The ontology of the anatomy and development of the solitary ascidian Ciona: the swimming larva and its metamorphosis

Ciona robusta (Ciona intestinalis type A), a model organism for biological studies, belongs to ascidians, the main class of tunicates, which are the closest relatives of vertebrates. In Ciona, a project on the ontology of both development and anatomy is ongoing for several years. Its goal is to standardize a resource relating each anatomical structure to developmental stages. Today, the ontology is codified until the hatching larva stage. Here, we present its extension throughout the swimming larva stages, the metamorphosis, until the juvenile stages. For standardizing the developmental ontology, we acquired different time-lapse movies, confocal microscope images and histological serial section images for each developmental event from the hatching larva stage (17.5 h post fertilization) to the juvenile stage (7 days post fertilization). Combining these data, we defined 12 new distinct developmental stages (from Stage 26 to Stage 37), in addition to the previously defined 26 stages, referred to embryonic development. The new stages were grouped into four Periods named: Adhesion, Tail Absorption, Body Axis Rotation, and Juvenile. To build the anatomical ontology, 203 anatomical entities were identified, defined according to the literature, and annotated, taking advantage from the high resolution and the complementary information obtained from confocal microscopy and histology. The ontology describes the anatomical entities in hierarchical levels, from the cell level (cell lineage) to the tissue/organ level. Comparing the number of entities during development, we found two rounds on entity increase: in addition to the one occurring after fertilization, there is a second one during the Body Axis Rotation Period, when juvenile structures appear. Vice versa, one-third of anatomical entities associated with the embryo/larval life were significantly reduced at the beginning of metamorphosis. Data was finally integrated within the web-based resource "TunicAnatO", which includes a number of anatomical images and a dictionary with synonyms. This ontology will allow the standardization of data underpinning an accurate annotation of gene expression and the comprehension of mechanisms of differentiation. It will help in understanding the emergence of elaborated structures during both embryogenesis and metamorphosis, shedding light on tissue degeneration and differentiation occurring at metamorphosis.

www.nature.com/scientificreports/ anatomical entities. An the anatomical entity, it constitutes the "structural organization" of an individual member of a biological species. At cellular level, "the structural organization" is easy to determine, as it is limited by the cell membrane. At structural level, an entity can be defined thanks to anatomical particularities (e.g. tail, trunk, atrial cavity) or to a specific function (e.g. heart, brain). Once an anatomical entity is recognized and defined in a hierarchical way (i.e., organized in an Anatomical Ontology, AO) and put in relationship with a developmental time-table specifying the developmental stage features (i.e., a Developmental Ontology, DO), it constitutes the basis upon which to build an Anatomical and Developmental Ontology (ADO). The latter is a powerful instrument to standardize different kinds of biological data and an irreplaceable tool associated with model species [1][2][3][4][5] . Among tunicates, the sister group of vertebrates 6,7 , the solitary ascidians Ciona intestinalis, is a recognized model species for evolutionary, developmental, and ecological studies [8][9][10][11] . Recently, it was shown that there were two cryptic species under the name C.intestinalis, called types A and B [12][13][14][15][16][17][18][19][20] . A taxonomic study 21 proposed to rename C.intestinalis type A as Ciona robusta, and C.intestinalis type B as C.intestinalis [21][22][23] . From an anatomical point of view, very few differences in adults 22 and in larvae 22 were reported between the two types. In addition, C. intestinalis type A (now C. robusta) and type B (now C. intestinalis) were used as indistinguishable models until 2015. Therefore, all anatomical entities used in this study are common to Ciona robusta (C. intestinalis type A) and Ciona intestinalis (type B), so here the term Ciona refers to both species.
In the ascidian larva, the typical chordate body plan can easily be recognized and studied: muscles for tail deflection during swimming flank a notochord; a hollow nerve cord is dorsal to the notochord, whereas an endodermal strand is ventral to it. This makes ascidians a privileged model for understanding the evolution of more complex vertebrates.
For Ciona, the ADO so far available regards 26 early developmental stages, from the unfertilized egg (Stage 0) to the hatching larva (Stage 26) 24 . This ontology is registered in the Bioportal web portal 25 .
