characterized the scaffold chain arrangements and the morphology of the GBM of hagfish, shark, frog, and salamander. We found that α4 and α5 chains in shark GBM and α1 and α5 chains in amphibian GBM are spatially separated. Scaffolds are distinct from one another and from the mammalian Col-IVα345 scaffold, and the GBM morphologies are distinct. Our findings revealed that the evolutionary emergence of the Col-IVα345 scaffold enabled the genesis of a compact GBM that functions as an ultrafilter. Findings shed light on the conundrum, defined decades ago, whether the GBM or slit diaphragm is the primary filter.

Molecular basis of the unique osmoregulatory strategy in the inshore hagfish, Eptatretus burgeri.

Hagfishes are characterized by omo- and iono-conforming nature similar to marine invertebrates. Conventionally, hagfishes had been recognized as the most primitive living vertebrate that retains plesiomorphic features. However, some of the "ancestral" features of hagfishes, such as rudimentary eyes and the lack of vertebrae, have been proven to be deceptive. Similarly, by the principle of maximum parsimony, the unique body fluid regulatory strategy of hagfishes seems to be apomorphic, since the lamprey, another cyclostome, adopts osmo- and iono-regulatory mechanisms as in jawed vertebrates. Although hagfishes are unequivocally important in discussing the origin and evolution of the vertebrate osmoregulatory system, the molecular basis for the body fluid homeostasis in hagfishes has been poorly understood. In the present study, we explored this matter in the inshore hagfish, Eptatretus burgeri, by analyzing the transcriptomes obtained from the gill, kidney, and muscle of the animals acclimated to distinct environmental salinities. Together with the measurement of parameters in the muscular fluid compartment, our data indicate that the hagfish possesses an ability to conduct free amino acid (FAA)-based osmoregulation at a cellular level, which is in coordination with the renal and branchial FAA absorption. We also revealed that the hagfish does possess the orthologs of the known osmoregulatory genes and that the transepithelial movement of inorganic ions in the hagfish gill and kidney is more complex than previously thought. These observations pose a challenge to the conventional view that the physiological features of hagfishes have been inherited from the last common ancestor of the extant vertebrates.

Biphasic burrowing in Atlantic hagfish (Myxine limosa).

Myxine limosa is a burrowing species of hagfish that occurs in the western North Atlantic in areas with muddy substrate and at depths generally greater than 100 meters. Burrowing of M. limosa has been observed from submersibles, but little is known about the behavior of these animals within the substrate or the biomechanical mechanisms involved. Here, we investigated burrowing in M. limosa by observing individuals as they burrowed through transparent gelatin. A photoelastic setup using crossed polarizers allowed us to visualize stress development in the gelatin as the hagfish moved through it. We found that M. limosa created U-shaped burrows in gelatin using a stereotyped, two-phase burrowing behavior. In the first ('thrash') phase, hagfish drove their head and their anterior body into the substrate using vigorous sinusoidal swimming movements, with their head moving side-to-side. In the second ('wriggle') phase, swimming movements ceased, with propulsion coming exclusively from the anterior, submerged portion of body. The wriggle phase involved side-to-side head movements and movements of the submerged part of the body that resembled the internal concertina strategy used by caecilians and uropeltid snakes. The entire burrowing process took on average 7.6 min to complete and ended with the hagfish's head protruding from the substrate and the rest of its body generally concealed. Understanding the burrowing activities of hagfishes could lead to improved understanding of sediment turnover in marine benthic habitats, new insights into the reproductive behavior of hagfishes, or even inspiration for the design of burrowing robots.

Leveraging the biotechnological promise of the hagfish variable lymphocyte receptors: tools for aquatic microbial diseases.

