Yuichi Narita

Optogenetic dissection of neuronal circuits in zebrafish using viral gene transfer and the Tet system

The conditional expression of transgenes at high levels in sparse and specific populations of neurons is important for high-resolution optogenetic analyses of neuronal circuits. We explored two complementary methods, viral gene delivery and the iTet-Off system, to express transgenes in the brain of zebrafish. High-level gene expression in neurons was achieved by Sindbis and Rabies viruses. The Tet system produced strong and specific gene expression that could be modulated conveniently by doxycycline. Moreover, transgenic lines showed expression in distinct, sparse and stable populations of neurons that appeared to be subsets of the neurons targeted by the promoter driving the Tet-activator. The Tet system therefore provides the opportunity to generate libraries of diverse expression patterns similar to gene trap approaches or the thy-1 promoter in mice, but with the additional possibility to pre-select cell types of interest. In transgenic lines expressing channelrhodopsin-2, action potential firing could be precisely controlled by two-photon stimulation at low laser power, presumably because the expression levels of the Tet-controlled genes were high even in adults. In channelrhodopsin-2-expressing larvae, optical stimulation with a single blue LED evoked distinct swimming behaviors including backward swimming. These approaches provide new opportunities for the optogenetic dissection of neuronal circuit structure and function.

Evolution of the Turtle Body Plan by the Folding and Creation of New Muscle Connections

Shelling Turtles In almost all vertebrates, the shoulder girdle (scapula) lies outside the ribs. The turtle is unique in that the carapace, the dorsal part of the shell, which is formed from the ribs, encapsulates the scapula. To understand the origin of the turtle-specific body plan, Nagashima et al. (p. 193; see the cover; see the Perspective by Rieppel) compared chicken, mouse, and the Chinese soft shelled-turtle, Pelodiscus sinensis. Modern embryos were studied via whole-mount immunostaining, three-dimensional reconstructions, and with markers for early skeletal precursors and compared with previously reported fossils. Initially, embryos of the three animals share a common developmental pattern, one that is likely to have been shared with their last common ancestor. This pattern, however, is modified in the turtle by a specific folding of its body wall during embryogenesis. This folding preserves some of the connectivity between skeletal and muscle elements but also produces new connections. The turtle body plan, unique among amniotes, is based on the folding of an ancestral pattern during embryogenesis. The turtle shell offers a fascinating case study of vertebrate evolution, based on the modification of a common body plan. The carapace is formed from ribs, which encapsulate the scapula; this stands in contrast to the typical amniote body plan and serves as a key to understanding turtle evolution. Comparative analyses of musculoskeletal development between the Chinese soft-shelled turtle and other amniotes revealed that initial turtle development conforms to the amniote pattern; however, during embryogenesis, lateral rib growth results in a shift of elements. In addition, some limb muscles establish new turtle-specific attachments associated with carapace formation. We propose that the evolutionary origin of the turtle body plan results from heterotopy based on folding and novel connectivities.

Thoracolumbar vertebral number: The first skeletal synapomorphy for afrotherian mammals

Abstract There is overwhelming molecular support for the monophyly of a supra‐ordinal clade of living African placental mammals, the Afrotheria, but there is not a single unequivocal morphological synapomorphy for this group. We conducted a survey of thoraco‐lumbar vertebral numbers across mammals, based on the examination of specimens representing 86 living and 12 fossil species and a thorough review of the anatomical literature. A total of 19 thoraco‐lumbar vertebrae is plesiomorphic for mammals, eutherians and metatherians. Metatherians show no variation in this plesiomorphic condition, suggesting the presence of a developmental constraint, in view of the contrasting variability of the equally old eutherian clade. Several deviations from the plesiomorphic condition have evolved independently in the course of placental phylogeny. Optimization of the information on a published phylogenetic framework based on molecular sequence data reveals that an increase in the number of thoracolumbar vertebrae is the first unambiguous skeletal synapomorphy of Afrotheria.