Moreover, representative 3D morphological reconstructions and optic cross-section images implement the ontology and are available in the web-based database FABA (https ://www.bpni.bio.keio.ac.jp/chord ate/faba/1.4/ top.html). In FABA, information about cell lineages in early development was annotated based on previous investigations [26][27][28][29] . Considering that the ascidian embryogenesis is stereotyped, this ontology provides a standardized resource of spatial and temporal information for both C. robusta and C. intestinalis, as well as other solitary ascidians.
After larval hatching, ascidian larvae disperse, swimming freely and searching for a suitable substrate on which to metamorphose. The metamorphosis is deep and transforms the larva with the chordate body plan into a sessile, filter-feeding adult ( Supplementary Fig. S1) 30 . In the latter, the chordate body plan is no longer recognizable, even if some other chordate features, such as the pharyngeal fissures (the stigmata) and the endostyle in the ventral pharynx (homologous to the vertebrate thyroid gland) 31 , are now visible.
To cover these further developmental phases, we decided to extend the ADO to the post-hatching larva development and metamorphosis. To build up this new part of the ontology, we conducted an anatomical investigation based on complementary methods. The method of phalloidin-staining, successfully used for visualizing anatomical structures until the hatching larva stage 24 , unfortunately was revealed as less useful, as cells shrink as development proceeds, thereby becoming hardly recognizable. Moreover, in differentiated individuals, low actinbased structures, such as the tunic or pigment cells (otolith and ocellus), are difficult to recognize. Consequently, we produced a comprehensive collection of both confocal scanning laser microscopy (CLSM) of whole-mount specimens, and light microscope images of 1-µm-thick histological serial sections of whole samples, for each developmental stage. For histology, specimens were cut according to the classical planes: transverse, sagittal, and frontal. This allowed us to build a complete anatomical atlas and was necessary for collecting the morphological information related to internal organs as well as the body shape and external surface. Lastly, for each anatomical entity, we annotated its definition, carefully checking the literature since 1893 and considering, in particular, some milestones of ascidian literature, such as the exhaustive description of C. intestinalis published by Millar 32 . Because the same anatomical structure was sometimes called with different names by researchers in different periods or belonging to different biological fields, we also annotated synonyms.
All this information, together with stereomicroscopy time-lapse movies, are consultable in the web-based resource called TunicAnatO (Tunicate Anatomical and developmental Ontology) (https ://www.bpni.bio.keio. ac.jp/tunic anato /3.0/). TunicAnatO includes the former FABA database 24 , therefore covering, in total, 37 developmental stages of Ciona development, from the unfertilized egg to the juvenile. Features exhibited in the newly defined Stages 26 to 37 are described in Supplementary Data S2. TunicAnatO is also reachable via the Bioportal (https ://biopo rtal.bioon tolog y.org/), which is the most comprehensive repository of biomedical ontologies, and via the Tunicate Web Portal (https ://www.tunic ate-porta l.org/), which is the main web tool for the Tunicate Community.

Results
The DO and AO from the post-hatching larva stage to the Juvenile stage: working method. To construct the DO referring to the developmental stages of Ciona following the hatching larva stage, time-lapse imaging and CLSM imaging of sequentially fixed specimens of Ciona robusta (Ciona intestinalis type A) were performed (Fig. 1, Supplementary Video S4). The DO presents the developmental stages grouped in Periods, which in turn are grouped into Meta-Periods, following the conventional nomenclature of ontologies (Table 1).  Table S3). The 12 new distinct stages correspond to six stages previously described by Chiba and collaborators 33 . Representative images of individuals belonging to each stage, at both stereomicroscopy and CLSM, were chosen as reference ( Fig. 1; Supplementary Video S4 and Supplementary Fig. S5). www.nature.com/scientificreports/ Table 1. Ciona developmental stages from the Larva Period to the Juvenile Period. Stages 1-41 were defined in the paper, Stages 1-37 described. 1 The average time course of Tail absorption period and Body axis rotation period in same batch was set-up once again after hatching. 2 The duration of larval swimming differs among individuals. Matsunobu et al. (2015) showed that the hatched larva requires at least three or four hours to get competence to commence metamorphosis. So the time after fertilization during Larva Period was broad. www.nature.com/scientificreports/ From now on, each entity, both developmental and anatomical, is written in bold when introduced for the first time; relations between entities appear in italics, while entity definitions appear between quotation marks. In the ontology, an identification (ID) code has been assigned to each anatomical and developmental entity. ID, which here is in brackets, is a set of numbers preceded by two prefixes. The first prefix is "Cirobu", referred to the species name C. robusta. The second prefix follows the first one and is "A" when the ID is referring to the anatomy and "D" when it is referring to development.
