Deep homologies in chordate caudal central nervous systems.

Invertebrate chordates, such as the tunicate Ciona, can offer insight into the evolution of the chordate phylum. Anatomical features that are shared between invertebrate chordates and vertebrates may be taken as evidence of their presence in a common chordate ancestor. The central nervous systems of Ciona larvae and vertebrates share a similar anatomy despite the Ciona CNS having ~180 neurons. However, the depth of conservation between the Ciona CNS and those in vertebrates is not resolved. The Ciona caudal CNS, while appearing spinal cord-like, has hitherto been thought to lack motor neurons, bringing into question its homology with the vertebrate spinal cord. We show here that the Ciona larval caudal CNS does, in fact, have functional motor neurons along its length, pointing to the presence of a spinal cord-like structure at the base of the chordates. We extend our analysis of shared CNS anatomy further to explore the Ciona "motor ganglion", which has been proposed to be a homolog of the vertebrate hindbrain, spinal cord, or both. We find that a cluster of neurons in the dorsal motor ganglion shares anatomical location, developmental pathway, neural circuit architecture, and gene expression with the vertebrate cerebellum. However, functionally, the Ciona cluster appears to have more in common with vertebrate cerebellum-like structures, insofar as it receives and processes direct sensory input. These findings are consistent with earlier speculation that the cerebellum evolved from a cerebellum-like structure, and suggest that the latter structure was present in the dorsal hindbrain of a common chordate ancestor.

Erratum: Automatic classification and neurotransmitter prediction of synapses in electron microscopy - CORRIGENDUM.

[This corrects the article DOI: 10.1017/S2633903X2200006X.].

Automatic classification and neurotransmitter prediction of synapses in electron microscopy.

This paper presents a deep-learning-based workflow to detect synapses and predict their neurotransmitter type in the primitive chordate Ciona intestinalis (Ciona) electron microscopic (EM) images. Identifying synapses from EM images to build a full map of connections between neurons is a labor-intensive process and requires significant domain expertise. Automation of synapse classification would hasten the generation and analysis of connectomes. Furthermore, inferences concerning neuron type and function from synapse features are in many cases difficult to make. Finding the connection between synapse structure and function is an important step in fully understanding a connectome. Class Activation Maps derived from the convolutional neural network provide insights on important features of synapses based on cell type and function. The main contribution of this work is in the differentiation of synapses by neurotransmitter type through the structural information in their EM images. This enables the prediction of neurotransmitter types for neurons in Ciona, which were previously unknown. The prediction model with code is available on GitHub.

Neuronal identity: the neuron types of a simple chordate sibling, the tadpole larva of Ciona intestinalis.

Neurons of the sparsely populated nervous system of the tadpole larva in the tunicate Ciona intestinalis, a chordate sibling, are known from sporadic previous studies but especially two recent reports that document the connectome of both the central and peripheral nervous systems at EM level. About 330 CNS cells comprise mostly ciliated ependymal cells, with ∼180 neurons that constitute about 50 morphologically distinguishable types. The neurons reveal various chordate characters amid many features that are idiosyncratic. Most neurons are ciliated and lack dendrites, some even lack an axon. Synapses mostly form en passant between axons, and resemble those in basal invertebrates; some are dyads and all have heterogenous synaptic vesicle populations. Each neuron has on average 49 synapses with other cells; these constitute a synaptic network of unpredicted complexity.

The peripheral nervous system of the ascidian tadpole larva: Types of neurons and their synaptic networks.

Physical and chemical cues from the environment are used to direct animal behavior through a complex network of connections originating in exteroceptors. In chordates, mechanosensory and chemosensory neurons of the peripheral nervous system (PNS) must signal to the motor circuits of the central nervous system (CNS) through a series of pathways that integrate and regulate the output to motor neurons (MN); ultimately these drive contraction of the tail and limb muscles. We used serial-section electron microscopy to reconstruct PNS neurons and their hitherto unknown synaptic networks in the tadpole larva of a sibling chordate, the ascidian, Ciona intestinalis. The larva has groups of neurons in its apical papillae, epidermal neurons in the rostral and apical trunk, caudal neurons in the dorsal and ventral epidermis, and a single tail tip neuron. The connectome reveals that the PNS input arises from scattered groups of these epidermal neurons, 54 in total, and has three main centers of integration in the CNS: in the anterior brain vesicle (which additionally receives input from photoreceptors of the ocellus), the motor ganglion (which contains five pairs of MN), and the tail, all of which in turn are themselves interconnected through important functional relay neurons. Some neurons have long collaterals that form autapses. Our study reveals interconnections with other sensory systems, and the exact inputs to the motor system required to regulate contractions in the tail that underlie larval swimming, or to the CNS to regulate substrate preference prior to the induction of larval settlement and metamorphosis.

Circuit Homology between Decussating Pathways in the Ciona Larval CNS and the Vertebrate Startle-Response Pathway.

Comparing synaptic circuits and networks between brains of different animal groups helps us derive an understanding of how nervous systems might have evolved. The circuits of the startle response pathway in the brains of tailed vertebrates are known from electrophysiological studies on the giant reticulospinal Mauthner cells (M-cells). To identify morphological counterparts in chordate tunicates, a sister group of vertebrates [1, 2], we have compiled a densely reconstructed connectome (defined in [3]) for the CNS in the tadpole larva of Ciona intestinalis (L.), using ssEM [4]. The dorsal, tubular CNS of the ∼1-mm tadpole larva is built on a similar plan to vertebrates, its neurons distributed rostrocaudally in three centers, a brain vesicle, motor ganglion, and caudal nerve cord [5]. A single pair of descending decussating neurons, ddNs, found in the motor ganglion, have similarities to reticulospinal neurons descending from the vertebrate hindbrain to the spinal cord. The pre- and postsynaptic connections and circuits of these ddNs support their homology with decussating vertebrate M-cells. Network analysis reveals that, like M-cells, ddNs receive mechanosensory input from the peripheral nervous system and provide input to motoneurons, premotor interneurons, and ascending commissural inhibitory neurons (ACINs). These circuits uncover a putative homologous startle network in the Ciona tadpole. However, differences in circuits, including a lack of bilateral symmetry in their network, and convergence of inputs from left and right sides, raise questions about the relationship between form and function, and are a possible outcome of the tiny number of neurons in ascidian larvae.

