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. Author manuscript; available in PMC: 2026 Jul 18.
Published in final edited form as: Development. 2026 Mar 18;153(6):dev205294. doi: 10.1242/dev.205294

A stage and anatomy ontology for embryogenesis in indirect-developing echinoderms

Charles A Ettensohn 1, Macie M Chess 1, Laurent Formery 2,*, Troy J Pells 3, Erik Segerdell 4, Peter D Vize 3, Jenifer C Croce 2,‡
PMCID: PMC13378604  NIHMSID: NIHMS2192372  PMID: 41717723

Abstract

Developmental and anatomical ontologies are fundamental tools for standardizing biological data. They facilitate the reproducibility of experiments and the integration of information across laboratories and animal species. They underpin digital curation, dissemination and analysis of diverse biological data, including gene expression patterns and phenotypes associated with pharmacological, molecular and genetic perturbations. As such, ontologies are cornerstones of model organism knowledgebases. Echinoderms have been prominent developmental biology models for more than a century and are widely used to address evolutionary questions; however, no formalized ontology has been produced for any echinoderm. Here, based on direct observations of living embryos of five species, representing euechinoids, cidaroids and sea stars, we provide developmental and anatomical ontologies for the embryogenesis of indirect-developing echinoderms. Moreover, we leverage these ontologies to create a ‘pan-echinoderm’ developmental and anatomical ontology, which we show will support comparative analyses across the phylum. These resources will be important for the curation of data from multiple species by Echinobase and Marimba, two public knowledgebases of echinoderm genomics and invertebrate marine models, respectively. They will greatly enhance the use and value of echinoderms as model organisms for biological research.

Keywords: Echinoderm, Euechinoid, Cidaroid, Asteroid, Embryo, Development, Ontology

INTRODUCTION

For more than a century, sea urchins, sea stars and other echinoderms have been prominent experimental models for the analysis of development (Davidson, 1986; Ettensohn et al., 2004; Ernst, 2011; McClay, 2011). Their popularity stems from their external mode of development and rapid embryogenesis, the ease with which large numbers of gametes and of synchronously developing embryos can be obtained, and the optical transparency and anatomical simplicity of their embryos. Thousands of published studies have examined echinoderm development, with a strong emphasis on species that exhibit indirect development; i.e. species with a feeding larva (Wray, 2022). These studies have provided fundamental insights regarding oocyte polarity, fertilization, gene activity in early development, cell specification, morphogenesis and gene regulatory networks (GRNs) (Angerer and Angerer, 2003; Ernst, 2011; Ettensohn and Sweet, 2000; McClay, 2016; (Cary and Hinman, 2017; Lowe et al., 2017; Martik et al., 2016; Peter and Davidson, 2016; Shashikant et al., 2018).

When considering all echinoderm classes, i.e. echinoids (sea urchins), asteroids (sea stars), holothuroids (sea cucumbers), ophiuroids (brittle stars) and crinoids (feather stars) (Telford et al., 2014), species currently used by the research community have divergence times ranging from a few million years to almost half a billion years. There are examples of developmental processes that have been conserved across the longest of these time scales, as well as striking examples of developmental novelties that have evolved relatively recently (e.g. Czarkwiani et al., 2022; Davidson et al., 2022; Dylus et al., 2018; Gildor et al., 2019; Hinman and Davidson, 2007; Khor and Ettensohn, 2020, 2022; Levin et al., 2022; Morino et al., 2016; Paganos et al., 2022; Semmens and Elphick, 2017; Thompson et al., 2015; Valencia et al., 2021; Wray, 2022; Yamazaki et al., 2021). Comparative studies using echinoderms have been facilitated by a rapidly expanding collection of genomic datasets of all kinds, including chromosome-level genome assemblies from many echinoderm species (Davidson et al., 2020; Galià-Camps et al., 2024; Han et al., 2024; Marlétaz et al., 2023; Parey et al., 2024; Warner et al., 2021; Zhong et al., 2024). The accumulation of gene- and development-centric data from diverse echinoderm clades has created an urgent need to develop tools that facilitate comparison and integration of experimental data across species.

Ontologies are essential tools in biological research (Bard and Rhee, 2004; Strömbäck et al., 2007). They standardize experimental data, enhancing the reproducibility and the integration of information across laboratories and species. They are crucial for the digital curation and dissemination of biological data, including gene expression patterns and developmental phenotypes. Ontologies standardize nomenclature by providing a controlled vocabulary for each entity they include, and define parental, anatomical and developmental relationships among entities. As such, they facilitate thorough, structured and formal descriptions of developing organs or animals (e.g. Richter et al., 2010; van Slyke et al., 2014). Among ontologies, developmental ontologies correspond to developmental staging series that standardize developmental phases in a manner independent of external variables such as temperature, culture medium or embryo batch. They describe and categorize the sequential steps of development based on observable morphological or structural changes. Anatomical ontologies consist of a ‘parts list’ of the developing organism, which includes cell types, germ layers, cell lineages, extracellular layers, tissues, organs and other anatomical entities, organized and classified in a formal, computationally accessible manner.

Given the usefulness of ontologies, it is not surprising that they are integral components of most major model organism knowledgebases (Segerdell et al., 2008; van Slyke et al., 2014). Over the past few decades, developmental staging and anatomical series have been constructed for several major developmental models, including mouse (Hayamizu et al., 2013; Kaufman, 1992; Theiler, 1972), zebrafish (Belmamoune and Verbeek, 2007; Kimmel et al., 1995) and frog (Nieuwkoop and Faber, 1994; Segerdell et al., 2008; 2013). Developmental staging and anatomical series have also recently been produced for the ascidian Ciona intestinalis (Hotta et al., 2020) and the lancelet Branchiostoma lanceolatum (Bertrand et al., 2021; Carvalho et al., 2021). Surprisingly, despite the rich history and continued prominence of sea urchins, sea stars and other echinoderms as developmental model organisms, no comprehensive formalized ontologies for embryonic development have been published for any echinoderm, even though developmental descriptions are available for four echinoids, Paracentrotus lividus (Formery et al., 2022), Lytechinus pictus (Nesbit and Hamdoun, 2020), Lytechinus variegatus (Morrill and Marcus, 2005) and Strongylocentrotus purpuratus (Cameron et al. (1991).

