Development of the vertebra and fin skeleton in the lamprey and its implications for the homology of vertebrate vertebrae.

BACKGROUND: Although vertebrae are the defining character of vertebrates, they are found only in rudimentary form in extant agnathans. In addition, the vertebrae of agnathans possess several unique features, such as elastin-like molecules as the main matrix component and late (post-metamorphosis) differentiation of lamprey vertebrae. In this study, by tracing the developmental process of vertebrae in lamprey, we examined the homology of vertebrae between lampreys and gnathostomes. RESULTS: We found that the lamprey somite is first subdivided mediolaterally, with myotome cells differentiating medially and non-myotome cells emerging laterally. Subsequently, collagen-positive non-myotome cells surround the myotome. This pattern of somitogenesis is rather similar to that in amphioxi and sheds doubt on the presence of a sclerotome, in terms of mesenchyme cells induced by a signal from the notochord, in lamprey. Further tracing of non-myotome cell development revealed that fin cartilage develops in ammocoete larvae approximately 35 mm in body length. The development of the fin cartilage occurs much earlier than that of the vertebra whose development proceeds during metamorphosis. CONCLUSION: We propose that the homology of vertebrae between agnathans and gnathostomes should be discussed carefully, because the developmental process of the lamprey vertebra is different from that of gnathostomes.

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.

Comparative analysis of Hmx expression and the distribution of neuronal somata in the trigeminal ganglion in lamprey and shark: insights into the homology of the trigeminal nerve branches and the evolutionary origin of the vertebrate jaw.

The evolutionary origin of the jaw remains one of the most enigmatic events in vertebrate evolution. The trigeminal nerve is a key component for understanding jaw evolution, as it plays a crucial role as a sensorimotor interface for the effective manipulation of the jaw. This nerve is also found in the lamprey, an extant jawless vertebrate. The trigeminal nerve has three major branches in both the lamprey and jawed vertebrates. Although each of these branches was classically thought to be homologous between these two taxa, this homology is now in doubt. In the present study, we compared expression patterns of Hmx, a candidate genetic marker of the mandibular nerve (rV3, the third branch of the trigeminal nerve in jawed vertebrates), and the distribution of neuronal somata of trigeminal nerve branches in the trigeminal ganglion in lamprey and shark. We first confirmed the conserved expression pattern of Hmx1 in the shark rV3 neuronal somata, which are distributed in the caudal part of the trigeminal ganglion. By contrast, lamprey Hmx genes showed peculiar expression patterns, with expression in the ventrocaudal part of the trigeminal ganglion similar to Hmx1 expression in jawed vertebrates, which labeled the neuronal somata of the second branch. Based on these results, we propose two alternative hypotheses regarding the homology of the trigeminal nerve branches, providing new insights into the evolutionary origin of the vertebrate jaw.

Unravelling the functional development of vertebrate pathways controlling gaze.

Animals constantly redirect their gaze away or towards relevant targets and, besides these goal-oriented responses, stabilizing movements clamp the visual scene avoiding image blurring. The vestibulo-ocular (VOR) and the optokinetic reflexes are the main contributors to gaze stabilization, whereas the optic tectum integrates multisensory information and generates orienting/evasive gaze movements in all vertebrates. Lampreys show a unique stepwise development of the visual system whose understanding provides important insights into the evolution and development of vertebrate vision. Although the developmental emergence of the visual components, and the retinofugal pathways have been described, the functional development of the visual system and the development of the downstream pathways controlling gaze are still unknown. Here, we show that VOR followed by light-evoked eye movements are the first to appear already in larvae, despite their burrowed lifestyle. However, the circuits controlling goal-oriented responses emerge later, in larvae in non-parasitic lampreys but during late metamorphosis in parasitic lampreys. The appearance of stabilizing responses earlier than goal-oriented in the lamprey development shows a stepwise transition from simpler to more complex visual systems, offering a unique opportunity to isolate the functioning of their underlying circuits.

