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.     

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.     

Induction of ascidian peripheral neuron by vegetal blastomeres.

Ascidian tadpole larvae have a similar dorsal tubular nervous system as vertebrates. The induction of brain formation from a4.2-derived (a-line) cells requires signals from the A4.1-derived (A-line) cells. However, little is known about the mechanism underlying the development of the larval peripheral nervous system due to the lack of a suitable molecular marker. Gelsolin, an actin-binding protein, is specifically expressed in epidermal sensory neurons (ESNs) that mainly constitute the entire peripheral nervous system of the ascidian young tadpoles. Here, we address the role of cell interactions in the specification of ESNs using immunostaining with an anti-gelsolin antibody. Animal half (a4.2- and b4.2-derived) embryos did not give rise to any gelsolin-positive neurons, indicating that differentiation of ESNs requires signals from vegetal cells. Cell isolation experiments showed that A4.1 blastomeres induce gelsolin-positive neurons from a-line cells but not from b4.2-derived (b-line) cells. On the other hand, B4.1 blastomeres induce gelsolin-positive neurons both from b-line cells and a-line cells. This is in sharp contrast to the specification of brain cells which is not affected by the ablation of B4.1-derived (B-line) cells. Furthermore, basic fibroblast growth factor (bFGF) induced ESNs from the a-line cells and b-line cells in the absence of vegetal cells. Their competence to form ESNs was lost between the 110-cell stage and the neurula stage. Our results suggested that the specification of the a-line cells and b-line cells into ESNs is controlled by distinct inducing signals from the anterior and posterior vegetal blastomeres. ESNs in the trunk appear to be derived from the a8.26 blastomeres aligning on the edge of presumptive neural region where ascidian homologue of Pax3 is expressed. These findings highlight the close similarity of ascidian ESNs development with that of vertebrate placode and neural crest.     

Nervous network in larvae of the ascidian Ciona intestinalis

 With the use of the monoclonal antibody UA301, which specifically recognizes the nervous system in ascidian larvae, the neuronal connections of the peripheral and central nervous systems in the ascidian Ciona intestinalis were observed. Three types of peripheral nervous system neurons were found: two located in the larval trunk and the other in the larval tail. These neurons were epidermal and their axons extended to the central nervous system and connected with the visceral ganglion directly or indirectly. The most rostral system (rostral trunk epidermal neurons, RTEN) was distributed bilateral-symmetrically. In addition, presumptive papillar neurons in palps were found which might be related to the RTEN. Another neuron group (apical trunk epidermal neurons, ATEN) was located in the apical part of the trunk. The caudal peripheral nervous system (caudal epidermal neurons, CEN) was located at the dorsal and ventral midline of the caudal epidermis. In the larval central nervous system, two major axon bundles were observed: one was of a photoreceptor complex and the other was connected with RTEN. These axon bundles joined in the posterior sensory vesicle, ran posteriorly through the visceral ganglion and branched into two caudal nerves which ran along the lateral walls of the caudal nerve tube. In addition, some immunopositive cells existed in the most proximal part of the caudal nerve tube and may be motoneurons. Received: 8 September 1997 / Accepted: 14 December 1997     

Neuronal form in the central nervous system of the tadpole larva of the ascidian Ciona intestinalis.

The dorsal tubular central nervous system (CNS) of the ascidian tadpole larva is a diagnostic feature by which the chordate affinities of this group, as a whole, are recognized. We have used two methods to identify larval neurons of Ciona intestinalis. The first is serial electron microscopy (EM), as part of a dedicated study of the visceral ganglion (1), and the second is the transient transfection of neural plate progeny with green fluorescent protein (GFP) (2), to visualize the soma and its neurites of individual neurons in whole-mounted larvae of C. intestinalis. Our observations reveal that ascidian larval neurons are simple inform, with a single axonal neurite arising from a soma that is either monopolar or has only very few, relatively simple neurites arising from it, as part of a presumed dendritic arbor. Somata in the visceral ganglion giving rise to axons descending in the caudal nerve cord are presumed to be those of motor neurons.     