The jawless vertebrates (agnathans/cyclostomes) are ancestral animals comprising lampreys and hagfishes as the only extant representatives. They possess an alternative adaptive immune system (AIS) that uses leucine-rich repeats (LRR)-based variable lymphocyte receptors (VLRs) instead of the immunoglobulin (Ig)-based antigen receptors of jawed vertebrates (gnathostomes). The different VLR types are expressed on agnathan lymphocytes and functionally resemble gnathostome antigen receptors. In particular, VLRB is functionally similar to the B cell receptor and is expressed and secreted by B-like lymphocytes as VLRB antibodies that bind antigens with high affinity and specificity. The potential repertoire scale of VLR-based antigen receptors is believed to be at least comparable to that of Ig-based systems. VLR proteins inherently possess characteristics that render them excellent candidates for biotechnological development, including tractability to recombinant approaches. In recent years, scientists have explored the biotechnological development and utility of VLRB proteins as alternatives to conventional mammalian antibodies. The VLRB antibody platform represents a non-traditional approach to generating a highly diverse repertoire of unique antibodies. In this review, we first describe some aspects of the biology of the AIS of the jawless vertebrates, which recognizes antigens by means of unique receptors. We then summarize reports on the development of VLRB-based antibodies and their applications, particularly those from the inshore hagfish (Eptatretus burgeri) and their potential uses to address microbial diseases in aquaculture. Hagfish VLRB antibodies (we call Ccombodies) are being developed and improved, while obstacles to the advancement of the VLRB platform are being addressed to utilize VLRBs effectively as tools in immunology. VLRB antibodies for novel antigen targets are expected to emerge to provide new opportunities to tackle various scientific questions. We anticipate a greater interest in the agnathan AIS in general and particularly in the hagfish AIS for greater elucidation of the evolution of adaptive immunity and its applications to address microbial pathogens in farmed aquatic animals and beyond.

Evolution of variable lymphocyte receptor B antibody loci in jawless vertebrates.

Three types of variable lymphocyte receptor (VLR) genes, VLRA, VLRB, and VLRC, encode antigen recognition receptors in the extant jawless vertebrates, lampreys and hagfish. The somatically diversified repertoires of these VLRs are generated by serial stepwise copying of leucine-rich repeat (LRR) sequences into an incomplete germline VLR gene. Lymphocytes that express VLRA or VLRC are T cell-like, while VLRB-expressing cells are B cell-like. Here, we analyze the composition of the VLRB locus in different jawless vertebrates to elucidate its configuration and evolutionary modification. The incomplete germline VLRB genes of two hagfish species contain short noncoding intervening sequences, whereas germline VLRB genes in six representative lamprey species have much longer intervening sequences that exhibit notable genomic variation. Genomic clusters of potential LRR cassette donors, fragments of which are copied to complete VLRB gene assembly, are identified in Japanese lamprey and sea lamprey. In the sea lamprey, 428 LRR cassettes are located in five clusters spread over a total of 1.7 Mbp of chromosomal DNA. Preferential usage of the different donor cassettes for VLRB assemblage is characterized in our analysis, which reveals evolutionary modifications of the lamprey VLRB genes, elucidates the organization of the complex VLRB locus, and provides a comprehensive catalog of donor VLRB cassettes in sea lamprey and Japanese lamprey.

Phylogenetic and functional properties of hagfish neurohypophysial hormone receptors distinct from their jawed vertebrate counterparts.

Vertebrate neurohypophysial hormones, i.e., vasopressin- and oxytocin-family peptides, exert versatile physiological actions via distinct G protein-coupled receptors. The neurohypophysial hormone receptor (NHR) family was classically categorized into four subtypes (V1aR, V1bR, V2R and OTR), while recent studies have identified seven subtypes (V1aR, V1bR, V2aR, V2bR, V2cR, V2dR and OTR; V2aR corresponds to the conventional V2R). The vertebrate NHR family were diversified via multiple gene duplication events at different scales. Despite intensive research effort in non-osteichthyes vertebrates such as cartilaginous fish and lamprey, the molecular phylogeny of the NHR family has not been fully understood. In the present study, we focused on the inshore hagfish (Eptatretus burgeri), another group of cyclostomes, and Arctic lamprey (Lethenteron camtschaticum) for comparison. Two putative NHR homologs, which were previously identified only in silico, were cloned from the hagfish and designated as ebV1R and ebV2R. In vitro, ebV1R, as well as two out of five Arctic lamprey NHRs, increased intracellular Ca2+ in response to exogenous neurohypophysial hormones. None of the examined cyclostome NHRs altered intracellular cAMP levels. Transcripts of ebV1R were detected in multiple tissues including the brain and gill, with intense hybridization signals in the hypothalamus and adenohypophysis, while ebV2R was predominantly expressed in the systemic heart. Similarly, Arctic lamprey NHRs showed distinct expression patterns, underscoring the multifunctionality of VT in the cyclostomes as in the gnathostomes. These results and exhaustive gene synteny comparisons provide new insights into the molecular and functional evolution of the neurohypophysial hormone system in vertebrates.