Consciousness in Jawless Fishes.

Jawless fishes were the first vertebrates to evolve. It is thus important to investigate them to determine whether consciousness was acquired in the common ancestor of all vertebrates. Most jawless fish lineages are extinct, and cyclostomes (lampreys and hagfish) are the sole survivors. Here, I review the empirical knowledge on the neurobiology of cyclostomes with special reference to recently proposed "markers" of primary, minimal consciousness. The adult lamprey appears to meet the neuroanatomical criteria but there is a practical limitation to behavioral examination of its learning ability. In addition, the consciousness-related neuroarchitecture of larvae and its reconstruction during metamorphosis remain largely uninvestigated. Even less is known of hagfish neurobiology. The hagfish forebrain forms the central prosencephalic complex, and the homology of its components to the brain regions of other vertebrates needs to be confirmed using modern techniques. Nevertheless, as behavioral responses to olfactory stimuli in aquariums have been reported, it is easier to investigate the learning ability of the hagfish than that of the lamprey. Based on these facts, I finally discuss the potential future directions of empirical studies for examining the existence of consciousness in jawless fishes.

Generation of knock-in lampreys by CRISPR-Cas9-mediated genome engineering.

The lamprey represents the oldest group of living vertebrates and has been a key organism in various research fields such as evolutionary developmental biology and neuroscience. However, no knock-in technique for this animal has been established yet, preventing application of advanced genetic techniques. Here, we report efficient generation of F0 knock-in lampreys by CRISPR-Cas9-mediated genome editing. A donor plasmid containing a heat-shock promoter was co-injected with a short guide RNA (sgRNA) for genome digestion, a sgRNA for donor plasmid digestion, and Cas9 mRNA. Targeting different genetic loci, we succeeded in generating knock-in lampreys expressing photoconvertible protein Dendra2 as well as those expressing EGFP. With its simplicity, design flexibility, and high efficiency, we propose that the present method has great versatility for various experimental uses in lamprey research and that it can also be applied to other "non-model" organisms.

Development of the lamprey velum and implications for the evolution of the vertebrate jaw.

BACKGROUND: The vertebrate jaw is thought to have evolved through developmental modification of the mandibular arch. An extant jawless vertebrate, the lamprey, possesses a structure called "velum"-a mandibular arch derivative-in addition to the oral apparatus. This leads us to assess the velum's possible contribution to the evolution of jaws. RESULTS: The velar muscles develop from progenitor cells distinct from those from which the oral muscles develop. In addition, the oral and velar regions originate from the different sub-population of the trigeminal neural crest cells (NCCs): the former region receives NCCs from the midbrain, whereas the latter region receives NCCs from the anterior hindbrain. The expression of patterning genes (eg, DlxA and MsxA) is activated at a later developmental stage in the velum compared to the oral region, and more importantly, in different cells from those in the oral region. CONCLUSION: The lamprey mandibular arch consists of two developmental units: the anterior oral unit and the posterior velar unit. Because structural elements of the lamprey velum may be homologous to the jaw, the evolution of vertebrate jaws may have occurred by the velum being released from its functional roles in feeding or respiration in jawless vertebrates.

The role of the optic tectum for visually evoked orienting and evasive movements.

As animals forage for food and water or evade predators, they must rapidly decide what visual features in the environment deserve attention. In vertebrates, this visuomotor computation is implemented within the neural circuits of the optic tectum (superior colliculus in mammals). However, the mechanisms by which tectum decides whether to approach or evade remain unclear, and also which neural mechanisms underlie this behavioral choice. To address this problem, we used an eye-brain-spinal cord preparation to evaluate how the lamprey responds to visual inputs with distinct stimulus-dependent motor patterns. Using ventral root activity as a behavioral readout, we classified 2 main types of fictive motor responses: (i) a unilateral burst response corresponding to orientation of the head toward slowly expanding or moving stimuli, particularly within the anterior visual field, and (ii) a unilateral or bilateral burst response triggering fictive avoidance in response to rapidly expanding looming stimuli or moving bars. A selective pharmacological blockade revealed that the brainstem-projecting neurons in the deep layer of the tectum in interaction with local inhibitory interneurons are responsible for selecting between these 2 visually triggered motor actions conveyed through downstream reticulospinal circuits. We suggest that these visual decision-making circuits had evolved in the common ancestor of vertebrates and have been conserved throughout vertebrate phylogeny.

