what is the term given to the swimming larval stage of the sea urchin?

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Planktonic body of water urchin larvae alter their swimming management in response to strong photoirradiation

• Shunsuke Yaguchi,
• Yuri Taniguchi,
• Haruka Suzuki,
• Mai Kamata,
• Junko Yaguchi

ten

• Published: February 10, 2022
• https://doi.org/10.1371/periodical.pgen.1010033

Figures

Abstract

To survive, organisms need to precisely respond to various environmental factors, such as light and gravity. Amid these, calorie-free is so important for most life on Earth that calorie-free-response systems have become extraordinarily developed during evolution, especially in multicellular animals. A combination of photoreceptors, nervous organisation components, and effectors allows these animals to answer to lite stimuli. In nearly macroscopic animals, muscles function as effectors responding to light, and in some microscopic aquatic animals, cilia play a function. It is probable that the cilia-based response was the first to develop and that it has been substituted by the muscle-based response along with increases in body size. Nonetheless, although the function of musculus appears prominent, it is poorly understood whether ciliary responses to lite are present and/or functional, specially in deuterostomes, because information technology is possible that these responses are likewise subtle to be observed, different muscle responses. Hither, nosotros testify that planktonic bounding main urchin larvae reverse their swimming management due to the inhibitory upshot of light on the cholinergic neuron signaling>forward pond pathway. We found that potent photoirradiation of larvae that stay on the surface of seawater immediately drives the larvae away from the surface due to backward swimming. When Opsin2, which is expressed in mesenchymal cells in larval artillery, is knocked down, the larvae do non show backward swimming nether photoirradiation. Although Opsin2-expressing cells are non neuronal cells, immunohistochemical analysis revealed that they directly attach to cholinergic neurons, which are thought to regulate forward swimming. These data indicate that lite, through Opsin2, inhibits the activity of cholinergic signaling, which normally promotes larval forwards swimming, and that the light-dependent ciliary response is nowadays in deuterostomes. These findings shed light on how light-responsive tissues/organelles have been conserved and diversified during evolution.

Author summary

The importance of lite for organisms on Earth has led to the extraordinary development of sophisticated light-response systems during development. It is likely that low-cal-dependent ciliary responses were initially acquired in unicellular and small multicellular organisms, merely the pathway is poorly understood in deuterostomes, whose behavior more often than not depends on responses involving musculus. Therefore, information technology is unclear whether ciliary responses to light are present and/or functional in deuterostomes since these responses may exist too subtle for observation, unlike muscle responses. This raises the questions of how light-response systems were established and how they diversified during deuterostome development. Here, we provide clear evidence that planktonic larvae of body of water urchin species, which belong to the deuterostome group, brandish backward swimming when calorie-free inhibits cholinergic signal-dependent forward pond.

Citation: Yaguchi Due south, Taniguchi Y, Suzuki H, Kamata M, Yaguchi J (2022) Planktonic sea urchin larvae change their pond direction in response to stiff photoirradiation. PLoS Genet eighteen(2): e1010033. https://doi.org/x.1371/periodical.pgen.1010033

Editor: Claude Desplan, New York University, UNITED STATES

Received: August 3, 2021; Accustomed: January 12, 2022; Published: February x, 2022

Copyright: © 2022 Yaguchi et al. This is an open access article distributed under the terms of the Creative Eatables Attribution License, which permits unrestricted use, distribution, and reproduction in whatever medium, provided the original author and source are credited.

Data Availability: Source data for all Figures and supporting information are provided inside the paper. Sequence data can be found in the genome database of Hemicentrotus pulcherrimus, HpBase (http://jail cell-innovation.nig.ac.jp/Hpul/).

Funding: This piece of work is supported, in office, by JST PRESTO Grant number JPMJPR194C, the Toray Science Foundation and Takeda Science Foundation to S.Y., and JSPS KAKENHI Grant number JP19K16199 to J.Y. H.Southward. was a JSPS Research Fellow with research grant (DC1: 19J20629). The funders had no role in written report design, data collection and analysis, conclusion to publish, or training of the manuscript.

Competing interests: The authors have alleged that no competing interests exist.