The CNS connectome of a tadpole larva of Ciona intestinalis (L.) highlights sidedness in the brain of a chordate sibling.

Left-right asymmetries in brains are usually minor or cryptic. We report brain asymmetries in the tiny, dorsal tubular nervous system of the ascidian tadpole larva, Ciona intestinalis. Chordate in body plan and development, the larva provides an outstanding example of brain asymmetry. Although early neural development is well studied, detailed cellular organization of the swimming larva's CNS remains unreported. Using serial-section EM we document the synaptic connectome of the larva's 177 CNS neurons. These formed 6618 synapses including 1772 neuromuscular junctions, augmented by 1206 gap junctions. Neurons are unipolar with at most a single dendrite, and few synapses. Some synapses are unpolarised, others form reciprocal or serial motifs; 922 were polyadic. Axo-axonal synapses predominate. Most neurons have ciliary organelles, and many features lack structural specialization. Despite equal cell numbers on both sides, neuron identities and pathways differ left/right. Brain vesicle asymmetries include a right ocellus and left coronet cells.

Migratory neuronal progenitors arise from the neural plate borders in tunicates.

The neural crest is an evolutionary novelty that fostered the emergence of vertebrate anatomical innovations such as the cranium and jaws. During embryonic development, multipotent neural crest cells are specified at the lateral borders of the neural plate before delaminating, migrating and differentiating into various cell types. In invertebrate chordates (cephalochordates and tunicates), neural plate border cells express conserved factors such as Msx, Snail and Pax3/7 and generate melanin-containing pigment cells, a derivative of the neural crest in vertebrates. However, invertebrate neural plate border cells have not been shown to generate homologues of other neural crest derivatives. Thus, proposed models of neural crest evolution postulate vertebrate-specific elaborations on an ancestral neural plate border program, through acquisition of migratory capabilities and the potential to generate several cell types. Here we show that a particular neuronal cell type in the tadpole larva of the tunicate Ciona intestinalis, the bipolar tail neuron, shares a set of features with neural-crest-derived spinal ganglia neurons in vertebrates. Bipolar tail neuron precursors derive from caudal neural plate border cells, delaminate and migrate along the paraxial mesoderm on either side of the neural tube, eventually differentiating into afferent neurons that form synaptic contacts with both epidermal sensory cells and motor neurons. We propose that the neural plate borders of the chordate ancestor already produced migratory peripheral neurons and pigment cells, and that the neural crest evolved through the acquisition of a multipotent progenitor regulatory state upstream of multiple, pre-existing neural plate border cell differentiation programs.

An endocrine disruptor, bisphenol A, affects development in the protochordate Ciona intestinalis: hatching rates and swimming behavior alter in a dose-dependent manner.

Bisphenol A (BPA) is widely used industrially to produce polycarbonate plastics and epoxy resins. Numerous studies document the harmful effects caused by low-dose BPA exposure especially on nervous systems and behavior in experimental animals such as mice and rats. Here, we exposed embryos of a model chordate, Ciona intestinalis, to seawater containing BPA to evaluate adverse effects on embryonic development and on the swimming behavior of subsequent larvae. Ciona is ideal because its larva develops rapidly and has few cells. The rate of larval hatching decreased in a dose-dependent manner with exposures to BPA above 3 µM; swimming behavior was also affected in larvae emerging from embryos exposed to 1 µM BPA. Adverse effects were most severe on fertilized eggs exposed to BPA within 7 h post-fertilization. Ciona shares twelve nuclear receptors with mammals, and BPA is proposed to disturb the physiological functions of one or more of these.

Developmental and morphological variation in the teleost craniofacial skeleton reveals an unusual mode of ossification.

We investigated the morphology and development of the scleral ossicles within the eyes of three species from three basal teleost orders, namely, the alewife (Alosa pseudoharengus; Clupeiformes), the surface morph of the Mexican tetra (Astyanax mexicanus; Characiformes) and zebrafish (Danio rerio; Cypriniformes). Two morphologies, circular and elongated, and one variation, fused elements, were identified. Zebrafish have small circular ossicles, whereas the alewife and the Mexican tetra have elongated ossicles. Surprisingly in the Mexican tetra these elements fuse at one end forming a continuous element with an antero-ventral opening; this may be typical for the Order Characiformes. Regardless of morphology, the ossicles develop via unilateral perichondral ossification of the scleral cartilage from two centers opposite one another in the eye. This unilateral type of ossification, in which only the perichondrium furthest from the retina contributes to the ossicles, has not previously been reported in any vertebrate. Because either the perichondrium and/or an extension of the perichondrium can transform into the scleral ossicle, we refer to the transitional tissue as periskeletal. Although the functional significance of the different shaped ossicles is unclear, skeletal muscle attaches directly to these bones, implying voluntary control. The morphological and developmental variation of teleost scleral ossicles makes them an ideal system for determining the genetic basis underlying phenotypic variation as well as for studying mechanisms underlying osteogenic and chondrogenic processes in teleosts. These data support our previous finding that scleral ossicles in teleosts may not be homologous to those in other vertebrates, such as reptiles.