To fill this knowledge gap, we present a developmental and anatomical ontology to support developmental studies with echinoderms. Several years ago, an initial Echinoderm Anatomy and Development Ontology (ECAO) (J. Croce, unpublished) covering the developmental stages and some anatomical features of indirect-developing euechinoids, the most widely studied echinoderm clade, was assembled based on information made available by the sea urchin community. The ECAO was deposited and accepted by the Open Biological and Biomedical Ontology (OBO) Foundry, a worldwide consortium of ontology developers and curators in the life sciences (Smith et al., 2007; Jackson et al., 2021). In the present study, we have elaborated on the original ECAO through direct detailed observations of the embryogenesis of five indirect-developing echinoderm species that, together, provide a wide sampling of the diversity of the phylum and represent most of the published studies on echinoderm development. These species include three euechinoids, (L. variegatus, P. lividus and S. purpuratus), one cidaroid (Eucidaris tribuloides) and one asteroid (Patiria miniata). We have significantly enhanced the ontology by refining developmental stages and by adding many new anatomical features. In addition, by comparing information from these five species, we developed a ‘pan-echinoderm’ developmental and anatomical ontology version of the ECAO subset related to embryogenesis in indirect-developing echinoderms, which we show will be useful to support comparative analyses in other euechinoids, as well as in holothuroids and ophiuroids. Taken together, our work provides a comprehensive framework for standardizing, integrating and curating biological data related to the embryogenesis of indirect-developing echinoderms.

RESULTS

ECAO ontology design

The developmental and anatomical ontology ECAO (unpublished) was originally built following community conventions and best practices recommended by the OBO foundry (Smith et al., 2007). As such, each term (or entity), whether a developmental stage or an anatomical structure, was assigned a unique IDENTIFIER (ID) (or accession number) in the form of ECAO_0XXXXXX. This identifier is uniquely associated with a given term, even if this term is subsequently eliminated through updates to the ECAO, which can readily be modified as new information emerges. New terms and identifiers can also be added to the ECAO as the need arises. Consequently, the identifiers do not convey any temporal or relationship information.

Following OBO foundry recommendations, each term was further provided a generic, or echinoderm-specific, LABEL (or name), usually in singular but, if mandatory, in plural. This label was chosen, when possible, based on usage of the term in the context of other ontologies accessible via the EMBL-EBI Ontology Lookup Service (https://www.ebi.ac.uk/ols4) [e.g. Cell Ontology (CL), Gene Ontology (GO) and Xenopus Anatomical Ontology (XAO)], to enable comparisons between echinoderms and other animals and to facilitate computer recognition and comprehension by non-specialists (e.g. ‘8-cell stage’ or ‘blastomere’). Alternatively, the label was chosen based on developmental stage and anatomical terms commonly used in other animals or in the echinoderm literature (e.g. ‘early mesenchyme blastula stage’ or ‘micromere’).

To further assist users, each term, whether a developmental stage or an anatomical structure, was supplied with a DEFINITION, describing the main criteria for identifying the related term. Moreover, whenever applicable, each term was provided with SYNONYMS found in the echinoderm literature and with CITATIONS (i.e. peer-reviewed articles) mentioning the related term.

Another important feature of ECAO was its partonomy. The data model used to build ECAO was a directed acyclic graph, in which the terms are organized as tree-shaped hierarchies (Fig. 1). Within these hierarchies, general, comprehensive terms (e.g. ‘life cycle stage’ and ‘anatomical structure’) are located near the top of the tree, while selective terms (e.g. ‘late gastrula stage’ and ‘animal blastomere’) are positioned near the bottom (Fig. 1A,B). In this configuration, ontological relationships (or partonomy) between parent and child terms, whether for developmental stages or anatomical structures, are revealed by either an IS_A or a PART_OF relationship. IS_A highlights that the child term is a specific type of the parent term. For example, a ‘4-cell stage’ IS_A ‘life cycle stage’ (Fig. 1C) or an ‘archenteron’ IS_A ‘anatomical structure’ (Fig. 1D). PART_OF indicates that the child term is a component of the parent term. In the same examples, a ‘4-cell stage’ is thus PART_OF ‘cleavage period’ itself part of ‘embryogenesis phase’, which IS-A ‘life cycle phase’ (Fig. 1C), while an ‘archenteron’ is PART_OF ‘vegetal hemisphere’, which is itself PART_OF ‘embryo’ (Fig. 1D). It should be highlighted, however, that a specific entity is often classified by both relationships, IS_A and PART_OF, which further provides a hierarchical classification for the IS_A relationship and a spatial classification for the PART_OF relationship.

Fig. 1. Schematic representations of ontological relationships in ECAO.

Fig. 1.

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(A,B) Examples of developmental staging relationships (A) and anatomical entity relationships (B) in tree representations. In A, lines between depth nodes correspond to PART_OF relationships. In B, lines between terms are IS_A relationships. (C,D) Examples of another ontological representation for the developmental stage ‘4-cell stage’ (C) and the anatomical entity ‘archenteron’ (D). In C,D, the representations include related terms as well as partonomy relationships (IS_A, PART_OF) and temporal grading (PRECEDED_BY, DEVELOPS_FROM, STARTS_AT, ENDS_AT).