Introduction

Information technology is essential for organisms to precisely reply to extrinsic signals, such as light, gravity, and temperature. Among these signals, light is very important for most organisms on Earth because it provides energy, visual information, and cues for circadian rhythms. Therefore, motile organisms accept adult photoreception, motor organs/organelles, and signaling systems that collaborate to promote positive or negative phototaxis/reflexes. For example, even single-celled organisms, such every bit Chlamydomonas, modify the beating pattern of cilia/flagella when photoirradiated [one]. As another example, another group of nonmetazoans, the choanoflagellates, besides evidence response behaviors to low-cal input [2]. Because of their small torso size and lack of a nervous system, the light-response network of these organisms is established mainly inside the intracellular space. On the other hand, along with the increase in body size and the acquisition of a nervous organization during animal evolution, it is expected that organisms started to establish and deport with an intercellular network involving photoreceptors, nervous organisation components and motor organs/organelles. For case, diel vertical migration (DVM) is a well-known behavior through which zooplankton stay at the surface at night and sink securely into the ocean during the daytime. This behavior has been explained every bit a ways by which zooplankton escape from diurnal predators such as fish and harmful ultraviolet (UV) low-cal during the daytime [three, 4], and it has been suggested that the behavior is driven by an intercellular network. 1 of the most studied model organisms for DVM and then far is Daphnia, and analyses of the migrations of Daphnia away from predators and UV light have been well reported. For case, stronger UV light induces deeper migration of Daphnia [iii–five]. As photoreceptors for these metazoan behaviors, opsin family members function mainly to receive light of a specific wavelength and transmit the betoken to downstream systems [half dozen, seven]. Opsins, which are establish just in the metazoan grouping [8], vest to the grouping of sensory K-poly peptide-coupled receptors (GPCRs) and are categorized into visual/nonvisual types [7]. The motor organs/organelles, every bit the effectors of light responses, are musculus in well-nigh macroscopic animals and cilia in relatively pocket-size aquatic animals. Light-induced muscle activities, such as those involved in the pupillary light reflex of our eyes, the light avoidance behavior of earthworms, and the phototaxis of insects, have been well investigated [nine–eleven]. Muscle-based light responses have been reported throughout the bilaterians [6, 9–15]. On the other hand, in the planktonic larvae of flatworms, Annelida, and Mollusca, responses to lite are supported mainly by changes in ciliary beating, with which the larvae show reflex behavior and/or phototaxis [xvi–18]. This cilia-based response to light is as well found in cnidarian and sponge larvae [19, twenty], although it is expected that the sponge lineage lost Opsin genes [viii]. Even though ctenophores and placozoa have motile cilia, they do not show the cilia-based response to light [21, 22]. Although nematodes and arthropods have lost motile cilia in their clades and of class accept no cilia-based responses to low-cal, information technology is obvious that the light-induced response of cilia is a shared feature within protostomes and cnidarians. However, it is unclear whether this pathway is conserved among neuralians because cilia-based responses to low-cal are poorly reported and understood in deuterostomes, resulting in a lack of understanding of the signaling pathway from light to cilia in these groups. This lack of clarity might exist because the body size of deuterostomes is relatively big, which makes the behaviors of these animals dependent on muscle activities and masks subtle ciliary activity if it is present.

Among deuterostomes, some echinoderms and hemichordates accept free-living planktonic embryos/larvae, which are the only groups that motion with mainly cilia rather than muscle. Therefore, they are candidates from which we might obtain information on the presence and mechanisms of cilia-based responses to lite input in deuterostomes. In detail, sea urchin larvae are ideal experimental model organisms for this purpose because they do non apply muscle at all for their swimming behavior and are suitable for genetic analysis. The ocean urchin genomes comprise a maximum of 9 opsin genes (e.g., Opsin1, Opsin2, Opsin3.1, Opsin3.2, Opsin4, Opsin5, Opsin6, Opsin7, and Opsin8 in Strongylocentrotus purpuratus), which belong to a group of sensory GPCRs, and almost of them are categorized into reported opsin groups, except for Opsin2 and Opsin5 [23]. Opsin2 and Opsin5 have been found only in echinoderms; therefore, they are thought to exist specific to the group. Because some of the expression patterns of these genes take been reported in both embryos/larvae and adults [24–27], it is reasonably expected that sea urchins have the ability to react to light stimuli from a genomic perspective, every bit reported in adult behavioral studies [28, 29]. Notwithstanding, the functions of photoreceptor genes have never been confirmed by using genetic modification except for the role of the recently reported gut-regulatory Go-Opsin [12], and the neural pathway regulating cilia-based larval beliefs has not notwithstanding been identified. Therefore, in this study, we sought to describe how the swimming behavior of ocean urchin larvae responds to light and to identify the neural pathway involved in the response.

Results

To investigate whether sea urchin larvae respond to light, we irradiated the larvae with strong light in a dish in which they had previously stayed at the surface of the seawater. We then observed their behaviors. Intriguingly, the larvae dropped from the surface immediately upon photoirradiation, and some of them swam backward (S1 Fig, S1 Video). Larvae of dissimilar species, such equally Hemicentrotus pulcherrimus and Temnopleurus reevesii, showed like behaviors (S1 Fig, S2 Video), suggesting that this response is common in some body of water urchin groups. To visualize and quantify this larval beliefs, we measured the velocities of diatom particles in front of the larvae that were attached and immobile on glass earlier and later on photoirradiation (Fig 1A, see Materials and Methods). The diatom movements reflected the water currents that were produced by the ciliary beating of the larvae [xxx]. Although the larvae swim in a three-dimensional earth, we measured their behaviors under a microscope in a 2-dimensional airplane in this study to obtain information that can exist reproduced with conventional equipment in ordinary laboratories, merely nosotros will try in hereafter analyses to detect swimming behavior with a loftier-speed tracking 3D microscope. In the absenteeism of strong light, the larvae generally swam forward (Fig 1B), but after stiff photoirradiation, a weakly reversed h2o current was produced, indicating that the larvae had stopped pond and/or swum astern in response to light (Fig 1B, S3 Video). The average velocities of the water current toward the larvae, which reflected larval forward swimming, were 90.63 μm/sec (before low-cal, N = 6, n = x each) and -12.19 μm/sec (afterward light, North = vi, north = 10 each).

Fig one. Bounding main urchin larvae stop swimming forward and swim backward in response to a lite stimulus.

