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.

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 Ca 2+ dynamics from pre-to post-metamorphosis. Ca 2+ 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 Ca 2+ transient in the papillae (adhesive organ of the anterior trunk) was coupled with the Ca 2+ transient in dorsally localized cells and endoderm cells just after mechanical stimulation. The Ca 2+ transients in Phase I were also observed when applying only short stimulation. In the second phase, Phase II, the Ca 2+ transient in papillae was observed again and lasted for approximately 5-11 min just after the Ca 2+ transient in Phase I continued for a few minutes. The impaired papillae by Foxg-knockdown failed to induce the second Ca 2+ transient in Phase II and tail regression. In Phase II, a wave-like Ca 2+ propagation was also observed across the entire epidermis. Our results indicate that the papillae sense a mechanical cue and tworound Ca 2+ 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.
© 2021 The Author(s) Published by the Royal Society. All rights reserved.
Ascidian papillae form a transient sensory adhesive organ that serves to attach the larva to a substrate, thereby ensuring settlement and the onset of metamorphosis into the filterfeeding adult [20]. After the papillae-mediated adhesion to a substrate, ascidian metamorphosis is characterized by tail regression [7,21].
Papillae seem to permit a larva to assess a substrate's suitability for settlement and metamorphosis. Papillae-mediated larval adhesion is essential for tail regression and tail regression is reportedly abolished in papillae-cut larva [7]. In the larval stage, the ascidian Ciona has three papillae. Foxg is expressed at the papillae under the ERK pathway [22]. Each papilla contains four glutamatergic neurons [8], which are considered sensory neurons [23,24]. Although papillary sensory neurons have been proposed to have both chemosensory and mechanosensory functions [25,26], there is no direct evidence that papillae can perceive external cues.
We have previously reported the Ca 2+ dynamics of Ciona embryo from gastrulation to the tailbud stages [27], but Ca 2+ dynamics in later developmental stages have not been reported before. Here, we present direct evidence of mechanical cues to the papillae playing a role in initiating Ciona metamorphosis via two-round Ca 2+ transients.

Material and methods (a) Samples
Ciona adults were obtained from Maizuru Fisheries Research Station (Kyoto University), Misaki Marine Biological Station (University of Tokyo) through the National Bioresource Project (NBRP), and Onagawa Field Center (Tohoku University), Japan. Eggs were collected by dissecting gonoducts. Fertilized eggs were incubated at 18°C until observation. As Ciona larvae acquire competence of tail regression after 29.5 h post-fertilization (hpf ) [28], we opted to use the stage 29 larvae [29] after 30 hpf.

(b) Preparation of reporter constructs and microinjection and pharmacological treatment
GCaMP6s mRNA was obtained as previously described [27] (see electronic supplementary material).

(c) Fixation of swimming larva and artificial papillae stimulation
The larvae were fixed to a Petri dish coated with poly-Dlysine (PDL) or poly-L-lysine (PLL), referring to practical tips for imaging ascidian embryos [30] (see electronic supplementary material).

(d) Artificial mechanical stimulation of papillae
After establishing the trunk fixation method, we designed a device that consists of a mechanical stimulator, its manipulator, and a holder to apply mechanical stimuli artificially to the papillae of Ciona larva. We called this device an 'artificial papillae stimulator' (see electronic supplementary material).

(e) Microscopy
Samples were observed with three different microscopy methods, namely fluorescence microscopy (FM) with a 3CCD camera, confocal laser scanning microscopy (CLSM), and light-sheet microscopy (LSM). For imaging by FM, we followed previous methods [27] (see electronic supplementary material). For LSM, we employed dual inverted selective plane illumination microscopy (diSPIM) with 40 x water immersion lens (NA 0.8, Nikon) mounted on objective piezos with fibre-coupled digital micro-mirror scanners (Applied Scientific Instrumentation, USA) for light-sheet generation. Bidirectional stack images measuring 512 × 512 pixels were acquired by an sCMOS camera (Zyla 4.2, Andor) at a time interval of 7 s using dispim plugin running on ImageJ Micromanager. Images from two directions were registered and fused using MIPAV (NIH) generate fusion plugin.