ECAO also included a developmental grading for each term, which was defined differently for developmental stages and anatomical structures. For developmental stages, the temporal ordering of the stages was revealed by a single relationship, i.e. PRECEDED_BY. As an example, the ‘4-cell stage’ is PRECEDED_BY the ‘2-cell stage’ (Fig. 1C). In contrast, for anatomical structures, representation of time was provided by three distinct relationships, which are DEVELOPS_FROM, STARTS_AT and ENDS_AT (Fig. 1D). Each anatomical structure is thus not only defined in terms of what previous structure it develops from, but also during which developmental stages it exists. Regarding the STARTS_AT and ENDS_AT relationships, a structure is pinpointed as STARTS_AT when it becomes first visible during development and as ENDS_AT as the last developmental stage at which it is still present. As such, an ‘archenteron’ can be depicted as DEVELOPS_FROM the ‘vegetal plate’ and STARTS_AT the ‘blastopore formation stage’ while ENDS_AT the ‘late organogenesis stage’ (Fig. 1D).

Extension of ECAO to the embryogenesis of multiple taxa

In typical, indirect-developing echinoderms (the focus of this work), three major phases define the life cycle: embryogenesis, larval development and adulthood (Fig. 2). The embryogenesis phase is usually considered to be the period during which the developing organism relies exclusively on maternal sources of energy, i.e. a phase during which there is little or no growth of the organism. The larval development phase is characterized by the utilization of external sources of energy, acquired through feeding, to support the growth of tissues and the organism. As such, the transition from the embryogenesis to the larval development phase is marked by the opening of the mouth, the onset of feeding and the beginning of growth (Gobala Krishnan et al., 2020; Nesbit and Hamdoun, 2020), although in some indirect-developing echinoderms, feeding and growth may be delayed following mouth opening due to a lag in esophageal muscle contractions [e.g. P. lividus (Formery et al., 2022) and Salmacis sphaeroides (Rahman et al., 2012)]. In indirect-developing echinoderms, the end of the larval development phase is unambiguously defined by metamorphosis, which marks the transition of the bilaterally symmetrical, planktonic larva into a radially symmetrical, benthic juvenile.

Fig. 2. Major stages in the life cycle of a representative indirectly developing echinoderm.

Fig. 2.

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Indirect development is the most common and ancestral echinoderm life history strategy. Diagrams are not drawn to scale. Fertilization is followed by a short embryogenesis phase (1–4 days) during which the zygote develops into a swimming and feeding larva. The opening of the mouth, onset of feeding and beginning of growth mark the beginning of a longer period of development during which the larva grows and develops new anatomical structures. A key event during larval development is the formation of the echinus adult rudiment, which is the progenitor of the juvenile. At metamorphosis, the larva settles on a substrate and a benthic juvenile emerges from the larval body. This marks the beginning of the adulthood phase, during which the juvenile grows (usually over several months) and becomes sexually mature. The life cycle is then repeated.

Initially, ECAO was designed to describe all developmental stages and anatomical structures associated with euechinoid development from oogenesis to adulthood. It was built primarily from observations and published data from the sea urchin P. lividus. In this study, we expanded the ECAO to generate a far more comprehensive ontology that can be used to curate information related to the embryogenesis of indirect-developing echinoderms across all clades. For this, we gathered data on developmental stages and anatomical structures through direct observations of live embryos (i.e. from oogenesis to the open mouth stage) of five indirect-developing species, including three euechinoids (L. variegatus, P. lividus and S. purpuratus), one cidaroid (E. tribuloides) and one asteroid (P. miniata). This led us to substantially revise the initial ECAO and add many new terms for both developmental stages and anatomical structures related to echinoderm embryogenesis.

In the updated version of ECAO, all developmental stage and anatomical terms related to embryogenesis have been attributed a new IDENTIFIER in the form of ECAO:1XXXXXX for developmental stages and ECAO:2XXXXXX for anatomical structures (Table S1). This new nomenclature was chosen to ensure that none of the embryonic terms in this study duplicates any already present in ECAO and to facilitate future addition of new terms following this new convention. For each term, we used the same data model that was originally used for ECAO and provided an appropriate LABEL, SYNONYMS, Ontology correspondences, DEFINITION, CITATIONS, partonomy and developmental grading (Table S1, see the tabs labeled ‘Terms’). For each term, we also indicated whether the term is present or absent, or not yet reported as present or absent in each of the five echinoderms we examined, or in closely related species (Table S1, see ‘TaxonDistribution’ tabs). For each developmental stage, we also defined the PRECEDED_BY relationship in each echinoderm (Table S1, see ‘Period_Species’ tab) and provided not only a generic definition for each stage but also taxon-specificities (Table S1, see ‘Dev_Terms’ tab). For the anatomical terms, we further determined for each species the STARTS_AT and ENDS_AT relationships (Table S1, see ‘Anat. Period_Species’ tab).

Finally, given that we had information from five distinct echinoderm species, we generated a ‘pan-echinoderm’ version of the ECAO that focused on the embryogenesis of indirect-developing species. For this, we developed a ‘pan-echinoderm’ staging series that included the same attributes as the species-specific staging series (IDENTIFIER, LABEL, partonomy, developmental grading and DEFINITION) (Table S1, see ‘Dev_Pan-Echino’ tab). For each anatomical structure, we also defined a ‘pan-echinoderm’ STARTS_AT and ENDS_AT developmental period, based on a union model (Table S1, see ‘Anat_Period_Species’ tab). Taken together, the updated ECAO developmental and anatomical ontology related to the embryogenesis phase now comprises a total of 58 developmental stage terms and 622 anatomical terms, each of which is assigned various ontological features and relationships, and is present in at least one of the five echinoderm species we examined.