Once we defined the DO, we constructed the AO (Fig. 1). We carefully studied our anatomical data, comparing stage-by-stage information from CLSM and histology. This allowed us to recognize all the organs/tissues and follow their differentiation over time. We then listed, in an Excel file, the terms referring to the recognized anatomical entities (including synonyms, when present) reported in the literature and used by researchers since 1893 (Supplementary Data S6). We listed 203 entities (Supplementary Data S7, column F, "Further specification 4"), assigning to each one its ID. Moreover, we detailed, for each anatomical entity, the following characteristics: the definition (column "Definition" in Supplementary Data S7, Supplementary Data S8) based on the literature; the anatomical hierarchical level, specifying to which superior entity each belongs (Part of); the tissue from which it derives (Develops from); the developmental stage of its disappearance (End stage); and the developmental stage in which it is first recognizable (Start stage). Therefore, the Start stage and End stage relationships link the AO to the DO, providing the precise description of the timing of development. If necessary, we took note of a specific feature (column Comment in Supplementary Data S7) and listed the bibliographic references (Supplementary Data S6 and column Literature in Supplementary Data S7). We also built a complete anatomical atlas in which most of the anatomical entities were efficiently annotated (Figs. 2-5; Supplementary Figs. S9-S15). Lastly, all the curated data were incorporated into a computable OBO format 34 (Fig. 1).
Below, we first describe how, where, and when the complex anatomical structures of C. robusta emerge and change during the Periods defined in this study (see Supplementary Data S2 for detailed description of stages). Then we present an overview of the number of anatomical entities and their appearance throughout the entirety of ontogenesis.
The embryonic development, pre-metamorphosis meta-period (stages 26 to 29). For the Embryonic Development, Pre-Metamorphosis Meta-Period, we described the last Period, called the Swimming Larva Period, during which the hatched larva (17.5 hpf) swims actively, beating its tail. Although larvae belonging to this Period are generally defined as "swimming" larvae, their internal structures change significantly over time. Therefore, the Period (17.5-24 h after fertilization at 18 °C) was divided into four anatomically distinguishable Stages, from Stage 26 to Stage 29, until the end of the locomotion phase (  Number of anatomical entities and their appearance during ontogenesis. All the anatomical entities annotated in the ontology, from both present results and previously reported data 24 , were analyzed in whole during the complete ontogenesis of Ciona. Figure 6 presents, stage by stage, their number from Stage 0 (unfertilized egg) to animal death. Entities associated to the embryo/larval life are 88/203 (Fig. 6, yellow column), those associated to the juvenile/adult life are 93/203 (Fig. 6, red column) and those persistent in biphasic life are 22/203 (Fig. 6, blue column). The graph shows that there are two rounds of tissue/organ increase. The first one is marked and occurs after fertilization; it reaches maximum number at Stage 25 (Mid-gastrula), when about 141 entities are recognizable. At Stage 34 (corresponding to the conclusion of the Body Axis Rotation Period),  www.nature.com/scientificreports/  www.nature.com/scientificreports/   24 and is devoted to anatomy and development. Moreover, the ADO built here is combined with the ANISEED database (https ://www.anise ed.cnrs.fr), which provides high-throughput data and in situ experiment data from the literature for ascidian species. Therefore, we integrate the panorama of actual databases and offer a tool that will help researchers in the recognition the anatomical structures of their interests. This will allow for the standardization of data underpinning an accurate annotation of gene expression and the comprehension of mechanisms of differentiation.
