Neural projections in planarian brain revealed by fluorescent dye tracing

The planarian brain has an inverted-U shaped structure with functional regionalization. To investigate how each region in the brain connects to each other, we traced neural projections by microinjection of fluorescence dye tracers. We found that external light and olfactory/taste signals received in the head region are conveyed in the main lobes (sponge region) of the brain. Chemosensory neurons distributed in the lateral branches project to the peripheral region of the sponge and visual neurons project to the medial region of the sponge. Parts of the sensory neurons project directly to the corresponding sensory neurons on the opposite side of the brain. However, all of the dye labeled brain neurons in the left and right lobes connect to each other via commissural neurons in the central region of the sponge. In addition to these observations, we detected regional differences in the planarian visual neurons. Posterior visual neurons have ipsilateral projection, but anterior visual neurons project to the contralateral side of the brain. A pair of longitudinal ventral nerve cords (VNC) connect to the brain on the ventral side, suggesting that they transmit signals which are integrated and processed in the brain. We also detected the direct connection of neurons in the brain and those of the pharynx, even though most pharynx neurons connect to VNC neurons. Here, we report for the first time on neural connections in the planarian central nervous system after overcoming technical difficulties specific to flatworms.     

Dissecting planarian central nervous system regeneration by the expression of neural-specific genes

The planarian central nervous system (CNS) can be used as a model for studying neural regeneration in higher organisms. Despite its simple structure, recent studies have shown that the planarian CNS can be divided into several molecular and functional domains defined by the expression of different neural genes. Remarkably, a whole animal, including the molecularly complex CNS, can regenerate from a small piece of the planarian body. In this study, a collection of neural markers has been used to characterize at the molecular level how the planarian CNS is rebuilt. Planarian CNS is composed of an anterior brain and a pair of ventral nerve cords that are distinct and overlapping structures in the head region. During regeneration, 12 neural markers have been classified as early, mid-regeneration and late expression genes depending on when they are upregulated in the regenerative blastema. Interestingly, the results from this study show that the comparison of the expression patterns of different neural genes supports the view that at day one of regeneration, the new brain appears within the blastema, whereas the pre-existing ventral nerve cords remain in the old tissues. Three stages in planarian CNS regeneration are suggested.     

The expression of neural-specific genes reveals the structural and molecular complexity of the planarian central nervous system

Planarians are attractive animals in which various questions related to the central nervous system (CNS) can be addressed, such as its origin and evolution, its degree of functional conservation among different organisms, and the plasticity and regenerative capabilities of neural cells and networks. However, it is first necessary to characterize at the gene expression level how this CNS is organized in intact animals. Previous studies have shown that the planarian brain can be divided into at least three distinct domains based on the expression of otd/Otx-related genes. In order to further characterize the planarian brain, we have recently isolated a large number of planarian neural-specific genes through DNA microarrays and ESTs projects. Here, we describe new molecular domains within the brain of intact planarians by the expression of 16 planarian neural-specific genes, including the putative homologues of protein tyrosine phosphatase receptor, synaptotagmin VII, slit, G protein and glutamate and acetylcholine receptors, by in situ hybridization in both whole-mount and transverse sections. Our results indicate that planarian otd/Otx-positive domains can be further subdivided into distinct molecular regions according to the expression of different neural genes. We found differences at the gene expression level between the dorsal and ventral sides of the brain, along its antero-posterior axis and also between the proximal and distal parts of the brain lateral branches. This high level of regionalization in the planarian brain contrasts with its apparent simplicity at the morphological level.     

