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.

Gross anatomy of the Pacific hagfish, Eptatretus burgeri, with special reference to the coelomic viscera.

Hagfish (Myxinoidea) are a deep-sea taxon of cyclostomes, the extant jawless vertebrates. Many researchers have examined the anatomy and embryology of hagfish to shed light on the early evolution of vertebrates; however, the diversity within hagfish is often overlooked. Hagfish have three lineages, Myxininae, Eptatretinae, and Rubicundinae. Usually, textbook illustrations of hagfish anatomy reflect the morphology of the Myxininae lineage, especially Myxine glutinosa, with its single pair of external branchial pores. Here, we instead report the gross anatomy of an Eptatretinae, Eptatretus burgeri, which has six pairs of branchial pores, especially focusing on the coelomic organs. Dissections were performed on fixed and unfixed specimens to provide a guide for those doing organ- or tissue-specific molecular experiments. Our dissections revealed that the ventral aorta is Y-branched in E. burgeri, which differs from the unbranched morphology of Myxine. Otherwise, there were no differences in the morphology of the lingual apparatus or heart in the pharyngeal domain. The thyroid follicles were scattered around the ventral aorta, as has been reported for adult lampreys. The hepatobiliary system more closely resembled those of jawed vertebrates than those of adult lampreys, with the liver having two lobes and a bile duct connecting the gallbladder to each lobe. Overall, the visceral morphology of E. burgeri does not differ significantly from that of the known Myxine at the level of gross anatomy, although the branchial morphology is phylogenetically ancestral compared to Myxine.

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.

A new species of six-gilled hagfish (Myxinidae: Eptatretus) from the Lakshadweep Sea.

A new species of hagfish, Eptatretus wadgensis sp. nov., is described from the Wadge Bank, Lakshadweep Sea, India, obtained from a depth of ~250300 m through deep-sea trawling. It is diagnosed by having six pairs of gill pouches and gill apertures, 3/3 multicusp teeth, total slime pores 6769, six branchial slime pores, and ventral aorta bifurcating at the 4th or between 4th and 5th gill pouch. The new species has significant morphological differences in total dental cusps, total slime pores, body proportions and the absence of the nasal-sinus papilla when compared to congeners and formed a distinct clade in phylogenetic reconstruction and a genetic distance of 3.414.00% when comparing K2P parameters with the nearest species. A key to the Eptatretus species of the Indian Ocean is provided.

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.

Evo-devo studies of cyclostomes and the origin and evolution of jawed vertebrates.

Modern vertebrates consist of two sister groups: cyclostomes and gnathostomes. Cyclostomes are a monophyletic jawless group that can be further divided into hagfishes and lampreys, which show conspicuously different developmental and morphological patterns. However, during early pharyngula development, there appears to be a stage when the embryos of hagfishes and lampreys resemble each other by showing an "ancestral" craniofacial pattern; this pattern enables morphological comparison of hagfish and lamprey craniofacial development at late stages. This cyclostome developmental pattern, or more accurately, this developmental pattern of the jawless grade of vertebrates in early pharyngula was very likely shared by the gnathostome stem before the division of the nasohypophyseal placode led to the jaw and paired nostrils. The craniofacial pattern of the modern jawed vertebrates seems to have begun in fossil ostracoderms (including galeaspids), and was completed by the early placoderm lineages. The transition from jawless to jawed vertebrates was thus driven by heterotopy of development, mainly caused by separation and shift of ectodermal placodes and resultant ectomesenchymal distribution, and shifts of the epithelial-mesenchymal interactions that underlie craniofacial differentiation. Thus, the evolution of the jaw was not a simple modification of the mandibular arch, but a heterotopic shift of the developmental interactions involving not only the mandibular arch, but also the premandibular region rostral to the mandibular arch.

Powering the hagfish "bite": The functional morphology of the retractor complex of two hagfish feeding apparatuses.

