Author response: Ciliary and rhabdomeric photoreceptor-cell circuits form a spectral depth gauge in marine zooplankton
2018-05-16

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Ciliary and rhabdomeric photoreceptor cells represent two main lines of photoreceptor-cell evolution in animals. The two cell types coexist in some animals, however how these cells functionally integrate is unknown. We used connectomics to map synaptic paths between ciliary and rhabdomeric photoreceptors in the planktonic larva of the annelid Platynereis and found that ciliary photoreceptors are presynaptic to the rhabdomeric circuit. The behaviors mediated by the ciliary and rhabdomeric cells also interact hierarchically. The ciliary photoreceptors are UV-sensitive and mediate downward swimming in non-directional UV light, a behavior absent in ciliary-opsin knockout larvae. UV avoidance overrides positive phototaxis mediated by the rhabdomeric eyes such that vertical swimming direction is determined by the ratio of blue/UV light. Since this ratio increases with depth, Platynereis larvae may use it as a depth gauge during vertical migration. Our results revealed a functional integration of ciliary and rhabdomeric photoreceptor cells in a zooplankton larva. https://doi.org/10.7554/eLife.36440.001 eLife digest The animal kingdom contains many different types of eyes, but all share certain features in common. All detect light using specialized cells called photoreceptors, of which there are two main kinds: ciliary and rhabdomeric. Crustaceans and their relatives, including insects, have rhabdomeric photoreceptors; while animals with backbones, including humans, have ciliary photoreceptors. There are also several groups of animals, mostly sea-dwellers, that inherited both types of photoreceptors from their ancestors that lived millions of years ago. These include the marine ragworm, Platynereis dumerilii. The larvae of Platynereis are free-swimming plankton. Each has a transparent brain and six small, pigmented eyes. The eyes contain rhabdomeric photoreceptors. These enable the larvae to detect and swim towards light sources. Yet the larval brain also contains ciliary photoreceptors, the role of which was unknown. Verasztó, Gühmann et al. now show that ultraviolet light activates ciliary photoreceptors, whereas cyan, or blue-green, light inhibits them. Shining ultraviolet light onto Platynereis larvae makes the larvae swim downwards. By contrast, cyan light makes the larvae swim upwards. In the ocean, ultraviolet light is most intense near the surface, while cyan light reaches greater depths. Ciliary photoreceptors thus help Platynereis to avoid harmful ultraviolet radiation near the surface. Though if the larvae swim too deep, cyan light inhibits the ciliary photoreceptors and activates the rhabdomeric pigmented eyes. This makes the larvae swim upwards again. Using high-powered microscopy, Verasztó, Gühmann et al. confirm that neural circuits containing ciliary photoreceptors exchange messages with circuits containing rhabdomeric photoreceptors. This suggests that the two work together to form a depth gauge. By enabling the larvae to swim at a preferred depth, the depth gauge influences where the worms end up as adults. Its discovery should also stimulate new ideas about the evolution of eyes and photoreceptors. https://doi.org/10.7554/eLife.36440.002 Introduction Bilaterian animals have two major photoreceptor cell-types, the rhabdomeric- and the ciliary-type photoreceptor cells (rPRC and cPRC, respectively) (Arendt, 2003; Arendt et al., 2004; Eakin, 1979; Erclik et al., 2009). These cells have distinct morphologies and express different classes of opsins (light-sensitive proteins). Rhabdomeric PRCs have apical microvillar specializations (rhabdom) that store the opsin photopigments. The visual photoreceptor cells in most protostome eyes, including the compound eyes of arthropods, the pigment-cup eyes of annelids and the camera or stalk eyes of mollusks, are rhabdomeric and express rhadomeric (r-) opsins (Arendt et al., 2002; Cowman et al., 1986; Katagiri et al., 2001; Katagiri et al., 1995; Ovchinnikov et al., 1988; Pollock and Benzer, 1988; Randel et al., 2013). Rhabdomeric PRCs also exist in the pigmented eyespots of both