We provide the first evidence for the presence of i-cells in siphonophores and describe their distribution using whole-mount in situ hybridization and histology. Broadly sampled phylogenetic analyses indicate that we could identify vasa-1, pl10, piwi, nanos-1, and nanos-2 orthologs in Nanomia bijuga (Additional file 1A–C). Negative controls with sense probes were performed for in situ hybridizations of all genes in all zooids, and none were positive. Key findings are presented in the figures of the main manuscript. All in situ results and negative controls are summarized in Additional files 2, 3, 4, 5, and 6. The whole-mount in situ hybridization techniques used in this study enabled us to identify tissue regions with co-expression of genes but lacked the spatial resolution to confirm if these genes were co-expressed in the same cells within these regions. We interpret the co-localized expression of all examined genes as a broad proxy for the presence of i-cells, though domains of expression may be supersets of domains with i-cells due to expression in other cell types, including germ cells, progenitor cells, and potentially somatic cells (e.g., [15, 18, 20, 21]). It will require follow-up studies to tie expression to particular cell types, which we have recently described in greater detail . We confirmed the presence of cells with i-cell morphology in select regions that showed expression. We interpret the absence of expression of examined genes as evidence of the absence of i-cells. In some cases we find clear differences between the expression domains of different genes, which we describe in greater detail below. These differences are likely due to some expression of some genes in cell types other than i-cells, including germ cells and nematoblasts. In cases where expression appeared co-localized across all five examined genes, we only feature exemplary results for vasa-1 in some of the main figures.
Evidence for the presence of i-cells in the horn of the siphosomal growth zone
The siphosomal growth zone produces most zooids in Nanomia bijuga (Fig. 1a, b). The general structure of the N. bijuga siphosomal growth zone, as well as its budding process, has previously been described . The zooids are arranged in repeating groups, known as cormidia. The budding sequence that produces cormidia and the zooid arrangement within them are highly organized (Fig. 1a, b, ). The siphosomal growth zone has a protrusion at its anterior end—the horn (labeled h in Fig. 1b). Pro-buds form at the tip of the horn and then subdivide into zooid buds as they mature and are carried to the posterior. These buds give rise to five different zooid types—gastrozooids (feeding polyps), palpons (polyps with function in circulation, defense, and digestion), bracts (defense), and female and male gonophores (gamete production) .
All examined genes were found to be expressed at the tip of the siphosomal horn and in all buds and young zooids within the siphosomal growth zone, with nanos-1 showing the lowest signal (Fig. 2a–f). Semi-thin sections and TEM analysis confirmed the presence of two types of cells within the ectoderm of the siphosomal horn, epithelial cells, and undifferentiated cells with i-cell morphology (Fig. 2g–i). Within the horn, cells with i-cell morphology were also found in the endoderm (Fig. 2h). The mesoglea within the horn appeared discontinuous suggesting that there may be migratory activity of i-cells between ectoderm and endoderm (Fig. 2h). In the endoderm of young zooid buds, however, no cells with i-cell morphology were observed. Nuclei of endodermal cells were located close to the mesoglea (Fig. 2j). Both epithelial cells and cells with i-cell morphology were found in the ectoderm of young zooids (Fig. 2j).
Co-localized vasa-1, pl10, piwi, nanos-1, and nanos-2 expression suggests spatial restriction of i-cells during zooid development
The distal portion of the pro-bud gives rise to the gastrozooid—the feeding zooid (Figs. 1b, 2b, and 3). Young gastrozooid buds had expression of all genes (Figs. 2b–f and 3a). The basigaster, a specialized region of nematocyst formation in siphonophores , was evident in young gastrozooid buds as a thickening of the proximal ectoderm (Fig. 3b). In the course of basigaster development, expression of all examined genes, except nanos-2 (Fig. 3l), became restricted to deep basigaster ectoderm (Fig. 3b–g, Additional file 3C, D) and then decreased until a signal was no longer detectable in mature gastrozooids (Fig. 3h–k, Additional file 4F). nanos-2 expression persisted in the basigaster region of gastrozooids of all ontogenetic stages (Figs. 2f and 3l, Additional file 6C, E, G, H). This finding was consistent with previous studies that indicated a nanos-2 function in nematocyst formation [15, 30]. Within the basigaster, nanos-2 seemed to be co-localized to the same region as minicollagen (see ), which is known to be involved in capsule formation . Though vasa-1, pl10, piwi, and nanos-1 transcripts were not detected in basigasters of mature gastrozooids (Fig. 3h–k), undifferentiated cells were still found along the mesoglea (Fig. 3m) indicating the presence of a determined progenitor cell population which gives rise to nematocytes but has lost interstitial stem cell transcriptional signatures. Immature nematocysts were observed in the outer layers of the mature basigaster (Fig. 3m). The gene vasa-1 was expressed in the same regions of the young gastrozooids as pl10, piwi, and nanos-1. In addition, it was expressed in both the ectoderm and endoderm of the tips of young gastrozooids (Figs. 2a and 3b–e).
