Large-scale characterization of genes specific to the larval nervous system in the ascidian Ciona intestinalis

The central and peripheral nervous systems (CNS and PNS) of the ascidian tadpole larva are comparatively simple, consisting of only about 350 cells. However, studies of the expression of neural patterning genes have demonstrated overall similarity between the ascidian CNS and the vertebrate CNS, suggesting that the ascidian CNS is sufficiently complex to be relevant to those of vertebrates. Recent progress in the Ciona intestinalis genome project and cDNA project together with considerable EST information has made Ciona an ideal model for investigating molecular mechanisms underlying the formation and function of the chordate nervous system. Here, we characterized 56 genes specific to the nervous system by determining their full-length cDNA sequences and confirming their spatial expression patterns. These genes included those that function in the nervous systems of other animals, especially those involved in photoreceptor-mediated signaling and neurotransmitter release. Thus, the nervous system-specific genes in Ciona larvae will provide not only probes for determining their function but also clues for exploring the complex network of nervous system-specific genes.     

Isolation of an early neural maker gene abundantly expressed in the nervous system of the ascidian, Halocynthia roretzi

Ascidian tadpole larvae possess a primitive nervous system, which is a prospective prototype of the chordate nervous system. It is composed of relatively few cells but sufficient for complex larval behavior. Here we report on HrETR-1, a gene zygotically expressed in a large proportion of the developing neural cells of the ascidian, Halocynthia roretzi. HrETR-1 is an early neural marker which can be used for analyzing neural differentiation. HrETR-1 expression intensified in most neural cells of genes isolated to date, in both central and peripheral nervous systems including palps as early as the 110-cell stage. Using this gene as a probe, we characterized neural cells in the nervous system as well as confirming their origins. Also, we recognized three types of peripheral epidermal neurons which presumably correlate to the larval neurons previously reported for another ascidian. Among these, five bilateral neurons located in the anterior region of the trunk appeared to be derived from a8.26 blastomeres.     

Ascidians as excellent chordate models for studying the development of the nervous system during embryogenesis and metamorphosis

The swimming larvae of the chordate ascidians possess a dorsal hollowed central nervous system (CNS), which is homologous to that of vertebrates. Despite the homology, the ascidian CNS consists of a countable number of cells. The simple nervous system of ascidians provides an excellent experimental system to study the developmental mechanisms of the chordate nervous system. The neural fate of the cells consisting of the ascidian CNS is determined in both autonomous and non-autonomous fashion during the cleavage stage. The ascidian neural plate performs the morphogenetic movement of neural tube closure that resembles that in vertebrate neural tube formation. Following neurulation, the CNS is separated into five distinct regions, whose homology with the regions of vertebrate CNS has been discussed. Following their larval stage, ascidians undergo a metamorphosis and become sessile adults. The metamorphosis is completed quickly, and therefore the metamorphosis of ascidians is a good experimental system to observe the reorganization of the CNS during metamorphosis. A recent study has shown that the major parts of the larval CNS remain after the metamorphosis to form the adult CNS. In contrast to such a conserved manner of CNS reorganization, most larval neurons disappear during metamorphosis. The larval glial cells in the CNS are the major source for the formation of the adult CNS, and some of the glial cells produce adult neurons.     

Ascidian embryos as a model system to analyze expression and function of developmental genes

Ascidians have served as an appropriate experimental system in developmental biology for more than a century. The fertilized egg develops quickly into a tadpole larva, which consists of a small number of organs including epidermis, central nervous system with two sensory organs, endoderm and mesenchyme in the trunk, and notochord and muscle in the tail. This configuration of the ascidian tadpole is thought to represent the most simplified and primitive chordate body plan. Their embryogenesis is simple, and lineage of embryonic cells is well documented. The ascidian genome contains a basic set of genes with less redundancy compared to the vertebrate genome. Cloning and characterization of developmental genes indicate that each gene is expressed under discrete spatio-temporal pattern within their lineage. In addition, the use of various molecular techniques in the ascidian embryo system highlights its advantages as a future experimental system to explore the molecular mechanisms underlying the expression and function of developmental genes as well as genetic circuitry responsible for the establishment of the basic chordate body plan. This review is aimed to highlight the recent advances in ascidian embryology.     