protostomes (e.g. annelids, flatworms) and some non-vertebrate deuterostomes (hemichordates, cephalochordates) (Arendt and Wittbrodt, 2001; Braun et al., 2015; Nakao, 1964). In contrast, the visual eyes of vertebrates have cPRCs (rods and cones) where the ciliary (c-) opsin photopigment is stored in specialized ciliary membrane compartments (Jan and Revel, 1974; Nir et al., 1984). Ciliary PRCs also occur in some invertebrates, where they can be part of pigmented eyespots (e.g. some mollusks and flatworms), in simple eyes (e.g. some nemerteans), or as brain photoreceptors not associated with pigment cells (e.g. some annelids) (Arendt et al., 2004; Barber et al., 1967; Döhren and Bartolomaeus, 2018; Randel and Jékely, 2016). The class of opsin expressed in a PRC generally correlates with the cell’s morphological type, with cPRCs usually expressing c-opsins and rPRCs expressing r-opsins (Arendt, 2003; Arendt et al., 2004; Randel et al., 2013; Vopalensky et al., 2012). However some exceptions to this rule are known, such as the occasional coexpression of melanopsin (an r-opsin) with c-opsins or r-opsins with Go- or xenopsins (Davies et al., 2011; Gühmann et al., 2015; Vöcking et al., 2017). Given their broad phylogenetic distribution and shared opsin expression, both photoreceptor cell types likely coexisted in the last common ancestor of bilaterians. The two cell types still coexist in several marine animals, including cephalochordates, some mollusks, flatworms, and annelids (Arendt et al., 2004; McReynolds and Gorman, 1970; Randel and Jékely, 2016; Vopalensky et al., 2012) and form parts of pigmented or non-pigmented light-sensitive structures. Understanding how the two photoreceptor cell types integrate at the functional and circuit levels in these animals will help to clarify the history of eyes and photoreceptor cells. Here we study the planktonic larva of Platynereis dumerilii, a marine annelid that has both photoreceptor cell types. In Platynereis, non-pigmented brain cPRCs with ramified cilia express a ciliary type opsin (c-opsin1) (Arendt et al., 2004) and coexist with r-opsin-expressing rPRCs that are part of the pigmented visual eyes (adult eyes) and eyespots (Arendt et al., 2002; Jékely et al., 2008; Randel et al., 2014, 2013). The pigmented larval eyespots and the adult eyes mediate early- and late-stage larval phototaxis, respectively (Gühmann et al., 2015; Jékely et al., 2008; Randel et al., 2014). The mechanism and neuronal circuitry of both early- and late-stage larval phototaxis is well understood. Trochophore larvae (approximately 1–2.5 days post fertilization) have a pair of pigmented eyespots with a rPRC that directly innervates the adjacent ciliary band. This rPRC is cholinergic and expresses r-opsin3 (Jékely et al., 2008; Randel et al., 2013). When the rPRC is activated during helical swimming, the ciliary beating changes on the illuminated side, so that the larva reorients its trajectory towards the light source. Nectochaete larvae (approximately 3–5 days post fertilization) develop two pairs of adult eyes with several rPRCs coexpressing r-opsin1, r-opsin3 and Go-opsin (Gühmann et al., 2015; Randel et al., 2013). These eyes mediate visual phototaxis by comparing the intensity of light on the two sides of the body. The adult eyes and their downstream neuronal circuitry regulate the contraction of the longitudinal muscles of the trunk to steer the larva towards or away from a light source (Randel et al., 2014). The function of cPRCs in Platynereis is much less clear. These and the surrounding cells (the ‘cPRC region’) have been proposed to produce melatonin and to entrain the circadian clock (Arendt et al., 2004; Tosches et al., 2014). C-opsin1 was recently shown to absorb UV light. This suggests that the c-opsin1-expressing cPRCs mediate circadian entrainment by ambient UV light (Tsukamoto et al., 2017). However, the precise function of the cPRCs in Platynereis larvae and how they interact with rPRCs is still unknown. Results Synaptic pathways between rhabdomeric and ciliary photoreceptors To identify synaptic connections between the cPRC and rPRC circuits, we used a serial-section transmission electron microscopy (ssTEM) dataset spanning the entire body of a 72 hr post fertilization (hpf) Platynereis