(A) Schematic images showing a change in swimming behavior subsequently photoirradiation. The images captured from S3 Video show the changes in the motion of diatom particles effectually larvae subsequently photoirradiation (cf. Aa to Ab). Ac and Advertisement show the superimposed images for ii seconds each before (Air-conditioning) and afterward (Advertisement) photoirradiation. The sky-blue rectangle indicates the region of involvement (roi) nosotros used for the calculation of particle velocity. Ae and Af show the temporal-color-lawmaking mode for the superimposed images. As shown in the indicator, dark blue and bright yellow marker the showtime and end of the 2 sec movie before (Ae) and after (Af) photoirradiation, respectively. (B) Particle velocity measured before and after photoirradiation. N = 6 in each experiment. The velocities of 10 particles were scored for each N. ****, p≤0.0001. (C) Larval arms are required for photoreception. The graph shows the percentages of the larval responses to photoirradiation. The images on each bar graph indicate the micromanipulated larvae used for each experiment. ANE, anterior neuroectoderm.

https://doi.org/10.1371/journal.pgen.1010033.g001

Because it is reasonable to speculate that the cessation of forrad swimming and/or the reversal of swimming direction after photoirradiation is mediated by a complex organization including photoreceptors, neurons, and ciliary cells, we attempted to identify the most important torso parts for this response by removing the larval anterior neuroectoderm (ANE) and/or postoral arms and and then observing larval swimming behaviors. Information technology has already been shown that this microdissection method is useful for investigating the essential parts of the body for a targeted phenomenon [12]. The abeyance of frontwards swimming or reversal of swimming direction later photoirradiation was observed in 80% of the normal H. pulcherrimus larvae and in the same percentage of T. reevesii larvae (Fig 1C). This quantitatively supports the thought that this phenomenon is common among some sea urchin groups. When we removed the postoral arms, the manipulated larvae showed a response to photoirradiation similar to that of the control larvae (Fig 1C). This issue suggested that the postoral artillery, which comprise ciliary bands and some neurons, do not seem to be important for the response to photoirradiation. Larvae from which the ANE and preoral arms were removed stopped swimming forward and/or swam astern upon photoirradiation, but the timing was delayed (>two sec) (Fig 1C). This result indicates that the ANE is more important for the response to photoirradiation than the postoral artillery. However, compared with that of command larvae, the response of One-removed larvae was weak, suggesting that the I is non the merely tissue essential for the response to low-cal. Intriguingly, when both the ANE and postoral arms were removed, the larvae swam only forward and did not strongly respond to photoirradiation (Fig 1C). These data indicate that some tissues, such as the nervous system almost the ANE and/or postoral artillery, play an important part in the photoirradiation response.

Among the members of the circuitous system that is expected to be critical for this lite-dependent phenomenon, neurons and cilia are present mainly in the ciliary band and throughout the entire trunk [31, 32], respectively, merely the spatial expression patterns of photoreceptors in H. pulcherrimus had not been reported until recently. Since the temporal and spatial patterns of the larval Opsins Opsin2 and Go-Opsin have been reported in Strongylocentrotus purpuratus [25, 33], nosotros tried to detect the expression patterns of these molecules in H. pulcherrimus. Similar to the blueprint in S. purpuratus, we establish that the Become-Opsin gene was expressed exclusively around the ANE; it was non expressed in the arms of H. pulcherrimus [25, 33], indicating that it is non a stiff candidate as the factor that focuses pond behavior. In addition, we accept already reported that Become-Opsin is involved in the calorie-free-dependent gut regulatory system [12]. On the other paw, Opsin2 was expressed in mesenchymal cells at the tips of the postoral arms (Fig 2A and 2B, arrows) and the preoral artillery adjacent to the ANE (Fig 2A and 2B, arrowheads). Identical expression patterns were observed in T. reevesii (Fig 2B, arrows, although the preoral artillery are out of focus in this image). Considering the mesenchymal cells at the tips of the arms include both primary mesenchymal cells (PMCs) and secondary mesenchymal cells (SMCs), to confirm which lineage expresses Opsin2, we blocked the specification of SMCs by attenuating the function of the specifier gene, glial cells missing (gcm) [34], with a specific morpholino. Opsin2 mRNA was non detected in gcm morphants (Fig 2C). In addition, in larvae treated with a gamma-secretase inhibitor (DAPT: Northward-[N-(3,5-difluorophenacetyl-Fifty-alanyl)]-(Southward)-phenylglycine t-butyl ester), in which the SMC specification signal transmitted via Delta/Notch signaling was blocked during early embryogenesis, Opsin2-positive cells were not present (Fig 2nd). These data strongly bespeak that Opsin2-positive mesenchymal cells vest to the SMC lineage. These patterns are dissimilar from those in S. purpuratus [33], peradventure because of species differences. To confirm our in situ hybridization information showing targeted Opsin2 mRNA expression, we generated a specific antibody confronting Opsin2 protein produced in Escherichia coli (meet Materials and Methods). The specificity of the antibody is shown in Figs 3C and S2. Immunohistochemistry using this antibody showed that the Opsin2 protein was present in the aforementioned mesenchymal cells that expressed Opsin2 mRNA (Fig 2E). This finding strongly indicates that Opsin2 mRNA and protein are expressed in the SMC lineage at the tips of both the postoral and preoral arms in sea urchin larvae.

Fig two. Opsin2 expression during embryogenesis.