Thyroid and endostyle development in cyclostomes provides new insights into the evolutionary history of vertebrates.

BACKGROUND: The endostyle is an epithelial exocrine gland found in non-vertebrate chordates (amphioxi and tunicates) and the larvae of modern lampreys. It is generally considered to be an evolutionary precursor of the thyroid gland of vertebrates. Transformation of the endostyle into the thyroid gland during the metamorphosis of lampreys is thus deemed to be a recapitulation of a past event in vertebrate evolution. In 1906, Stockard reported that the thyroid gland in hagfish, the sister cyclostome group of lampreys, develops through an endostyle-like primordium, strongly supporting the plesiomorphy of the lamprey endostyle. However, the findings in hagfish thyroid development were solely based on this single study, and these have not been confirmed by modern molecular, genetic, and morphological data pertaining to hagfish thyroid development over the last century. RESULTS: Here, we showed that the thyroid gland of hagfish undergoes direct development from the ventrorostral pharyngeal endoderm, where the previously described endostyle-like primordium was not found. The developmental pattern of the hagfish thyroid, including histological features and regulatory gene expression profiles, closely resembles that found in modern jawed vertebrates (gnathostomes). Meanwhile, as opposed to gnathostomes but similar to non-vertebrate chordates, lamprey and hagfish share a broad expression domain of Nkx2-1/2-4, a key regulatory gene, in the pharyngeal epithelium during early developmental stages. CONCLUSIONS: Based on the direct development of the thyroid gland both in hagfish and gnathostomes, and the shared expression profile of thyroid-related transcription factors in the cyclostomes, we challenge the plesiomorphic status of the lamprey endostyle and propose an alternative hypothesis where the lamprey endostyle could be obtained secondarily in crown lampreys.

A new species of the hagfish genus Eptatretus (Myxinidae) from the Bahamas, western North Atlantic.

A new species of the hagfish genus Eptatretus (Myxinidae) is described based on two specimens (407-433 mm total length) collected off the northern Bahamas, between depths of 910 and 1153 m. The new species is distinguished from its congeners by having seven pairs of gill apertures well-spaced and arranged in a near straight line, a 3/2 multicusp pattern of teeth, 10-11 anterior unicusps, 50-51 total cusps, 12-14 prebranchial pores, 48-52 trunk pores, 79-84 total pores, and no nasal-sinus papillae. An identification key for the species of Eptatretus from the western Atlantic Ocean is also provided.

A glimpse into the past: phylogenesis and protein domain analysis of the group XIV of C-type lectins in vertebrates.

BACKGROUND: The group XIV of C-type lectin domain-containing proteins (CTLDcps) is one of the seventeen groups of CTLDcps discovered in mammals and composed by four members: CD93, Clec14A, CD248 and Thrombomodulin, which have shown to be important players in cancer and vascular biology. Although these proteins belong to the same family, their phylogenetic relationship has never been dissected. To resolve their evolution and characterize their protein domain composition we investigated CTLDcp genes in gnathostomes and cyclostomes and, by means of phylogenetic approaches as well as synteny analyses, we inferred an evolutionary scheme that attempts to unravel their evolution in modern vertebrates. RESULTS: Here, we evidenced the paralogy of the group XIV of CTLDcps in gnathostomes and discovered that a gene loss of CD248 and Clec14A occurred in different vertebrate groups, with CD248 being lost due to chromosome disruption in birds, while Clec14A loss in monotremes and marsupials did not involve chromosome rearrangements. Moreover, employing genome annotations of different lampreys as well as one hagfish species, we investigated the origin and evolution of modern group XIV of CTLDcps. Furthermore, we carefully retrieved and annotated gnathostome CTLDcp domains, pointed out important differences in domain composition between gnathostome classes, and assessed codon substitution rate of each domain by analyzing nonsynonymous (Ka) over synonymous (Ks) substitutions using one representative species per gnathostome order. CONCLUSIONS: CTLDcps appeared with the advent of early vertebrates after a whole genome duplication followed by a sporadic tandem duplication. These duplication events gave rise to three CTLDcps in the ancestral vertebrate that underwent further duplications caused by the independent polyploidizations that characterized the evolution of cyclostomes and gnathostomes. Importantly, our analyses of CTLDcps in gnathostomes revealed critical inter-class differences in both extracellular and intracellular domains, which might help the interpretation of experimental results and the understanding of differences between animal models.