larva (Randel et al., 2015). Previously, we reported the synaptic connectome of the rPRCs from the visual eyes and eyespots, (Randel et al., 2015, 2014) and the direct postsynaptic circuit of the four cPRCs with ramified cilia (Williams et al., 2017a). Briefly, the glutamatergic rPRCs of the adult eyes connect through three layers of interneurons to cholinergic motoneurons in the ventral head (vMN) that innervate longitudinal trunk muscles. The main premotor interneurons in this circuit are the Schnörkel interneurons (INsn) (Figure 1C,G) (Randel et al., 2015; Randel et al., 2014). The rPRCs of the eyespot synapse directly on the ciliary band and the vMNs (Figure 1G) (Jékely et al., 2008; Randel et al., 2015, 2013). The four cholinergic cPRCs send axons to the neurosecretory plexus in the anterior nervous system and synapse on four peptidergic/cholinergic RGW interneurons (labelled INRGW, and named after the expression of the RGWamide neuropeptide [Shahidi et al., 2015; Williams et al., 2017]) and four NOS interneurons (labelled INNOS, and named after the expression of the nitric oxide synthase (NOS) gene; unpublished). The cPRCs also synapse on a group of flask-shaped sensory-neurosecretory neurons that are part of the neurosecretory anterior nervous system. These cells express diverse neuropeptides but have no postsynaptic partners (Williams et al., 2017). The RGW cells synapse on two serotonergic cells (Ser-h1) that, together with their postsynaptic partner, the cholinergic MC neuron, are part of the ciliomotor circuitry of the larva (Verasztó et al., 2017). Figure 1 with 2 supplements see all Download asset Open asset Wiring diagram of cPRC and rPRC circuits in the Platynereis larval head. (A) All cells of the cPRC and rPRC circuits in the larva (in color). The body outline is shown in grey. (B) All cells of the cPRC and rPRC circuits (in color), anterior view. All other neurons are shown in grey. Circuits were reconstructed from a whole-body ssTEM volume of a 72 hpf larva. (C) All neurons of the rPRC circuit. (D) The four cPRCs and all neurons directly postsynaptic to them. (E) Connections between the cPRC and rPRC circuits. Top panel: the four RGW interneurons (INRGW) are presynaptic to the INsn cells. Bottom panel: the six INpreMN cells and the two MS cells are presynaptic to the ventral motoneurons. (F) Grouped connectivity matrix of the cPRC and rPRC circuits. The connections from the cPRC to the rPRC circuit are outlined in red. (G) Wiring diagram of the cPRC and rPRC circuits. Nodes represent groups of neurons (number indicated in square brackets), arrows represent synaptic connections. Synaptic connections from the cPRC to the rPRC circuit are in red. Edge width is a function of log synaptic count. https://doi.org/10.7554/eLife.36440.003 Figure 1—source data 1 Grouped connectivity matrix of the cPRC and rPRC circuits. https://doi.org/10.7554/eLife.36440.006 Download elife-36440-fig1-data1-v2.txt Figure 1—source data 2 Full ungrouped connectivity matrix of the cPRC and rPRC circuits. https://doi.org/10.7554/eLife.36440.007 Download elife-36440-fig1-data2-v2.xls To analyze the possible synaptic integration of the rPRC and cPRC circuits, we searched for all synaptic connections between neurons of these circuits (Figure 1) in a synapse-level skeleton reconstruction of all cells in the larval head (2359 cells of which approximately 1230 are neurons; unpublished data). We did a systematic network search in Catmaid (Schneider-Mizell et al., 2016) for all possible synaptic paths (two hops) between the rPRC or the cPRC circuit. We identified three sites of contact. First, the RGW interneurons synapse on the Schnörkel premotor interneurons (INsn)(Figure 1E–G and Video 1). Second, we identified six interneurons (INpreMN) that are postsynaptic to the RGW interneurons and presynaptic to the ventral motoneurons (vMNs) of the visual circuit (Figure 1E–G). Third, two putative mechanosensory neurons bearing a sensory cilium and a collar of microvilli and located in the median head (MS1 and MS2)(Bezares-Calderon et al., 2018) are postsynaptic to the RGW cells and presynaptic to the vMNs and INpro interneurons of the visual circuit (Figure 1E–G and Video 1). Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Wiring diagram of cPRC and rPRC circuits in the Platynereis larval head. The anatomy of the reconstructed neurons is shown together with the position of the same cells in the wiring diagram. https://doi.org/10.7554/eLife.36440.008 Our graph search did not reveal any neurons that were directly postsynaptic to the rPRC circuit (from rPRC to vMN) and presynaptic to any neuron of the cPRC circuit. Thus, the cPRC circuit feeds hierarchically into the visual rPRC circuit (Figure 1G). This suggests that the cPRCs could influence phototaxis, a behavior mediated by the rhabdomeric eyes and eyespots (Gühmann et al., 2015; Jékely et al., 2008; Randel et al., 2014). Acute UV-violet sensitivity of the ciliary photoreceptors in Platynereis What is the role of cPRCs in larval behavior and how do they influence phototaxis? First, to estimate the light sensitivity of the cPRCs, we reconstructed the morphology with ssTEM and measured the total sensory membrane surface-area of a cPRC (Figure 2A–D). Each cPRC has 12–15 basal bodies each with a root and an extensively branched sensory cilium. The branches are supported by single microtubule doublets (Figure 2—figure supplement 3). Based on the average diameter and total length of all branches in one cPRC we estimated a membrane area of 276 μm2, which is approximately 10 times smaller than the total disk membrane surface area of rat rods (Mayhew and Astle, 1997). This suggests that Platynereis cPRCs are sensitive enough to mediate acute light sensation. Figure 2 with 3 supplements see all Download asset Open asset Light responses of brain ciliary photoreceptors and their downstream circuitry in Platynereis larvae. (A) Acetylated tubulin staining of a 72 hpf larva. The ramified sensory cilia of cPRCs are marked with dashed lines. (B) TEM image of a section with a cPRC. Cell body in green, sensory cilia outlined in dashed white. (C) TEM image of a cPRC with sensory cilia. Yellow arrows mark the basal bodies of a cPRC. (D) Serial TEM reconstruction of the sensory cilia of a cPRC. (E) Absorption spectrum of purified Platynereis c-opsin1. Inset: dark-light difference spectrum. (F) Top: high GCaMP6s signal in the cPRCs during imaging conditions. Asterisks mark cPRC nuclei. Bottom: activation of two sensory neurons (SNearly) upon violet stimulation of cPRCs. (G) Top: representative example of cPRC response to prolonged local 488 nm and 405 nm stimulation. The colored boxes show the duration of the stimulation. Bottom: average cPRC response during continuous 488 nm and 405 nm stimulation. Data show mean and s.d. of mean, 488 nm N = 8, 405 nm N> 30. (H) Responses of a cPRC to repeated 405 nm and 488 nm (duration: 20 s) stimulation. (I) Responses of SNearly and SNlate sensory neurons to cPRC 405 nm stimulation. Correlation images are shown for SNearly and SNlate. Asterisks mark cPRC nuclei. (J) Responses of RGW cells to UV stimulation of a cPRC. Scale bars: (A) 50 μm (B) 10 μm, (F) 20 μm. https://doi.org/10.7554/eLife.36440.009 Figure 2—source data 1 Light and dark-light difference spectrum of Platynereis c-opsin1 and calcium imaging traces for panels G-J. https://doi.org/10.7554/eLife.36440.014 Download elife-36440-fig2-data1-v2.zip Next, we expressed Platynereis c-opsin1 in COS1 cells, reconstituted it with 11-cis-retinal and purified it. The reconstituted pigment absorbed in the UV range with a λ-max of 384 nm in the dark spectrum and a λ-max of 370 nm in the dark-light difference spectrum (Figure 2E), in agreement with a recent report (Tsukamoto et al., 2017). To investigate how the cPRCs respond to light, we did calcium imaging with larvae ubiquitously expressing the calcium sensor GCaMP6s (Chen et al., 2013; Verasztó et al., 2017). When imaged with a low-intensity 488 nm laser, the cPRCs had a high resting calcium level and the GCaMP6s signal highlighted their sensory cilia. We could thus identify the four cPRCs without stimulation (Figure 2F). When the cPRC cilia were locally stimulated (Figure 2—figure supplement 1) for 5 min with 405 nm light (140–250 times more photons on the sensory cilia than by the imaging laser), the calcium level dropped transiently at the cPRC somata and then increased strongly. This indicates transient cPRC hyperpolarization followed by depolarization. However, when