The stepwise development of the lamprey visual system and its evolutionary implications.

Lampreys, which represent the oldest group of living vertebrates (cyclostomes), show unique eye development. The lamprey larva has only eyespot-like immature eyes beneath a non-transparent skin, whereas after metamorphosis, the adult has well-developed image-forming camera eyes. To establish a functional visual system, well-organised visual centres as well as motor components (e.g. trunk muscles for locomotion) and interactions between them are needed. Here we review the available knowledge concerning the structure, function and development of the different parts of the lamprey visual system. The lamprey exhibits stepwise development of the visual system during its life cycle. In prolarvae and early larvae, the 'primary' retina does not have horizontal and amacrine cells, but does have photoreceptors, bipolar cells and ganglion cells. At this stage, the optic nerve projects mostly to the pretectum, where the dendrites of neurons in the nucleus of the medial longitudinal fasciculus (nMLF) appear to receive direct visual information and send motor outputs to the neck and trunk muscles. This simple neural circuit may generate negative phototaxis. Through the larval period, the lateral region of the retina grows again to form the 'secondary' retina and the topographic retinotectal projection of the optic nerve is formed, and at the same time, the extra-ocular muscles progressively develop. During metamorphosis, horizontal and amacrine cells differentiate for the first time, and the optic tectum expands and becomes laminated. The adult lamprey then has a sophisticated visual system for image-forming and visual decision-making. In the adult lamprey, the thalamic pathway (retina-thalamus-cortex/pallium) also transmits visual stimuli. Because the primary, simple light-detecting circuit in larval lamprey shares functional and developmental similarities with that of protochordates (amphioxus and tunicates), the visual development of the lamprey provides information regarding the evolutionary transition of the vertebrate visual system from the protochordate-type to the vertebrate-type.

Direct Dopaminergic Projections from the SNc Modulate Visuomotor Transformation in the Lamprey Tectum.

Dopamine neurons in the SNc play a pivotal role in modulating motor behavior via striatum. Here, we show that the same dopamine neuron that targets striatum also sends a direct branch to the optic tectum (superior colliculus). Whenever SNc neurons are activated, both targets will therefore be affected. Visual stimuli (looming or bars) activate the dopamine neurons coding saliency and also elicit distinct motor responses mediated via tectum (eye, orienting or evasive), which are modulated by the dopamine input. Whole-cell recordings from tectal projection neurons and interneurons show that dopamine, released by SNc stimulation, increases or decreases the excitability depending on whether they express the dopamine D1 or the D2 receptor. SNc thus exerts its effects on the visuomotor system through a combined effect directly on tectum and also via striatum. This direct SNc modulation will occur regardless of striatum and represents a novel mode of motor control.

A Phenomenological and Dynamic View of Homology: Homologs as Persistently Reproducible Modules.

Homology is a fundamental concept in biology. However, the metaphysical status of homology, especially whether a homolog is a part of an individual or a member of a natural kind, is still a matter of intense debate. The proponents of the individuality view of homology criticize the natural kind view of homology by pointing out that homologs are subject to evolutionary transformation, and natural kinds do not change in the evolutionary process. Conversely, some proponents of the natural kind view of homology argue that a homolog can be construed both as a part of an individual and a member of a natural kind. They adopt the Homeostatic Property Cluster (HPC) theory of natural kinds, and the theory seems to strongly support their construal. Note that this construal implies the acceptance of essentialism. However, looking back on the history of the concept of homology, we should not overlook the fact that the individuality view was proposed to reject the essentialist interpretation of homology. Moreover, the essentialist notions of natural kinds can, in our view, mislead biologists about the phenomena of homology. Consequently, we need a non-essentialist view of homology, which we name the "persistently reproducible module" (PRM) view. This view highlights both the individual-like and kind-like aspects of homologs while stripping down both essentialist and anti-essentialist interpretations of homology. In this article, we articulate the PRM view of homology and explain why it is recommended over the other two views.

