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.

The evolution of basal progenitors in the developing non-mammalian brain.

The amplification of distinct neural stem/progenitor cell subtypes during embryogenesis is essential for the intricate brain structures present in various vertebrate species. For example, in both mammals and birds, proliferative neuronal progenitors transiently appear on the basal side of the ventricular zone of the telencephalon (basal progenitors), where they contribute to the enlargement of the neocortex and its homologous structures. In placental mammals, this proliferative cell population can be subdivided into several groups that include Tbr2(+) intermediate progenitors and basal radial glial cells (bRGs). Here, we report that basal progenitors in the developing avian pallium show unique morphological and molecular characteristics that resemble the characteristics of bRGs, a progenitor population that is abundant in gyrencephalic mammalian neocortex. Manipulation of LGN (Leu-Gly-Asn repeat-enriched protein) and Cdk4/cyclin D1, both essential regulators of neural progenitor dynamics, revealed that basal progenitors and Tbr2(+) cells are distinct cell lineages in the developing avian telencephalon. Furthermore, we identified a small population of subapical mitotic cells in the developing brains of a wide variety of amniotes and amphibians. Our results suggest that unique progenitor subtypes are amplified in mammalian and avian lineages by modifying common mechanisms of neural stem/progenitor regulation during amniote brain evolution.

Evolutionary mechanisms that generate morphology and neural-circuit diversity of the cerebellum.

The cerebellum is derived from the dorsal part of the anterior-most hindbrain. The vertebrate cerebellum contains glutamatergic granule cells (GCs) and gamma-aminobutyric acid (GABA)ergic Purkinje cells (PCs). These cerebellar neurons are generated from neuronal progenitors or neural stem cells by mechanisms that are conserved among vertebrates. However, vertebrate cerebella are widely diverse with respect to their gross morphology and neural circuits. The cerebellum of cyclostomes, the basal vertebrates, has a negligible structure. Cartilaginous fishes have a cerebellum containing GCs, PCs, and deep cerebellar nuclei (DCNs), which include projection neurons. Ray-finned fish lack DCNs but have projection neurons termed eurydendroid cells (ECs) in the vicinity of the PCs. Among ray-finned fishes, the cerebellum of teleost zebrafish has a simple lobular structure, whereas that of weakly electric mormyrid fish is large and foliated. Amniotes, which include mammals, independently evolved a large, foliated cerebellum, which contains massive numbers of GCs and has functional connections with the dorsal telencephalon (neocortex). Recent studies of cyclostomes and cartilaginous fish suggest that the genetic program for cerebellum development was already encoded in the genome of ancestral vertebrates. In this review, we discuss how alterations of the genetic and cellular programs generated diversity of the cerebellum during evolution.

Reconstructing the ancestral vertebrate brain.

Highly complicated morphologies and sophisticated functions of vertebrate brains have been established through evolution. However, the origin and early evolutionary history of the brain remain elusive, owing to lack of information regarding the brain architecture of extant and fossil species of jawless vertebrates (agnathans). Comparative analyses of the brain of less studied cyclostomes (only extant agnathan group, consisting of lampreys and hagfish) with the well-known sister group of jawed vertebrates (gnathostomes) are the only tools we have available to illustrate the ancestral architecture of the vertebrate brain. Previous developmental studies had shown that the lamprey lacked well-established brain compartments that are present in gnathostomes, such as the medial ganglionic eminence and the rhombic lip. The most accepted scenario suggested that cyclostomes had fewer compartments than that of the gnathostome brain and that gnathostomes thus evolved by a stepwise addition of innovations on its developmental sequence. However, recent studies have revealed that these compartments are present in hagfish embryos, indicating that these brain regions have been acquired before the split of cyclostomes and gnathostomes. By comparing two cyclostome lineages and gnathostomes, it has become possible to speculate about a more complex ancestral state of the brain, excluding derived traits in either of the lineages. In this review, we summarize recent studies on the brain development of the lamprey and hagfish. Then, we attempt to reconstruct the possible brain architecture of the last common ancestor of vertebrates. Finally, we discuss how the developmental plan of the vertebrate brain has been modified independently in different vertebrate lineages.