the cPRCs were stimulated with a 488 nm laser of equal photon flux, they showed prolonged hyperpolarization only, without depolarization (Figure 2G). When the 405 nm stimulation was switched off during the hyperpolarization phase (after 20 s), the cPRCs still depolarized afterwards (Figure 2H). UV-violet-specific activation of neurons postsynaptic to the ciliary photoreceptors 405 nm stimulation of the cPRCs also changed the activity of other neurons in the larval brain. Four neurons followed the activity pattern of the cPRCs (Figure 2J; Figure 2—figure supplement 2). These cells correspond by position to the four RGW interneurons, which together with the four NOS interneurons are direct postsynaptic targets of the cPRCs (Figure 1G)(Williams et al., 2017a). Additionally, two flask-shaped sensory neurons (SNearly) in the middle of the anterior nervous system depolarized upon stimulus onset. Two further sensory cells (SNlate) flanking the SNearly cells depolarized later, in parallel with the rising phase of cPRC activity (Figure 2I). These four SN cells correspond by position to four sensory-neurosecretory neurons that are postsynaptic to the cPRCs in the anterior nervous system (SNMIP1l, SNASTC1r and two SNNS20 cells)(Figure 1D)(Williams et al., 2017a). We next compared the responses of the RGW interneurons and the four SN neurons to 405 and 488 nm stimulation of the cPRCs. The RGW interneurons and the SNearly sensory neurons only responded to 405 nm stimulation (with opposite sign) but not to 488 nm stimulation. The SNlate neurons in some larvae also responded to 488 nm stimulation but these responses were weaker (Figure 2—figure supplement 2). Thus, the cPRCs and their postsynaptic neurons respond differentially to violet and blue stimulation, with only violet light inducing cPRC depolarization and consistent changes in the activity of postsynaptic neurons. UV-violet avoidance behavior in Platynereis larvae To characterize how Platynereis larvae react to UV-violet light, we assayed larval swimming behavior in a vertical column setup. Since Platynereis larvae show strong directional phototaxis to a broad spectrum of light (between 380–540 nm)(Gühmann et al., 2015; Jékely et al., 2008), we illuminated the setup equally from two opposite sides with non-directional UV light so that the larvae could not respond with directional phototaxis (Figure 3A). Figure 3 Download asset Open asset UV-violet avoidance and phototaxis form a ratio-chromatic depth gauge in Platynereis larvae. (A) Larval trajectories recorded in a vertical column in the dark (left) and under illumination with UV light from the side (right). (B) Action spectrum of non-directional light avoidance in 48 hpf larvae (n = 20 batches). (C) Developmental onset of UV-avoidance behavior and phototaxis in Platynereis larvae (n > 7 batches for each stage). (D) Time course (30 s bins) of vertical swimming in different larval stages following 380 nm illumination from above (n > 11 batches for each stage). (E) Action spectrum of vertical swimming in early- and late-stage larvae, under stimulus light coming from the top of the column. The responses between 1.5–3.5 min after stimulus onset are shown (n > 7 batches for each stage). (F) Repeated switching between upward and downward swimming in different larval stages under 380 and 520 nm stimulus light coming from above. The responses between 1.5–4.5 min after stimulus onset are shown (n = 4 batches for 53 hpf, >11 for the other stages). (G) Vertical displacement of 3-day-old larvae swimming in a column and stimulated from above with different ratios of 380/480 nm or nm light (n > 7 batches for each with = were are indicated Figure data 1 data of Figure Download When the larvae were stimulated with non-directional UV light, they to swim To characterize the of this we assayed larvae in a vertical and stimulated with light of different from two The larvae to UV-violet light (between but not to Figure This downward swimming UV-avoidance behavior to non-directional UV-violet light has not been reported in The that UV-avoidance can be by non-directional light and has an spectrum that the spectrum of c-opsin1 (Figure that the response is mediated by the non-pigmented cPRCs. the cPRCs mediate then the onset