Gtwnt-5 a member of the wnt family expressed in a subpopulation of the nervous system of the planarian Girardia tigrina

Wnt proteins are a family of highly conserved secreted glycoproteins that regulate cell-to-cell interactions during embryogenesis. They act as signaling molecules and take part in many crucial decisions throughout the development of organisms ranging from Hydra to human. We have isolated and characterized the expression of a member of the Wnt family, Gtwnt-5 gene in the planarian Girardia tigrina. Planarians are free-living members (Class Turbellaria) of the Phylum Platyhelminthes. They are best known for their high regenerative capabilities. These organisms have an apparently simple central nervous system (CNS) from a morphological perspective, with cephalic ganglia in the dorsal anterior region and two ventral main nerve cords along the body. However, a large number of planarian neural genes have recently been identified and therefore it is possible to define different molecular and functional domains in the planarian brain. The present study shows expression of Gtwnt-5 in a subpopulation of the whole CNS of intact organisms, being activated during regeneration. Gtwnt-5 reveals a differential spatial pattern: the expression is preferentially found in the most external region of the CNS. In addition, a kind of iterative pattern has been observed at the ganglia level, suggesting that the planarian brain might not be a continuous structure but compartmented or regionalized. Gtwnt-5 signal is also detected at the sensors of the worm: at the auricle level and all around the cephalic periphery. All these data provide us with a new neural marker for the planarian brain, and can be used to follow regeneration of the CNS.     

Are protochordates chordates?

This paper challenges the widely accepted view that protochordates (lancelets and tunicates) should be included together with vertebrates within the monophyletic assemblage of the chordates since they share a few distinguishing characters, such as a dorsally located notochord and central nervous system (CNS). The homology of these axial structures is not supported convincingly by morphology and molecular biology. Besides, for notochord and CNS to be dorsal, the embryos of protochordates, unlike those of vertebrates, should be orientated with the blastopore coincident with the dorsal side. This embryonic orientation is never reported in other bilaterians and is inconsistent with the genetic control of the body axes. Alternatively, protochordates could be orientated just as the vertebrates according to the regulation of axial patterning. In this case, the notochord and CNS appear to be located on the ventral side. As suggested by molecular and structural data, they may correspond to the stomodaeal/ventral midline cells and CNS of gastroneuralians. This conclusion may have far-reaching implications concerning the origin of the vertebrates and the evolution of nervous systems and neural crest/placodes.  © 2006 The Linnean Society of London, Biological Journal of the Linnean Society , 2006, 87 , 261–284.     

Dorsal–ventral patterning of the neural tube: A tale of three signals

Development of the vertebrate nervous system begins with the acquisition of neural identity from the midline dorsal‐ectodermal cells of the gastrulating embryos. The subsequent progressive specification of the neural plate along its anterior–posterior and dorsal–ventral (DV) axes allows the generation of the tremendous variety of neuronal and glial cells that compose the vertebrate central nervous system (CNS). Studies on the development of the spinal cord, the anatomically simplest part of the CNS, have generated most of our current knowledge on the signaling events and the genetic networks that orchestrate the DV patterning of the neural plate. In this review, we discuss the recent advances in our understanding of these events and highlight unresolved questions. We focused our attention on the activity and the integration of the three main instructive cues: Sonic hedgehog, the Wnts and the Bone Morphogenetic Proteins, giving particular attention to the less well understood dorsal signaling events. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2012     

PAC1 receptor localization in a model nervous system: light and electron microscopic immunocytochemistry on the earthworm ventral nerve cord ganglia

The presence and pattern of pituitary adenylate cyclase activating polypeptide (PACAP) type I (PAC1) receptors were identified by means of pre- and post-embedding immunocytochemical methods in the ventral nerve cord ganglia (VNC) of the earthworm Eisenia fetida. Light and electron microscopic observations revealed the exact anatomical positions of labeled structures suggesting that PACAP mediates the activity of some interneurons, a few small motoneurons and certain sensory fibers that are located in ventrolateral, ventromedial and intermediomedial sensory longitudinal axon bundles of the VNC ganglia. No labeling was located on large interneuronal systems such as dorsal medial and lateral giant axon systems and ventral giant axons. At the ultrastructural level labeling was mainly restricted to endo- and plasma membranes showing characteristic unequal distribution in various neuron parts. An increasing abundance of PAC1 receptors located on both rough endoplasmic reticulum and plasma membranes was seen from perikarya to neural processes, indicating that intracellular membrane traffic might play a crucial role in the transportation of PAC1 receptors. High number of PAC1 receptors was found in both pre- and postsynaptic membranes in addition to extrasynaptic sites suggesting that PACAP acts as neurotransmitter and neuromodulator in the earthworm nervous system.     