The developmental ontology. In this work, merging the previously reported developmental stages 24,33 , with new data from stereomicroscopy, CLSM, and histology, we implemented the description of the whole life cycle of Ciona, from fertilization to juvenile. The whole development has been divided into Meta-Periods, Periods, and Stages, following the canonical temporal subdivision of developmental ontologies 33 . Using a lowresolution microscope for dissection to examine larvae, metamorphosing individuals, and juveniles, we defined the new subdivision into stages. The simplicity of stage recognition is a prerequisite for a good staging method. Researchers will easily be able to discriminate stages, using a simple instrument, a stereomicroscope, when checking the development of their living samples in the laboratory after in vitro fertilization, or when analyzing fixed whole-mount specimens. We introduced 12 new stages (from Stage 26, Hatching Larva, to Stage 37, Early Juvenile I) that add to those already reported up to the Larva Stage 24 . Therefore, 37 stages are now described in detail and documented with original images. Considering that we also defined (without describing) Stage 38 (Early Juvenile II) and Stage 39 (Mid Juvenile I), once they are described, the whole Juvenile Period will be completed. The lacking steps are, then, the Young Adult Period (Stage 40) and the Mature Adult Period (Stage 41). However, for the latter Period, the exhaustive anatomical description by Millar 32 is still an essential reference. In summary, the whole life cycle of Ciona is almost described and annotated. This is an important result, considering that ontologies regarding other model organisms are limited to embryogenesis 1, [3][4][5]40 .
It should be noted that, in annotating the progressive organ appearance and degeneration, we could also describe in detail the metamorphosis process, whose general reports for ascidians are dated, not so accurate and timed, and limited to a few species (see for review: 29,41 ). Only some specific processes occurring during metamorphoses, such as tail regression 39,42 or papillae retraction 42 , have been described in detail in C. robusta.
The anatomical ontology. This study underlines the importance of a combined analysis of data. In fact, for each stage, we examined corresponding high-resolution images thanks to CLSM and histology. The two methods have advantages and disadvantages in terms of studying anatomy. CLSM provides high tissue resolution and relatively rapid processing, so it allows for the analysis of multiple samples. Moreover, in automatically making z-stacks, we can quickly obtain 3D reconstructions. However, tissues/organs can be difficult to recognize due to the limited number of fluorochromes that can be simultaneously used. Moreover, a low laser penetration can be a limit for the study of thick specimens. On the other hand, histology offers (other than a high tissue resolution) an easy tissue/organ recognition thanks to the different tissue affinities to labeling. However, the method is time-consuming, which means that few samples can be analyzed, and 3D reconstructions are not automatically generated. Therefore, we used, in combination, the complementary information coming from these two working methods, making it possible to identify, with precision, the inner structures as well as the outer surface of individuals, annotating in total 203 anatomical territories.  (D III ) sides. Toluidine blue. Enlargement is the same in D-D III . brc: branchial chamber; cil duc: ciliated duct of neural gland; ds: dorsal sinus; dst: dorsal strand; es: endostyle; hc: haemocytes; ht: heart; lbr: larval brain remnant; mi: medium intestine; mc: myocardium; ng: neural gland; oes: oesophagus; os: oral siphon; osm: oral siphon muscle; pb: peripharyngeal band; pc: pericardium; pg: pyloric gland; prox int: proximal intestine; rph: raphe; rpsm: right protostigmata; stom: stomach; tail remn: tail remnants; tun: runic; tunc: tunic cells; ve: velum.

Scientific Reports
| (2020) 10:17916 | https://doi.org/10.1038/s41598-020-73544-9 www.nature.com/scientificreports/ To build the hierarchical tree of anatomical entities (specified by the relation Part of in Supplementary Data S7) and to define each of them (complete with synonyms), we consulted several publications covering over 120 years of literature on Ciona and other ascidians, from 1893 31 to today. Fundamental references for creating the dictionary were, among others, those published by Millar 32 , Kott 43 , Burighel and Cloney 41 , Chiba and collaborators 33 , and the last description of the species by Brunetti and collaborators 21 . We also consulted the glossary TaxaGloss (https ://stric ollec tions .org/porta l/index .php).