(A) Fluorescent in situ hybridization of Opsin2 mRNA. Although 48-hour-erstwhile larvae showed no mRNA signal, 72-60 minutes-old larvae clearly had Opsin2 expression at the tips of their arms. VV, ventral view; DV, dorsal view. (B) Chromogenic in situ hybridization of Opsin2 mRNA in pluteus larvae of H. pulcherrimus and T. reevesii. Bar = 20 μm. The arrows and arrowheads show Opsin2-expressing cells in the postoral artillery and preoral arms, respectively. The Opsin2-expressing cells in the preoral arms of T. reevesii are out of focus in this epitome. (C, D) The expression of Opsin2 was abrogated in larvae in which the secondary mesenchymal jail cell specifier Gcm (C) or Delta-Notch signal (D) was adulterate. (Eastward) Opsin2 protein was expressed in the same cells expressing Opsin2 mRNA. Light-green shows serotonergic neurons. Blue in the left epitome shows the nuclei. The fluorescent image is merged with the brightfield image on the right. (F) Opsin2-expressing cells were close to the ciliary band and neurons. Major neuronal axons, which express the panneural marker synaptotagmin B (SynB), were located beneath the ciliary band.

https://doi.org/10.1371/journal.pgen.1010033.g002

Fig 3. Opsin2, which is expressed in mesenchymal cells located in the arms, is required for photoreception.

(A) Immunohistochemical assay of the Conversation poly peptide. The ciliary ring neurons in the postoral artillery are cholinergic. (inset) In situ hybridization of choline acetyltransferase (Conversation) mRNA in pluteus larvae. (D) The Opsin2 protein was not translated in Opsin2-MO2 morphants. (B) Details of the relationship between Opsin2-expressing cells and cholinergic neurons. The prison cell processes from Opsin2-expressing cells appear to attach or exist closely adjacent to neurons (arrowhead). (C) The Opsin2 protein was expressed in mesenchymal cells in the postoral arms (sky-blueish arrow) and preoral arms (sky-blue arrowheads), and it disappeared afterward morpholino injection (white arrows and arrowheads). (D) The graph shows the deviation in particle velocity earlier vs. later on photoirradiation in the presence or absence of the Opsin2 protein. The particle velocity was measured earlier and after photoirradiation. N = vii in each experiment. The velocities of x particles were measured for each N. *, p≤0.05, **, p≤0.01, ***, p≤0.001, ****, p≤0.0001, Due north.S., not significant. (E) Schematic images summarizing the results of this report. Under nighttime or weak-lite weather condition (left), the cholinergic system drives forward swimming, but strong photoirradiation (right) stops the cholinergic system via Opsin2.

https://doi.org/x.1371/journal.pgen.1010033.g003

It has been suggested that swimming behaviors of sea urchin larvae are regulated mainly by a nervous system underlying the ciliary band (Fig 2F) [xxx, 32], and Opsin2-expressing cells are located close to the ciliary ring. Nonetheless, since no papers have reported experimental results indicating the interest of ciliary band neurons in pond behaviors, nosotros adjacent investigated whether ciliary ring neurons regulate larval movements under normal conditions. As shown in Fig 2F, Synaptotagmin B (SynB)-positive neurons were distributed under the unabridged ciliary band. A recent paper reported that some ciliary ring neurons are cholinergic in larvae of the sea urchin Lytechinus variegatus [35]. To ostend whether the same type of neuron was present nether the ciliary band in our model sea urchin, H. pulcherrimus, we performed in situ hybridization using an RNA probe specific for choline acetyltransferase (Conversation) mRNA. The results showed that Chat-expressing cells were likely located nether the ciliary band, as reported in Fifty. variegatus (Fig 3A, inset). Yet, because of the shrunken shapes of the pluteus larvae, it was difficult to identify the detailed expression patterns. Therefore, we adult a specific antibody against the Chat protein (see Materials and Methods) and observed the expression patterns. The specificity of the antibody is shown in S3 Fig. The results of this observation showed that most ciliary ring neurons seemed to be cholinergic, especially in larval arms (Fig 3A). These results suggest that ciliary beating controlling larval swimming behaviors involves the activeness of cholinergic neurons. To confirm this supposition, we tried to knock down the action of ChAT using a specific morpholino and succeeded in blocking translation, as shown in S3A Fig. However, this occludent had multiple effects on larval behaviors (which will be described in a afterward publication), leading us to conclude that simply knocking downwardly the cholinergic neural pathway is not suitable for analyzing swimming behavior. Thus, we applied inhibitors of acetylcholine receptors and assessed pond behavior. In sea urchin genomes, two types of acetylcholine receptors have been annotated: muscarinic acetylcholine receptor (mAChR) and nicotinic acetylcholine receptor (nAChR) [36]. When we blocked mAChR using the specific inhibitor atropine, the larvae did not bear witness a normal response to calorie-free due to their slight forward swimming (S4A Fig); some of them exhibited only backward swimming. To ostend the findings, we applied a lower concentration of atropine and traced and measured the swimming behavior. Compared with the altitude of movement over 45 sec of the control larvae, that of the atropine-treated larvae was significantly shorter (S4B and S4C Fig). In addition, some other inhibitor of mAChR, scopolamine, had a similar consequence on larvae equally atropine treatment (S4B and S4C Fig). In contrast, when we blocked nAChR using the specific inhibitor d-tubocurarine, the larvae showed a normal response to light (S4A Fig). These results suggest that cholinergic neurons and mAChR are required for normal forwards swimming. Based on the temporal expression patterns of a member of annotated mAChRs in S. purpuratus, mAChR-M5 (SPU_016177) is expressed during embryonic and larval stages [37]. In situ hybridization using an antisense probe for HPU_21265, a homolog of SPU_016177 in H. pulcherrimus [27], revealed that mAChR was expressed in the inductive half and mainly at the oral ectoderm, which was surrounded by the ciliary band (S3D Fig). Because of the presence of cilia on all ectodermal cells, it has been suggested that larval swimming patterns and directions are likely regulated by complex combinations of ciliary beating in dissimilar areas [30, 38]. These patterns of forrad pond and the move of the h2o in front of the larvae, on which nosotros focused in this study, reverberate a process involving cholinergic neurons and mAChR.