Expression patterns of Sema3A in developing amniote limbs: With reference to the diversification of peripheral nerve innervation.

Paired limbs were acquired in the ancestor of tetrapods and their morphology has been highly diversified in amniotes in relation to the adaptive radiation to the terrestrial environment. These morphological changes may have been induced by modification of the developmental program of the skeletal or muscular system. To complete limb modification, it is also important to change the neuronal framework, because the functions of the limbs rely on neural circuits that involve coordinated movement. Previous studies have shown that class 3 semaphorins (Sema3 semaphorins), which act as repulsive axonal guidance cues, play a crucial role in the formation of the peripheral nerves in mice. Here, we studied the expression pattern of Sema3A orthologues in embryos of developing amniotes, including mouse, chick, soft-shelled turtle, and ocelot gecko. Sema3A transcripts were expressed in restricted mesenchymal parts of the developing limb primordium in all animals studied, and developing spinal nerves appeared to extend through Sema3A-negative regions. These results suggest that a Sema3A-dependent guidance system plays a key role in neuronal circuit formation in amniote limbs. We also found that Sema3A partially overlapped with the distribution of cartilage precursor cells. Based on these results, we propose a model in which axon guidance and skeletogenesis are linked by Sema3A; such mechanisms may underlie functional neuron rearrangement during limb diversification.

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.

Mapping the face in the somatosensory brainstem.

The facial somatosensory map in the cortex is derived from facial representations that are first established at the brainstem level and then serially 'copied' at each stage of the somatosensory pathway. Recent studies have provided insights into the molecular mechanisms involved in the development of somatotopic maps of the face and whiskers in the trigeminal nuclei of the mouse brainstem. This work has revealed that early molecular regionalization and positional patterning of trigeminal ganglion and brainstem target neurons are established by homeodomain transcription factors, the expression of which is induced and maintained by signals from the brain and face. Such position-dependent information is fundamental in transforming the early spatial layout of sensory receptors into a topographic connectivity map that is conferred to higher brain levels.

Development of the thalamo-dorsal ventricular ridge tract in the Chinese soft-shelled turtle, Pelodiscus sinensis.

With the exception of that from the olfactory system, the vertebrate sensory information is relayed by the dorsal thalamus (dTh) to be carried to the telencephalon via the thalamo-telencephalic tract. Although the trajectory of the tract from the dTh to the basal telencephalon seems to be highly conserved among amniotes, the axonal terminals vary in each group. In mammals, thalamic axons project onto the neocortex, whereas they project onto the dorsal pallium and the dorsal ventricular ridge (DVR) in reptiles and birds. To ascertain the evolutionary development of the thalamo-telencephalic connection in amniotes, we focused on reptiles. Using the Chinese soft-shelled turtle (Pelodiscus sinensis), we studied the developmental course of the thalamic axons projecting onto the DVR. We found, during the developmental period when the thalamo-DVR connection forms, that transcripts of axon guidance molecules, including EphA4 and Slit2, were expressed in the diencephalon, similar to the mouse embryo. These results suggest that the basic mechanisms responsible for the formation of the thalamo-telencephalic tract are shared across amniote lineages. Conversely, there was a characteristic difference in the expression patterns of Slit2, Netrin1, and EphrinA5 in the telencephalon between synapsid (mammalian) and diapsid (reptilian and avian) lineages. This indicates that changes in the expression domains of axon guidance molecules may modify the thalamic axon projection and lead to the diversity of neuronal circuits in amniotes.

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

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

Reptiles: a new model for brain evo-devo research.

Vertebrate brains exhibit vast amounts of anatomical diversity. In particular, the elaborate and complex nervous system of amniotes is correlated with the size of their behavioral repertoire. However, the evolutionary mechanisms underlying species-specific brain morphogenesis remain elusive. In this review we introduce reptiles as a new model organism for understanding brain evolution. These animal groups inherited ancestral traits of brain architectures. We will describe several unique aspects of the reptilian nervous system with a special focus on the telencephalon, and discuss the genetic mechanisms underlying reptile-specific brain morphology. The establishment of experimental evo-devo approaches to studying reptiles will help to shed light on the origin of the amniote brains.

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.

Evolution of motor innervation to vertebrate fins and limbs.