The AO is documented by the database TunicAnatO, which is an anatomical atlas of original images readily available via the internet and easily accessible from any standard web browser (https ://www.bpni.bio.keio.ac.jp/ tunic anato /3.0/). This database contains information from both z-slice sections and 3D reconstruction images, and histological sections at each time point along the developmental course of C. robusta. In images, the anatomical entities were labeled, providing a guide for tissue recognition.

Data integration in anatomical and developmental ontology.
In this work, we linked DO and AO in a comprehensive DAO, as we defined, for each entity, the relations Develops from, Start stage, and End stage. These relations were determined at the cell level when cell lineage data were available and at the tissue/organ level where complexity did not allow for the following of cell genealogy. Lastly, when possible, we also annotated features linked to organ functionality (swimming, feeding, respiration, or heart beating). Some entities showed multiple possibilities to be defined, while others had uncertain/controversial definitions.
In some cases, and where possible, we defined the anatomical entities according to multiple organizational levels. For example, the "atrial siphon muscle" Start stage is Stage 10 if we refer to cell lineage [44][45][46] , while it is Stage 33 if we refer to histology (muscles recognizable on sections) and Stage 36 if we refer to the functional state of the muscles (ability to contract). Similarly, the Start stage for the "endostyle" is Stage 27 if we refer to the cell lineage 47 , while it is Stage 34 if we consider the presence of its main histological features (its subdivision into eight symmetrical zones, visible on sections) and Stage 36 if we refer to its physiological activity during feeding (mucus production trapping food particles). These and other, similar examples, exhibiting multiple tissue recognition levels, are all annotated in the ontology (among the Comments in Supplementary Data S7), for a comprehensive view of development. It is to note that in Ciona the cell lineage is not known for a few anatomical entities. In these cases (referred mainly to larval pharynx, tail epidermis and some larval brain components), we referred to data from Halocynthia roretzi 35,47,48   www.nature.com/scientificreports/ presence of comments associated with each entity in this ontology, and the huge number of citations reported, assure users of a comprehensive view of Ciona anatomy and development. An analysis of other ontologies currently available shows that the ontology of Ciona presented here is very rich in information. Sixty-one ontologies deal with anatomy on FAIR sharing (https ://tinyu rl.com/ybhhf d8c) 49 . Among them, 12 describe the anatomy of animal model organisms (e.g., Drosophila, Caenorabditis elengans, mosquito, mouse, zebrafish, Xenopus, planaria, the ascidian Botryllus schlosseri). In terms of a comparison of the ontology of C. robusta presented here with the latter (Supplementary Table S16), the first one possesses very rich lineage information compared to other ones. Moreover, among the 12 above-mentioned ontologies, four combine developmental stages and anatomical terms, eight include the relation Develops from, and 10 use references as a source of data. The ontology of Ciona exhibits all these features. It is to be considered, however, that ontologies are never-ending tools. They must be continuously updated when new information becomes available.
An overview of the ontogenesis of Ciona. Thanks to the annotation of the relations, Start stage and End stage, we could verify, in Ciona, the progressive emergence-and, where appropriate, disappearance-of its unique features. Looking at them as a whole, we obtained a global view of ontogenesis.
Our results show that ascidians have two rounds of increasing complexity: the first one during cleavage until gastrulation, and the second one during metamorphosis. This can reflect the development of structures associated with the larval life (88 in total; for example, the larval nervous system, the tail with associated notochord and muscles) and with the juvenile/adult life (93 in total; for example, the branchial basket, the gonad, the gut). Other structures (22 in total) are, of course, persistent throughout the whole ontology: they are, for example, the epidermis, the tunic, and the hemocytes.
Moreover, we show that almost one-third of the anatomical structures disappear from stage 33 (134 entities) to stage 34 (90 entities) (Fig. 6). This occurs during the Tail Absorption Period and the beginning of the Body Axis Rotation Period, when structures exclusively formed for the larval life degenerate. This drastic event was not previously documented quantitatively. It should also occur in other invertebrate species, such as barnacles and sea urchins 50,51 . In fact, there is the loss of many organs associated with the motile larva that metamorphoses in a stationary form. Such a sharp decrease in anatomical structures was not reported in other chordate animals.