Early specification of ascidian larval motor neurons

In the tadpole larvae of the ascidian Halocynthia roretzi, six motor neurons, Moto-A, -B, and -C (a pair of each), are localized proximal to the caudal neural tube and show distinct morphology and innervation patterns. To gain insights into early mechanisms underlying differentiation of individual motor neurons, we have isolated an ascidian homologue of Islet, a LIM type homeobox gene. Earliest expression of Islet was detected in a pair of bilateral blastomeres on the dorsal edge of the late gastrula. At the neurula stage, this expression began to disappear and more posterior cells started to express Islet. Compared to expression of a series of motor neuron genes, it was confirmed that early Islet-positive blastomeres are the common precursors of Moto-A and -B, and late Islet-positive cells in the posterior neural tube are the precursors of Moto-C. Overexpression of Islet induced ectopic expression of motor neuron markers, suggesting that Islet is capable of regulating motor neuron differentiation. Since early expression of Islet colocalizes with that of HrBMPb, the ascidian homologue of BMP2/4, we tested a role of BMP in specification of the motor neuron fate. Overexpression of HrBMPb led to expansion of Lim and Islet expression toward the central area of the neural plate, and microinjection of mRNA coding for a dominant-negative BMP receptor weakened the expression of these genes. Our results suggest that determination of the ascidian motor neuron fate takes place at late gastrula stage and local BMP signaling may play a role in this step.     

Trunk lateral cells are neural crest-like cells in the ascidian Ciona intestinalis: insights into the ancestry and evolution of the neural crest

Neural crest-like cells (NCLC) that express the HNK-1 antigen and form body pigment cells were previously identified in diverse ascidian species. Here we investigate the embryonic origin, migratory activity, and neural crest related gene expression patterns of NCLC in the ascidian Ciona intestinalis. HNK-1 expression first appeared at about the time of larval hatching in dorsal cells of the posterior trunk. In swimming tadpoles, HNK-1 positive cells began to migrate, and after metamorphosis they were localized in the oral and atrial siphons, branchial gill slits, endostyle, and gut. Cleavage arrest experiments showed that NCLC are derived from the A7.6 cells, the precursors of trunk lateral cells (TLC), one of the three types of migratory mesenchymal cells in ascidian embryos. In cleavage arrested embryos, HNK-1 positive TLC were present on the lateral margins of the neural plate and later became localized adjacent to the posterior sensory vesicle, a staging zone for their migration after larval hatching. The Ciona orthologues of seven of sixteen genes that function in the vertebrate neural crest gene regulatory network are expressed in the A7.6/TLC lineage. The vertebrate counterparts of these genes function downstream of neural plate border specification in the regulatory network leading to neural crest development. The results suggest that NCLC and neural crest cells may be homologous cell types originating in the common ancestor of tunicates and vertebrates and support the possibility that a putative regulatory network governing NCLC development was co-opted to produce neural crest cells during vertebrate evolution.     

The development of three identified motor neurons in the larva of an ascidian, Halocynthia roretzi

