During animal development, numerous cells move around the embryo to form and shape the growing tissues. As these cells move, they are guided to their destination by molecular cues. The embryo’s tissues produce these cues and the cues can either repel or attract migrating cells. Ephrins are a large and well-studied family of proteins that serve as guidance cues and are found on the surface of certain types of cells. Some migrating cells have receptors for Ephrin and are repelled from tissues that contain Ephrin proteins. In these cases, the repulsive interaction between Ephrins and cells with receptors ensures that migrating cells avoid certain locations and reach the correct final destination.

The sea urchin is an important model organism for studying how animals develop and in particular how genes control animal development. This is in part because these animals can be easily manipulated in the laboratory and are more closely related to animals with backbones than many other model organisms. Sea urchins also have a relatively simple set of genes; many of which are similar to the human form of the gene. In sea urchin embryos, pigmented cells called immunocytes are known to migrate from one region of the embryo to another where they form part of its immune system. However it was not clear what guides this migration.

Sea urchins produce one type of Ephrin protein and its associated receptor, and now Krupke et al. show that immunocytes carry the receptor for Ephrin and migrate to embryonic tissues that produce high levels of this Ephrin. This finding suggested that the Ephrin is actually attracting the immunocytes to their final destination rather than repelling them. Further experiments supported this idea and revealed that immunocytes that lack the Ephrin receptor fail to enter the right tissue. Similarly, altering the pattern of Ephrin in the embryo’s tissues altered immunocyte migration in a predictable way.

These findings of Krupke et al. suggest that Ephrin and its receptor have changed their biological functions during evolution of animals. This raises a number of questions for future research including whether the molecular mechanisms used by Ephrin and its receptor to attract immunocytes in sea urchins is the same as that used to repel cells in other species.

Dispersal of cells from a site of origin to form a predictable pattern throughout an organism is a spectacular demonstration of directed migration, an essential component of the metazoan patterning toolkit. The complex movements of neural crest or the extensions of central nervous system axons are familiar vertebrate examples that are brought about by a relatively small number of cellular mechanisms. Attraction to a chemical source, permissive substrates, and mechanisms of exclusion or repulsion appear to the principal means of controlling even complex cell migrations (Davies, 2005).

In many situations Ephrin and Eph are important components of the signaling that regulates migration of cells (Pasquale, 2005, 2008; Xu and Henkemeyer, 2012; Poliakov et al., 2004). Eph receptors are a family of receptor tyrosine kinases that are activated by cell surface ligands, Ephrins. Ephrins are bound to membranes through a glycosylphosphatidylinositol linkage (Ephrin A) or a transmembrane domain (Ephrin B). The receptors and ligands are found throughout metazoans and they are thought to accompany the evolution of multicellularity (Srivastava et al., 2010; Tischer et al., 2013). Vertebrate receptors and ligands are diverse with as many as 16 Eph receptors and 8 Ephrins. The principal developmental functions of Eph/Ephrin signaling include defining tissue domains and guiding migrating cells or growth cones (Klein, 2012; Cayuso et al., 2015). In the best-understood models, Eph receptors repulse cells from regions expressing Ephrin, or with reverse signaling, where ligand bearing cells respond to receptor binding, Ephrin-expressing cells are repulsed from cells expressing Eph receptors. Eph and Ephrins regulate cytoskeletal dynamics and often integrate activity of other receptor ligand systems to coordinate adhesive and migratory responses to the environment of the cell (Bashaw and Klein, 2010; Bush and Soriano, 2012).

In vertebrates Eph and Ephrins have complex patterns of expression and interaction, which complicates functional studies. Genomic studies of sea urchins reveal that there is a single Eph receptor (Sp-Eph) and a single Ephrin ligand (Sp-Efn), making them an attractive model for studies of function (Whittaker et al., 2006). In addition, urchins are basal deuterostomes, so they provide an opportunity to study the range of morphogenetic mechanisms employed by embryos that share a common ancestor with vertebrates (Drescher, 2002; Mellott and Burke, 2008). In S. purpuratus, embryos express Sp-Eph and Sp-Efn in two overlapping domains of ectoderm and Eph/Ephrin signaling appears to have a role in morphogenesis of the ciliary band (Krupke and Burke, 2014).