Results
(a) Artificial induction of tail regression by a new experimental system At first, we verified that the Ca 2+ indicator GCaMP6 s can sense Ca 2+ dynamics even at later developmental stages (electronic supplementary material, figure S1, stage 36 [29], late body axis rotation period; see electronic supplementary material). To observe Ca 2+ dynamics in larva up to the tail regression stage controlling the timing of adhesion, we established a new experimental system for applying artificial mechanical stimulation to the papillae of an individual swimming larva [31]. The larval trunk is immobilized using a PDL-coated glass base to avoid vibration caused by swimming (figure 1a). In the absence of stimulation, 94.6% of immobilized larva retained the tail after 29.5 hpf. At this time, Ciona larvae can undergo tail regression if they receive adhesive stimulation (figure 1b,c). By contrast, when the artificial mechanical stimulus was applied to immobilized larvae (figure 1d), tail regression was induced in 83% of 12 larvae (figure 1e). These results indicate that trunk immobilization prevented tail regression and that our new experimental system can induce tail regression by controlling the timing of mechanical stimulation. Interestingly, we found that posterior trunk epidermal cells moved towards the posterior at the onset of tail regression (electronic supplementary material, figure S2; Video S1; Video S2; Video S3 from 0:09:41.360), which was the first observable change in the initiation of Ciona metamorphosis earlier than the onset of tail regression. Thus, we defined this backward movement of the posterior trunk epidermis as the timing of the start of tail regression, and we could align the time axis for metamorphosis among individuals, thereby linking each developmental stage.
(b) Two-round Ca 2+ transients in papillae were observed prior to tail regression  After Phase I, Phase II including the second Ca 2+ transient in papillae without coupling with the endoderm was observed. The second Ca 2+ transient reached a peak at 3.2 ± 1.0 min after the maximum peak of the first Ca 2+ transient (electronic supplementary material, table S1, c). It took 5.3 ± 0.8 min from the peak to stable state (electronic supplementary material, table S1, d). After 8.9 ± 3.3 min lasting of the second Ca 2+ transient, larvae started tail regression (electronic supplementary material, table S1, f). During Phase II, various tissues, including the epidermis and epidermal sensory neurons, as well as CNS, endoderm, and papillae, exhibited increased Ca 2+ activity (see electronic supplementary material, Video S3). In particular, wave-like propagations by strong Ca 2+ increases were observed at whole epidermal cells (n = 6/6, figure 2f; see electronic supplementary material, Video S3, time point 0:03:17-0:06:21) before tail regression. Compared with before tail regression, the Ca 2+ activity of the extraembryonic region located in the larval tunic, including motile test cells and extracellular ciliated structure, ASNET [32,33], increased. royalsocietypublishing.org/journal/rspb Proc. R. Soc. B 288: 20203207 As two-round Ca 2+ transients (Phase I and Phase II) at the papillae were observed in all tail-regressed larvae (     (figure 4a,c). The second Ca 2+ transient did not occur in the absence of stimulation (figure 4a and table 1, column of 0 s). By contrast, all larvae that underwent metamorphosis displayed the second Ca 2+ transient (figure 4c and table 1, column of continuous). Phase II occurred only when tail regression was observed. We therefore propose that the second Ca 2+ transient of papillae is essential for tail regression.
Second, to determine if the second Ca 2+ transient specifically occurs when the larva is stimulated for a sufficiently long duration to induce tail regression, we applied 10 s of continuous stimulation after 29.5 hpf (figure 4b,c). Following brief stimulation, the Ca 2+ transients in Phase I was observed but the Ca 2+ transient in Phase II and tail regression did not occur (figure 4b and table 1, column for 10 s stimulus). By contrast, all larvae subject to longer stimulation metamorphosed and showed Ca 2+ transient in both Phase I and Phase II ( figure 4c and table 1 (n = 6)). Since the second Ca 2+ transient was observed only when continuous stimulation was applied, continuous stimulation (12 min average from adhesion to start of tail regression in this study) appears to induce the second Ca 2+ transient. Moreover, only larvae that showed the second Ca 2+ transient began tail regression (table 1) suggesting that the second Ca 2+ transient induces tail regression. These results suggest that Ca 2+ transients in Phase II are essential for the start of tail regression.
In addition, to clarify how the papillae differentiation is associated with the induction of Ca 2+ transient in Phase I and Phase II and subsequent tail regression, we examined the dynamics of Ca 2+ transient and tail regression in Foxgknockdown larva. It has been reported that Foxg is expressed in larval papillae where it functions to specify the papillae as sensory neurons [22]. During embryogenesis, Foxg expression in neural plate cells is controlled by the mitogen-activated protein kinase (MAPK)/ERK. In Foxg-knockdown larva, short Ca 2+ transients were observed at the anterior trunk epidermis under continuous stimulation. However, neither the second Ca 2+ transient in Phase II nor tail regression was observed ( figure 4d and table 1). This result suggests that Foxg-specified sensory neurons are required for the generation of the second Ca 2+ transient and tail regression. Comparing these locations with our phalloidin staining results (figure 5b), the Ca 2+ transient in the papillae include the more posterior part called the preoral lobe (figure 5b, orange). The dorsal subregion corresponded to cells located dorsally above the neck region of the central nervous system and the epidermal region (figure 5b, white arrowheads) [23,32,33,37]. The Ca 2+ transient in the endodermal region (figure 2) was identified as endoderm surrounding the anterior tip of the notochord (figure 5b, blue). Because this is the first report of a Ca 2+ transient being observed in the endoderm at the beginning of metamorphosis, we focused on the region expressing the Ca 2+ transient in response to the mechanical cue. To derive more anatomical information about this region, we performed in vivo Ca 2+ imaging using LSM. Consistent with our phalloidin staining results, a simultaneous Ca 2+ transient was observed in both the papillae and the endodermal cells contacting the anterior part of the notochord This region was identified as the future digestive tract, in agreement with a previous study that identified the same region as the future digestive tract [38]. These results indicate that the papillary Ca 2+ transient was coupled with that of the dorsally localized cells in the posterior trunk and the primordial digestive tract in Phase I.