The evolution and diversification of vertebrate behaviors associated with locomotion depend highly on the functional transformation of paired appendages. Although the evolution of fins into limbs has long been a focus of interest to scientists, the evolution of neural control during this transition has not received much attention. Recent studies have provided significant progress in the understanding of the genetic and developmental bases of the evolution of fin/limb motor circuitry in vertebrates. Here we compare the organization of the motor neurons in the spinal cord of various vertebrates. We also discuss recent advances in our understanding of these events and how they can provide a mechanistic explanation for the evolution of fin/limb motor circuitry in vertebrates.

Allometric growth of the trunk leads to the rostral shift of the pelvic fin in teleost fishes.

The pelvic fin position among teleost fishes has shifted rostrally during evolution, resulting in diversification of both behavior and habitat. We explored the developmental basis for the rostral shift in pelvic fin position in teleost fishes using zebrafish (abdominal pelvic fins) and Nile tilapia (thoracic pelvic fins). Cell fate mapping experiments revealed that changes in the distribution of lateral plate mesodermal cells accompany the trunk-tail protrusion. Presumptive pelvic fin cells are originally located at the body wall adjacent to the anterior limit of hoxc10a expression in the spinal cord, and their position shifts rostrally as the trunk grows. We then showed that the differences in pelvic fin position between zebrafish and Nile tilapia were not due to changes in expression or function of gdf11. We also found that hox-independent motoneurons located above the pelvic fins innervate into the pelvic musculature. Our results suggest that there is a common mechanism among teleosts and tetrapods that controls paired appendage positioning via gdf11, but in teleost fishes the position of prospective pelvic fin cells on the yolk surface shifts as the trunk grows. In addition, teleost motoneurons, which lack lateral motor columns, innervate the pelvic fins in a manner independent of the rostral-caudal patterns of hox expression in the spinal cord.

[A family with attenuated familial adenomatous polyposis identified because of fundic gland polyposis].

We report a 41-year-old woman who had fundic gland polyposis without a colorectal polyp. Because her father had colon cancer, multiple colorectal polyps and the gastric polyposis, we suspected AFAP and researched the APC gene. There was 1 base pair deletion of guanine at codon 99-100 in exon 3 of the APC gene, and its frame shift mutation made stop codon at codon 124. It was proved that the gene carrier of AFAP can be discovered based on the family-history and the presence of fundic gland polyposis, even when no colorectal polyps exist.

Genetic Mechanism for the Cyclostome Cerebellar Neurons Reveals Early Evolution of the Vertebrate Cerebellum.

The vertebrate cerebellum arises at the dorsal part of rhombomere 1, induced by signals from the isthmic organizer. Two major cerebellar neuronal subtypes, granule cells (excitatory) and Purkinje cells (inhibitory), are generated from the anterior rhombic lip and the ventricular zone, respectively. This regionalization and the way it develops are shared in all extant jawed vertebrates (gnathostomes). However, very little is known about early evolution of the cerebellum. The lamprey, an extant jawless vertebrate lineage or cyclostome, possesses an undifferentiated, plate-like cerebellum, whereas the hagfish, another cyclostome lineage, is thought to lack a cerebellum proper. In this study, we found that hagfish Atoh1 and Wnt1 genes are co-expressed in the rhombic lip, and Ptf1a is expressed ventrally to them, confirming the existence of r1's rhombic lip and the ventricular zone in cyclostomes. In later stages, lamprey Atoh1 is downregulated in the posterior r1, in which the NeuroD increases, similar to the differentiation process of cerebellar granule cells in gnathostomes. Also, a continuous Atoh1-positive domain in the rostral r1 is reminiscent of the primordium of valvula cerebelli of ray-finned fishes. Lastly, we detected a GAD-positive domain adjacent to the Ptf1a-positive ventricular zone in lampreys, suggesting that the Ptf1a-positive cells differentiate into some GABAergic inhibitory neurons such as Purkinje and other inhibitory neurons like in gnathostomes. Altogether, we conclude that the ancestral genetic programs for the formation of a distinct cerebellum were established in the last common ancestor of vertebrates.

Development of the central and peripheral nervous systems in the lamprey.