Pigmented immunocytes are a well-studied cell lineage in urchin embryos because of their distinctive reddish granules and their early specification within the nested rings of endomesodermal cells in the vegetal plate. Specification of pigmented immunocyte precursors begins in cleavage stages when micromeres express Delta, which initiates Notch-mediated specification of a non-skeletogenic mesoderm domain in an adjacent ring of veg2-derived blastomeres (Sherwood and McClay, 1999; Sweet et al., 2002). This domain is first demarcated by expression of the transcription factor glial cells missing (GCM), which is critical to the specification of pigmented immunocytes in the dorsal half of the domain (Ransick and Davidson, 2006, 2012; Ruffins and Ettensohn, 1996). During gastrulation, the pigmented immunocytes transition to mesenchyme and migrate to the ectoderm (Gibson and Burke, 1985, 1987; Takata and Kominami, 2004). Once in the ectoderm the cells adopt a distinctive dendritic form and mediate innate responses to pathogens (Solek et al., 2013). Although many aspects of the lineage have been well studied (Barsi et al., 2015; Beeble and Calestani, 2012; Buckley and Rast, 2015), we know little about the dispersal and insertion into epithelium of pigmented immunocytes.

Here we report immunocyte precursors express the receptor, Sp-Eph, while migrating over a gradient of Sp-Efn ligand on the basal surfaces of dorsal ectodermal cells. Manipulations of Sp-Eph and Sp-Efn concentrations reveal that suppression of Sp-Eph signaling interferes with insertion of immunocytes into the ectoderm. Altering ligand concentration indicates that pigmented immunocytes are more likely to insert in ectoderm expressing high levels of Sp-Efn. We propose Sp-Eph and Sp-Efn function initially in the dispersion of immunocytes precursors, and subsequently they are necessary for the insertion of mesenchyme into epithelium.

Ephrins and Eph receptors are typically expressed on cell surfaces and signaling is mediated by direct cellular contact (Klein, 2012). Soluble Ephrins have been shown to function as competitive inhibitors and details of receptor activation indicate that clustering is an essential part of the activation process (Davis et al., 1994; Himanen et al., 2010; Seiradake et al., 2010). Our data show that Sp-Efn is abundant on cytonemal processes on the basal surfaces of dorsal ectodermal cells. This is a distinctive mechanism of Ephrin presentation and may indicate that the epithelial cells could expand their range of Ephrin signaling by distances of up to 15 µm from the cell surface. An implication of this manner of ligand presentation is that cells within the length of cytonemes could potentially activate receptors on a pigmented immunocyte without making direct contact with the cell – a form of paracrine signaling. Live imaging indicates that immunocytes migrate from the vegetal plate and contact the basal surface of the ectoderm as they migrate. Presentation of Ephrin on cytonemes suggests an active presentation of ligand in a context where immunocyte precursors move through a shag carpet of Ephrin. However, at this time there is no experimental evidence supporting a need for Sp-Efn expression on cytonemes in order for it to function. Cytonemes on the ectoderm of sea urchin embryos may have additional roles in presentation of ligands and receptors.

Thin filopodia have not previously been termed cytonemes in sea urchin embryos. Andrews (1897) described filopodia on the basolateral membranes of the epithelial cells of blastulae of sea urchin embryos that extend, retract, branch and fuse. He suggested that the processes are connections between blastomeres and provided cytoplasmic continuity. The existence of these fine processes was confirmed with phase contrast imaging, time-lapse, and transmission EM (Dan, 1960; Gustafson, 1961; Vacquier, 1968). The filopodia of blastomeres were suggested to function in cell adhesion or to maintain cytoplasmic continuity of blastomeres. Miller et al. (1995) described ectodermal filopodia using DIC optics and noted that they are shorter and less abundant than filopodia of mesenchyme. McClay (1999) distinguished thin filopodia from other forms of filopodia and emphasized the potential role they may have as structures detecting patterning information. In Drosophila, thin filopodia have been demonstrated to be involved in signaling; either in presentation of ligands or receptors, and are distinguished by calling them cytonemes (Ramirez-Weber and Kornberg, 1999). Numerous examples of signaling are now known to involve the subset of filopodia called cytonemes (Gradilla and Guerrero, 2013; Kornberg, 2014; Kornberg and Roy, 2014). The demonstration that thin ectodermal filopodia of sea urchin embryos present the ligand Sp-Efn indicates that they are cytonemes that function in presentation of patterning information.

