Admp regulates tail bending by controlling ventral epidermal cell polarity via phosphorylated myosin localization in Ciona.

Ventral tail bending, which is transient but pronounced, is found in many chordate embryos and constitutes an interesting model of how tissue interactions control embryo shape. Here, we identify one key upstream regulator of ventral tail bending in embryos of the ascidian Ciona. We show that during the early tailbud stages, ventral epidermal cells exhibit a boat-shaped morphology (boat cell) with a narrow apical surface where phosphorylated myosin light chain (pMLC) accumulates. We further show that interfering with the function of the BMP ligand Admp led to pMLC localizing to the basal instead of the apical side of ventral epidermal cells and a reduced number of boat cells. Finally, we show that cutting ventral epidermal midline cells at their apex using an ultraviolet laser relaxed ventral tail bending. Based on these results, we propose a previously unreported function for Admp in localizing pMLC to the apical side of ventral epidermal cells, which causes the tail to bend ventrally by resisting antero-posterior notochord extension at the ventral side of the tail.

Calcium-induced upregulation of energy metabolism heats neurons during neural activity.

Cellular temperature affects every biochemical reaction, underscoring its critical role in cellular functions. In neurons, temperature not only modulates neurotransmission but is also a key determinant of neurodegenerative diseases. Considering that the brain consumes a disproportionately high amount of energy relative to its weight, neural circuits likely generate a lot of heat, which can increase cytosolic temperature. However, the changes in temperature within neurons and the mechanisms of heat generation during neural excitation remain unclear. In this study, we achieved simultaneous imaging of Ca2+ and temperature using the genetically encoded indicators, B-GECO and B-gTEMP. We then compared the spatiotemporal distributions of Ca2+ responses and temperature. Following neural excitation induced by veratridine, an activator of the voltage-gated Na+ channel, we observed an approximately 2 °C increase in cytosolic temperature occurring 30 s after the Ca2+ response. The temperature elevation was observed in the non-nuclear region, while Ca2+ increased throughout the cell body. Moreover, this temperature increase was suppressed under Ca2+-free conditions and by inhibitors of ATP synthesis. These results indicate that Ca2+-induced upregulation of energy metabolism serves as the heat source during neural excitation.

Distribution changes of non-self-test cells and self-tunic cells surrounding the outer body during Ciona metamorphosis.

BACKGROUND: Ascidians significantly change their body structure through metamorphosis, but the spatio-temporal cell dynamics in the early metamorphosis stage has not been clarified. A natural Ciona embryo is surrounded by maternally derived non-self-test cells before metamorphosis. However, after metamorphosis, the juvenile is surrounded by self-tunic cells derived from mesenchymal cell lineages. Both test cells and tunic cells are thought to be changed their distributions during metamorphosis, but the precise timing is unknown. RESULTS: Using a metamorphosis induction by mechanical stimulation, we investigated the dynamics of mesenchymal cells during metamorphosis in a precise time course. After the stimulation, two-round Ca2+ transients were observed. Migrating mesenchymal cells came out through the epidermis within 10 min after the second phase. We named this event "cell extravasation." The cell extravasation occurred at the same time as the backward movement of posterior trunk epidermal cells. Timelapse imaging of transgenic-line larva revealed that non-self-test cells and self-tunic cells temporarily coexist outside the body until the test cells are eliminated. At the juvenile stage, only extravasated self-tunic cells remained outside the body. CONCLUSIONS: We found that mesenchymal cells extravasated following two-round Ca2+ transients, and distributions of test cells and tunic cells changed in the outer body after tail regression.

Different strategies for tissue scaling in dwarf tailbud embryos revealed by single-cell analysis.