The BMP pathway is essential for re-specification and maintenance of the dorsoventral axis in regenerating and intact planarians

The bone morphogenetic protein (BMP) pathway has been shown to play an important role in the establishment of the dorsoventral axis during development in both vertebrate and invertebrate species. In an attempt to unravel the role of BMPs in pattern formation during planarian regeneration, we studied this signaling pathway in Schmidtea mediterranea. Here, we functionally characterize planarian homologues of two key elements of the pathway: Smed-BMP and Smed-Smad1. Whole-mount in situ hybridization showed that Smed-BMP is expressed at the planarian dorsal midline, suggesting a role in dorsoventral patterning, while Smed-Smad1 is widely expressed throughout the mesenchyme and in the central nervous system. RNA interference (RNAi) knockdowns of Smed-BMP or Smed-Smad1 led to the disappearance of dorsal markers along with the ectopic expression of ventral markers on the dorsal side of the treated animals. In almost all cases, a duplicated central nervous system differentiated dorsally after Smed-BMP or Smed-Smad1 RNAi. These defects were observed not only during regeneration but also in intact non-regenerating animals. Our results suggest that the BMP signaling pathway is conserved in planarians and that it plays a key role in the regeneration and maintenance of the dorsoventral axis.     

Prospective Neural Areas and their Morphogenetic Movements during Neural Plate Formation in the Xenopus Embryo. II. Disposition of Transplanted Ectoderm Pieces of X. borealis Animal Cap in Prospective Neural Areas of Albino X. laevis gastrulae.

Small pieces of the animal cap of X. borealis gastrulae were transplanted into various regions of the noninvoluting marginal zone of albino X. laevis gastrulae, and the distribution of the donor cells was analyzed by quinacrine fluorescence staining.
The present study indicated that the prospective central nervous system (CNS) lies as a belt-shaped area in the noninvoluting marginal zone of early gastrulae. This belt-shaped prospective neural area extends as far as 0.7 mm (115° to the vegetal pole) above the blastopore in the dorsal midline and 1.3 mm lateral (130° to the dorsal midline) to the dorsal midline. The ectoderm of the dorsal region extends in the animal-vegetal direction and forms the ventral side of the CNS. The dorsalateral and lateral regions converge toward the dorsal midline and extended in the animal-vegetal direction. The former constitutes the lateral side of the anterior CNS, and the latter the dorso-lateral side of the posterior CNS.
The outer layer of ectoderm which was transplanted onto the inner layer of the host gastrula differentiated into neural tissues.
The prospective areas of the CNS and their morphogenetic movement during Xenopus embryogenesis are also discussed with regard to neural induction.     

Morphogenesis defects are associated with abnormal nervous system regeneration following roboA RNAi in planarians

The process by which the proper pattern is restored to newly formed tissues during metazoan regeneration remains an open question. Here, we provide evidence that the nervous system plays a role in regulating morphogenesis during anterior regeneration in the planarian Schmidtea mediterranea. RNA interference (RNAi) knockdown of a planarian ortholog of the axon-guidance receptor roundabout (robo) leads to unexpected phenotypes during anterior regeneration, including the development of a supernumerary pharynx (the feeding organ of the animal) and the production of ectopic, dorsal outgrowths with cephalic identity. We show that Smed-roboA RNAi knockdown disrupts nervous system structure during cephalic regeneration: the newly regenerated brain and ventral nerve cords do not re-establish proper connections. These neural defects precede, and are correlated with, the development of ectopic structures. We propose that, in the absence of proper connectivity between the cephalic ganglia and the ventral nerve cords, neurally derived signals promote the differentiation of pharyngeal and cephalic structures. Together with previous studies on regeneration in annelids and amphibians, these results suggest a conserved role of the nervous system in pattern formation during blastema-based regeneration.     