Developmental staging series

Based on direct observations of living embryos (Figs S1–S6) and taking into consideration historical terminology that continues to be widely used by the research community, we defined an individual embryonic developmental staging series for each of the five species we studied (Fig. 3A, Table S1). These individual series were developed with a depth of four nodes (Fig. 3A, Table S1). The first node encompassed the major life cycle phases: embryogenesis, larval development and adulthood. The embryogenesis phase was then divided into four periods: cleavage, blastula, gastrula and organogenesis. We further subdivided the gastrula period into two episodes: mesenchyme blastula and invagination. Finally, each period (or episode) was divided into several embryonic stages, each of which constitutes a terminal node of the hierarchy. For example, the ‘8-cell stage’ is PART_OF the ‘cleavage period’, while the ‘mid-gastrula stage’ is PART_OF the ‘invagination episode’, which is PART_OF the ‘gastrulation period’ (Fig. 3A).

Fig. 3. Embryonic staging series for five representative indirectly developing echinoderm species and integration into a ‘pan-echinoderm’ staging series.

Fig. 3.

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(A) Species-specific embryonic staging series, depicted using four depth nodes (phase, period, episode and stage). (B,C) A ‘pan-echinoderm’ staging series, shown as a table (B) and a graphic (C), that integrates the five individual schemes into a single series widely applicable across indirectly developing echinoderms (see Table S3). Definitions of all developmental phases, periods, episodes and stages are provided in Table S1.

Within the five developmental staging series established, some stages were species or taxon specific (Table S1). This was the case, for instance, for the ‘motile blastula stage’ specific to E. tribuloides, the ‘very late organogenesis stage’ specific to P. miniata, and the ‘early prism stage’ specific to L. variegatus, P. lividus and S. purpuratus (Fig. 3A). Because different echinoderm species develop at different temperatures and rates, similar stages took place at different times after fertilization. For example, the ‘8-cell stage’ took place at about 2.25 h post-fertilization for L. variegatus (at 22.5°C) but at 4.5 h post-fertilization for P. miniata (at 15°C) (Table S2). Moreover, the characteristics of a specific stage sometimes varied among species. For example, the ‘16-cell stage’ was characterized in euechinoids by an embryo composed of three cell tiers exhibiting three distinct cell sizes (Figs S1–S3), while in cidaroids the embryo at this stage was composed of 16 cells of various sizes (Fig. S4) and in asteroids very subtle cell size asymmetry was observed at this stage (Fig. S5) (Barone et al., 2022). As a result, the updated ECAO developmental staging series now includes a total of 39 developmental species-specific stage terms (Table S1), for each of which a general definition, applicable to all indirect-developing echinoderms and taxon specificities, is provided in Table S1 (see ‘Dev_Terms’ tab). This creates a standardized controlled vocabulary that allows consistent descriptions and comparisons of developmental stages in any echinoderms related to the five species we examined (Fig. 4A,B).

Fig. 4. Developmental stage annotation using ECAO.

Fig. 4.

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(A) Schematic representations of mid-gastrula stage euechinoid, cidaroid and asteroid embryos. (B) Annotation of mid-gastrula stage embryos using the species-specific developmental and anatomical series of ECAO. Gray shading indicates information common to all three echinoderm classes. Species-specific information is color-coded as in A. (C) Annotation of mid-gastrula stage embryos using the ‘pan-echinoderm’ developmental and anatomical series of ECAO.

By leveraging the species-specific staging series, we developed a ‘pan-echinoderm’ staging series that integrated the individual stages of all five species into a single series (Fig. 3B,C, Table S1). This integrated series allows direct cross-species comparisons, and makes possible the digital curation and computational integration of datasets across multiple echinoderm species (Fig. 4C). A challenge in constructing the ‘pan-echinoderm’ staging series was the variability in the timing of some developmental events across species, independent of differences in rearing temperatures. These include, for example, the time of hatching (Table S1). E. tribuloides embryos hatch early in development and undergo a prolonged period as a swimming blastula before the onset of gastrulation (Fig. S4). By contrast, most euechinoid embryos, including L. variegatus and P. lividus, hatch immediately before the ingression of skeletogenic primary mesenchyme cells (PMCs), an event that marks the onset of gastrulation (Figs S1, S2). In S. purpuratus, however, hatching is usually delayed until after PMC ingression is under way (Fig. S3). There is also significant interspecies variability in the timing of appearance of mesenchyme cells, which occurs prior to the invagination of the vegetal plate in euechinoid sea urchins, but much later in gastrulation in cidaroids and asteroids (Figs S1–S5, Table S1). Likewise, the formation of the hydropore canal rudiment occurs concomitantly with the opening of the mouth in S. purpuratus, E. tribuloides and P. miniata (Figs S3–S5, Table S1), but after the onset of feeding in L. variegatus and P. lividus (Figs S1, S2, S6) (Formery et al., 2022). Interspecies variability in the timing of these developmental events precluded their use as reference points for the ‘pan-echinoderm’ staging series.

Nonetheless, many other major features of embryonic development were shared by all the indirect-developing echinoderms we studied and could be used to ground the ‘pan-echinoderm’ staging series. For example, in all echinoderms that have been examined, the zygote undergoes initially two holoblastic cleavages and gives rise to a spherical, ciliated blastula with a central blastocoel (Figs S1–S5). Gastrulation is preceded by the formation of a thickened vegetal plate that then invaginates, and mesenchymal cells are released from the tip of the archenteron as it elongates (Figs S1–S5). The primitive gut undergoes an anterior-to-posterior wave of morphogenesis that begins with an expansion of the anterior tip followed by the outpocketing of paired (left and right) coelomic pouches (Figs S1–S5). Gut morphogenesis continues with the formation of two constrictions that give rise in the larva to two sphincters: first the cardiac constriction at the foregut-midgut boundary and then the pyloric constriction at the midgut-hindgut boundary (Figs S1–S5). Concomitant with later events of gut morphogenesis, an invagination of the oral ectoderm produces the stomodeum, and morphogenetic interactions between the foregut and the stomodeal invagination bring these two tissues together, leading to the opening of the mouth and the onset of feeding (Figs S1–S5). We therefore used all these features, universal to the five indirect-developing echinoderms we studied, to anchor the ‘pan-echinoderm’ staging series, which includes 19 stages (Fig. 3B,C, Table S2) defined as indicated in Table S1.