Neural marker genes differently expressed in subsets of embryonic neural cells of the ascidian Halocynthia roretzi

Ascidians are lower chordates that possess a possible prototype of the vertebrate nervous system. The central and peripheral nervous systems of ascidian larvae are composed of only a few hundred cells (Nicol and Meinertzhagen, 1991). To investigate how these ascidian nervous systems develop, dissection at the molecular level using subset-specific markers is essential. Here we describe four new genes zygotically expressed in subsets of the ascidian neural cells. The spatial expression domains of these genes overlap in some parts but not in other parts of the nervous systems. Our results suggest that there are functionally different regions in the nervous systems owing to the gene expression differences. Further analyses of these genes will enable us to determine the molecular neuro-developmental characteristics of various clusters of neural cells.     

Formation of the ascidian epidermal sensory neurons: insights into the origin of the chordate peripheral nervous system

The vertebrate peripheral nervous system (PNS) originates from neural crest and placodes. While its developmental origin is the object of intense studies, little is known concerning its evolutionary history. To address this question, we analyzed the formation of the larval tail PNS in the ascidian Ciona intestinalis. The tail PNS of Ciona is made of sensory neurons located within the epidermis midlines and extending processes in the overlying tunic median fin. We show that each midline corresponds to a single longitudinal row of epidermal cells and neurons sharing common progenitors. This simple organization is observed throughout the tail epidermis, which is made of only eight single-cell rows, each expressing a specific genetic program. We next demonstrate that the epidermal neurons are specified in two consecutive steps. During cleavage and gastrula stages, the dorsal and ventral midlines are independently induced by FGF9/16/20 and the BMP ligand ADMP, respectively. Subsequently, Delta/Notch–mediated lateral inhibition controls the number of neurons formed within these neurogenic regions. These results provide a comprehensive overview of PNS formation in ascidian and uncover surprising similarities between the fate maps and embryological mechanisms underlying formation of ascidian neurogenic epidermis midlines and the vertebrate median fin.     

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.     

The selector gene cut represses a neural cell fate that is specified independently of the Achaete-Scute-Complex and atonal.

The peripheral nervous system (PNS) of Drosophila offers a powerful system to precisely identify individual cells and dissect their genetic pathways of development. The mode of specification of a subset of larval PNS cells, the multiple dendritic (md) neurons (or type II neurons), is complex and still poorly understood. Within the dorsal thoracic and abdominal segments, two md neurons, dbd and dda1, apparently require the proneural gene amos but not atonal (ato) or Achaete-Scute-Complex (ASC) genes. ASC normally acts via the neural selector gene cut to specify appropriate sensory organ identities. Here, we show that dbd- and dda1-type differentiation is suppressed by cut in dorsal ASC-dependent md neurons. Thus, cut is not only required to promote an ASC-dependent mode of differentiation, but also represses an ASC- and ato-independent fate that leads to dbd and dda1 differentiation.     

Structure of the caudal neural tube in an ascidian larva: Vestiges of its possible evolutionary origin from a ciliated band

Ultrastructural analysis and differential immunocytochemical staining with two antitubulin monoclonal antibodies were used to reexamine the organization and development of the neural tube in the larva of an ascidian, Ciona intestinalis, in appraisal of a theory that the dorsal tubular nervous system of the chordates evolved from two halves of a ciliated band in an auricularia-like larva of the kind found in echinoderms and hemichordates. One of the antibodies stained cilia in the nervous system and elsewhere; the other reacted primarily with neuronal axons. The caudal neural tube consists of four rows of large ciliated ependymal-glial cells enclosing an axial neural canal into which their single cilia extend. Two ventrolateral nerve tracts, containing axons, arise in the posterior brain region and extend along the length of the caudal tube, partially surrounded by the ependymal cells. The nonnervous, ciliated, ependymal neural tube of the ascidian larva with its two associated nerve tracts survives as a primitive early condition that could result from a ciliated band transformation. Tissues in the distal-most part of the ascidian larval tail have cell lineage origins that indicate an evolutionary history different from those in the proximal majority of the tail. The ependymal cells in this presumed later addition to the tail are not ciliated, although all of the others in the caudal ependymal tube appear to be.     

Structure of the caudal neural tube in an ascidian larva: vestiges of its possible evolutionary origin from a ciliated band.