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.     

Characterization of tyramine beta-hydroxylase in planarian Dugesia japonica: cloning and expression

The planarian Dugesia japonica has a relatively well-organized central nervous system (CNS) consisting of a brain and ventral nerve cords (VNCs), and can completely regenerate it CNS utilizing pluripotent stem cells present in the mesenchymal space. This remarkable capacity has begun to be exploited for research on neural regeneration. Recently, several kinds of molecular markers for labeling of neural subtypes have been reported in planarians. These molecular markers are useful for visualizing the distinct neural populations in planarians. In this study, we isolated a cDNA encoding tyramine beta-hydroxylase (TBH), an octopamine (OA) biosynthetic enzyme, by degenerate PCR in the planarian D. japonica, and named it DjTBH (D. japonica tyramine beta-hydroxylase). In order to examine whether DjTBH contributes to OA biosynthesis, we measured the OA content in DjTBH-knockdown planarians created by RNA interference. In addition, to examine the specificity of DjTBH for OA biosynthesis, we measured not only OA content but also noradrenaline (NA) content, because NA is synthesized by a pathway similar to that for OA. According to high-performance liquid chromatography analysis, the amount of OA, but not NA, was significantly decreased in DjTBH-knockdown planarians. In addition, we produced anti-DjTBH antibody to visualize the octopaminergic neural network. As shown by immunofluorescence analysis using anti-DjTBH antibody, DjTBH-immunopositive neurons were mainly distributed in the head region, and elongated their dendrites and/or axons along the VNCs. In order to visualize octopaminergic and dopaminergic nervous systems (phenolamine/catecholamine nervous system) in the planarian CNS, double-immunofluorescence analysis was carried out using both anti-DjTBH antibody and anti-DjTH (a planarian tyrosine hydroxylase) antibody. DjTBH-immunopositive neurons and DjTH-immunopositive neurons mainly formed distinct neural networks in the head region. Here, we demonstrated that DjTBH clearly contributes to OA biosynthesis, and DjTBH antibody is a useful tool for detecting octopaminergic neurons in planarians.     

The central nervous system, its cellular organisation and development, in the tadpole larva of the ascidian Ciona intestinalis

From its numerical composition, the central nervous system (CNS) of the ascidian larva is one of the simplest known nervous systems having a chordate plan. Fewer than 350 cells together constitute a caudal nerve cord, an interposed visceral ganglion containing motor circuits for swimming and, rostrally, an expanded sensory vesicle containing major sensory and interneuron regions of the CNS. Some cells are ependymal, with ciliated surfaces lining the neural canal, while others are clearly either sensory receptors or motoneurons, but most are distinguishable only on cytological grounds. Although reassignments between categories are still being made, there is evidence for determinancy of total cell number. We have made three-dimensional cell maps either from serial semithin sections, or from confocal image stacks of whole-mounted embryos and larvae stained with nuclear markers. Comparisons between the maps of neural tubes in embryos of successive ages, that is, between cells in one map and their progeny in older maps, enable us to follow the line of mitotic descent through successive maps, at least for the caudal neural tube. Details are clear for the lateral cell rows in the neural tube, at least until the latter contains approximately 320 cells, and somewhat for the dorsal cell row, but the ventral row is more complex. In the hatched larva, serial-EM reconstructions of the visceral ganglion reveal two ventrolateral fibre bundles at the caudalmost end, each of 10-12 axons. These tracts include at least five pairs of presumed motor axons running into the caudal nerve cord. Two pairs of axons decussate. Complementing this vertebrate feature in the CNS of the larval form of Ciona, we confirm that synapses form upon the somata and dendrites of its neurons, and that its motor tracts are ventral.     