of this response should with the morphological of cPRCs. To we assayed UV-avoidance nm light from the as well as phototaxis nm light from the at different larval stages (Figure and the behaviors to photoreceptor but not UV-avoidance was at hpf, at a when the larval eyespots are functional (Jékely et al., UV avoidance at hpf, approximately with the morphological of cPRCs (after hpf, as by the of the ramified but the of the adult eyes 72 et al., Jékely et al., 2008; Randel et al., 2014). Thus, Platynereis larvae show avoidance that is of phototaxis, can be by non-directional stimulus light, and is likely mediated by the non-pigmented cPRCs. UV avoidance and phototaxis behaviors form a To study how UV avoidance with phototaxis, we stimulated the larvae in the vertical column with directional light from above. When we used 380 nm stimulus light, both early- and late-stage and larvae upward towards the light approximately one and then downward (Figure Video 2). This upward phase was not when the larvae were illuminated from the side not These results that the phase is phototaxis, which is then by the UV-avoidance Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg responses of Platynereis larvae to 380 nm illumination from above. The square in the top when the UV stimulus light was switched The are on direction The Video is up To the of this we measured larval responses to different of directional light coming from the top of the vertical column. We responses 1.5–3.5 min after stimulus onset to on the phase when UV-avoidance has phototaxis (Figure In response to illumination with nm light, larvae downward after prolonged stimulation. In response to illumination with nm light or nm light larvae upward (Figure nm light did not the vertical displacement of the larvae. The swimming direction could be switched several times by the of the light from 380 nm to 520 nm (Figure that this does not after to light. These results that directional UV-violet light phototaxis (the pigmented eyes are sensitive in the UV-violet range et al., 2008; Gühmann et al., which is then by the UV-avoidance response likely mediated by the cPRCs. light only the switching in response cannot be as a between positive and phototaxis, larvae are (Jékely et al., 72 hpf et al., show the in swimming direction (Figure we if the phototaxis and UV-avoidance behaviors could form a mechanism to depth by the larvae. Since blue and UV-violet light in the ratio of blue to UV-violet light increases with depth data from the where Platynereis were shown et al., UV-violet light at the surface is to larvae to swim downward as an avoidance and strong blue light in is to upward switching could form a ratio-chromatic 2009). To we larvae to light containing different photon ratios of UV and blue light coming from the top of the vertical column. high 380/480 larvae while at ratios larvae a 380/480 larvae at a average depth (Figure to a directional light the same intensity UV light as with nm light to which larvae do not respond at the intensity used (Figure did not upward swimming at any ratio (Figure This that a in UV intensity does not a in swimming These results that UV avoidance and phototaxis and each other under certain in no vertical This is consistent with the of a ratio-chromatic depth gauge in Platynereis larvae. c-opsin1 knockout larvae have UV-violet and avoidance behavior To c-opsin1 the UV-violet response in Platynereis larvae, we used a c-opsin1 Platynereis knockout by The the of c-opsin1 and an (Figure In larvae, the cPRCs had resting calcium level and depolarized upon 405 nm stimulation (Figure Thus, c-opsin1 in the Platynereis cPRCs is for an resting calcium level in the dark and for hyperpolarization and depolarization upon 405 nm The high resting calcium level in the cPRCs indicates a dark a of rods and et al., Figure 4 Download asset Open asset c-opsin1 knockout larvae UV-violet (A) of the Platynereis c-opsin1 and the (B) GCaMP6s signal during calcium imaging in type and larvae. (C) responses to s 405 nm stimulation in type and larvae (n > larvae for both The stimulus light was switched on at (D) Vertical swimming in type and larvae stimulated with blue and UV light from above (n = for both The data are shown in s with Figure data 1 data of Figure