Detailed observations revealed that ChAT-positive ciliary band neurons appeared to exist directly associated with or closely side by side to Opsin2-expressing cells in both the preoral and postoral arms (Fig 3B, arrowheads). To examine whether Opsin2 plays an essential role as a photoreceptor in the observed phenomenon, we knocked it downwards with specific morpholino oligonucleotides (Opsin2-MO) (Fig 3C [white arrows and arrowheads], S2) and observed larval swimming behavior. The control larvae normally stopped pond forward or swam astern in response to photoirradiation, whereas the Opsin2 morphants showed only weakened forward pond (Fig 3D, S4 Video). The average velocities of the water current toward the larvae were 84.29 μm/sec (before light in the control group, N = 7, n = 10 each), -13.49 μm/sec (afterward lite in the command grouping, North = 7, northward = 10 each), 113.12 μm/sec (before low-cal in the Opsin2 morphant group, N = 7, n = 10 each), and 51.43 μm/sec (subsequently lite in the Opsin2 morphant group, Northward = 7, n = 10 each). This issue strongly suggests that Opsin2 is the photoreceptor that mediates light-dependent arrest/reversal of swimming, peradventure by inhibiting the activity of cholinergic neurons. In addition, considering all Opsin2-positive mesenchymal cells are located at the tips of the postoral and preoral artillery, the finding that Opsin2 morphants continued pond forward supports the finding that the armless larvae kept swimming forrad under photoirradiation conditions (Fig 1C). On the other mitt, the fact that weakened frontwards swimming was observed in Opsin2 morphants suggests that some other components, such as other opsins and/or nervous system pathways, might be involved in the change in swimming behavior upon photoirradiation. The detailed mechanisms past which these cells communicate with each other, including by directly or indirect contact, will be studied in the time to come.

Discussion

Based on the data shown hither, under normal atmospheric condition, sea urchin larvae use cholinergic neurons to bulldoze forward swimming; however, when the larvae face strong photoirradiation, the activated photoreceptor Opsin-ii seems to suppress the activity of cholinergic neurons, causing forward swimming to be stopped/weakened and promoting backward swimming (Fig 3E). Although the details of this indicate transduction pathway from Opsin-ii cells to cholinergic neurons have not yet been revealed, our data clearly show that sea urchin larvae reply to calorie-free stimuli, equally reflected past their swimming behavior. The presence of this pathway from light to stopping/reversal of pond through secondary mesenchymal photoreceptors and the ciliary band nervous system was strongly supported by multiple knockdown experiments using MOs and pharmacological experiments. In addition, this phenomenon is likely common, at least in some body of water urchin species, since both H. pulcherrimus and T. reevesii showed the aforementioned response to photoirradiation and the same localization of Opsin2 cells (Figs 1C and 2B). Because all echinoderm larvae mainly use cilia, non muscles, in their pond behavior and considering the ciliary ectoderm of these larvae is associated with the nervous system [32, 39, twoscore], it is probable that all echinoderm groups take a similar light-induced ciliary response. In fact, the larvae of brittle stars show DVM behavior, in which they swim downwardly to the deep ocean during the daytime and swim up to the surface during the nighttime [41], suggesting that their ciliary chirapsia is controlled in response to lite. Since information technology has rarely been proven that a cilia-based response to light input is present in other deuterostomes by experiments thus far [42, 43], it is of interest that bounding main urchins have the ability to answer to calorie-free with cilia via an opsin-nervous arrangement network. Since the appearance of opsin genes at the metazoan stem [8], information technology is speculated that about photoreception systems take get dependent on opsin proteins in metazoans. In improver, information technology is also expected that the advent of neurons helped enable long-distance intercellular communication between photoreceptors and motor organs/organelles, especially in neuralians. Considering cnidarians and ciliated protostome larvae utilize mainly the lite-induced response of cilia in the regulation of their behaviors [8, 16, 17], the light-induced response of cilia was likely already present prior to the advent of the mesodermal muscles that play essential roles in bilaterian movements. Without experimental or detailed observation data in chordates, it is still unclear whether the low-cal-induced response of cilia is commonly present or absent-minded in the deuterostome lineage, although the pathway has now been found in the sea urchin group. One scenario is that the low-cal-induced response of cilia is conserved amid deuterostomes only even so unidentified. In hemichordates, which belong to Ambulacraria together with the echinoderms, it has been reported that adults respond to lite and movement to avoid it with muscle [13], merely larval behaviors away from light have rarely been an experimental inquiry focus. Indirectly developing larvae swim with neuron-associated cilia similar echinoderm larvae [44], and some of them have clearly pigmented eyespots [45]. In addition to the presence of opsin genes in their genomes [23, 46] and the similarity of their developmental steps to those of echinoderms, these morphological characteristics propose that the larvae of hemichordates might have cilia-based responses to light stimuli. On the other hand, because muscular movement is so conspicuous in the chordate lineage, information technology is possible that the subtle responses of tiny cilia are masked and/or accept never been focused on even if cilia-based responses to light are present. Although it is yet unclear whether the light-cilia relationship observed in sea urchin larvae is conserved in vertebrates, in human behavior, there is a miracle that might exist comparable: the photic sneeze reflex. This is a miracle in which humans have a sneezing reflex when they are exposed to stiff photoirradiation, such every bit sunlight. Although the detailed molecular mechanisms responsible for the photic sneeze reflex take not however been investigated [47], it is possible that dysfunction of ciliary beating is induced past photoirradiation, as in body of water urchin systems, and causes the sneezing reflex in the absence of any debris or irritants. In detail, ciliary beating in the nose/nasal crenel is controlled by cholinergic neurons, and it has been suggested that dysfunction of these neurons is the reason for the photic sneeze reflex [48]. It is of involvement if the cessation of cholinergic neuron signaling and the consistent sneeze reflex are induced by photoirradiation, since our data might provide hints to help determine whether the light-induced response of cilia is nowadays in vertebrates and whether the involved organisation is conserved between echinoderms and vertebrates. In add-on, cilia are present within of the ventricular system in human brain, and cerebrospinal fluid flows via cilia to send neurotransmitters/neuromodulators, meaning that cilia are one of the key factors for the functions of the brain [49]. If similar responses of cilia to lite occur in the ventricular system, the signaling pathway found in bounding main urchin larvae might aid elucidate the mechanisms of human behaviors and/or feelings in response to light.