Conclusions
In this study, we present the ADO of the ascidian C. robusta (C. intestinalis type A), from the swimming larva stage, through metamorphosis and until the juvenile stages. We define 12 stages that, together with the previously described stages related to embryogenesis, extend our knowledge to almost the whole ontogenesis. This ontology, providing the hierarchical description of more than 203 anatomical entities, complete with definitions, synonyms, and bibliographic references, provides the guideline for several functional studies on tunicate cell biology, development, and evolution. It allows for the standardization of data underpinning the accurate annotation of gene expression and the comprehension of mechanisms of differentiation. It will help in understanding the emergence of elaborated structures during both embryogenesis and metamorphosis, shedding light on tissue degeneration and differentiation occurring at metamorphosis.

Methods
Biological materials. C. intestinalis type A (C. robusta) adults for time-lapse imaging and for confocal scanning laser microscopy (CLSM) were provided by NBRP from the Maizuru bay and Tokyo bay areas in Japan. For histology, adults were obtained from the Lagoon of Venice, Italy. Species determination was performed checking the discriminating factor "trunk shape" of late larvae 22 . Specimens collected in different sites possessed the same anatomical and developmental features.
Preparation of embryos for time-lapse imaging. Eggs and sperm were obtained surgically from gonoducts. After insemination, eggs were maintained in agarose-coated dishes with Millipore-filtered seawater (MFSW) containing 50 µg/ml streptomycin sulfate, and the early cleavages were uniformly synchronized (data not shown). To keep the temperature stable, we used a Peltier-type incubator (CN-25B, Mitsubishi, Japan) without any vibration to prevent embryo fusion. Embryos developed in hatched larvae approximately 18 h after insemination.
The naturally hatched larvae derived from egg with chorion were maintained in plastic dishes on the thermoplate at 20 °C to acquire images. Using a digital camera (Olympus SP-350) mounted on a microscope, images were acquired every 3 to 10 min for 7 days (Supplementary Video S4). After 3 days post-fertilization, food was given (vegetal plankton, sun culture).

Scientific Reports
| (2020) 10:17916 | https://doi.org/10.1038/s41598-020-73544-9 www.nature.com/scientificreports/ (LSM image browser, Zeiss, Germany). The focus interval depended on the sample (from 0.5 to 1.2 µm). The resulting stacks were then exported to raw image series or to 3D image data for database integration. Although the timing of metamorphosis showed a huge deviation depending on the timing of adhesion, we considered an average timing, looking at animals exhibiting a representative morphology. Lastly, these stacks were integrated into the database TunicAnatO.
Histology. After in vitro fertilization, larvae, metamorphosing individuals, and juveniles were fixed in 1.5% glutaraldehyde buffered with 0.2 M sodium cacodylate, pH 7.4, plus 1.6% NaCl. After being washed in buffer and postfixated in 1% OsO 4 in 0.2 M cacodylate buffer, specimens were dehydrated and embedded in Araldite. Sections (1 µm) were counterstained with Toluidine blue. Transverse, frontal, and sagittal serial sections were cut. Images were recorded with a digital camera (Leica DFC 480) mounted on a Leica DMR compound microscope. All photos were typeset in Corel Draw X5.
AO/DO ID curation section. The anatomical and developmental terms with synonyms, definitions, and information about developmental events and anatomical entities were accumulated from textbooks, journals, and scientific observations. This information has been collected and formatted in two Excel files: one file on anatomy, the other on development. TunicAnatO was built in OBO format by using the open-source graphical ontology editor Open Biological and Biomedical Ontologies (OBO) edit 34 .

Data availability
The datasets generated and/or analyzed during the current study are available in the following repositories, as well as our database, TunicAnatO (https ://www.bpni.bio.keio.ac.jp/tunic anato /3.0/). www.nature.com/scientificreports/ Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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