Each gastrozooid has a single tentacle attached at its base. The tentacle has side branches, known as tentilla, which bear packages of nematocysts at their termini (Fig. 1a, ). All examined genes were expressed in the tentacle bases throughout all ontogenetic stages of gastrozooids (Fig. 3b, d–g, h–l). The expression domains, however, differed between genes. Whereas nanos-2 expression was restricted to the very proximal end of the tentacle and very early tentilla buds (Fig. 3l, Additional file 6H, I), signal for the other four genes persisted in developing tentilla as well (Fig. 3d, e, j, k). None of the examined genes were expressed in mature tentilla (e.g., in Fig. 3j, k).
Anterior to each gastrozooid, a series of buds develop into palpons—zooids thought to have a function in circulation of gastrovascular content, digestion, and defense (Fig. 1b, ). Like gastrozooids, each palpon has a single tentacle (Fig. 1a), which is known as a palpacle . The palpacle is, in contrast to the gastrozooid tentacle, unbranched and nematocysts can be found along its entire length. As in gastrozooids, strong expression was detected for all examined genes in young palpons within the growth zone, and expression disappeared from later developmental stages (e.g. Fig. 2a–d, f). Expression was absent from mature palpons (Figs. 2a and 4a–d, f), except for nanos-2, which remained expressed in a small domain at the proximal end of the palpon (Fig. 4f, Additional file 6J). Unlike in gastrozooids, this nanos-2 expression domain did not extend around the entire zooid but was restricted to a small patch close to the palpacle base (Fig. 4f). Semi-thin sections indicated this patch as a site of nematogenesis (Fig. 4g), suggesting that it is equivalent to the basigaster of gastrozooids. These similarities between gastrozooids and palpons were consistent with the hypothesis that palpons are derived gastrozooids that lost the ability to feed, i.e., they lack a mouth opening . Expression of all examined genes was found at the proximal end of the palpacle (Fig. 4a–f, Additional file 2G). Densely packed, undifferentiated cells with i-cell morphology were present within palpacle bases (Fig. 4g). Additional secondary palpons are added at the anterior end of mature cormidia, and gonodendra form laterally from these secondary palpons . We frequently found small buds anteriorly from the youngest primary palpon, which were at the sites where these secondary structures arise. All examined genes were found to be expressed in such buds (Fig. 4h–j).
Bracts are protective zooids, which can be found laterally along the siphosomal stem but also associated with palpons and gastrozooids (Fig. 1b, ). They are of scale-like morphology and function as protective shields. As in gastrozooids and palpons, all examined genes were expressed in early developing bract buds (shown for vasa-1, Fig. 4e), but expression was absent in older bracts once the typical bract morphology became obvious (shown for vasa-1, Fig. 2a).
vasa-1, pl10, piwi, nanos-1 and nanos-2 expression in sexual zooids
While some siphonophore species are dioecious, a colony of Nanomia bijuga produces gametes of both sexes . Gametes are produced by gonophores, each of which is either male or female. These gonophores are arranged into groups called gonodendra , which each exclusively bear male or female gonophores. Gonodendra are attached directly to the stem and develop laterally at the base of the palpon peduncle. There are gonodendra of both sexes associated with each palpon, one male and up to two female gonodendra. The locations of these male and female gonodendra alternate between adjacent palpons (Figs. 1a and 5a, ).
Female gonodendron formation has been described previously  as follows. Female gonodendra start to form as small buds protruding at the base of the palpon peduncle. Germ cells develop in between endoderm and ectoderm. Each gonophore within the female gonodendron contains a single egg. The egg is enclosed by a thin ectodermal layer within the developing female gonophore. Two lateral canals form from endodermal epithelial cells within the gonophore. The mature gonophore is attached to the blind-ending central stalk of the gonodendron by a delicate peduncle.