Genetic and functional analysis of a Pacific hagfish opioid system.

The actions of endogenous opioids and nociceptin/orphanin FQ are mediated by four homologous G protein-coupled receptors that constitute the opioid receptor family. However, little is known about opioid systems in cyclostomes (living jawless fish) and how opioid systems might have evolved from invertebrates. Here, we leveraged de novo transcriptome and low-coverage whole-genome assembly in the Pacific hagfish (Eptatretus stoutii) to identify and characterize the first full-length coding sequence for a functional opioid receptor in a cyclostome. Additionally, we define two novel endogenous opioid precursors in this species that predict several novel opioid peptides. Bioinformatic analysis shows no closely related opioid receptor genes in invertebrates with regard either to the genomic organization or to conserved opioid receptor-specific sequences that are common in all vertebrates. Furthermore, no proteins analogous to vertebrate opioid precursors could be identified by genomic searches despite previous claims of protein or RNA-derived sequences in several invertebrate species. The presence of an expressed orthologous receptor and opioid precursors in the Pacific hagfish confirms that a functional opioid system was likely present in the common ancestor of all extant vertebrates some 550 million years ago, earlier than all previous authenticated accounts. We discuss the premise that the cyclostome and vertebrate opioid systems evolved from invertebrate systems concerned with antimicrobial defense and speculate that the high concentrations of opioid precursors in tissues such as the testes, gut, and activated immune cells are key remnants of this evolutionary role.

Molecular identification and postprandial regulation of glucose carrier proteins in the hindgut of Pacific hagfish, Eptatretus stoutii.

Hagfish are an excellent model species in which to draw inferences on the evolution of transport systems in early vertebrates owing to their basal position in vertebrate phylogeny. Glucose is a ubiquitous cellular energy source that is transported into cells via two classes of carrier proteins: sodium-glucose-linked transporters (Sglt; Slc5a) and glucose transporters (Glut; Slc2a). Although previous pharmacological evidence has suggested the presence of both sodium-dependent and -independent transport mechanisms in the hagfish, the molecular identities were heretofore unconfirmed. We have identified and phylogenetically characterized both a Slc5a1-like and Slc2a-like gene in the Pacific hagfish (Eptatretus stoutii), the latter sharing common ancestry with other glucose-transporting isoforms of the Slc2a family. To assess the potential postprandial regulation of these glucose transporters, we examined the abundance and localization of these transporters with qPCR and immunohistochemistry alongside functional studies using radiolabeled d-[14C]glucose. The effects of glucose or insulin injection on glucose transport rate and transporter expression were also examined to determine their potential role(s) in the regulation of intestinal glucose carrier proteins. Feeding prompted an increase in glucose uptake across the hindgut at both 0.5 mM (∼84%) and 1 mM (∼183%) concentrations. Concomitant increases were observed in hindgut Slc5a1 protein expression. These effects were not observed following either of glucose or insulin injection, indicating these postprandial factors are not the driving force for transporter regulation over this timeframe. We conclude that Pacific hagfish utilize evolutionarily conserved mechanisms of glucose uptake and so represent a useful model to understand early-vertebrate evolution of glucose uptake and regulation.

Scalable purification of recombinant structural proteins, hagfish intermediate filament α and É£, from inclusion bodies for fiber formation.

The purpose of this study was to determine a method to purify recombinant hagfish intermediate filament proteins, alpha and gamma, in a scalable manner. The study succeeded by having an increase in protein recovery of up to 35% when comparing centrifuge purification and the developed tangential flow purification. The proteins were approximately the same purity of 70% pure but further purification increased the purity of the proteins by 16%, based on ImageJ analysis. The developed tangential flow filtration purification and final purification methods could be easily scaled up to meet industry scale purification needs. The scaled-up processes described in this study did not interfere with fiber production or formation, indicating the methods can produce usable proteins for material development.

Breathing versus feeding in the Pacific hagfish.