The tailbud stage is part of the organogenesis period-an evolutionarily conserved developmental period among chordates that is essential for determining the characteristics of the chordate body plan. When the volume of the egg is artificially decreased by cutting, ascidians produce a normal-looking but miniature (dwarf) tailbud embryo. Although cell lineages during ascidian embryogenesis are invariant, the number of cell divisions in the dwarf embryo is altered by a different mechanism in each tissue (Yamada and Nishida, 1999). Here, to elucidate the size-regulation strategies of the Ciona robusta dwarf tailbud embryo, we compared anatomical structure, developmental speed, and cell number/volume in each tissue between dwarf and wild type (WT) embryos. To do this, we constructed a 3D virtual mid-tailbud embryo (Nakamura et al., 2012). We could make a Ciona dwarf tailbud embryo from eggs with a diameter over 108 â€‹µm (correspond to â€‹> â€‹40% of the wild type egg volume). The timings of cleavage (~St. 12) and subsequent morphogenesis were nearly the same but blastomeres of animal hemisphere slightly delayed the timing of mitosis in the early cleavage period. Intriguingly, the tissue-to-tissue volume ratios of dwarf tailbud embryos were similar to those of wild type embryos suggesting that the ratio of tissue volumes is essential for maintaining the proper shape of the tailbud embryo. The number of cells in the epidermis, nervous system, and mesenchyme was significantly reduced in the dwarf embryos whereas the cell volume distribution of these tissues was similar in the dwarf and wild type. In contrast, the number of cells in the notochord, muscle, heart, and endoderm were maintained in the dwarf embryos; cell volumes were significantly reduced. Neither parameter changed in germline precursors. These results indicate that each tissue uses different scaling strategies to coordinate cell number and cell volume in accordance with the embryo size.

High responsiveness of auditory neurons to specific combination of acoustic features in female songbirds.

Zebra finch (Taeniopygia guttata) is a songbird species in which males sing their unique songs to attract females who then select their preferred male. Acoustic features in the songs of individual males are important features for female auditory perception. While the male of this species is a classic model of vocal production, it has been little known about auditory processing in female. In the higher auditory brain regions, the caudomedial mesopallium (CMM) and nidopallium (NCM) contribute to female's sound recognition, we, therefore, extracted acoustic features that induce neural activities with high detection power on both regions in female finches. A multiple linear regression analysis revealed that neurons were sensitive to mean frequency and Wiener entropy. In addition, we performed an experiment with modified artificial songs and harmonic songs to directly investigate neural responsiveness for deriving further evidence for the contribution of these two acoustic features. Finally, we illustrated a specific ratio combining these two acoustic features that showed highest sensitivity to neural responsiveness, and we found that properties of sensitivity are different between CMM and NCM. Our results indicate that the mixture of the two acoustic features with the specific ratio is important in the higher auditory regions of female songbirds, and these two regions have differences in encoding for sensitivity to these acoustic features.

Mechanical stimulus-evoked signal transduction between keratinocytes and sensory neurons via extracellular ATP.

The skin is exposed to various external stimuli. Keratinocytes, which are the main cell type in the epidermis, interact with peripheral sensory neurons and modulate neuronal activity. Recent studies have revealed that keratinocytes play crucial roles in nociception, and that ATP is one of the main mediators of signal transduction from keratinocytes to sensory neurons. However, no quantitative cellular level analyses of ATP-mediated information flow from keratinocytes to sensory dorsal root ganglion (DRG) neurons have been conducted. In this study, we performed simultaneous imaging of cell surface ATP and intracellular Ca2+ signals using both iATPSnFR, a genetically encoded ATP probe localized to the outside of the cell membrane, and the Ca2+ probe, Fura-red. Upon mechanical stimulation of the keratinocyte with a glass needle, an increase in Ca2+ and ATP release were observed around the stimulated area, and these phenomena were positively correlated. In cultured DRG neurons and keratinocytes neighboring the stimulated keratinocyte, increased intracellular Ca2+ concentration and levels of cell surface ATP on the side closer to the stimulated cell were detected. The ratio of Ca2+ response to input ATP signal was significantly larger in DRG neurons than in keratinocytes. We found that DRG neurons were more sensitive to ATP than keratinocytes, and therefore, only DRG neurons responded to ATP at 1 µM or lower concentrations when in co-culture with keratinocytes. Moreover, signals caused by moderate mechanical stimulation of keratinocytes were transmitted predominantly to DRG neurons. These findings would be important in the further determination of the detailed mechanism of nociception in the epidermis.

Two-Round Ca2+ transient in papillae by mechanical stimulation induces metamorphosis in the ascidian Ciona intestinalis type A.