Hagfish use forceful retractions of a dental plate to shear and ingest food. Retractile force is generated by the retractor muscle complex of the posterior hagfish feeding apparatus (HFA). While gross morphological descriptions exist, the organization of muscle and connective tissue fibers that form the soft tissue retractor complex do not. In this study, we used paraffin histology to prepare serial sections of Pacific (Eptatretus stoutii, Lockington, 1879) and Atlantic (Myxine glutinosa, Linnaeus, 1758) hagfishes in order to describe constituent soft tissue anatomy and fiber orientations. We generated 3D reconstructions in which digitized sections were segmented and fitted to volumetric scans of retractor complexes taken prior to microtomy. These models confirmed that the retractor complex is composed of a perpendicularis muscle that fits within the eye of a needle-shaped clavatus muscle, which anteriorly bears the dental plate tendon, and in turn fits within a sleeve-like tubulatus muscle. Analysis of fiber orientations within these muscles resulted in novel functional hypotheses: (a) The tubulatus muscle represents a novel tubular bipennate muscle with a considerable physiological cross-sectional area. Its activation may indirectly create tension in the dental plate tendon: as the tubulatus muscle forcefully extends, it displaces the terminal bulb and the clavatus muscle posteriorly. (b) Within the HFA terminal bulb, the muscle fibers of the clavatus and perpendicularis muscles are mutually perpendicular and may cocontract to form a swelling stopper knot-like muscular complex that resists being pulled through the tubulatus muscle. (c) While overall feeding apparatus muscle morphology is conserved, the physiological cross-sectional area of the tubulatus muscle in E. stoutii, is relatively larger than that of M. glutinosa, suggesting a more forceful retraction. The tubular bipennate construction of the tubulatus may represent a novel soft robotic actuator design.

Hagfish from the Cretaceous Tethys Sea and a reconciliation of the morphological-molecular conflict in early vertebrate phylogeny.

Hagfish depart so much from other fishes anatomically that they were sometimes considered not fully vertebrate. They may represent: (i) an anatomically primitive outgroup of vertebrates (the morphology-based craniate hypothesis); or (ii) an anatomically degenerate vertebrate lineage sister to lampreys (the molecular-based cyclostome hypothesis). This systematic conundrum has become a prominent case of conflict between morphology- and molecular-based phylogenies. To date, the fossil record has offered few insights to this long-branch problem or the evolutionary history of hagfish in general, because unequivocal fossil members of the group are unknown. Here, we report an unequivocal fossil hagfish from the early Late Cretaceous of Lebanon. The soft tissue anatomy includes key attributes of living hagfish: cartilages of barbels, postcranial position of branchial apparatus, and chemical traces of slime glands. This indicates that the suite of characters unique to living hagfish appeared well before Cretaceous times. This new hagfish prompted a reevaluation of morphological characters for interrelationships among jawless vertebrates. By addressing nonindependence of characters, our phylogenetic analyses recovered hagfish and lampreys in a clade of cyclostomes (congruent with the cyclostome hypothesis) using only morphological data. This new phylogeny places the fossil taxon within the hagfish crown group, and resolved other putative fossil cyclostomes to the stem of either hagfish or lamprey crown groups. These results potentially resolve the morphological-molecular conflict at the base of the Vertebrata. Thus, assessment of character nonindependence may help reconcile morphological and molecular inferences for other major discords in animal phylogeny.

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.

The ventilation mechanism of the Pacific hagfish Eptatretus stoutii.

We made anatomical and physiological observations of the breathing mechanisms in Pacific hagfish Eptatretus stoutii, with measurements of nostril flow and pressure, mouth and pharyngo-cutaneous duct (PCD) pressure and velum and heart impedance and observations of dye flow patterns. Resting animals frequently exhibit spontaneous apnea. During normal breathing, water flow is continuous at a high rate (~125 ml kg-1 min-1 at 12°C) powered by a two-phase unidirectional pumping system with a fast suction pump (the velum, ~22 min-1 ) for inhalation through the single nostril and a much slower force pump (gill pouches and PCD ~4.4 min-1 ) for exhalation. The mouth joins the pharynx posterior to the velum and plays no role in ventilation at rest or during swimming. Increases in flow up to >400 ml kg-1 min-1 can be achieved by increases in both velum frequency and stroke volume and the ventilatory index (product of frequency x nostril pressure amplitude) provides a useful proxy for ventilatory flow rate. Two types of coughing (flow reversals) are described. During spontaneous swimming, ventilatory pressure and flow pulsatility becomes synchronised with rhythmic body undulations.

Post-prandial physiology and intestinal morphology of the Pacific hagfish (Eptatretus stoutii).