The expression of Sp-Eph on pigmented immunocyte precursors, the expression of Sp-Efn on basal cytonemes, and the pattern of abundance of Sp-Efn provide a strong correlative basis for the hypothesis that Eph/Ephrin signaling is a component of the mechanisms determining the distribution of pigmented immunocytes. The principal effect of suppressing expression of Sp-Eph or inhibiting Eph kinase function is the pigmented immunocytes not adopting a characteristic dendritic morphology. The treatments result in a small increase the number of pigmented immunocyte precursors in the blastocoel, which suggests attachment to the ectoderm is independent of Eph/Ephrin signaling. Suppressing expression of Sp-Eph or inhibiting Eph kinase function results in a 20% reduction in the number of pigmented immunocytes in 72 hr embryos. There is no difference in the number of pigmented immunocytes at 48 hr when Eph is either knocked down or inhibited and activated caspase-3 and morphological indications of apoptosis occur in a small proportion of the immunocytes. As well, apoptosis appears to occur relatively late in the process of immunocyte migration (72 hr) and is almost completely absent in control embryos. We have concluded that the 20% reduction in the number of pigmented immunocytes is in part due to immunocytes becoming apoptotic. If Eph functioned solely as a survival factor selecting among randomly dispersed immunocyte precursors, apoptosis would be detected in control embryos. As well, using Sp1 as a differentiation marker, it is apparent that immunocyte precursors are distributed throughout the dorsal ectoderm before the end of gastrulation, which indicates patterning arises during migration. Although there is a single ligand for Eph in urchins, morpholinos directed at the receptor result in a reduction of pigmented immunocytes, and morpholinos directed at the ligand (SpEphrin) have no effect on cell numbers. This outcome may result from other interactions, both extra- and intra-cellular, with Sp-Eph, or from differences in efficiencies of the morpholinos that target the receptor or the ligand. Complete suppression of either the ligand or the receptor is necessary to fully explain this observation. Our loss-of-function data support an essential role for Eph/Ephrin signaling in the insertion of mesenchyme into epithelium, however we are unable to distinguish the precise step in the transition requiring Eph activation. The morphology of cells changes as they insert into the ectoderm, but the underlying mechanisms are not clear. Loss-of-function data does not distinguish between a sequential process and a process that is directly dependent on Eph/Ephrin. In one situation, insertion and the change in morphology are independent pathways that are sequentially activated, and only one pathway is dependent on Eph/Ephrin signaling. An alternative is that insertion and the change in morphology are independent processes, but they are directly dependent on Eph/Ephrin signaling.

By injecting Sp-Efn RNA we are able to express ligand throughout the embryo, including the ventral ectoderm, where this ligand is not normally expressed. The ventral ectoderm does have cytonemes, at similar densities to dorsal ectoderm, but they do not express Sp-Efn. Expression of Sp-Efn in ventral ectoderm is sufficient for pigmented immunocytes to disperse beneath ventral ectoderm and insert in the epithelium. As well, a high level of Sp-Efn expression in half of an embryo enhances immunocyte insertion in dorsal ectoderm. Overall, loss-of-function and gain-of-function outcomes indicate that Sp-Eph and Sp-Efn are components of the pathways that facilitate insertion of immunocytes into epithelium.

It is also clear from our data that there are additional factors that influence the distribution of Sp-Efn and the ability of pigmented immunocytes to insert in an epithelium. When Sp-Efn is ectopically expressed, early gastrula stages have uniform expression of Sp-Efn. However, by the completion of gastrulation, Sp-Efn cannot be detected in endoderm (Figure 4L). This may account for pigmented immunocytes not inserting into endoderm or coelomic epithelia, however the mechanism by which Sp-Efn is excluded from the endoderm is not known.

