Regeneration has been hypothesized to evolve multiple times in all the different animal phyla by re-using pre-existent developmental regulatory modules (Brockes and Kumar, 2008; Smith et al., 2011). Here, I present the comparison of skeletogenesis during embryogenesis and adult regeneration in the same species revealing vast similarities between the two developmental processes.
6.2.1 Morphological similarities of skeletogenic cells and spicules between the embryo and regenerating arm
Skeletogenesis of echinoderm embryos has been studied extensively in sea urchins and, more recently, in brittle stars providing an excellent basis for comparing the development of the skeleton with adult regeneration. In sea urchin and brittle star embryos skeletogenic cells become specified at late cleavage stage (Davidson et al., 1998; Dylus et al., 2016; Okazaki, 1975;
Wilt and Ettensohn, 2008), which is exemplified by expression of the key activator of the skeletogenic GRN - alx1 (Dylus et al., 2016; Ettensohn et al., 2003; Koga et al., 2016). These cells then undergo an epithelial-to-mesenchymal transition and remain epithelial-to-mesenchymal throughout skeletal development. In chapter 3 I show that the cells, which express skeletogenic TFs including Afi-alx1 and Afi-jun, and downstream genes like Afi-c-lectin,
are also mesenchymal cells located in the dermal, collagenous layer of connective tissue in the early regenerate (Figure 3.5). My observations of the formation of the skeleton during early regenerating stages suggest that cells undergo specification and differentiation events very early during the regeneration process (Figure 3.7). The spicule primordia observed at stage 3 resemble the granule-like skeletal rudiment of A. filiformis embryos (Dylus et al., 2016), and both sea urchin embryos (Okazaki, 1975; Wilt and Ettensohn, 2008) and juveniles (Yajima and Kiyomoto, 2006) at the earliest step in the development of the skeleton, which then branch into tri-radiated and tetra-radiated spicules.
6.2.2 Late differentiation and patterning of the skeleton
The 50% differentiated arm shows the developmental progress of the skeleton from single spicules up to the formation of complex mesh-like structures of the dermal plates (lateral, oral and aboral shields), spines and vertebrae (Figure 3.8). The program used to form these intricate patterns of skeletal elements in the adult might be similar to the mechanism of patterning of the pluteus shape skeletal rods in the larva. Several studies have shown that the interaction of the skeletogenic mesoderm with its adjacent ectoderm is crucial for the correct patterning of the larval skeleton in sea urchins and brittle stars (Mcintyre et al., 2014). FGF and VEGF ligands are expressed in the ectoderm and their interaction with their respective receptors, expressed by skeletogenic cells, mediate correct migration of SM cells, as well as localization and elongation of the skeletal rods (Adomako-Ankomah and Ettensohn, 2013; Duloquin et al., 2007; Morino et al., 2012; Röttinger et al.,
2008). pax2/5/8 and wnt5 expressed in the ectoderm overlying the SM both in sea urchin and brittle star larvae have also been implicated in the development of the pluteus skeleton (McIntyre et al., 2013; Morino et al., 2016). I found that FGF and VEGF signalling genes are expressed in a topologically conserved relationship during adult arm regeneration in A.
filiformis at late stages (Figure 5.2), and thus could play a role in patterning of the different skeletal elements differentiating in proximal segments. This will need to be confirmed by future analysis of the effects of FGF/VEGF perturbation at late stages of regeneration.
Interestingly, the vertebral spicules, which are internal skeletal elements, appear much later than those involved in the formation of the lateral shields and spines (Figure 3.8). As seen in SEM images, the complete vertebrae in adult non-regenerating arms of ophiuroids are composed of two conjoined ambulacral plates (Gage, 1990; Irimura and Fujita, 2003; Stöhr et al., 2012), which could explain why during regeneration the vertebrae appear to form by a fusion of skeletal elements from bilateral halves of each segment (Figure 3.8). The same SEM studies also show that they are clearly the most complex and dense skeletal elements in the ophiuroid arms (Gage, 1990; Irimura and Fujita, 2003; Stöhr et al., 2012). Taken together, these data suggest molecular differences and possibly that a separate developmental program might be involved in the formation of those internal-most skeletal structures compared to the sparser stereom constituting the lateral shields and spines. This is supported by differences in expression of genes present in all skeletal territories compared to those localized preferentially in only some skeletal elements (Figure 4.7). Interestingly, a
combination of different sets of differentiation genes has also been observed in skeletogenic cells of the late pluteus larva in sea urchins (Sun and Ettensohn, 2014). Certainly different positional cues must be required to shape different skeletal elements. While it is conceivable that the epidermis acts as a signalling centre for the underlying dermal layer of skeletogenic cells, as the ectoderm provides essential positional cues in the sea urchin embryos (Duloquin et al., 2007), this is unlikely to be the case for the skeletogenic cells forming the vertebrae. Nevertheless, the presence of FGF and VEGF ligand expression in patches of expression deeper in the tissue (Figure 5.1) might suggest these signalling pathways are also involved in vertebrae formation, though either through a different set of receptors or the receptor expression in the vertebral skeletogenic cells is undetectable. It would be interesting to test what potential signalling pathways might be involved in the formation of individual skeletal ossicles during brittle star arm regeneration.