Hagfishes are unique to the vertebrate lineage in that they acquire dissolved nutrients across multiple epithelia including the intestine, gill, and skin. This feat has been attributed to their immersive feeding behavior that likely simultaneously provides benefits (nutrient rich) and potentially adverse (hypercapnia, hypoxia, high environmental ammonia) physiological effects. Examinations have been conducted of the ex vivo transport capabilities of specific nutrients as well as in vivo effects of the hypothesized feeding environments, yet the physiological effects of feeding itself have never been elucidated. We examined the post-prandial physiology of Pacific hagfish (Eptatretus stoutii), identifying changes in oxygen consumption, acid-base balance, ammonia waste excretion, and intestinal morphology following feeding in captivity. Following voluntary feeding, post-prandial oxygen consumption was significantly elevated (1868 ± 272 µmol kg-1 h-1) 8 h following feeding when compared to control resting metabolic oxygen consumption (642 ± 51 µmol kg-1 h-1) and resulted in a factorial metabolic scope of 2.92. Changes in acid-base status were not observed following feeding in either the excreted components or the caudal blood samples; however, a significant alkalosis was observed 8 h post-feeding in the major intestinal blood vein. Significant increases (16-fold) in ammonia excretion were recorded in 36 h post-fed hagfish. Finally, significant post-prandial increases in intestinal mucosal thickness and microvilli length were observed, with mucosal thickness remaining significantly increased throughout 36 h and the microvilli length returning to fasted lengths by 36 h. These results demonstrate the post-feeding physiology of the earliest diverging extant craniate and identify correlations between physiology and hindgut morphology 8 h following feeding.

Flaccid skin protects hagfishes from shark bites.

Hagfishes defend themselves from fish predators by releasing large volumes of gill-clogging slime when they are attacked. Slime release is not anticipatory, but is only released after an attack has been initiated, raising the question of how hagfishes survive the initial attack, especially from biting predators such as sharks. We tested two hypotheses that could explain how hagfishes avoid damage from shark bites: puncture-resistant skin, and a loose and flaccid body design that makes it difficult for teeth to penetrate body musculature and viscera. Based on data from skin puncture tests from 22 fish species, we found that hagfish skin is not remarkably puncture resistant. Simulated shark bites on hagfish and their closest living relatives, lamprey, as well as whole animal inflation tests, revealed that the loose attachment of hagfish skin to the rest of the body and the substantial 'slack volume' in the subcutaneous sinus protect hagfish musculature and viscera from penetrating teeth. While recent work has found evidence that the capacious subcutaneous sinus in hagfishes is important for behaviours such as knot-tying and burrowing, our work demonstrates that it also plays a role in predator defence.

No support for Heincke's law in hagfish (Myxinidae): lack of an association between body size and the depth of species occurrence.

This study tests for interspecific evidence of Heincke's law among hagfishes and advances the field of research on body size and depth of occurrence in fishes by including a phylogenetic correction and by examining depth in four ways: maximum depth, minimum depth, mean depth of recorded specimens and the average of maximum and minimum depths of occurrence. Results yield no evidence for Heincke's law in hagfishes, no phylogenetic signal for the depth at which species occur, but moderate to weak phylogenetic signal for body size, suggesting that phylogeny may play a role in determining body size in this group.

Meristic and morphometric analysis of two hagfish species (Myxine affinis and Notomyxine tridentiger) from the Magellan Strait, Chile / Análisis nerístico y morfométrico de dos especies de anguila babosa (Myxine affinis y Notomyxine tridentiger) en el Estrecho de Magallanes, Chile

Myxinoids in Chile are represented by the subfamilies Eptatretinae and Myxininae, with a total of 14 species, the identification is complex due to the low level of morphological differentiation that characterizes this taxonomic group. Worldwide, hagfish are species of commercial value, and in Chile many attempts have been reported to initiate small-scale fisheries. The aim of the present study is describe the hagfish species caught in an incipient fishery of the Magellan Strait. Samples were collected in the Magellan Strait during eight fishing expeditions from June 2009 to October 2010 in Bahía Lomas (5348`S; 70°46'W) and Agua Fresca (5323`S; 70°45'W). The samples were taken at two depths, 0-70 meters and 71-140 meters. Taxonomic keys were used to identify the species. All specimens were individuals from the Myxininae subfamily. From a total of 3946 hagfishes, 99 % (n=3905) were the species Myxine affinis and the remaining 1 % were Notomyxine tridentiger, both reported for Chilean and Argentinean Patagonia. The range of variation for meristic variables recorded in this research was wider than those reported in the literature. This could be explained by differences in sample size between the present study and those previously published. Body proportions and meristic variables were not species specific in Myxine sp, so there is a large overlap of ranges between species, which makes their diagnostic use not applicable. This research updates the information and extends the meristic ranges for both species. The esophageocutaneous duct (in N. tridentiger) and the number of fused teeth (bicuspid in M. affinis and tricuspid in N. tridentiger) are the morphological characters that allow a clear identification in the field of the two species.