Marine invertebrate larvae are known to begin metamorphosis in response to environmentally derived cues. However, little is known about the relationships between the perception of such cues and internal signalling for metamorphosis. To elucidate the mechanism underlying the initiation of metamorphosis in the ascidian, Ciona intestinalis type A (Ciona robusta), we artificially induced ascidian metamorphosis and investigated Ca2+ dynamics from pre- to post-metamorphosis. Ca2+ transients were observed and consisted of two temporally distinct phases with different durations before tail regression which is the early event of metamorphosis. In the first phase, Phase I, the Ca2+ transient in the papillae (adhesive organ of the anterior trunk) was coupled with the Ca2+ transient in dorsally localized cells and endoderm cells just after mechanical stimulation. The Ca2+ transients in Phase I were also observed when applying only short stimulation. In the second phase, Phase II, the Ca2+ transient in papillae was observed again and lasted for approximately 5-11 min just after the Ca2+ transient in Phase I continued for a few minutes. The impaired papillae by Foxg-knockdown failed to induce the second Ca2+ transient in Phase II and tail regression. In Phase II, a wave-like Ca2+ propagation was also observed across the entire epidermis. Our results indicate that the papillae sense a mechanical cue and two-round Ca2+ transients in papillae transmits the internal metamorphic signals to different tissues, which subsequently induces tail regression. Our study will help elucidate the internal mechanism of metamorphosis in marine invertebrate larvae in response to environmental cues.

14-3-3εa directs the pulsatile transport of basal factors toward the apical domain for lumen growth in tubulogenesis.

The Ciona notochord has emerged as a simple and tractable in vivo model for tubulogenesis. Here, using a chemical genetics approach, we identified UTKO1 as a selective small molecule inhibitor of notochord tubulogenesis. We identified 14-3-3εa protein as a direct binding partner of UTKO1 and showed that 14-3-3εa knockdown leads to failure of notochord tubulogenesis. We found that UTKO1 prevents 14-3-3εa from interacting with ezrin/radixin/moesin (ERM), which is required for notochord tubulogenesis, suggesting that interactions between 14-3-3εa and ERM play a key role in regulating the early steps of tubulogenesis. Using live imaging, we found that, as lumens begin to open between neighboring cells, 14-3-3εa and ERM are highly colocalized at the basal cortex where they undergo cycles of accumulation and disappearance. Interestingly, the disappearance of 14-3-3εa and ERM during each cycle is tightly correlated with a transient flow of 14-3-3εa, ERM, myosin II, and other cytoplasmic elements from the basal surface toward the lumen-facing apical domain, which is often accompanied by visible changes in lumen architecture. Both pulsatile flow and lumen formation are abolished in larvae treated with UTKO1, in larvae depleted of either 14-3-3εa or ERM, or in larvae expressing a truncated form of 14-3-3εa that lacks the ability to interact with ERM. These results suggest that 14-3-3εa and ERM interact at the basal cortex to direct pulsatile basal accumulation and basal-apical transport of factors that are essential for lumen formation. We propose that similar mechanisms may underlie or may contribute to lumen formation in tubulogenesis in other systems.

Neural properties of fundamental function encoding of sound selectivity in the female avian auditory cortex.

Zebra finches (Taeniopygia guttata) use their voices for communication. Song structures in the songs of individual males are important for sound recognition in females. The caudomedial mesopallium (CMM) and nidopallium (NCM) are known to be essential higher auditory regions for sound recognition. These two regions have also been discussed with respect to their fundamental functions and song selectivity. To clarify their functions and selectivity, we investigated latencies and spiking patterns and also developed a novel correlation analysis to evaluate the relationship between neural activity and the characteristics of acoustic factors. We found that the latencies and spiking patterns in response to song stimuli differed between the CMM and NCM. In addition, our correlation analysis revealed that amplitude and frequency structures were important temporal acoustic factors for both regions. Although the CMM and NCM have different fundamental functions, they share similar encoding systems for acoustic factors.

Cellular thermogenesis compensates environmental temperature fluctuations for maintaining intracellular temperature.

Temperature governs states and dynamics of all biological molecules, and several cellular processes are often heat sources and/or sinks. Technical achievement of intracellular thermometry enables us to measure intracellular temperature, and it can offer novel perspectives in biology and medicine. However, little is known that changes of intracellular temperature throughout the cell-cycle and the manner of which cells regulates their thermogenesis in response to fluctuation of the environmental temperature. Here, cell-cycle-dependent changes of intracellular temperature were reconstructed from the snapshots of cell population at single-cell resolution using ergodic analysis for asynchronously cultured HeLa cells expressing a genetically encoded thermometry. Intracellular temperature is highest at G1 phase, and it gradually decreases along cell-cycle progression and increases abruptly during mitosis. Cells easily heated up are harder to cool down and vice versa, especially at G1/S phases. Together, intracellular thermogenesis depends on cell-cycle phases and it maintains intracellular temperature through compensating environmental temperature fluctuations.

Developmental Roles and Evolutionary Significance of AMPA-Type Glutamate Receptors.