To date, some characteristics of the swimming behaviors of sea urchin larvae have been reported. For example, the larvae swim against gravity, and this negative gravitaxis is managed by serotonergic neurons, which are exclusively located in the Ane region [thirty]. Notwithstanding, because of the lack of knowledge on how sea urchin larvae detect gravity, the signaling pathway that is initiated by gravity and is transmitted through serotonergic neurons remains unknown. In addition, previous works have analyzed larval behaviors by considering larvae equally particles moving confronting gravity [50]; withal, it is hard to draw the detailed signaling pathway in the nervous system based on detection of gravity in combination with ciliary beating. Similarly, although ocean urchins answer to external and ecology stimuli, the detectors and critical nervous arrangement components have not been well described for a long fourth dimension, except for the Go-Opsin>serotonergic neurons>nitric oxide pathway, which was recently described in a publication [12]. Another study has reported that dopamine can induce backward swimming in body of water urchin larvae [51]. This neurotransmitter may be a candidate controller of swimming abeyance and/or backward swimming under photoirradiation; however, larvae defective the postoral artillery, in which dopaminergic neurons are present [52], had a normal response (Fig 1), suggesting that dopamine is not primal to this behavior. Although the details regarding how lite information is transmitted to and stops the activity of cholinergic neurons are all the same unclear, the data presented in this newspaper show that light-exposed Opsin2-expressing cells inhibit constitutively activated cholinergic signaling. Opsin2-expressing cells appear to directly communicate with cholinergic neurons, but we cannot completely exclude the possibility that Opsin2-expressing cells secrete unknown factors that inhibit mAChR. Considering the beating of respiratory cilia in mammals and palate/esophageal cilia in frogs is driven by an activated cholinergic system through mAChR and considering antagonists of mAChR inhibit ciliary beating [53–56], some parts of the human relationship between the mAChR-based cholinergic system and cilia seem to exist conserved in vertebrates and sea urchins.

Materials and methods

Animals (sea urchins)

Adults of Hemicentrotus pulcherrimus were nerveless around the Shimoda Marine Research Middle of the Academy of Tsukuba and around the Marine and Coastal Research Middle of Ochanomizu University. Adults of Temnopleurus reevesii were collected effectually the Shimoda Marine Research Heart of the University of Tsukuba. Both species were nerveless nether the special harvest permission of the prefectures and Nippon fishery cooperatives. They were kept in temperature-controlled aquariums (13°C and 24°C for H. pulcherrimus and T. reevesii, respectively) until apply, and the used adults were kept until the side by side breeding season or released to the place from which they were collected. Gametes were collected by intrablastocoelic injection of 0.5 Chiliad KCl, and the embryos/larvae of H. pulcherrimus and T. reevesii were cultured at 15°C and 22°C, respectively, in glass beakers or plastic dishes that contained filtered natural seawater (FSW) with 50 μg/ml kanamycin. In some experiments, we fed 10 μl of SunCulture algae (Chaetoceros calcitrans, Marinetech, Aichi, Japan, approx. xxx,000 cells/μl) to the larvae equally forage in 3.0 ml of FSW almost every mean solar day.

Photoirradiation process and ascertainment of larvae

In each experiment, pluteus larvae at appropriate stages (4–5 days mail service-fertilization) were attached to poly-Fifty-lysine (Merck)-coated glass slides with a drop (v.0 μl) of seawater. To visualize the h2o current, 0.5 μl of SunCulture algae (Chaetoceros calcitrans) was added to the seawater drop. The specimen was moved onto an 9-70 microscope (Olympus, Tokyo, Nippon) equipped with a DP-71 digital camera (Olympus) under ambient room light (7.5–xiii.0 μmol m^-2 s^-1), which does not induce cessation of forward pond. Photoirradiation of the specimens was performed by turning on the calorie-free of a microscope without whatsoever neutral density filters. The photon flux density was adjusted to 1425 μmol m^-2 south^-i.