The accumulation of pigmented immunocytes in regions of abundant Sp-Efn and experiments in which Sp-Efn levels are perturbed in half of the embryo indicate that pigmented immunocytes move into regions expressing high levels of Sp-Efn. Several mechanisms could potentially account for the apparent attraction of immunocyte precursors to ectoderm expressing Sp-Efn. Given the established role for Eph/Ephrin signaling in regulating cellular adhesion, we propose a mechanism in which Sp-Eph activation leads to a localized increase in adhesion. Although soluble attractants cannot be eliminated as possible co-factors, haptotaxis may be sufficient to account for the observations that we report. Haptotaxis guides by means of a gradient of adhesion and is dependent upon adhesive bonds that are continuously made and broken. Stronger adhesions resist disruption better than weaker adhesions, with the result that random motions will move a cell up an adhesive gradient (Davies, 2005). Our data indicate that Ephrin abundance is not uniform and pigmented immunocytes continuously extend and retract short processes. If Eph activation leads to a localized enhancement of adhesion, random movements of cells would cause them to disperse and move to regions in which the level of Ephrin is higher. Eph activation has been demonstrated to promote integrin mediated cell adhesion in mammalian cells (Huynh-Do et al., 1999) and the kinase NIK (Nck-Interacting Ste20 Kinase) is activated by Eph receptors, which leads to integrin activation (Becker et al., 2000). As well, Eph receptors have been demonstrated to interact with Ig Superfamily receptors, which are also expressed by pigmented immunocytes (Barsi et al., 2015). Gradients of Ephrin have been previously demonstrated to be critical to retinotectal and corticospinal patterning. However, the predominant mechanism is for high levels of ligand to suppress projection of axons (Suetterlin et al., 2012; Triplett and Feldheim, 2012). Thus, our data suggest that Sp-Eph and Sp-Efn may also function in the dispersion of immunocyte precursors from their site of origin, in addition to their role in insertion into the ectoderm.

Phylogenetic analyses suggest that Eph receptors and ephrin ligands diverged into A- and B-types at different points in their evolutionary history, such that primitive chordates likely possessed an ancestral ephrin-A and an ancestral ephrin-B, but only a single Eph receptor (Drescher, 2002; Mellott and Burke, 2008). No extant groups of deuterostomes are known to have retained this primitive condition. However, urchins have the simplest set: a single receptor and ligand. In urchins, Eph and Ephrin are expressed in broad domains of ectoderm, where they function at the interface to mediate apical contractility (Krupke and Burke, 2014). We now add to this a role in mediating aspects of dispersal and epithelial transition of migratory cells. Summaries of the diverse functions of Eph and Ephrin in vertebrates emphasize broad categories of functions in establishing domains within epithelia such as we find in developing hindbrains, somites, intestinal villi, and vascular remodelling (Klein et al., 2009, 2012; Pasquale, 2005, 2008; Poliakov et al., 2004). In addition, Eph and Ephrin have a second broad set of functions in guiding migrating cells, or axonal outgrowths (Nievergall et al., 2012; Klein, 2004; Triplett and Feldheim, 2012: Suetterlin et al., 2012). This parallel suggests that urchin embryos are a model that reveals a basal range of Eph and Ephrin functions, which has expanded and diversified such that Eph and Ephrin are the principal molecular determinants of cellular positioning and tissue patterning in vertebrates.

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Author details

1. Oliver A Krupke

   Department of Biology, University of Victoria, Victoria, Canada

   Contribution

   OAK, Developed materials, Concepts and approaches, Performed experiments, Analyzed data, Prepared parts of the manuscript, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

2. Ivona Zysk

   Department of Biochemistry and Microbiology, University of Victoria, Victoria, Canada

   Contribution

   IZ, Developed materials and performed experiments, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

3. Dan O Mellott

   Department of Biochemistry and Microbiology, University of Victoria, Victoria, Canada

   Contribution

   DOM, Developed materials and performed experiments, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

4. Robert D Burke

   Department of Biochemistry and Microbiology, University of Victoria, Victoria, Canada

   Contribution

   RDB, Developed concepts and approaches, Performed experiments, Analyzed data, Prepared parts of the manuscript, Contributed unpublished essential data or reagents

   For correspondence

   rburke@uvic.ca

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0001-5527-4410

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