Vertebrate brains are highly organized structures that show remarkable diversity throughout the animal groups. Among the vertebrates, the agnathan animals, which diverged from the gnathostomes early in the evolution of the vertebrates, occupy a key phylogenetic position in order to clarify the origin and evolution of the brain. We found that the lamprey brain has the basic molecular mechanisms necessary to form brain compartments. Conversely, the telencephalon and cerebellum display gnathostome-specific developmental mechanisms. We also propose that, in contrast to those of gnathostomes, the maxillary ramus of the trigeminal nerve and buccal ramus of the nerve on the anterior lateral line are not fused in the developing lamprey. Thus, the development of the central nervous system and the framework of the peripheral nerve around the oral region are thought to have improved in the course of the agnathan-gnathostome transition.

FGF- and SHH-based molecular signals regulate barbel and craniofacial development in catfish.

BACKGROUND: Catfish (Siluriformes) are characterized by unique morphologies, including enlarged jaws with movable barbels and taste buds covering the entire body surface. Evolution of these characteristics was a crucial step in their adaptive radiation to freshwater environments. However, the developmental processes of the catfish craniofacial region and taste buds remain to be elucidated; moreover, little is known about the molecular mechanisms underlying the morphogenesis of these structures. RESULTS: In Amur catfish (Silurus asotus), three pairs of barbel primordia are formed by 2 days post-fertilization (dpf). Innervation of the peripheral nerves and formation of muscle precursors are also established during early development. Taste buds from the oral region to the body trunk are formed by 4 dpf. We then isolated catfish cognates Shh (SaShh) and Fgf8 (SaFgf8), which are expressed in maxillary barbel primordium at 1-2 dpf. Further, SHH signal inhibition induces reduction of mandibular barbels with abnormal morphology of skeletal elements, whereas it causes no apparent abnormality in the trigeminal and facial nerve morphology. We also found that mandibular barbel lengths and number of taste buds are reduced by FGF inhibition, as seen in SHH signal inhibition. However, unlike with SHH inhibition, the abnormal morphology of the trigeminal and facial nerves was observed in FGF signal-inhibited embryos. CONCLUSION: The developmental processes of Amur catfish are consistent with those reported for other catfish species. Thus, developmental aspects of craniofacial structures and taste buds may be conserved in Siluriformes. Our findings also suggest that SHH signaling plays a crucial role in the formation of barbels and taste buds, without affecting nerve projection, while FGF signaling is required for the development of barbels, taste buds, and branchial nerves. Thus, SHH and FGF signaling plays key roles in the ontogenesis and evolution of some catfish-specific characteristics.

Atlas of the telencephalon based on cytoarchitecture, neurochemical markers, and gene expressions in Rhinogobius flumineus [Mizuno, 1960].

Gobiida is a basal subseries of percomorphs in teleost fishes, holding a useful position for comparisons with other orders of Percomorpha as well as other cohort of teleosts. Here, we describe a telencephalic atlas of a Gobiida species Rhinogobius flumineus (Mizuno, Memoirs of the College of Science, University of Kyoto, Series B: Biology, 1960; 27, 3), based on cytoarchitectural observations, combined with analyses of the distribution patterns of neurochemical markers and transcription factors. The telencephalon of R. flumineus shows a number of features distinct from those of other teleosts. Among others, the followings were of special note. (a) The lateral part of dorsal telencephalon (Dl), which is known as a visual center in other teleosts, is composed of as many as seven regions, some of which are conspicuous, circumscribed by cell plates. These subdivisions of the Dl can be differentiated clearly by differential soma size and color with Nissl-staining, and distribution patterns of neural markers. (b) Cell populations continuous with the ventral region of dorsal part of ventral telencephalon (vVd) exhibit extensive dimension. Especially, portion 1 of the central part of ventral telencephalon appears to represent a cell population laterally translocated from the vVd, forming a large cluster of small cells that penetrate deep into the central part of dorsal telencephalon. (c) The magnocellular subdivision of dorsal part of dorsal telencephalon (Ddmg) contains not only large cells but also vglut2a-positive clusters of small cells that cover a wide range of the caudal Ddmg. Such clusters of small cells have not been observed in the Ddmg of other teleosts.