Chapter 5 Hox Genes in Neural Patterning and Circuit Formation in the Mouse Hindbrain

The mammalian hindbrain is the seat of regulation of several vital functions that involve many of the organ systems of the body. Such functions are controlled through the activity of intricate arrays of neuronal circuits and connections. The establishment of ordered patterns of neuronal specification, migration, and axonal topographic connectivity during development is crucial to build such a complex network of circuits and functional connectivity in the mature hindbrain. The early development of the vertebrate hindbrain proceeds according to a fundamental metameric partitioning along the anteroposterior axis into cellular compartments known as rhombomeres. Such an organization has been highly conserved in vertebrate evolution and has a fundamental impact on the hindbrain adult structure, nuclear organization, and connectivity. Here, we review the cellular and molecular mechanisms underlying hindbrain neuronal circuitry in the mouse, with a specific focus on the role of the homeodomain transcription factors of the Hox gene family. The Hox genes are crucial determinants of rhombomere segmental identity and anteroposterior patterning. However, recent findings suggest that, in addition to their well-known roles at early embryonic stages, the Hox genes may play important roles also in later aspect of neuronal circuit development, including stereotypic neuronal migration, axon pathfinding, and topographic mapping of connectivity.

Assembly of the Auditory Circuitry by a Hox Genetic Network in the Mouse Brainstem

Rhombomeres (r) contribute to brainstem auditory nuclei during development. Hox genes are determinants of rhombomere-derived fate and neuronal connectivity. Little is known about the contribution of individual rhombomeres and their associated Hox codes to auditory sensorimotor circuitry. Here, we show that r4 contributes to functionally linked sensory and motor components, including the ventral nucleus of lateral lemniscus, posterior ventral cochlear nuclei (VCN), and motor olivocochlear neurons. Assembly of the r4-derived auditory components is involved in sound perception and depends on regulatory interactions between Hoxb1 and Hoxb2. Indeed, in Hoxb1 and Hoxb2 mutant mice the transmission of low-level auditory stimuli is lost, resulting in hearing impairments. On the other hand, Hoxa2 regulates the Rig1 axon guidance receptor and controls contralateral projections from the anterior VCN to the medial nucleus of the trapezoid body, a circuit involved in sound localization. Thus, individual rhombomeres and their associated Hox codes control the assembly of distinct functionally segregated sub-circuits in the developing auditory brainstem.

On the carapacial ridge in turtle embryos: its developmental origin, function and the chelonian body plan

The chelonian carapace is composed of dorsolaterally expanded ribs; an evolutionary change in the rib-patterning program is assumed to be related to this novelty. Turtle embryos exhibit a longitudinal ridge called the carapacial ridge (CR) on the flank, and its histological resemblance to the apical ectodermal ridge of the limb bud implies its inductive activity in the unique patterning of the ribs. We studied the Chinese soft-shelled turtle, Pelodiscus sinensis, and confirmed by labeling with a lipophilic dye, DiI, that the CR contains the somite-derived dermis and that it is a unique structure among amniotes. Using electroporation of a dominant-negative form of LEF-1, the CR-specific gene, we showed that CR-specific genes function in the growth and maintenance of the CR. Microcauterization or implantation of the CR did not change the dorsoventral pattern of the ribs, and only their fan-shaped pattern was arrested by CR removal. We conclude that the CR is a true embryonic novelty among amniotes and, because of the specific expression of regulatory genes, it functions in the marginal growth of the carapacial primordium, thereby inducing the fan-shaped arrangement of the ribs.