The generation of distinct classes of motor neurons underlies the development of complex motile behavior in all animals and is well characterized in chordates. Recent molecular studies indicate that the ascidian larval central nervous system (CNS) exhibits anteroposterior regionalization similar to that seen in the vertebrate CNS. To extend the understanding about the diversity of motor neurons in the ascidian larva, we have identified the number, position, and projection of individual motor neurons in Halocynthia roretzi, using a green fluorescent protein under the control of a neuron-specific promoter. Three pairs of motor neurons, each with a distinct shape and innervation pattern, were identified along the anteroposterior axis of the neural tube: the anterior and posterior pairs extend their axons toward dorsal muscle cells, whereas the middle pair project their axons toward ventral muscle. Overexpression of a dominant-negative form of a potassium channel in these cells resulted in paralysis on the injected side, thus these cells must constitute the major population of motor neurons responsible for swimming behavior. Lim class homeobox genes have been known as candidate genes that determine subtypes of motor neurons. Therefore, the expression pattern of Hrlim, which is a Lim class homeobox gene, was examined in the motor neuron precursors. All three motor neurons expressed Hrlim at the tailbud stage, although each down-regulated Hrlim at a different time. Misexpression of Hrlim in the epidermal lineage led to ectopic expression of TuNa2, a putative voltage-gated channel gene normally expressed predominantly in the three pairs of motor neurons. Hrlim may control membrane excitability of motor neurons by regulating ion channel gene expression.     

Notch signaling is involved in nervous system formation in ascidian embryos

Notch signaling plays crucial roles during embryogenesis in various metazoans. HrNotch, a Notch homologue in the ascidian Halocynthia roretzi, has been previously cloned, and its expression pattern suggests that HrNotch signaling is involved in nervous system formation. To determine the function of HrNotch signaling, in the present study we examined the effects of the constitutively activated forms of HrNotch. Overexpression resulted in larvae with defects in neural tube closure and brain vesicle formation. In embryos expressing the activated HrNotch, the expression of a neural marker gene, HrETR-1, was enhanced and expanded in the central nervous system, although ectopic expression decreased during the tailbud stage. The activated HrNotch also suppressed the formation of the adhesive organ (palps) and the peripheral nervous system, which consists of ciliary mechanosensory neurons, whereas it promoted epidermal differentiation. The suppression and promotion of the formation of these respective cell types were confirmed by examination of the expression of relevant tissue-specific markers. We also cloned Hrdelta, an ascidian homologue of DSL family genes, which encode ligands for which Notch acts as a receptor. The expression of Hrdelta was observed in the precursors of palps and peripheral neurons in addition to the CNS. These results suggest that Notch signaling is important for ascidian nervous system formation and that it affects the fate choice between palps and epidermis and between peripheral neurons and epidermis within the neurogenic regions of the surface ectoderm by suppressing the formations of palps and peripheral neurons and promoting epidermal differentiation.     

Distinct Neuronal Lineages of the Ascidian Embryo Revealed by Expression of a Sodium Channel Gene

The ascidian larva contains tubular neural tissue, one of the prominent anatomical features of the chordates. The cell-cleavage pattern and cell maps of the nervous system have been described in the ascidian larva in great detail. Cell types in the neural tube, however, have not yet been defined due to the lack of a suitable molecular marker. In the present work, we identified neuronal cells in the caudal neural tube of theHalocynthiaembryo by utilizing a voltage-gated Na⁺channel gene, TuNa I, as a molecular marker. Microinjection of a lineage tracer revealed that TuNa I-positive neurons in the brain and in the trunk epidermis are derived from the a-line of the eight-cell embryo, which includes cell fates to epidermal and neural tissue. On the other hand, TuNa I-positive cells in the more caudal part of the neural tissue were not stained by microinjection into the a-line. These neurons are derived from the A-line, which contains fates of notochord and muscle, but not of epidermis. Electron microscopic observation confirmed that A-line-derived neurons consist of motor neurons innervating the dorsal and ventral muscle cells. Isolated A-line blastomeres have active membrane excitability distinct from those of the a-line-derived neuronal cells after culture under cleavage arrest, suggesting that the A-line gives rise to a neuronal cell distinct from that of the a-lineage. TuNa I expression in the a-line requires signals from another cell lineage, whereas that in the A-line occurs without tight cell contact. Thus, there are at least two distinct neuronal lineages with distinct cellular behaviors in the ascidian larva: the a-line gives rise to numerous neuronal cells, including sensory cells, controlled by a mechanism similar to vertebrate neural induction, whereas A-line cells give rise to motor neurons and ependymal cells in the caudal neural tube that develop in close association with the notochord or muscle lineage, but not with the epidermal lineage.     