In situ hybridizations for all five genes yielded identical expression patterns in gonodendra. Findings for vasa-1 are summarized in Fig. 5 and are representative for the other four examined genes. Close to the growth zone, the first indication of gonodendron development was round clusters of cells with strong expression on the stem at the base of the young palpons (Figs. 1b and 5b, Additional file 6M). These clusters were visible before bud formation became obvious, and male and female clusters were morphologically indistinguishable from each other at this stage. In situ hybridization revealed expression of all five genes in a helical pattern in the mature female gonodendron. This pattern corresponds to a previously unobserved helical morphological organization (Fig. 5e, f, h, Additional files 3K, 4M, 5J, and 6G, P). The gonodendron buds started to twist early in development, and a stronger signal was observed on the outer side of the developing stalk away from the axis of the helix (Fig. 5c, d, Additional file 6N, O). This pattern persisted during the first turns until the gonodendron took on an appearance reminiscent of clusters of grapes. At this stage, all examined genes were strongly expressed in all gonophores along the gonodendron, and the helical organization was not apparent. Helical organization became obvious again in later ontogenetic stages (Fig. 5e, f, Additional files 3K, 4M, 5J, and 6G) when expression decreased in mature gonophores (Fig. 5e, f). The presence of signal in immature gonophores distributed in a helical pattern along the gonodendron indicated that new gonophores were produced along one side of the entire twisted stalk of the gonodendron. The chirality of the helices changed with the site of attachment. Gonodendra attached on the left side of a palpon showed a clockwise directionality of turns.
The male gonodendron starts with the formation of a primary gonophore, which is cone shaped. Secondary and tertiary gonophores bud off the delicate peduncle of the primary gonophore (Fig. 5g). The male gonophore is an elongated structure with a massive population of putative germ cells in the ectoderm (see ). All examined genes were strongly expressed in young and medium-sized gonophores, but signal intensity was lower or absent in gonophores close to or at maturity (Fig. 5h, Additional files 3M, 4N, 5L, and 6Q). The absence of graded signals along the proximal-distal axis suggests that sperm maturation took place along the entire gonophore.
Nectosomal growth zone has a similar structure as the siphosomal growth zone
Nanomia bijuga, like most other siphonophore species, has a nectosomal growth zone (Fig. 1c) near the anterior end that produces the swimming zooids, called nectophores, which propel the whole colony through the water . All examined genes were strongly expressed in the nectosomal growth zone at the tip of the horn, in nectophore buds, and in young developing nectophores (Fig. 6a–d, Additional file 5A). Co-localized expression of all genes and histological sections suggested the presence of i-cells in the thickened region of the nectosomal stem, the horn of the growth zone, and young nectophore buds (Fig. 6a–d, Additional file 5A). In case of vasa-1, the transcript persisted longest along the ridges of the nectophores (Fig. 6a). Older nectophores were free of gene transcripts in case of all examined genes (e.g., Fig. 6b–d, Additional file 5A). In contrast to the other four genes, nanos-2 expression was restricted to the very youngest nectophore buds (Fig. 6c). In addition, in the stem subtending the growth zone, the transcript was detected on the nectosomal stem in a salt and pepper pattern (Fig. 6c). Sections revealed developing nematocysts in this region of the stem (Fig. 6f). Undifferentiated cells with interstitial cell morphology were identified in the ectoderm of the horn and developing nectophores (Fig. 6e–g).
Co-localized vasa-1, pl10, piwi, nanos-1 and nanos-2 expression is found in a subset of regions with high rates of cell proliferation
A qualitative assessment of cell proliferation revealed high densities of EdU-labeled nuclei in domains with expression of all examined genes (compare Figs. 2a and 7a). Specifically, EdU-labeled nuclei were found in the horns of both growth zones as well as in young buds and developing zooids both within the growth zones and along the siphosomal stem (Fig. 7a–f). In all analyzed tissue samples, the palpacle bases consistently had strong EdU labeling in developing palpons as well as in mature palpons (Fig. 7f). Tentacle bases and developing tentilla were also strongly EdU labeled in gastrozooids (Fig. 7a, g). In addition, high densities of EdU-labeled nuclei were found in stem regions at the level and adjacent to both growth zones (Fig. 7c, d, i), where only a few or no EdU-labeled nuclei were identified in posterior regions of the nectosomal (Fig. 7h) and siphosomal (Fig. 7j) stem. These EdU-labeled regions in the stem are the main sites of stem elongation in Nanomia bijuga. Interestingly, these stem regions were devoid of vasa-1, pl10, piwi, nanos-1, and nanos-2 gene expression (compare Figs. 2b and 7c and Figs. 6a, b and 7d). Conspicuous cell division was occasionally observed along the dorsal midline of the stem (Fig. 7k), whereas no signal was obtained in these regions in the in situ hybridizations. The number of EdU-labeled cells in a particular zooid type decreased with level of maturity, and in many cases, no proliferative activity was found in mature zooids (Fig. 7l–t). In developing male gonophores, our EdU assay showed a large number of dividing cells in the ectoderm (Fig. 7u, v). In developing female gonodendra, EdU-labeled nuclei were consistently detected in developing gonophore bells (Fig. 7w).