Los Myxinoideos en Chile están representados por las subfamilias Eptatretinae y Myxininae, con un total de 14 especies, cuya identificación resulta compleja debido al bajo nivel de diferenciación morfológica que caracteriza a este grupo taxonómico. A nivel mundial las anguilas babosas constituyen especies de valor comercial, y en Chile se reportan varios intentos para iniciar pesquerías de pequeña escala. El presente estudio tuvo como propósito la descripción de las especies de anguila babosa capturadas en una pesquería incipiente del Estrecho de Magallanes. Los ejemplares fueron colectados durante ocho expediciones de pesca, desde junio de 2009 a octubre de 2010 en Bahía Lomas (5348`S; 70°46'W) y Agua Fresca (5323`S; 70°45'W). Las muestras fueron tomadas en dos rangos de profundidad, 0-70 metros y 71-140 metros. Todos los especímenes fueron pertenecientes a la subfamilia Myxininae. De un total de 3946 anguilas el 99 % (n=3905) pertenecieron a la especie Myxine affinis y el restante 1 % a la especie Notomyxine tridentiger, ambas reportadas para Chile y la Patagonia Argentina. El rango de variación para las variables merísticas, registradas en esta investigación, fue mayor a los reportados en literatura. Esto puede ser explicado por las diferencias en el tamaño de muestra entre el presente estudio y aquellos publicados previamente. Las proporciones corporales y las variables merísticas no fueron especie-específica en Myxine sp, por lo que existe una gran sobre posición de los rangos entre las especies, lo que hace que su uso como diagnóstico no sea aplicable. Esta investigación actualiza la información y extiende los rangos merísticos para ambas especies. El conducto esofágico-cutáneo (en N. tridentiger) y el número de dientes fusionados (bicúspide en M. affinis y tricúspide en N. tridentiger), son los caracteres morfológicos que permiten una identificación clara de ambas especies.

The Central Nervous System of Jawless Vertebrates: Encephalization in Lampreys and Hagfishes.

Lampreys and hagfishes are the sole surviving representatives of the early agnathan (jawless) stage in vertebrate evolution, which has previously been regarded as the least encephalized group of all vertebrates. Very little is known, however, about the extent of interspecific variation in relative brain size in these fishes, as previous studies have focused on only a few species, even though lampreys exhibit a variety of life history traits. While some species are parasitic as adults, with varying feeding behaviors, others (nonparasitic species) do not feed after completing their macrophagous freshwater larval phase. In addition, some parasitic species remain in freshwater, while others undergo an anadromous migration. On the basis of data for postmetamorphic individuals representing approximately 40% of all lamprey species, with representatives from each of the three families, the aforementioned differences in life history traits are reflected in variations in relative brain size. Across all lampreys, brain mass increases with body mass with a scaling factor or slope (α) of 0.35, which is less than those calculated for different groups of gnathostomatous (jawed) vertebrates (α = 0.43-0.62). When parasitic and nonparasitic species are analyzed separately, with phylogeny taken into account, the scaling factors of both groups (parasitic α = 0.43, nonparasitic α = 0.45) approach those of gnathostomes. The relative brain size in fully grown adults of parasitic species is, however, less than that of the adults of nonparasitic species, paralleling differences between fully grown adults and recently metamorphosed individuals of anadromous species. The average degree of encephalization is found in anadromous parasitic lampreys and might thus represent the ancestral condition for extant lampreys. These results suggest that the degree of encephalization in lampreys varies according to both life history traits and phylogenetic relationships.

Hagfish Houdinis: biomechanics and behavior of squeezing through small openings.

Hagfishes are able to squeeze through small openings to gain entry to crevices, burrows, hagfish traps and carcasses, but little is known about how they do this, or what the limits of this ability are. The purpose of this study was to describe this ability, and to investigate possible mechanisms by which it is accomplished. We investigated the hypothesis that the passive movement of blood within a hagfish's flaccid subcutaneous sinus allows it to squeeze through narrow apertures that it would not be able to if it were turgid. To test this hypothesis, we analyzed videos of Atlantic hagfish (Myxine glutinosa) and Pacific hagfish (Eptatretus stoutii) moving through narrow apertures in the lab. We measured changes in body width as the animals moved through these openings and documented the behaviors associated with this ability. We found that hagfishes are able to pass through narrow slits that are less than one half the width of their bodies. Our results are consistent with the idea that a flaccid subcutaneous sinus allows hagfish to squeeze through narrow apertures by facilitating a rapid redistribution of venous blood. In addition, we describe nine distinct behaviors associated with this ability, including a form of non-undulatory locomotion also seen in snakes and lampreys. Our results illuminate a behavior that may be a critical component of the hagfish niche, as a result of its likely importance in feeding and avoiding predators.

Pigmented anatomy in Carboniferous cyclostomes and the evolution of the vertebrate eye.