Anteroposterior patterning of the epidermis by inductive influences from the vegetal hemisphere cells in the ascidian embryo.

Patterning along the anteroposterior axis is a critical step during animal embryogenesis. Although mechanisms of anteroposterior patterning in the neural tube have been studied in various chordates, little is known about those of the epidermis. To approach this issue, we investigated patterning mechanisms of the epidermis in the ascidian embryo. First we examined expression of homeobox genes (Hrdll-1, Hroth, HrHox-1 and Hrcad) in the epidermis. Hrdll-1 is expressed in the anterior tip of the epidermis that later forms the adhesive papillae, while Hroth is expressed in the anterior part of the trunk epidermis. HrHox-1 and Hrcad are expressed in middle and posterior parts of the epidermis, respectively. These data suggested that the epidermis of the ascidian embryo is patterned anteroposteriorly. In ascidian embryogenesis, the epidermis is exclusively derived from animal hemisphere cells. To investigate regulation of expression of the four homeobox genes in the epidermis by vegetal hemisphere cells, we next performed hemisphere isolation and cell ablation experiments. We showed that removal of the vegetal cells before the late 16-cell stage results in loss of expression of these homeobox genes in the animal hemisphere cells. Expression of Hrdll-1 and Hroth depends on contact with the anterior-vegetal (the A-line) cells, while expression of HrHox-1 and Hrcad requires contact with the posterior-vegetal (the B-line) cells. We also demonstrated that contact with the vegetal cells until the late 32-cell stage is sufficient for animal cells to express Hrdll-1, Hroth and Hrcad, while longer contact is necessary for HrHox-1 expression. Contact with the A-line cells until the late 32-cell stage is also sufficient for formation of the adhesive papillae. Our data indicate that the epidermis of the ascidian embryo is patterned along the anteroposterior axis by multiple inductive influences from the vegetal hemisphere cells and provide the first insight into mechanisms of epidermis patterning in the chordate embryos.     

Neural expression of the Huntington's disease gene as a chordate evolutionary novelty

Huntington's disease is a progressive neuro-degenerative disorder in humans, which is scharacterized by onset of dementia, muscular ataxia, and death. Huntington's disease is caused by the expansion of the polyglutamine (polyQ) tract in the N-terminus of the HD protein (Huntingtin). CAG expansion is a dominant gain of function mutation that affects striated neurons in the brain (Cattaneo, 2003, News Physiol Sci 18:34). The evolutionary origins of the vertebrate Hd gene are not well understood. In order to address the evolutionary history of the Hd gene, we have cloned and characterized the expression of the Hd gene in two invertebrate deuterostomes, an echinoderm and an ascidian, and have examined the expression patterns in a phylogenetic context. Echinoderms are basal deuterostomes and ascidians are basal chordates; both are useful for understanding the origins of and evolutionary trends in genes important in vertebrates such as the Huntigton's disease gene. Expression of Hd RNA is detected at all stages of development in both the echinoderm and ascidian studied. In the echinoderm Heliocidaris erythrogramma, Hd is expressed in coelomic mesodermal tissue derivatives, but not in the central nervous system. In the ascidian Halocynthia roretzi expression is located in both mesoderm and nervous tissue. We suggest that the primitive deuterostome expression pattern is not neural. Thus, neural expression of the Hd gene in deuterostomes may be a novel feature of the chordate lineage, and the original role(s) of HD in deuterostomes may have been non-neural.     