Body plan of turtles: an anatomical, developmental and evolutionary perspective

The evolution of the turtle shell has long been one of the central debates in comparative anatomy. The turtle shell consists of dorsal and ventral parts: the carapace and plastron, respectively. The basic structure of the carapace comprises vertebrae and ribs. The pectoral girdle of turtles sits inside the carapace or the rib cage, in striking contrast to the body plan of other tetrapods. Due to this topological change in the arrangement of skeletal elements, the carapace has been regarded as an example of evolutionary novelty that violates the ancestral body plan of tetrapods. Comparing the spatial relationships of anatomical structures in the embryos of turtles and other amniotes, we have shown that the topology of the musculoskeletal system is largely conserved even in turtles. The positional changes seen in the ribs and pectoral girdle can be ascribed to turtle-specific folding of the lateral body wall in the late developmental stages. Whereas the ribs of other amniotes grow from the axial domain to the lateral body wall, turtle ribs remain arrested axially. Marginal growth of the axial domain in turtle embryos brings the morphologically short ribs in to cover the scapula dorsocaudally. This concentric growth appears to be induced by the margin of the carapace, which involves an ancestral gene expression cascade in a new location. These comparative developmental data allow us to hypothesize the gradual evolution of turtles, which is consistent with the recent finding of a transitional fossil animal, Odontochelys, which did not have the carapace but already possessed the plastron.

Hepatocyte growth factor is crucial for development of the carapace in turtles.

Turtles are characterized by their shell, composed of a dorsal carapace and a ventral plastron. The carapace first appears as the turtle‐specific carapacial ridge (CR) on the lateral aspect of the embryonic flank. Accompanying the acquisition of the shell, unlike in other amniotes, hypaxial muscles in turtle embryos appear as thin threads of fibrous tissue. To understand carapacial evolution from the perspective of muscle development, we compared the development of the muscle plate, the anlage of hypaxial muscles, between the Chinese soft‐shelled turtle, Pelodiscus sinensis, and chicken embryos. We found that the ventrolateral lip (VLL) of the thoracic dermomyotome of P. sinensis delaminates early and produces sparse muscle plate in the lateral body wall. Expression patterns of the regulatory genes for myotome differentiation, such as Myf5, myogenin, Pax3, and Pax7 have been conserved among amniotes, including turtles. However, in P. sinensis embryos, the gene hepatocyte growth factor (HGF), encoding a regulatory factor for delamination of the dermomyotomal VLL, was uniquely expressed in sclerotome and the lateral body wall at the interlimb level. Implantation of COS‐7 cells expressing a HGF antagonist into the turtle embryo inhibited CR formation. We conclude that the de novo expression of HGF in the turtle mesoderm would have played an innovative role resulting in the acquisition of the turtle‐specific body plan.

Origin of the Turtle Body Plan: The Folding Theory to Illustrate Turtle-Specific Developmental Repatterning

The turtle shell is comprised of a dorsal carapace and a ventral plastron, and is an autapomorphy of this group. The carapace consists of the vertebral column and ribs as well as a specialized dermis. The formation of the shell is accompanied by a change in the spatial relationship of the ribs and the pectoral girdle. Because of this rearrangement, the turtle shell has been regarded as an example of an evolutionary novelty. Understanding the changes behind this developmental repatterning will help us elucidate the evolutionary history of turtles. The change has been attributed to a deflected pattern of development of the ribs, which in normal tetrapods grow ventrally into the lateral body wall. In turtles, they grow laterally toward the primordium of the carapacial margin, called the carapacial ridge (CR), while remaining in the axial part of the embryonic body. Based on a similarity in histological configuration, the CR has been thought to possess inductive activity for rib growth, as seen in the apical ectodermal ridge of the amniote limb bud. The CR does not function as a guidance cue for rib progenitor cells but rather functions in the marginal growth of the carapacial primordium, resulting in fanned-out growth of the ribs. This peripheral and concentric expansion of the axial domain makes the lateral body wall fold inward, while the ribs cover the pectoral girdle. The turtle ribs develop along the muscle plate as in other amniotes, and do not take a different trajectory from that in other amniotes, unlike the scenario hypothesized previously. This folding enables turtles to change the apparent spatial relationships between the ribs and the pectoral girdle without altering their topological alignment and body plan as amniotes. This developmental sequence of the modern turtles aligns with a stepwise evolutionary process in the group, which is supported by the anatomy of a recently discovered fossil species, Odontochelys.