Discussion
How larvae perceive environmental cues, such as mechanical stimuli, chemical ligands, temperature, and light, before triggering metamorphosis had evaded researchers until now.
In this study, we revealed novel insights about the role of Ca 2+ transients in ascidian to link external cues to inner signals that control metamorphosis through precise timing and targeting of specific tissues.
We have identified a two-round Ca 2+ transient in the adhesive papillae that respond to mechanical cues to initiate metamorphosis in Ciona larvae. The observed Ca 2+ transients resulting from mechanical stimulation up until tail regression (electronic supplementary material, Video S3) are summarized as follows ( figure 6).  Phase I was strongly coupled to mechanosensation (figure 3, figure 4 and table 1) and Phase II was coupled to tail regression ( figure 4c and table 1). We considered that the two rounds of Ca 2+ transients in these phases were essential for Ciona tail regression during metamorphosis. According to a previous study, at least 28 min of adhesion is necessary for tail regression, and individuals more than 29.5 hpf start tail regression after an average adhesion time of 32 min [28]. By contrast, five out of six larvae start tail regression less than 28 min after adhesion in our experiments. The average time between adhesion and tail regression was 12 ± 4.3 min. These different response times may be due to differences in the substrate or in the strength of adhesion.
Is there a causal connection between Phase I and Phase II? We consider there is a temporal threshold of Ca 2+ in Phase I that is prerequisite for activation of Phase II. From our results, less than 10 s stimulation induced only Phase I whereas an average of 12 min continuous stimulation induced both Phase I and Phase II Ca 2+ transients. In our experimental system, Phase II is only observed after Phase I occurs (table 1). Therefore, we think Phases I and II are tightly coupled also in the natural condition. However, inhibition of the specification of the papilla sensory neurons by Foxg MO decoupled them (figure 4d). Further studies of papillary sensory neurons will provide a better understanding of the mechanisms that will cause Phase II.

(a) Papillary cells activated by mechanical stimulation
Our results suggest that the papillae include mechanically sensitive sensory cells. There are three types of cells in the papillae, namely four axial columnar cells (ACC), four lateral primary sensory neurons (PNS), and 12 central collocytes (CC). Their nuclei are in posterior papillae processes and tissues that make up the anterior trunk [8]. The mechanism      royalsocietypublishing.org/journal/rspb Proc. R. Soc. B 288: 20203207 metamorphosis. One of the surprising results in our study is the Ca 2+ transient in endodermal cells surrounding the notochord in response to mechanical stimulation (figure 3b). Ca 2+ transient has not been observed in the endoderm from gastrulation to the tailbud stage in previous studies [27]. Since this endodermal region differentiates into the digestive tract during metamorphosis, the Ca 2+ transient in the endoderm may be related to the promotion of digestive tract differentiation. It is also interesting that dorsally localized cells in the posterior trunk responded during Phase I (figure 2 and figure 5, white arrowhead). The dorsally localized cells are located beneath the epidermis and close to the central nervous system (CNS). Although these cells are unknown, they do not appear to be identical to posterior apical trunk epidermal neuron ( pATEN) nor anterior apical trunk epidermal neuron (aATEN) because the cells are more posterior (figure 5b). During Phase II, wave-like Ca 2+ propagations were observed in cells across the entire epidermis (figure 2f; electronic supplementary material, Video S3). This shows the direct evidence of metamorphic signals spread through epithelial conduction of Ca 2+ . A similar epithelial-conduction model of metamorphic signal propagation has been proposed for the hydrozoan cnidarian Mitrocomella polydiademata [15]. Our method developed in this study can be applied to other species to test whether the Ca 2+ transients that cause metamorphosis are evolutionarily conserved in other marine invertebrates.
Considering the backward movement of the epidermis that occurred following the Ca 2+ propagations, these Ca 2+ propagations might increase the tension within the entire epidermis to generate the pulling force required for tail regression. The tail epidermis has been proposed to generate a sufficiently strong force to absorb the axial organs into the trunk region [39] during tail regression. It was also confirmed that tail regression is inhibited by using cytochalasin B, indicating that actin fibres play an important role in tail regression [40]. Another hypothesis of the tail epidermis contraction to explain the tail regression during ascidian metamorphosis is based on apoptosis [21,41,42]. Krasovec et al. [41] reported that the tail regression depends on a postero-anterior wave of a caspase-dependent apoptosis coupled with a contraction event. This apoptosis wave might be triggered by the Phase II wave-like Ca 2+ propagations in the epidermis.
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