Microscopy and prototype analysis, including calculation of the velocity of diatoms in front of the larvae

Specimens were observed using a fluorescence microscope (IX70, Olympus, Tokyo, Nippon), a confocal laser scanning microscope (FV10i, Olympus), and a dissecting microscope (M165C, Leica Microsystems GmbH, Wetzlar, Germany). Superimposed images were made with Fiji (Z-projection with the standard deviation option), and the fourth dimension-encoding with color to bear witness the management of the particles was accomplished with the temporal-color-code program from Kota Miura (https://github.com/republic of the fiji islands/republic of the fiji islands/hulk/chief/plugins/Scripts/Epitome/Hyperstacks/Temporal-Color_Code.ijm). For Figs 1B and 3D, videos of the specimen were captured five sec before and ten sec after photoirradiation. The videos were opened with Fiji (https://imagej.net/Fiji) and analyzed with the Particle Track and Analysis (PTA) plugin (https://github.com/arayoshipta/projectPTAj). Initially, nosotros chose an roi at the front end of the larva (dotted-line rectangle, Fig 1Ac and 1Ad) and cropped it. Then, the PTA plugin was run for the roi. The velocity of the diatoms was calculated by dividing the travel distance (μm) past the time (sec). The velocity was calculated with the direction toward the larva every bit the positive management and the direction away from the larva every bit the negative direction. The velocities of the 10 diatoms with the longest migration in each sample were averaged to obtain the boilerplate velocity of each larva. To compare experimental specimens with controls, we used Welch'south t exam (two-tailed) or one-way ANOVA followed by Tukey's post hoc test with a significance level of 0.01 or 0.05. For Figs 1C and S4A, we counted the number of larvae that responded (stopped swimming frontwards or swimming astern) or did not respond to photoirradiation (continued pond forward), and the ratio was calculated. These samples were dissected past micromanipulation or drug-treated. The swimming distance was also measured for S4B and S4C Fig; Fiji was used for the superimposed images. The panels and drawings for the figures were made using Adobe Photoshop and Microsoft PowerPoint.

Reagent treatments

three,five-Difluorophenylacetyl-L-alanyl-50-Due south-phenylglycine T-butyl ester (DAPT; Sigma–Aldrich, St Louis, MO, USA), which was used as a γ-secretase inhibitor, was prepared equally a 20 mM stock in dimethyl sulfoxide (DMSO) and diluted in FSW to twenty μM before use [57]. To obtain larvae in which the secondary mesenchymal cells failed to develop completely, we treated the larvae with DAPT from i hour to 24 hours after fertilization [58]. The same volume of DMSO was applied every bit a command. Atropine sulfate and d-tubocurarine chloride were used as mAChR and nAChR inhibitors, respectively. The atropine and d-tubocurarine were dissolved in distilled water (DW) as 100 mM stocks and diluted into the larval culture medium 1 min earlier observation.

Whole-mountain in situ hybridization and immunohistochemistry

Whole-mount in situ hybridization was performed as described previously [59] with some modifications. A cDNA mix from several embryonic stages was used to isolate the targeting genes to make RNA probes based on the H. pulcherrimus genome and transcriptome [27]. Digoxygenin (Dig)-labeled RNA probes were generated from PCR amplicons with SP6 and T7 primers from pCS2+ChAT and pCS+mAChR. The samples were fixed with 3.7% formalin (Merck) containing FSW overnight at 4°C. They were washed with MOPS buffer (0.1 M MOPS pH 7.0, 0.5 Grand NaCl, 0.i% Tween-20) vii times for vii min each at room temperature (RT) and rinsed with hybridization buffer (70% formamide, 0.one M MOPS pH 7.0, 0.v Grand NaCl, one mg/ml BSA, 0.1% Tween-xx) iii times. And then, the samples were incubated in hybridization buffer with Dig-labeled RNA probes (0.iv ng/μl concluding concentration) of ChAT (HPU_01496) and mAChR (HPU_21265) at 50°C for 5–7 days. After washing with MOPS buffer seven times at RT, three times at 50°C, and twice at RT, the Dig-labeled probes were detected with an anti-Dig POD-conjugated antibiotic (Roche) for ChAT overnight at 4°C and treated with a Tyramide Signal Distension Plus System (TSA; PerkinElmer, Waltham, MA, USA) for viii minutes at RT. When observed, the samples were incubated in MOPS buffer containing 2.5% 1,iv-diazabicyclo-2-2-2-octane (Sigma–Aldrich) to prevent photobleaching. For mAChR, after washing, the Dig-labeled probes were detected with an anti-Dig AP-conjugated antibiotic (Roche) for 1 hour and washed with MOPS buffer overnight. The samples were done 3 times in alkaline phosphatase buffer (0.ane M Tris-HCl pH 9.5, 0.ane M NaCl, fifty mM MgCl_ii, 1.0 mM levamisole, and 0.1% Tween-20), and the betoken was detected with 5-bromo-4-chloro-iii-indolyl-phosphate/nitro blueish tetrazolium (BCIP/NBT) in AP buffer containing 10% dimethylformamide.