Anatomical series

Anatomical ontologies consist of a computable representation of the structures, regions, parts and elements of a developing organism (Fig. 5, Table S1). Based on direct observations of living embryos (Figs S1–S6) and taking into consideration available information from published literature, we assembled a comprehensive list of anatomical entities present during the embryogenesis phase of the five indirect-developing echinoderms we examined (Table S1). This list includes gamete-related entities, embryo-specific entities and embryonic entities that continue to be present during the larval development and adulthood phases of the life cycle. The anatomical series was developed with a depth of several nodes, with the anatomical structure, system, region, space and element representing the highest-level of the nodes. Obviously, not all anatomical entities were subject to the same node depth. For example, a general entity, such as ‘archenteron’, had a lesser node depth then a more specific entity, such as the ‘right coelomic pouch’ (Table S1). In these examples, ‘archenteron’ IS_A ‘anatomical structure’, while the ‘right coelomic pouch’ IS_A ‘coelomic pouch’, which itself IS_A ‘coelom’ that IS_A ‘anatomical structure’.

Fig. 5. Annotating embryonic morphology using ECAO.

Fig. 5.

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Schematic representations of mid-gastrula stage euechinoid, cidaroid and asteroid embryos, color-coded to highlight the position of each of the anatomical entities indicated on the right, with partonomy relationships shown on the left.

Similar to the developmental staging series, the taxonomic distribution of anatomical entities across the five species also revealed some species or taxon specificity (Table S1). For example, the ‘equatorial pigment band’ has only been reported to date in the euechinoid P. lividus, and the ‘preoral ciliary band’ and ‘postoral ciliary band’, which only apply to bilaterian larvae with two separate ciliary bands, are only present in the sea star P. miniata and other sea stars (Table S1). The term ‘micromere’ indicates a cell type found only in all euechinoids, and not in cidaroids or asteroids (Table S1). Similarly, an elaborate, calcified ‘endoskeleton’ is present in echinoids (both euechinoids and cidaroids), but is entirely absent from asteroids, while a ‘posterior enterocoel’ forms in P. miniata and other asteroids but is absent from echinoids (Table S1). Several other structures have also been described in euechinoids, such as ‘bottle cell’, ‘endoderm-associated neuron’ and sub-classes of blastocoelar cells, but these have not yet been reported in other, less well-studied, echinoderm taxa, precluding any conclusions about their presence or absence in these taxa. In total, the anatomical series related to embryogenesis that we developed as part of the updated ECAO encompasses a total of 622 entities, for each of which partonomy and species attributes are provided in Table S1. This series provides all the necessary standardized controlled vocabulary to identify and describe all anatomical entities associated with each developmental stage, and to carry out comparisons across species. As an example, Fig. 5 illustrates euechinoid, cidaroid and asteroid embryos at the mid-gastrula stage, using many of the anatomical terms relevant to this stage, whether their partonomy IS-A ‘germ layer’, ‘anatomical structure’, ‘embryonic structure’ or ‘embryonic region’. This codification will be key for annotating phenotypes of wild-type and experimentally perturbed embryos, and for highlighting other similarities and differences between echinoderm species.

Using the individual species-specific developmental staging series, we defined the developmental window during which each entity is present in each of the five species we examined (STARTS_AT and ENDS_AT relationships) (Table S1). While doing so, we noted some variability in the timing of appearance and/or disappearance of certain anatomical entities across species. For example, the ‘presumptive ciliary band’, a region of the embryo marked by the onset of onecut expression, is first apparent during the ‘mesenchyme blastula episode’ in S. purpuratus (Poustka et al., 2004; Barsi and Davidson, 2016) but does not arise until well after the onset of invagination in P. miniata (Yankura et al., 2013) and E. tribuloides (Bishop et al., 2013). Likewise, ‘germ cell’ emerges at the ‘32-cell stage’ in euechinoids such as S. purpuratus, P. lividus and L. variegatus, while this cell type becomes detectable at a molecular level only at the ‘late organogenesis stage’ in P. miniata (Fresques et al., 2014). While some of this interspecies variability is likely to be genuine, some may be attributable to variations in the experimental methods used in published studies and/or differences in the granularity of the analyses, thereby reinforcing the need to establish echinoderm anatomical ontologies.

Despite the variability across species, the taxonomic distribution of the anatomical entities revealed that the vast majority of the reported entities were found throughout the three echinoderm clades included in this study. More specifically, of all anatomical entities encompassed in the updated ECAO, more than 97% were present in all three euechinoid species, 93% were present in E. tribuloides and 49% were present in P. miniata (Table S1). In the last case, the differences were mostly due to the lack of a skeleton in asteroid embryos. In addition, in most cases, the time period during which each of these shared entities was present was similar across species (Table S1). These features thus reflected the evolutionary conservation of developmental processes across the phylum and enabled the establishment of a ‘pan-echinoderm’ time frame for each anatomical entity (Table S1, see ‘Anat. Period species’ tab). To develop this time frame, we employed a ‘union’ model, such that any anatomical entity present in at least one of the five species was assigned a ‘pan-echinoderm’ time window that was defined using the earliest STARTS_AT stage and latest ENDS_AT stage for that entity in any of the five species.