Characterization of an amphioxus paired box gene, AmphiPax2/5/8: developmental expression patterns in optic support cells, nephridium, thyroid-like structures and pharyngeal gill slits, but not in the midbrain-hindbrain boundary region

On the basis of developmental gene expression, the vertebrate central nervous system comprises: a forebrain plus anterior midbrain, a midbrain-hindbrain boundary region (MHB) having organizer properties, and a rhombospinal domain. The vertebrate MHB is characterized by position, by organizer properties and by being the early site of action of Wnt1 and engrailed genes, and of genes of the Pax2/5/8 subfamily. Wada and others (Wada, H., Saiga, H., Satoh, N. and Holland, P. W. H. (1998) Development 125, 1113-1122) suggested that ascidian tunicates have a vertebrate-like MHB on the basis of ascidian Pax258 expression there. In another invertebrate chordate, amphioxus, comparable gene expression evidence for a vertebrate-like MHB is lacking. We, therefore, isolated and characterized AmphiPax2/5/8, the sole member of this subfamily in amphioxus. AmphiPax2/5/8 is initially expressed well back in the rhombospinal domain and not where a MHB would be expected. In contrast, most of the other expression domains of AmphiPax2/5/8 correspond to expression domains of vertebrate Pax2, Pax5 and Pax8 in structures that are probably homologous - support cells of the eye, nephridium, thyroid-like structures and pharyngeal gill slits; although AmphiPax2/5/8 is not transcribed in any structures that could be interpreted as homologues of vertebrate otic placodes or otic vesicles. In sum, the developmental expression of AmphiPax2/5/8 indicates that the amphioxus central nervous system lacks a MHB resembling the vertebrate isthmic region. Additional gene expression data for the developing ascidian and amphioxus nervous systems would help determine whether a MHB is a basal chordate character secondarily lost in amphioxus. The alternative is that the MHB is a vertebrate innovation.     

Ascidian neural crest-like cells: phylogenetic distribution, relationship to larval complexity, and pigment cell fate

Migratory neural crest-like cells, which express the cell surface antigen HNK-1 and develop into pigment cells, have recently been identified in the ascidian Ecteinascidia turbinata. Here we use HNK-1 expression as a marker to determine whether neural crest-like cells are responsible for pigment development in diverse ascidian species. We surveyed HNK-1 expression and tyrosinase activity in 12 ascidian species, including those with different adult organizations, developmental modes, and larval sizes and complexities. We observed HNK-1 positive cells in every species, although the timing of HNK-1 expression varied according to the extent of larval complexity. HNK-1 expression was initiated during the late tailbud stage in species in which adult features are formed precociously in large complex larvae. In contrast, HNK-1 positive cells did not appear until the swimming tadpole or juvenile stage in species with small simple larvae in which most adult features appear after metamorphosis. Double labeling experiments indicated that HNK-1 and tyrosinase are expressed in the same subset of pigment-forming mesenchymal cells in species with complex or simple larvae. In addition, the absence of HNK-1 and tyrosinase expression in albino morphs of the colonial ascidian Botryllus schlosseri suggested that the major fate of neural crest-like cells is to become pigment cells. The results suggest that ascidian neural crest-like cells and vertebrate neural crest cells had a common origin during chordate evolution and that their primitive function was to generate body pigmentation.     

Nematostella vectensis achaete-scute homolog NvashA regulates embryonic ectodermal neurogenesis and represents an ancient component of the metazoan neural specification pathway

achaete-scute homologs (ash) regulate neural development in all bilaterian model animals indicating that they represent a component of the ancestral neurogenic pathway. We test this by investigating four ash genes during development of a basal metazoan, the cnidarian sea anemone Nematostella vectensis. Spatiotemporal expression of ash genes in the early embryo and larval stages suggests that they regulate neurogenesis. More specifically, NvashA is co-expressed with neural genes in the embryonic ectoderm. Knockdown of NvashA results in decreased expression of eight neural markers, including the six novel neural targets identified here. Conversely, overexpression of NvashA induces increased expression of all eight genes, but only within their normal axial domains. Overexpression of NvashB-D differentially increases expression of NvashA targets. The expression patterns and differential ability of ash genes to regulate neural gene expression reveals surprising molecular complexity in these 'simple' animals. These data suggest that achaete-scute homologs functioned in the ancestral metazoan neurogenic pathway and provide a foundation to investigate further the evolution of neurogenesis and the origin of complex central nervous systems.