Whole-mount immunohistochemistry was besides performed as described previously [59] with some modifications. Common cold methanol-fixed (10 min) samples were blocked with 1% skim milk in PBST for 1 hour at RT and incubated with primary antibodies (dilutions: mouse anti-Synaptotagmin B (SynB) [32] one:100, rabbit anti-acetylated-α-tubulin 1:800 (Cell Signaling Engineering), mouse anti-Opsin2 1:200 (see below), and rabbit anti-Opsin2 i:100 (run across below)) overnight at 4°C. The chief antibodies were detected with Alexa 488- or Alexa 555-conjugated secondary antibodies (Thermo Fisher Scientific, Waltham, MA, United states).

Chat and Opsin2 antibodies

cDNA encoding the first 86 amino acids of ChAT and amino acids 784–1395 of Opsin2 were cloned into the pET32a vector (Novagen, Darmstadt, Germany), and the histidine-tagged proteins were produced in ArcticExpress (DE3) leaner with a concluding concentration of 1.0 mM isopropyl ß-D-thiogalactopyranoside (IPTG) for iv hr at RT. The leaner were lysed with eight M urea, and the fusion proteins were purified past Ni-column chromatography (Cytiva, Tokyo, Japan) and used to immunize three mice under the permission of the Animal Intendance Committee of the University of Tsukuba (No. 19–65, 1040). Using the aforementioned purified Conversation protein, rabbit antisera were produced by Eurofins Genomics (Tokyo, Nihon). The antisera were screened by whole-mount immunohistochemistry, and IgG was purified with Melon gel (Thermo Fisher Scientific) and used for experiments.

Microinjection of morpholino antisense oligonucleotides (MOs)

For microinjection, we used injection buffer (24% glycerol, 20 mM HEPES pH eight.0 and 120 mM KCl). The morpholino (Gene Tools, Philomath, OR, Us) sequences are in the reagent tabular array, and the in-needle concentration with injection buffer was one.0 mM. Two nonoverlapping translation-blocking morpholinos for Opsin2 and Conversation were used to confirm the function specificity (S1 and S2 Figs). For negative control experiments, nosotros injected a random MO or only injection buffer. The concentrations of MOs in the needles were equally follows:

Opsin2-MO1 (0.4 mM), 5'-AGTTTGCCATCTTTGTGTTGCTTCG -iii'

Opsin2-MO2 (0.4 mM), 5'-CGCCAATAACCACTGATCACAGTCG -3'

ChAT-MO1 (0.2 mM), v'-ACGATTAGGCATGTGGTTCATGTAT -3'

Chat-MO2 (1.0 mM), 5'-TGGAACGTCCAATAGTGGTATTGTA -3'

Gcm-MO, v'-GCTTTGGACTAACCTTTTGCACCAT -3' [34], and

Random-MO (1.0 mM).

Microinjections into fertilized eggs were performed as previously described [60, 61]. Afterward microinjection, the embryos were done with FSW three times and stored with 50μk/ml kanamycin until the desired stages were reached.

Micromanipulation

Larvae were placed in 6 cm plastic dishes filled with seawater, and the postoral arms or I/preoral arms were cut off with an injection needle under a dissecting microscope (Leica M165). The dissected larvae were transferred to new seawater and used for experiments.

Supporting data

S1 Fig. Larvae of both Hemicentrotus pulcherrimus and Temnopleurus reevesii movement away from the water surface when illuminated.

These are captured images from S1 and S2 Videos. The times (sec) shown on the images indicate the timing before and after photoirradiation.

https://doi.org/10.1371/periodical.pgen.1010033.s001

(PDF)

S3 Fig. Specificity of the ChAT antibody.

(A) Two nonoverlapping morpholinos blocked the translation of ChAT. (B) Using the aforementioned antigen, nosotros made an anti-ChAT antibody in a rabbit. Although the background was slightly loftier, the antibiotic recognized ciliary band neurons to a similar caste as a mouse antibody. The middle paradigm is a magnified region of the dotted-line rectangle on the left. The right image shows the disappearance of the rabbit anti-Conversation antibiotic indicate in the Conversation-MO1 morphant, supporting the specificity of the antibody.

https://doi.org/x.1371/journal.pgen.1010033.s003

(PDF)

S4 Fig. Normal forward swimming of sea urchin larvae depends on cholinergic neurons.

(A) The graph shows the alter in particle management before and later photoirradiation in the presence of acetylcholine and antagonists of its receptors. Excess acetylcholine inhibited the larval response to light. Considering atropine-treated larvae basically did not testify frontwards swimming, they did not reply to light. The nicotinic acetylcholine inhibitor d-tubocurarine did not inhibit the larval response to light input. (B) Superimposed images of 45 sec of swimming behaviors of sea urchin larvae (control [water-], atropine-, and scopolamine-treated). (C) The graph shows the distance of larval swimming shown in (C). Water-treated larvae (n = xx) could swim significantly longer distances than atropine-treated (n = 20) or scopolamine-treated (n = 14) larvae. *, p≤0.05, ****, p≤0.0001, Due north.S., not significant. (D) In situ hybridization for mAChR. mRNA of mAChR was expressed in the anterior regions of the larvae. The ciliary band region and posterior end (asterisks) had no stiff expression.

https://doi.org/ten.1371/journal.pgen.1010033.s004

(PDF)

Acknowledgments

We thank Y. Nakajima and R.D. Burke for the essential reagents. Nosotros thank Chiliad. Kiyomoto, T. Sato, D. Shibata, M. Ooue, T. Kodaka, J. Takano, C. Nakamura, M. Yamaguchi, and JF Izu/Shimoda for collecting and keeping the developed sea urchins.

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