Curation of gene expression patterns using ECAO

The developmental and anatomical series we developed will also be pivotal for the annotation of embryonic gene expression profiles. As an example, we annotated the profiles of two transcription factors, glial cells missing (gcm) and brachyury (bra), in all five of the species we examined (Figs 6 and 7). This annotation was based primarily on data from previously published work but also included new observations (Fig. 6A). We annotated gene expression patterns using both species-specific entities (Fig. 6B–D) and ‘pan-echinoderm’ entities (Fig. 7). Species-specific entities enabled a more precise, fine-grained annotations of gene expression, while ‘pan-echinoderm’ entities provided more general information related to indirect-developing echinoderms as a whole and facilitated comparisons across species. When curating gcm expression at a given developmental stage (i.e. ‘late blastula stage’), it was not possible to assign a single ‘pan-echinoderm’ anatomical entity that accurately described expression in all five species examined (Figs 6C and 7A). Thus, our annotation reflected the variable pattern of expression of gcm across species. On the other hand, curation of bra captured the highly conserved pattern of expression of this gene across echinoderms (Figs 6D and 7B). Taken together, these examples demonstrate that gene expression can be effectively curated in a wide range of indirectly developing echinoderms using the revised ECAO. We acknowledge, however, that updates and refinements will be necessary as additional gene expression patterns and phenotypes are curated.

Fig. 6. Annotating gene expression profiles using species-specific developmental and anatomical entities.

Fig. 6.

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(A) Whole-mount in situ hybridization analysis of glial cells missing (gcm) expression during P. lividus embryogenesis. Scale bar: 30 μm. (B) Curation of gcm expression in P. lividus using ECAO. (C) Curation of gcm expression in L. variegatus, P. lividus, S. purpuratus, E. tribuloides and P. miniata based on data in A and on previously published work (Croce and McClay, 2010; Duboc et al., 2010; Erkenbrack, 2018; Erkenbrack et al., 2018; Hinman and Davidson, 2007; McCauley et al., 2010; Ransick and Davidson, 2006; Ransick et al., 2002; Spurrell et al., 2023; Valencia, 2018), using the species-specific subset of ECAO. (D) Curation of brachyury (bra) expression in L. variegatus, P. lividus, S. purpuratus, E. tribuloides and P. miniata based on previously published work (Croce et al., 2001; Erkenbrack, 2016; Erkenbrack et al., 2018; Gross and McClay, 2001; McCauley et al., 2015; Peter and Davidson, 2010), using the species-specific subset of ECAO. In B-D, dots or boxes indicate binary gene expression (expressed/not expressed) for the indicated developmental stage and anatomical structure. mes., mesenchyme.

Fig. 7. Annotating gene expression profiles using ‘pan-echinoderm’ developmental and anatomical entities.

Fig. 7.

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(A) Curation of glial cells missing (gcm) expression in late blastula stage euechinoid, cidaroid and asteroid embryos using the species-specific (top and middle rows) and ‘pan-echinoderm’ (bottom row) subsets of ECAO. (B) Curation of brachyury (bra) expression at the vegetal plate stage in euechinoids, cidaroids and asteroids using the species-specific (top and middle rows) and ‘pan-echinoderm’ (bottom row) subsets of ECAO. Expression profiles of gcm and bra are based on data and previously published work as indicated in Fig. 6 legend.

Applicability of ECAO across indirectly developing echinoderms

As the ‘pan-echinoderm’ ontology was based on direct observations of five representative species, we next assessed the degree of conservation of developmental stages and anatomical terms across three additional indirect-developing echinoderms, i.e. an additional euechinoid (L. pictus), a holothuroid (Apostichopus parvimensis) and an ophiuroid (Amphipholis kochii) (Table S3). This analysis was carried out using available published data. Indeed, we were able to readily map all developmental stages and anatomical entities of the ‘pan-echinoderm’ ECAO ontology to these additional species. As we observed when working on the original five species, we also detected some species-specific differences, but in the same proportion as before and for the same reasons (e.g. absence of endoskeleton or of multiple ciliary bands). This clearly demonstrates the broad utility of ECAO in annotating and comparing phenotypes and gene expression profiles across indirect-developing echinoderms. It also illustrates the flexibility of the ECAO ontology, which can easily be updated to incorporate new information and new species as knowledge increases.

DISCUSSION

Echinoderms, and in particular sea urchins and sea stars, have been prominent model organisms for developmental biology studies for more than a century. Despite this rich history, no comprehensive ontology of embryogenesis has been produced for any echinoderm, although atlases of development or fate maps have been published for four euechinoids: L. variegatus (Morrill and Marcus, 2005), P. lividus (Formery et al., 2022), L. pictus (Nesbit and Hamdoun, 2020) and S. purpuratus (Cameron et al., 1991). In the absence of a formal ontology, the interpretation of widely used terms such as ‘early gastrula’ and ‘prism’ varies greatly, even among experts. Consequently, it is very difficult to compare experimental data across studies, even within a single species. The rapid accumulation of biological data of many types, including mRNA expression patterns, protein localization data and embryonic phenotypes resulting from molecular, pharmacological and genetic manipulations has also created a need for tools to support the curation and integration of this information. The ontology we present here will provide a framework for the digital curation of these important types of experimental data by knowledgebases such as Echinobase, the knowledgebase of echinoderm gene-related information (Arshinoff et al., 2022; Telmer et al., 2024) and Marimba, the knowledgebase of invertebrate marine models (http://marimba.obs-vlfr.fr/). More generally, the ECAO developmental and anatomical ontology we have developed will significantly enhance the utility of echinoderms for biological research, by standardizing experimental data, increasing reproducibility, and facilitating comparisons and integration of information across laboratories, experiments and species.