Regeneration and maintenance of the planarian midline is regulated by a slit orthologue

Several families of evolutionarily conserved axon guidance cues orchestrate the precise wiring of the nervous system during embryonic development. The remarkable plasticity of freshwater planarians provides the opportunity to study these molecules in the context of neural regeneration and maintenance. Here we characterize a homologue of the Slit family of guidance cues from the planarian Schmidtea mediterranea. Smed-slit is expressed along the planarian midline, in both dorsal and ventral domains. RNA interference (RNAi) targeting Smed-slit results in the collapse of many newly regenerated tissues at the midline; these include the cephalic ganglia, ventral nerve cords, photoreceptors, and the posterior digestive system. Surprisingly, Smed-slit RNAi knockdown animals also develop morphologically distinguishable, ectopic neural structures near the midline in uninjured regions of intact and regenerating planarians. These results suggest that Smed-slit acts not only as a repulsive cue required for proper midline formation during regeneration but that it may also act to regulate the behavior of neural precursors at the midline in intact planarians.     

Origin of the chordate central nervous system – and the origin of chordates

 Contrary to traditional views, molecular evidence indicates that the protostomian ventral nerve cord plus apical brain is homologous with the vertebrates’ dorsal spinal cord plus brain. The origin of the protostomian central nervous system from a larval apical organ plus longitudinal areas along the fused blastopore lips has been documented in many species. The origin of the chordate central nervous system is more enigmatic. About a century ago, Garstang proposed that the ciliary band of a dipleurula-type larva resembling an echinoderm larva should have moved dorsally and fused to form the neural tube of the ancestral chordate. This idea is in contrast to a number of morphological observations, and it is here proposed that the neural tube evolved through lateral fusion of a ventral, postoral loop of the ciliary band in a dipleurula larva; the stomodaeum should move from the ventral side via the anterior end to the dorsal side, which faces the substratum in cephalo- chordates and vertebrates. This is in accordance with the embryological observations and with the molecular data on the dorsoventral orientation. The molecular observations further indicate that the anterior part of the insect brain is homologous with the anterior parts of the vertebrate brain. This leads to the hypothesis that the two organs evolved from the same area in the latest common bilaterian ancestor, just anterior to the blastopore, with the protostome brain developing from the anterior rim of the blastopore (i.e. in front of the protostome mouth) and the chordate brain from an area in front of the blastopore, but behind the mouth (i.e. behind the deuterostome mouth). Received: 28 August 1998 / Accepted: 14 November 1998     

Search for the evolutionary origin of a brain: planarian brain characterized by microarray

The origin of the brain remains a challenging problem in evolutionary studies. To understand when and how the structural brain emerged, we analyzed the central nervous system (CNS) of a lower invertebrate, planarian. We conducted a large-scale screening of the head part-specific genes in the planarian by constructing a cDNA microarray. Competitive hybridization of cDNAs between a head portion and the other body portion of planarians revealed 205 genes with head part-specific spikes, including essential genes in the vertebrate nervous system. The expression patterns of the top 30 genes showing the strongest spikes implied that the planarian brain has undergone functional regionalization. We demonstrate the complex cytoarchitecture of the planarian brain, despite its simple superficiality of the morphology.     