Evolutionary history of the extant amphioxus lineage with shallow-branching diversification.

Amphioxus or lancelets have been regarded as a key animal in understanding the origin of vertebrates. However, the evolutionary history within this lineage remains unexplored. As the amphioxus lineage has likely been separated from other chordates for a very long time and displays a marked left-right asymmetry, its evolutionary history is potentially helpful in better understanding chordate and vertebrate origins. We studied the phylogenetic relationships within the extant amphioxus lineage based on mitochondrial genomes incorporating new Asymmetron and Epigonichthys populations, and based on previously reported nuclear transcriptomes. The resulting tree patterns are consistent, showing the Asymmetron clade diverging first, followed by the Epigonichthys and Branchiostoma clades splitting. Divergence time estimates based on nuclear transcriptomes with vertebrate calibrations support a shallow diversification of the extant amphioxus lineage in the Tertiary. These estimates fit well with the closure of seaways between oceans by continental drift, ocean currents, and present geographical distributions, and suggest a long cryptic history from the origin of amphioxus to its most recent diversification. Deduced character polarities based on phylogenetic analyses suggest that the common ancestor of the extant amphioxus existed in a tiny epibenthic state with larva-like appearance of extant amphioxus, likely with ciliate epidermis.

Two-headed mutants of the lamprey, a basal vertebrate.

BACKGROUND: This is the first report of two-headed (bicephaly) lamprey twins. Although lampreys sit at a crucial phylogenetic position, there are only a few reports on their teratology and developmental abnormalities. RESULTS: Two-headed mutants were obtained by artificial fertilization in the laboratory as spontaneous occurrences. All mutants were derived from single fertilizations using single male and female gametes, suggestive of a genetic background. The anterio-posterior position of the axonal bifurcation and symmetricity varied in each mutant. Other malformations were coincidently observed, including pericardial edema, yolk sac edema and axial bending. Asymmetrical (lateral- branched) mutants displayed more severe abnormalities in the cranial nerves than symmetrical mutants. CONCLUSION: Two-headed mutants of the lamprey are described. These mutants have similar malformations to dorsal blastopore lip-transplanted lamprey embryos, suggesting that they could be generated by a disorder in head-organizing activity.

Comparative morphology and development of extra-ocular muscles in the lamprey and gnathostomes reveal the ancestral state and developmental patterns of the vertebrate head.

The ancestral configuration of the vertebrate head has long been an intriguing topic in comparative morphology and evolutionary biology. One peculiar component of the vertebrate head is the presence of extra-ocular muscles (EOMs), the developmental mechanism and evolution of which remain to be determined. The head mesoderm of elasmobranchs undergoes local epithelialization into three head cavities, precursors of the EOMs. In contrast, in avians, these muscles appear to develop mainly from the mesenchymal head mesoderm. Importantly, in the basal vertebrate lamprey, the head mesoderm does not show overt head cavities or signs of segmental boundaries, and the development of the EOMs is not well described. Furthermore, the disposition of the lamprey EOMs differs from those the rest of vertebrates, in which the morphological pattern of EOMs is strongly conserved. To better understand the evolution and developmental origins of the vertebrate EOMs, we explored the development of the head mesoderm and EOMs of the lamprey in detail. We found that the disposition of lamprey EOM primordia differed from that in gnathostomes, even during the earliest period of development. We also found that three components of the paraxial head mesoderm could be distinguished genetically (premandibular mesoderm: Gsc+/TbxA-; mandibular mesoderm: Gsc-/TbxA-; hyoid mesoderm: Gsc-/TbxA+), indicating that the genetic mechanisms of EOMs are conserved in all vertebrates. We conclude that the tripartite developmental origin of the EOMs is likely to have been possessed by the latest common ancestor of the vertebrates. This ancestor's EOM developmental pattern was also suggested to have resembled more that of the lamprey, and the gnathostome EOMs' disposition is likely to have been established by a secondary modification that took place in the common ancestor of crown gnathostomes.