One important application of developmental staging and anatomical series is to describe gene expression patterns and phenotypes with a controlled, fixed vocabulary, facilitating comparisons between normal and experimentally perturbed embryos, as well as between species (Bradford et al., 2023; Fisher et al., 2023; Öztürk-Çolak et al., 2024). In several model systems, anatomical series have been used to characterize the morphological and molecular phenotypes of wild-type embryos, genetic mutants and embryos that have been treated with pharmacological inhibitors or after targeted gene knockdowns (Beck et al., 2009; Finger et al., 2011; Knowlton et al., 2008; Osumi-Sutherland et al., 2013). These series and their use are therefore providing a way to annotate gene expression patterns (as shown here) and phenotypes in a form that is computationally accessible and query-able, and that can further be used to group annotations in biologically meaningful ways. Based on these considerations, we anticipate that a major application of the ontology we have produced will be the curation on Echinobase and Marimba of gene expression patterns and morphological phenotypes, during normal development and following experimental perturbations.

Another valuable application of the developmental stage and anatomical series we have produced will be the annotation of the expression patterns controlled by transcriptional drivers. Cis-regulatory elements (CREs) that drive transgene expression in specific spatiotemporal patterns are used by the echinoderm research community to control the expression of diverse molecules, including transcription factors, Cas9, inducible transcriptional activators (e.g. tTA) and fluorescent cell lineage markers (Damle and Davidson, 2012; Barsi et al., 2014; Cheatle Jarvela et al., 2016; Khor and Ettensohn, 2023). By using the ontology developed here to annotate CRE expression patterns, the research community will have access to a comprehensive searchable palette of transcriptional drivers for directing transgene expression to specific cell types or tissues.

We deliberately based our developmental staging scheme on morphological landmarks that can easily be observed in living embryos using only a light microscope. Conventional bright-field imaging is sufficient in this regard, although differential contrast (DIC) imaging is useful. The only developmental stages that are difficult to identify in this manner are the early to late blastula stages, when there are no anatomical changes in the spherical embryo other than an increase in cell number and a concomitant decrease in cell size, and when there are too many cells to count easily. To accurately stage such embryos, the most reliable approach is to label embryos with nuclear stains and count cell numbers in squash preparations. For P. lividus and L. variegatus in particular, other researchers have opted to sequentially name these stages one after the other with a time interval of 1 h (Croce and McClay, 2010; Formery et al., 2022). This approach is restricted to a standard temperature, however, and is difficult to extend across species.

The growing interest in comparative studies using echinoderms has created a need for an ontology that can integrate biological information across the phylum. This was the primary motivation for producing a ‘pan-echinoderm’ ontology. The five indirect-developing species that anchored our ontology were chosen because they account for most published studies on echinoderm development and represent diverse developmental patterns and deeply divergent clades within the phylum. A recent (January 2026) search of PubMed entries with the terms ‘<species name>’ and ‘embryo’ indicates that the three most widely used species are S. purpuratus (845 citations), P. lividus (600 citations) and L. variegatus (392 citations). The next-most widely cited species, the sea urchins H. pulcherrimus (222 citations) and L. pictus (189 citations), are very closely related to S. purpuratus and L. variegatus, respectively, and we have shown that our ontology is easily applicable to such closely related species (Table S3). Our inclusion of a cidaroid (E. tribuloides) and an asteroid (P. miniata) enabled us to develop an ontology that can be applied to indirect-developing species across the phylum. We confirmed this extendibility by applying ECAO to an indirect-developing ophiuroid and an indirect-developing holothuroid. Our focus on indirect-developing species precluded consideration of crinoids, as all species of crinoids that have been described to date exhibit direct development. The embryogenesis of direct-developing species is often so radically different from that of indirect developers (Wray, 2022) that the creation of a developmental and anatomical ontology that encompasses both direct and indirect developing species will be a challenge for the future. As it stands, our work appears to be among the first efforts to integrate the developmental and anatomical ontologies of diverse and deeply divergent species, although ontologies that encompass more closely related species are now appearing (Carvalho et al., 2021; Mungall et al., 2012). Thus, in addition to supporting developmental studies using echinoderms, our work provides a generalizable model for the construction of integrative animal ontologies.

MATERIALS AND METHODS

ECAO ontology

ECAO was originally built in the open-source graphical ontology editor OBO-Edit (Day-Richter et al., 2007). The revised ECAO will soon be available as OBO and Web Ontology Language (OWL) format files through GitHub (https://github.com/echinoderm-ontology/ecao_ontology) and the OBO foundry website (https://obofoundry.org/ontology/ecao.html) (Jackson et al., 2021). It will also shortly be accessible for downloads and user searches at EMBL-EBI Ontology Lookup Service (https://www.ebi.ac.uk/ols4/ontologies/ecao), Echinobase (https://www.echinobase.org/echinobase/) (Arshinoff et al., 2022; Telmer et al., 2024) and Marimba (http://marimba.obs-vlfr.fr/). The revised ECAO is intended to be updated as further knowledge on current and new species becomes available. Requests for additions or modifications may be submitted to the corresponding authors or through Echinobase.

All ECAO terms that were generated as part of this study, along with related synonyms, partonomy, developmental grading, definitions, time frames, and presence or absence, related to the five main echinoderms examined, were determined from direct observations made during this study, along with published data. By contrast, for L. pictus, A. parvimensis and A. kochii, the data were based primarily on published material. For all ECAO terms, whenever possible, developmental stage and anatomical term labels and definitions were adopted from those in UBERON (Mungall et al., 2012; Haendel et al., 2014). The information was compiled and formatted into Excel files containing separate tabs for developmental staging and anatomical entities series (Tables S1, S3). The developmental staging in the ‘pan-echinoderm’ ontology was built on a ‘union’ model; the ‘pan-echinoderm’ stage range for each anatomical entity used the earliest STARTS_AT stage for that entity in any of the five species and the latest ENDS_AT stage in any of the five species.