Organogenesis and metamorphosis require the intricate orchestration of multiple types of cellular interactions and signaling pathways. Glutamate (Glu) is an excitatory extracellular signaling molecule in the nervous system, while Ca2+ is a major intracellular signaling molecule. The first Glu receptors to be cloned are Ca2+ -permeable receptors in mammalian brains. Although recent studies have focused on Glu signaling in synaptic mechanisms of the mammalian central nervous system, it is unclear how this signaling functions in development. Our recent article demonstrated that Ca2+ -permeable AMPA-type Glu receptors (GluAs) are essential for formation of a photosensitive organ, development of some neurons, and metamorphosis, including tail absorption and body axis rotation, in ascidian embryos. Based on findings in these embryos and mammalian brains, we formed several hypotheses regarding the evolution of GluAs, the non-synaptic function of Glu, the origin of GluA-positive neurons, and the neuronal network that controls metamorphosis in ascidians.

AMPA glutamate receptors are required for sensory-organ formation and morphogenesis in the basal chordate.

AMPA-type glutamate receptors (GluAs) mediate fast excitatory transmission in the vertebrate central nervous system (CNS), and their function has been extensively studied in the mature mammalian brain. However, GluA expression begins very early in developing embryos, suggesting that they may also have unidentified developmental roles. Here, we identify developmental roles for GluAs in the ascidian Ciona intestinalis Mammals express Ca2+-permeable GluAs (Ca-P GluAs) and Ca2+-impermeable GluAs (Ca-I GluAs) by combining subunits derived from four genes. In contrast, ascidians have a single gluA gene. Taking advantage of the simple genomic GluA organization in ascidians, we knocked down (KD) GluAs in Ciona and observed severe impairments in formation of the ocellus, a photoreceptive organ used during the swimming stage, and in resorption of the tail and body axis rotation during metamorphosis to the adult stage. These defects could be rescued by injection of KD-resistant GluAs. GluA KD phenotypes could also be reproduced by expressing a GluA mutant that dominantly inhibits glutamate-evoked currents. These results suggest that, in addition to their role in synaptic communication in mature animals, GluAs also have critical developmental functions.

Compartmentalized cGMP Responses of Olfactory Sensory Neurons in Caenorhabditis elegans.

Cyclic guanosine monophosphate (cGMP) plays a crucial role as a second messenger in the regulation of sensory signal transduction in many organisms. In AWC olfactory sensory neurons of Caenorhabditis elegans, cGMP also has essential and distinctive functions in olfactory sensation and adaptation. According to molecular genetic studies, when nematodes are exposed to odorants, a decrease in cGMP regulates cGMP-gated channels for olfactory sensation. Conversely, for olfactory adaptation, an increase in cGMP activates protein kinase G to modulate cellular physiological functions. Although these opposing cGMP responses in single neurons may occur at the same time, it is unclear how cGMP actually behaves in AWC sensory neurons. A hypothetical explanation for opposing cGMP responses is region-specific behaviors in AWC: for odor sensation, cGMP levels in cilia could decrease, whereas odor adaptation is mediated by increased cGMP levels in soma. Therefore, we visualized intracellular cGMP in AWC with a genetically encoded cGMP indicator, cGi500, and examined spatiotemporal cGMP responses in AWC neurons. The cGMP imaging showed that, after odor exposure, cGMP levels in AWC cilia decreased transiently, whereas levels in dendrites and soma gradually increased. These region-specific responses indicated that the cGMP responses in AWC neurons are explicitly compartmentalized. In addition, we performed Ca2+ imaging to examine the relationship between cGMP and Ca2+ These results suggested that AWC sensory neurons are in fact analogous to vertebrate photoreceptor neurons.SIGNIFICANCE STATEMENT Cyclic guanosine monophosphate (cGMP) plays crucial roles in the regulation of sensory signal transduction in many animals. In AWC olfactory sensory neurons of Caenorhabditis elegans, cGMP also has essential and distinctive functions involving olfactory sensation and adaptation. Here, we visualized intracellular cGMP in AWC neurons with a genetically encoded cGMP indicator and examined how these different functions could be regulated by the same second messenger in single neurons. cGMP imaging showed that, after odor application, cGMP levels in cilia decreased transiently, whereas levels in dendrites and soma gradually increased. These region-specific responses indicated that the responses in AWC neurons are explicitly compartmentalized. In addition, by combining cGMP and Ca2+ imaging, we observed that AWC neurons are analogous to vertebrate photoreceptor neurons.