Distribution Pattern of γ-amino Butyric Acid Immunoreactive Neural Structures in the Central and Peripheral Nervous System of the Tubificid Worm, Limnodrilus hoffmeisteri

By means of whole-mount immunohistochemistry, putative inhibitory (GABAergic) neural structures were identified in the central and peripheral nervous system of the tubificid worm, Limnodrilus hoffmeisteri. In the supraoesophageal ganglion (brain) only few strongly labelled cells were observed. However, in its commissural part a high number of stained nerve fibres, arising mainly from the ventral nerve cord and prostomium, occurred. Except for the suboesophageal ganglion the arrangement of γ-amino butyric acid-immunoreactive (GABA-IR) structures proved to be identical in each VNC ganglion. Behind the first segmental nerves three pairs of heavily stained neurones were located. Their processes (both ipsi- and contralateral) form four bundles of fine-fibred polysegmental interneuronal tracts that run close to the dorsal giant axons from the terminal ganglion to the suboesophageal one without interruption. A few small motoneurons and a pair of large ones with contralateral processes were also identified. A bipolar (presumably sensory) neuron was located at the root of each second segmental nerve. GABA-IR neurons were also found in the stomatogastric ganglia and pharyngeal wall; however, the latter structure had a well-developed fibre network, as well. Present results suggest that GABA acts as a common neurotransmitter in sensory, interneuronal and motor system of L. hoffmeisteri. The possible functional role of the identified GABA-IR neural structures in locomotion, escape and withdrawal reflexes in tubificid worms is discussed.     

Neuroblast formation and patterning during early brain development in Drosophila

The Drosophila embryo provides a useful model system to study the mechanisms that lead to pattern and cell diversity in the central nervous system (CNS). The Drosophila CNS, which encompasses the brain and the ventral nerve cord, develops from a bilaterally symmetrical neuroectoderm, which gives rise to neural stem cells, called neuroblasts. The structure of the embryonic ventral nerve cord is relatively simple, consisting of a sequence of repeated segmental units (neuromeres), and the mechanisms controlling the formation and specification of the neuroblasts that form these neuromeres are quite well understood. Owing to the much higher complexity and hidden segmental organization of the brain, our understanding of its development is still rudimentary. Recent investigations on the expression and function of proneural genes, segmentation genes, dorsoventral-patterning genes and a number of other genes have provided new insight into the principles of neuroblast formation and patterning during embryonic development of the fly brain. Comparisons with the same processes in the trunk help us to understand what makes the brain different from the ventral nerve cord. Several parallels in early brain patterning between the fly and the vertebrate systems have become evident.     

Chordin is required for the Spemann organizer transplantation phenomenon in Xenopus embryos

We analyzed the Chordin requirement in Xenopus development. Targeting of both chordin Xenopus laevis pseudoalleles with morpholino antisense oligomers (Chd-MO) markedly decreased Chordin production. Embryos developed with moderately reduced dorsoanterior structures and expanded ventroposterior tissues, phenocopying the zebrafish chordino mutant. A strong requirement for Chordin in dorsal development was revealed by experimental manipulations. First, dorsalization by lithium chloride treatment was completely blocked by Chd-MO. Second, Chd-MO inhibited elongation and muscle differentiation in Activin-treated animal caps. Third, Chd-MO completely blocked the induction of the central nervous system (CNS), somites, and notochord by organizer tissue transplanted to the ventral side of host embryos. Unexpectedly, transplantations into the dorsal side revealed a cell-autonomous requirement of Chordin for neural plate differentiation.     

Bone morphogenetic protein is required for dorso-ventral patterning in the planarian Dugesia japonica

In order to clarify the function of the Djbmp (Dugesia japonica bone morphogenetic protein) gene in planarian body patterning, we carried out knockdown of this gene by RNA interference. When the planarians were treated with double-stranded RNA of Djbmp, a bulge formed on the dorsal side, with a dent in the middle of the bulge, and the body surface inside the dent was smoothened and less pigmented. In situ hybridization of the DjIFb gene, which is expressed in the body margin, revealed that the additional body margin was formed ectopically at the region surrounding the dent. The Djbmp-knockdown planarians often had a pair of incomplete nerve cords in the dorsal side, in addition to the original pair of ventral nerve cords. Taken together, we concluded that the Djbmp-knockdown induced formation of an ectopic ventral side, suggesting that Djbmp is required for the dorso-ventral body patterning in the planarian.