Animals and embryo culture

Five representative, indirect-developing echinoderms were used: three euechinoids (L. variegatus, P. lividus and S. purpuratus), one cidaroid (E. tribuloides) and one asteroid (P. miniata). Gravid adult sea urchins and sea stars were obtained from Patrick Leahy (California Institute of Technology, Pasadena, CA, USA; S. purpuratus and P. miniata), from Pelagic Corporation (Sugarloaf Key, FL, USA; E. tribuloides and L. variegatus) or from the IMEV Service Aquariologie (Institut de la Mer de Villefranche, Villefranchesur-Mer, France; P. lividus). For each species, light microscopic observations were carried out on at least four batches of embryos derived from separate mating pairs. Embryos were cultured in glass bowls or 6–10 cm plastic cell culture dishes, without stirring or feeding, at 15°C (P. miniata and S. purpuratus), 18°C (P. lividus) or 22.5° C (E. tribuloides and L. variegatus) using temperature-controlled incubators.

Imaging

For light microscopic observations, at the appropriate developmental time following fertilization, embryos were concentrated by low-speed centrifugation (E. tribuloides, L. variegatus, P. miniata and S. purpuratus) or collected by micropipetting (P. lividus) and mounted on glass slides. To prevent compression of the embryos, coverslips were supported by spacers that consisted of one or more layers of double-sided tape (Scotch 665, 3M) or clay. For examination of swimming stages, embryos that had been collected by centrifugation were re-suspended in 0.01% protamine sulfate (P4020, Sigma-Aldrich) diluted in sea water to slow ciliary beating. They were then mounted on coverslips that had been coated with poly-L-lysine hydrobromide (Ettensohn, 1985) (E. tribuloides, L. variegatus, P. miniata and S. purpuratus). For embryos that were collected by micropipetting, they were immobilized by adding a drop of 8% paraformaldehyde (P6148, Sigma-Aldrich) prepared in sea water to the cell culture dish (P. lividus). Mounted specimens were examined with a 20× dry or 40× oil immersion objective using DIC optics.

For counts of cell numbers at late cleavage stages, embryos were fixed in 100% methanol overnight at −20°C, rinsed five times in 1×PBS and stained for 10 min at room temperature with 10 μg/ml Hoechst 33342 (H3570, Invitrogen) diluted in 1×PBS. Squash preparations were imaged using epifluorescence optics and nuclei were counted.

All images were collected with either a LC30 CMOS color camera (Olympus) and a Xyla 4.2 sCMOS camera (Oxford Instruments) (E. tribuloides, L. variegatus, P. miniata and S. purpuratus) or an Axiocam 506 Color camera (Zeiss) (P. lividus). All acquired images were processed with cellSens imaging software (Olympus) and Adobe Photoshop (E. tribuloides, L. variegatus, P. miniata and S. purpuratus), or with ImageJ version 1.44o (Schneider et al., 2012) and Affinity Photo (Serif) (P. lividus).

Expression pattern analysis

The expression pattern of the transcription factor glial cells missing (gcm) in P. lividus was determined using whole-mount in situ hybridization (Lhomond et al., 2012).

Graphical representations

Graphical representations were generated using Microsoft Powerpoint or Affinity Designer (Serif), along with AirBrush (https://airbrush.com/) and Affinity Photo (Serif).

Supplementary Material

1

Acknowledgements

The authors thank Ina Arnone, Smadar Ben-Tabou-de-Leon, Robert Burke, Delphine Dauga, Evelyn Houliston, Simon Jupp, Thierry Lepage, Guy Lhomond, Nicolas Matentzoglu, Julia Morales, Isabelle Peter, and Shunsuke Yaguchi for their contributions. The authors also thank the EvoInSiDe team members, past and present, for their support, as well as the Service Aquariologie (SA) and the Plateforme d’Imagerie par Microscopie (PIM) of the Institut de la Mer de Villefranche (IMEV), two services supported by EMBRC-France (ANR-10-INBS-02) from the Agence Nationale pour la Recherche.

Funding

This work was supported by the National Institutes of Health (P41HD095831 to C.A.E. and P.D.V.), the Institut des Sciences Biologiques of the Centre national de la recherche scientifique (J.C.C.), the European project CORBEL through H2020 Research Infrastructures (H2020-INFRADEV-4-2014-2015 RIA to J.C.C.), the Ministère de l’Enseignement Supérieur, de la Recherche, de la Science et de la Technologie (2173/2015 to L.F.) and the Japan Society for the Promotion of Science (PE18758 to L.F.).

Footnotes

Competing interests

The authors declare no competing or financial interests.

Peer review history

The peer review history is available online at https://journals.biologists.com/dev/lookup/doi/10.1242/dev.205294.reviewer-comments.pdf

Data and resource availability

The revised ECAO is available as OBO and Web Ontology Language (OWL) format files through GitHub (https://github.com/echinoderm-ontology/ecao_ontology) and the OBO foundry website (https://obofoundry.org/ontology/ecao.html) (Jackson et al., 2021). It is also accessible for downloads and user searches at EMBL-EBI Ontology Lookup Service (https://www.ebi.ac.uk/ols4/ontologies/ecao), Echinobase (https://www.echinobase.org/echinobase/) (Arshinoff et al., 2022; Telmer et al., 2024) and Marimba (http://marimba.obs-vlfr.fr/). All other relevant data and details of resources can be found within the article and its supplementary information.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS2192372-supplement-1.pdf (11.6MB, pdf)

Data Availability Statement

The revised ECAO is available as OBO and Web Ontology Language (OWL) format files through GitHub (https://github.com/echinoderm-ontology/ecao_ontology) and the OBO foundry website (https://obofoundry.org/ontology/ecao.html) (Jackson et al., 2021). It is also accessible for downloads and user searches at EMBL-EBI Ontology Lookup Service (https://www.ebi.ac.uk/ols4/ontologies/ecao), Echinobase (https://www.echinobase.org/echinobase/) (Arshinoff et al., 2022; Telmer et al., 2024) and Marimba (http://marimba.obs-vlfr.fr/). All other relevant data and details of resources can be found within the article and its supplementary information.

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