Hagfish represent the oldest extant connection to the ancestral vertebrates, but their physiology is not well understood. Using behavioural (video), physiological (respirometry, flow measurements), classical morphological (dissection, silicone injection) and modern imaging approaches (micro-MRI, DICE micro-CT), we examined the interface between feeding and the unique breathing mechanism (nostril opening, high-frequency velum contraction, low-frequency gill pouch contraction and pharyngo-cutaneous duct contraction) in the Pacific hagfish, Eptatretus stoutii. A video tour via micro-MRI is presented through the breathing and feeding passages. We have reconciled an earlier disagreement as to the position of the velum chamber, which powers inhalation through the nostril, placing it downstream of the merging point of the food and water passage, such that the oronasal septum terminates at the anterior end of the velum chamber. When feeding occurs by engulfment of large chunks by the dental plates, food movement through the chamber may transiently interfere with breathing. Swallowing is accelerated by peristaltic body undulation involving the ventral musculature, and is complete within 5 s. After a large meal (anchovy, 20% body mass), hagfish remain motionless, defaecating bones and scales at 1.7 days and an intestinal peritrophic membrane at 5 days. O2 consumption rate approximately doubles within 1 h of feeding, remaining elevated for 12-24 h. This is achieved by combinations of elevated O2 utilization and ventilatory flow, the latter caused by varying increases in velar contraction frequency and stroke volume. Additional imaging casts light on the reasons for the trend for greater O2 utilization by more posterior pouches and the pharyngo-cutaneous duct in fasted hagfish.

Characterization of Hagfish (Eptatretus burgeri) Variable Lymphocyte Receptor-Based Antibody and Its Potential Role in the Neutralization of Nervous Necrosis Virus.

The variable lymphocyte receptor (VLR) mediates the humoral immune response in jawless vertebrates, including lamprey (Petromyzon marinus) and hagfish (Eptatretus burgeri). Hagfish VLRBs are composed of leucine-rich repeat (LRR) modules, conjugated with a superhydrophobic C-terminal tail, which contributes to low levels of expression in recombinant protein technology. In this study, we screened Ag-specific VLRBs from hagfish immunized with nervous necrosis virus (NNV). The artificially multimerized form of VLRB was constructed using a mammalian expression system. To enhance the level of expression of the Ag-specific VLRB, mutagenesis of the VLRB was achieved in vitro through domain swapping of the LRR C-terminal cap and variable LRR module. The mutant VLRB obtained, with high expression and secretion levels, was able to specifically recognize purified and progeny NNV, and the Ag binding ability of this mutant was increased by at least 250-fold to that of the nonmutant VLRB. Furthermore, preincubation of the Ag-specific VLRB with NNV reduced the infectivity of NNV in E11 cells in vitro, and in vivo experiment. Our results suggest that the newly developed Ag-specific VLRB has the potential to be used as diagnostic and therapeutic reagents for NNV infections in fish.

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.

Forebrain Architecture and Development in Cyclostomes, with Reference to the Early Morphology and Evolution of the Vertebrate Head.

The vertebrate head and brain are characterized by highly complex morphological patterns. The forebrain, the most anterior division of the brain, is subdivided into the diencephalon, hypothalamus, and telencephalon from the neuromeric subdivision into prosomeres. Importantly, the telencephalon contains the cerebral cortex, which plays a key role in higher order cognitive functions in humans. To elucidate the evolution of the forebrain regionalization, comparative analyses of the brain development between extant jawed and jawless vertebrates are crucial. Cyclostomes - lampreys and hagfishes - are the only extant jawless vertebrates, and diverged from jawed vertebrates (gnathostomes) over 500 million years ago. Previous developmental studies on the cyclostome brain were conducted mainly in lampreys because hagfish embryos were rarely available. Although still scarce, the recent availability of hagfish embryos has propelled comparative studies of brain development and gene expression. By integrating findings with those of cyclostomes and fossil jawless vertebrates, we can depict the morphology, developmental mechanism, and even the evolutionary path of the brain of the last common ancestor of vertebrates. In this review, we summarize the development of the forebrain in cyclostomes and suggest what evolutionary changes each cyclostome lineage underwent during brain evolution. In addition, together with recent advances in the head morphology in fossil vertebrates revealed by CT scanning technology, we discuss how the evolution of craniofacial morphology and the changes of the developmental mechanism of the forebrain towards crown gnathostomes are causally related.