Medusa tentacle branching
Our observations of medusa tentacle branch formation in the jellyfish C. pacificum reveal features of branch patterning and morphogenesis that are common to other well-studied branching systems in animal species, as well as ones that are unique to C. pacificum. While C. pacificum branched tentacles appear to be an elaborate structure, we found that they form through repeated applications of a simple rule: branching at the proximal part of the main tentacle. This mechanism of repeating a simple rule is widely used in other branching systems [7, 17, 19, 20, 23], and thus may represent a fundamental mechanism for generating complex structures, such as branched organs, across a wide range of animal species including non-bilaterians. However, we also found that the branching of C. pacificum tentacles differed from that in other branching systems, including those found in corals and colonial hydroids [16,17,18], in that it occurs at the proximal end of the branching structure. The proximal region of medusa tentacles has been shown to be the site of active cell proliferation in Aurelia and Clytia jellyfish species [30, 31], which suggests that cell proliferation may be involved in the proximal branching in C. pacificum. In examples of branching from mammalian and Drosophila models of airway formation and angiogenesis, branches form at the tips of branching tissues [21, 22]. This may be because the tissues are growing branches to find all possible target cells, thus branching at sites of cell searches, possibly in response to signals from these cells, is likely more efficient. Medusa tentacles, on the other hand, can flexibly move their branches by muscle contraction, even after the branch architecture has been established. Further, the adhesive branches may contribute to the unique method of tentacle branching in C. pacificum. The adhesive branches, which extend off of the adaxial side of the proximal main tentacles (Fig. 1b), enable the medusa to “stand up” on a substratum, such as seagrass. This allows for the medusa to secure a space between the mouth and the substratum, while the distally located nematocyst branches deliver prey into the mouth. However, as the main tentacles extend in length, the adhesive branches shift too far away to contribute to standing and no longer serve their original function. It might therefore be more efficient to recycle these established branches into hunting branches than to form new branches at distal regions. Despite the different branching methods between C. pacificum and other animals, the resulting branch structures are advantageous for expanding the epithelial surface areas and maximizing functions.
Acquisition of adhesive organs and nematocysts
The results from our ablation experiments indicate that the acquisition of nematocysts does not depend on formation of adhesive organs (Fig. 8). We speculate that this is because the nutrition-finding function of nematocysts was prioritized over adhesive organ formation after surgical removal of the distal parts of tentacles containing nematocyst clusters (Fig. 8a). During tentacle growth, nematocyst clusters appear on the main tentacle as early as on Day 2, even before the adhesive organ is formed for the first time on the first branch (Fig. 6u). In support of this notion, we found that limiting the amount of Artemia Nauplius prey to two individuals per day, or every other day, enhanced the formation of functional nematocysts in the absence of adhesive organ formation (0% with an excess amount of the prey every day (n = 12); 25% with two prey per day (n = 12); 61.1% with two prey every other day (n = 12)). Interestingly, however, functional nematocysts did not form earlier in the ablated tentacles than in controls (Fig. 8); thus, the timing of nematocyst acquisition may be tightly regulated.
RTK signaling and mesoderm origin
Branch formation occurs through local cellular movements, such as cell migration, proliferation, rearrangement, and deformation, which generate new branch buds [20,21,22,23]. We found that tentacle branching in C. pacificum may be initiated by extension of the epidermal epithelial cells along the apico-basal axis. This observation highlights a possibly important and conserved role of regulating epithelial cell shape in branch formation among a wide range of animals covering both non-bilaterian and bilaterian animals. In the mammalian pancreas and salivary gland, branch bud cells have a characteristic columnar shape [32, 33]. In stolons of hydroids, a plate of columnar ectodermal cells is formed at the site of branching [17].
At the molecular level, many of the cellular behaviors involved in branch formation require receptor tyrosine kinase (RTK) signaling [20,21,22]. For example, FGF signaling is required for specification of leading cells in cell migration in the Drosophila trachea [34] and mammary gland [35] and for regionalized cell proliferation in the mouse salivary gland [10], vascular endothelial growth factor (VEGF) signaling is required for leading cell specification in mammalian retinal blood vessels [36], and glial cell-derived neurotrophic factor (GDNF) signaling is required for cell proliferation in the mouse kidney [37]. The ligands for these RTKs are produced in the mesenchyme, which surrounds the core structure of branched organs made of epithelial cells. In this study, we found that inhibition of MEK in C. pacificum led to the absence of branch formation in the tentacles, suggesting that medusa tentacle branch formation in this species also requires RTK signaling. However, we note that jellyfish are diploblastic animals without mesoderm. Although this is debatable, as there is bilaterian-like striated muscle in the sub-umbrella region of most hydrozoan medusae and the striated muscle originates in the entocodon cell mass which develops between the ectoderm and endoderm [38, 39], to our knowledge there are no mesoderm-like cells in the tentacle region. Therefore, it is of particular interest to determine the source ligand for RTK signaling in tentacle branch formation.
In relation to the absence of mesoderm in the medusa tentacles, we would also like to note that the tentacle branches extend out towards the apical side of the epithelial layers. This contrasts with branching morphogenesis in Drosophila and mammals, where branches grow into the mesenchyme located on the basal side of the epithelial layers [20,21,22,23]. In this sense, the medusa tentacles as well as stolons [17] in hydrozoa species, may be more comparable to plant roots in terms of cellular processes of branch formation. Plant roots also extend their branches out towards the external environment and regionalized cell proliferation is involved in their branch formation [40].
Branched tentacles as a new trait in evolution
Cladonema pacificum belongs to the family Cladonematidae, which is characterized by a number of synapomorphic features including branched medusa tentacles with adhesive organs [27]. Therefore, studying Cladonema tentacle branch formation could provide clues to understand how a new trait might have been acquired in the course of evolution. Another genus that belongs to the family Cladonematidae, Staurocladia, also has branched tentacles [27, 41], which, unlike C. pacificum, branch only once. It would be interesting to examine how the Staurocladia species prevent further branch formation. The regulation of RTK signaling might be involved in this inter-genus difference.
The Staurocladia medusa main tentacles have nematocyst clusters with single branches bearing adhesive organs extending off the adaxial side of the main tentacle. Unlike C. pacificum, the branches do not seem to change their functions. In our study of C. pacificum medusae at Day 7, at which time the third branches (Branch③) are first observed (Fig. 2j) and the second branches (Branch②) have only adhesive organs (Fig. 6u), we tried to eliminate the effect of the third branches by cutting them on Day 7. Although this cutting resulted in regeneration of branches at the cut site on the next day, we cut them again, mimicking the situation in Staurocladia, which lacks any younger branches. We then examined whether the second branches change their function to nematocyst branches after the third branch ablation. We found that their function shifted following the normal time course (100%, n = 7), suggesting that the presence or absence of third branches does not determine whether the second branches change their function. Therefore, the lack of functional changes in Staurocladia branches may not be due to the absence of younger branches. Continued study of these two closely related species would further explain the developmental and evolutionary aspects of tentacle branch formation that may possibly apply to other species without branched tentacles.