Ultrastructural analysis and differential immunocytochemical staining with two antitubulin monoclonal antibodies were used to reexamine the organization and development of the neural tube in the larva of an ascidian, Ciona intestinalis, in appraisal of a theory that the dorsal tubular nervous system of the chordates evolved from two halves of a ciliated band in an auricularia-like larva of the kind found in echinoderms and hemichordates. One of the antibodies stained cilia in the nervous system and elsewhere; the other reacted primarily with neuronal axons. The caudal neural tube consists of four rows of large ciliated ependymal-glial cells enclosing an axial neural canal into which their single cilia extend. Two ventrolateral nerve tracts, containing axons, arise in the posterior brain region and extend along the length of the caudal tube, partially surrounded by the ependymal cells. The nonnervous, ciliated, ependymal neural tube of the ascidian larva with its two associated nerve tracts survives as a primitive early condition that could result from a ciliated band transformation. Tissues in the distal-most part of the ascidian larval tail have cell lineage origins that indicate an evolutionary history different from those in the proximal majority of the tail. The ependymal cells in this presumed later addition to the tail are not ciliated, although all of the others in the caudal ependymal tube appear to be.     

The peripheral nervous system supports blood cell homing and survival in the Drosophila larva

Interactions of hematopoietic cells with their microenvironment control blood cell colonization, homing and hematopoiesis. Here, we introduce larval hematopoiesis as the first Drosophila model for hematopoietic colonization and the role of the peripheral nervous system (PNS) as a microenvironment in hematopoiesis. The Drosophila larval hematopoietic system is founded by differentiated hemocytes of the embryo, which colonize segmentally repeated epidermal-muscular pockets and proliferate in these locations. Importantly, we show that these resident hemocytes tightly colocalize with peripheral neurons and we demonstrate that larval hemocytes depend on the PNS as an attractive and trophic microenvironment. atonal (ato) mutant or genetically ablated larvae, which are deficient for subsets of peripheral neurons, show a progressive apoptotic decline in hemocytes and an incomplete resident hemocyte pattern, whereas supernumerary peripheral neurons induced by ectopic expression of the proneural gene scute (sc) misdirect hemocytes to these ectopic locations. This PNS-hematopoietic connection in Drosophila parallels the emerging role of the PNS in hematopoiesis and immune functions in vertebrates, and provides the basis for the systematic genetic dissection of the PNS-hematopoietic axis in the future.     

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.     

Peripheral sensory neurons differentiate from neural precursors derived from human embryonic stem cells

Abstract Neural precursors have been derived from human embryonic stem cells (hESC) using the bone morphogenetic protein antagonist noggin. These neural precursors can be further differentiated to produce neural cells that express central nervous system (CNS) markers. We have recently shown that naïve hESC can be directed to differentiate into peripheral sensory (PS) neuron-like cells and putative neural crest precursors by co-culturing with PA6 stromal cells. In the present study, we examine whether hESC-derived neural precursors (NPC) can differentiate into the peripheral nervous system, as well as CNS cells. As little as 1 week after co-culture with PA6 cells, cells with the molecular characteristics of PS neurons and neural crest are observed in the cultures. With increased time in culture, more PS-like neurons appear, in parallel with a reduction in the neural crest-like cells. These results provide the first evidence that neural precursors derived from hESC have the potential to develop into PS neurons-like as well as CNS-like neuronal cells. About 10% of the cells in NPC-PA6 co-cultures express PS neuron markers after 3 weeks, compared with <1% of hESC cultured on PA6. This enrichment for peripheral neurons makes this an attractive system for generation of peripheral neurons for pathophysiology study and drug development for diseases of the peripheral nervous system such as Familial Dysautonomia and varicella virus infection.     

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.     

Identification of neuron-specific promoters in Ciona intestinalis

We isolated 5' flanking regions of four genes, Ci-Galphai1, Ci-arr, Ci-vAChTP, and Ci-vGAT, each of which is expressed in distinct sets of neurons in the central nervous system of the ascidian Ciona intestinalis, and we examined their function by introducing green fluorescent protein (GFP)-fusion constructs into Ciona embryos. The reporter gene driven by the 5' flanking region of Ci-Galphai1, Ci-arr, and Ci-vAChTP recapitulated the endogenous gene expression patterns, while that of Ci-vGAT can drive GFP expression in particular subsets of neurons expressing the endogenous gene. Deletion analysis revealed that the Ci-Galphai1 promoter consists of multiple regulatory modules controlling the expression in different types of cells. The GFP fluorescence enabled visualization of cell bodies and axons of different sets of neurons in ascidian larvae. These promoters can be a powerful tool for studying molecular mechanisms of neuronal development as well as neuron networks and functions in ascidians.