Characterization of calcium transients during early embryogenesis in ascidians Ciona robusta (Ciona intestinalis type A) and Ciona savignyi.

The calcium ion (Ca2+) is an important second messenger, and a rapid increase in Ca2+ level (Ca2+ transient) is involved in various aspects of embryogenesis. Although Ca2+ transients play an important role in early developmental stages, little is known about their dynamics throughout embryogenesis. Here, Ca2+ transients were characterized by visualizing Ca2+ dynamics in developing chordate embryos using a fluorescent protein-based Ca2+ indicator, GCaMP6s in combination with finely tuned microscopy. Ca2+ transients were detected in precursors of muscle cells in the late gastrula stage. In the neurula stage, repetitive Ca2+ transients were observed in left and right neurogenic cells, including visceral ganglion (VG) precursors, and the duration of Ca2+ transients was 39±4s. In the early tailbud stage, Ca2+ transients were observed in differentiating precursors of nerve cord neurons. A small population of VG precursors showed rhythmical Ca2+ transients with a duration of 22±4s, suggesting a central pattern generator (CPG) origin. At the mid tailbud stage, Ca2+transients were observed in a wide area of epidermal cells and named CTECs. The number and frequency of CTECs increased drastically in late tailbud stages, and the timing of the increase coincided with that of the relaxation of the tail bending. The experiment using Ca2+ chelator showed that the CTECs were largely depending on the extracellular Ca2+. The waveform analysis of Ca2+ transients revealed different features according to duration and frequency. The comprehensive characterization of Ca2+ transients during early ascidian embryogenesis will help our understanding of the role of Ca2+ signaling in chordate embryogenesis.

Effect of interactions among individuals on the chemotaxis behaviours of Caenorhabditis elegans.

In many species, individual social animals interact with others in their group and change their collective behaviours. For the solitary nematode Caenorhabditis elegans strain N2, previous research suggests that individuals can change the behaviour of other worms via pheromones and mechanosensory interactions. In particular, pheromones affect foraging behaviour, so that the chemotactic behaviours of individuals in a group (population) can be modulated by interactions with other individuals in the population. To investigate this, we directly compared the chemotactic behaviours of isolated (single) worms with those of individual animals within a population. We found that worms approached an odour source in a distinct manner depending on whether they were alone or in a population. Analysis of behaviours of the N2 worm and a pheromone production-defective mutant revealed that the 'pirouette' strategy was modulated by interaction of the worms via pheromones. Thus, pheromones play an important role in the characteristic collective behaviours seen in the population condition.

Altered expression of Mg(2+) transport proteins during Parkinson's disease-like dopaminergic cell degeneration in PC12 cells.

Mg(2+) is an essential cation to maintain cellular functions, and intracellular Mg(2+) concentration ([Mg(2+)]i) is regulated by Mg(2+) channels and transporters. In our previous study, we demonstrated that MPP(+) elicits Mg(2+) influx across the cell membrane and Mg(2+) mobilization from mitochondria, and the resulting [Mg(2+)]i is an important determinants of the cell viability in MPP(+) model of Parkinson's disease (PD). It indicates that cellular Mg(2+) transport is one of the important factors to determine the progress of PD. However, whether the expression levels of Mg(2+) transport proteins change in the progress of PD has still been obscure. In this study, we estimated the mRNA expression levels of Mg(2+) transport proteins upon the exposure to MPP(+). In thirteen Mg(2+) transport proteins examined, mRNA expression level of SLC41A2 was increased and that of ACDP2, NIPA1 and MMgT2 were decreased. Knockdown of SLC41A2, ACDP2 or NIPA1 accelerated the MPP(+)-induced cell degeneration, and overexpression attenuated it. The decrease in the mRNA expression levels of NIPA1 and MMgT2 were also elicited by rotenone, H2O2 and FCCP, indicating that mitochondrial dysfunction related to this down-regulation. The increase in that of SLC41A2 was induced by an uncoupler, FCCP, as well as MPP(+), suggesting that it is an intrinsic protection mechanism against depolarized mitochondrial membrane potential and/or cellular ATP depletion. Our results shown here indicate that alteration of Mg(2+) transport proteins is implicated in the MPP(+) model of PD, and it affects cell degeneration.