Expression patterns of Eph genes in the "dual visual development" of the lamprey and their significance in the evolution of vision in vertebrates.

Image-forming vision is crucial to animals for recognizing objects in their environment. In vertebrates, this type of vision is achieved with paired camera eyes and topographic projection of the optic nerve. Topographic projection is established by an orthogonal gradient of axon guidance molecules, such as Ephs. To explore the evolution of image-forming vision in vertebrates, lampreys, which belong to the basal lineage of vertebrates, are key animals because they show unique "dual visual development." In the embryonic and pre-ammocoete larval stage (the "primary" phase), photoreceptive "ocellus-like" eyes develop, but there is no retinotectal optic nerve projection. In the late ammocoete larval stage (the "secondary" phase), the eyes grow and form into camera eyes, and retinotectal projection is newly formed. After metamorphosis, this retinotectal projection in adult lampreys is topographic, similar to that of gnathostomes. In this study, we explored the involvement of Ephs in lamprey "dual visual development" and establishment of the image-form vision. We found that gnathostome-like orthogonal gradient expression was present in the retina during the "secondary" phase; i.e., EphB showed a gradient of expression along the dorsoventral axis, while EphC was expressed along the anteroposterior axis. However, no orthogonal gradient expression was observed during the "primary" phase. These observations suggest that Ephs are likely recruited de novo for the guidance of topographical "second" optic nerve projection. Transformations during lamprey "dual visual development" may represent "recapitulation" from a protochordate-like ancestor to a gnathostome-like vertebrate ancestor.

A comparative examination of neural circuit and brain patterning between the lamprey and amphioxus reveals the evolutionary origin of the vertebrate visual center.

Vertebrates are equipped with so-called camera eyes, which provide them with image-forming vision. Vertebrate image-forming vision evolved independently from that of other animals and is regarded as a key innovation for enhancing predatory ability and ecological success. Evolutionary changes in the neural circuits, particularly the visual center, were central for the acquisition of image-forming vision. However, the evolutionary steps, from protochordates to jaw-less primitive vertebrates and then to jawed vertebrates, remain largely unknown. To bridge this gap, we present the detailed development of retinofugal projections in the lamprey, the neuroarchitecture in amphioxus, and the brain patterning in both animals. Both the lateral eye in larval lamprey and the frontal eye in amphioxus project to a light-detecting visual center in the caudal prosencephalic region marked by Pax6, which possibly represents the ancestral state of the chordate visual system. Our results indicate that the visual system of the larval lamprey represents an evolutionarily primitive state, forming a link from protochordates to vertebrates and providing a new perspective of brain evolution based on developmental mechanisms and neural functions.

A genome-wide survey of genes encoding transcription factors in Japanese pearl oyster Pinctada fucata: II. Tbx, Fox, Ets, HMG, NFκB, bZIP, and C2H2 zinc fingers.

To gain a better understanding of molluscan development and its relation to the evolution of their unique body plan, we performed a genomic survey of genes encoding transcription factors, such as Tbx, Fox, Ets, HMG, NFκB, bZIP, and C2H2 zinc finger proteins in the Japanese pearl oyster, Pinctada fucata. We annotated 133 transcription factor genes. Together with the orthologs of known deuterostome genes, we found several orphan genes in each class of transcription factor. Some possessed clear orthologs in other species of lophotrochozoans, while no counterpart genes were found in the deuterostomes or ecdysozoans. These observations suggest that a number of transcription factor genes are unique to lophotrochozoans, and thus additional research frontiers remain to be explored with regard to such transcription factors.