The success of vertebrates is linked to the evolution of a camera-style eye and sophisticated visual system. In the absence of useful data from fossils, scenarios for evolutionary assembly of the vertebrate eye have been based necessarily on evidence from development, molecular genetics and comparative anatomy in living vertebrates. Unfortunately, steps in the transition from a light-sensitive 'eye spot' in invertebrate chordates to an image-forming camera-style eye in jawed vertebrates are constrained only by hagfish and lampreys (cyclostomes), which are interpreted to reflect either an intermediate or degenerate condition. Here, we report-based on evidence of size, shape, preservation mode and localized occurrence-the presence of melanosomes (pigment-bearing organelles) in fossil cyclostome eyes. Time of flight secondary ion mass spectrometry analyses reveal secondary ions with a relative intensity characteristic of melanin as revealed through principal components analyses. Our data support the hypotheses that extant hagfish eyes are degenerate, not rudimentary, that cyclostomes are monophyletic, and that the ancestral vertebrate had a functional visual system. We also demonstrate integument pigmentation in fossil lampreys, opening up the exciting possibility of investigating colour patterning in Palaeozoic vertebrates. The examples we report add to the record of melanosome preservation in Carboniferous fossils and attest to surprising durability of melanosomes and biomolecular melanin.

Morphological analysis of the hagfish heart. II. The venous pole and the pericardium.

The morphological characteristics of the venous pole and pericardium of the heart were examined in three hagfish species, Myxine glutinosa, Eptatretus stoutii, and Eptatretus cirrhatus. In these species, the atrioventricular (AV) canal is long, funnel-shaped and contains small amounts of myocardium. The AV valve is formed by two pocket-like leaflets that lack a papillary system. The atrial wall is formed by interconnected muscle trabeculae and a well-defined collagenous system. The sinus venosus (SV) shows a collagenous wall and is connected to the left side of the atrium. An abrupt collagen-muscle boundary marks the SV-atrium transition. It is hypothesized that the SV is not homologous to that of other vertebrates which could have important implications for understanding heart evolution. In M. glutinosa and E. stoutii, the pericardium is a closed bag that hangs from the tissues dorsal to the heart and encloses both the heart and the ventral aorta. In contrast, the pericardium is continuous with the loose periaortic tissue in E. cirrhatus. In all three species, the pericardium ends at the level of the SV excluding most of the atrium from the pericardial cavity. In M. glutinosa and E. stoutii, connective bridges extend between the base of the aorta and the ventricular wall. In E. cirrhatus, the connections between the periaortic tissue and the ventricle may carry blood vessels that reach the ventricular base. A further difference specific to E. cirrhatus is that the adipose tissue associated with the pericardium contains thyroid follicles. J. Morphol. 277:853-865, 2016. © 2016 Wiley Periodicals, Inc.

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.

Morphological analysis of the hagfish heart. I. The ventricle, the arterial connection and the ventral aorta.

We have studied the heart in three species of hagfish: Myxine glutinosa, Eptatretus stoutii, and Eptatretus cirrhatus and report about the morphology of the ventricle, the arterial connection and the ventral aorta. On the whole, the hagfish heart lacks outflow tract components, the ventricle and atrium adopt a dorso-caudal rather than a ventro-dorsal relationship, and the sinus venosus opens into the left side of the atrium. This may indicate a "defective" cardiac looping during embryogenesis. The ventral aorta is elongated in M. glutinosa and E. stoutii but sac-like in E. cirrhatus. The ventricles are entirely trabeculated. The myocytes show a low myofibrillar content and junctional complexes formed by fascia adherens and desmosomes. Gap junctions could not be demonstrated. Myocardial cells in M. glutinosa contain numerous lipid droplets. These droplets are less numerous in E. stoutii and practically absent in E. cirrhatus, suggesting different metabolic requirements. Other cell types present in the ventricle are chromaffin cells and granular leukocytes that contain rod-shaped granules. The ventricle-aorta connection is guarded by a bicuspid valve with left and right, pocket-like leaflets. The leaflets extend from the cranial end of the ventricle into the aorta but the junction is asymmetrical. This junction contains a ganglion-like structure in E. cirrhatus. The ventral aorta shows endothelial, media, and adventitial layers. The media contains smooth muscle cells surrounded by dense bands formed by tightly-packed extracellular filaments. In addition, a short number of elastic fibers are observed in M. glutinosa and E. stoutii. Cellular and extracellular elements are more loosely organized in the aorta of E. cirrhatus. The collagenous adventitia contains ganglion-like cells in the three species. In the absence of nerves, chromaffin and ganglion-like cells may control the activity of the myocardium and that of the aortic smooth muscle cells, respectively.