Intracellular magnesium level determines cell viability in the MPP(+) model of Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder resulting from mitochondrial dysfunction in dopaminergic neurons. Mitochondria are believed to be responsible for cellular Mg²âº homeostasis. Mg²âº is indispensable for maintaining ordinal cellular functions, hence perturbation of the cellular Mg²âº homeostasis may be responsible for the disorders of physiological functions and diseases including PD. However, the changes in intracellular Mg²âº concentration ([Mg²âº]i) and the role of Mg²âº in PD have still been obscure. In this study, we investigated [Mg²âº]i and its effect on neurodegeneration in the 1-methyl-4-phenylpyridinium (MPP⁺) model of PD in differentiated PC12 cells. Application of MPP⁺ induced an increase in [Mg²âº]i immediately via two different pathways: Mg²âº release from mitochondria and Mg²âº influx across cell membrane, and the increased [Mg²âº]i sustained for more than 16 h after MPP⁺ application. Suppression of Mg²âº influx decreased the viability of the cells exposed to MPP⁺. The cell viability correlated highly with [Mg²âº]i. In the PC12 cells with suppressed Mg²âº influx, ATP concentration decreased and the amount of reactive oxygen species (ROS) increased after an 8h exposure to MPP⁺. Our results indicate that the increase in [Mg²âº]i inhibited cellular ROS generation and maintained ATP production, which resulted in the protection from MPP⁺ toxicity.

Qualitative and quantitative estimation of comprehensive synaptic connectivity in short- and long-term cultured rat hippocampal neurons with new analytical methods inspired by Scatchard and Hill plots.

To investigate comprehensive synaptic connectivity, we examined Ca(2+) responses with quantitative electric current stimulation by indium-tin-oxide (ITO) glass electrode with transparent and high electro-conductivity. The number of neurons with Ca(2+) responses was low during the application of stepwise increase of electric current in short-term cultured neurons (less than 17 days in-vitro (DIV)). The neurons cultured over 17 DIV showed two-type responses: S-shaped (sigmoid) and monotonous saturated responses, and Scatchard plots well illustrated the difference of these two responses. Furthermore, sigmoid like neural network responses over 17 DIV were altered to the monotonous saturated ones by the application of the mixture of AP5 and CNQX, specific blockers of NMDA and AMPA receptors, respectively. This alternation was also characterized by the change of Hill coefficients. These findings indicate that the neural network with sigmoid-like responses has strong synergetic or cooperative synaptic connectivity via excitatory glutamate synapses.

Evaluation of drug toxicity profiles based on the phenotypes of ascidian Ciona intestinalis.

In vivo toxicity evaluation using model organisms is an important step for the development of new drugs. Here, we report that Ciona intestinalis, a chordate invertebrate, is beneficial to drug toxicity evaluation for the following reasons: rapid embryonic and larval development, resemblance to vertebrates, ease of management, low cost, transparent body, and low risk of ethical issues. The dynamic phenotypic change of Ciona larvae during metamorphosis prompted us to examine the effect of cytotoxic drugs on its development by quantifying six toxicity endpoints: degenerated tail size, ampulla length, rotation of body axis, stomach size, heart rate, and body size. As a result, mitochondrial respiratory inhibitors, tubulin polymerization/depolymerization inhibitors, or DNA/RNA synthesis inhibitors showed distinct toxicity profiles against these six endpoints, but drugs with the same targets showed a similar toxicity profile in Ciona. Our results suggest Ciona is an effective animal model for profiling drug toxicity and exploring the mechanisms of drugs with unknown targets.

Comprehensive morphological analysis of individual peripheral neuron dendritic arbors in ascidian larvae using the photoconvertible protein Kaede.

BACKGROUND: Ascidian larval epidermal sensory neurons (ESNs) each extend a single cilium into the outer body structure, or tunic, to form a unique external sensory network, the ASNET (ascidian dendritic network in tunic). The functions of ESNs and the ASNET are unknown, though it has been suggested that they play important roles in swimming larvae. In this study, we used transgenic larva expressing the photoconvertible protein Kaede pan-neuronally to identify the orientation and morphology of single ESN external processes within the ASNET. RESULTS: When individual ESN cell bodies were UV-irradiated, the photoconverted Kaede protein diffused into the external processes that projected to the edge of the tunic, indicating that these processes are cytoplasmically connected to the cell bodies of ESNs. We were, therefore, able to comprehensively catalog the morphology and orientation of each ESN external process. Most trunk ESNs appeared to extend neurites to the palp, but no processes were observed to emanate from palp neurons. ESN processes differed systematically in their morphology and orientation, suggesting that the ASNET is formed in a regulated, non-random fashion. CONCLUSIONS: This study reveals the organization of ESN sensory fields in the ascidian larval tunic.