From the above observations, it seems plausible to assume that all the hagfish somites form sclerotomes and dermomyotomes at all axial levels in positions corresponding to those in gnathostomes, and that these somatic components arise via molecular developmental mechanisms similar to those in gnathostome embryos (Fig. 4). At the post-cloacal level, the sclerotome-derived cells are distributed in regions prospectively occupied by the vestigial cartilaginous nodules in adults. Thus, in the hagfish, the somite appears to respond to signals derived from the notochord to specify cells destined towards skeletal differentiation. In this connection, the potential skeletogenic property of hagfish paraxial mesoderm is suggested in the chordal cranium (the caudal portion of the neurocranium known as the parachordals; Supplementary Fig. S6)23,24. Therefore, it seems likely that the notochordally derived signal is primarily capable of inducing chondrification in the paraxial mesoderm of the hagfish, and that this mechanism is lost secondarily from the pre-cloacal part of the hagfish trunk.

Our observations are consistent at the anatomical and developmental level with the presence of axial cartilage in hagfish that is homologous to gnathostome vertebrae. This indicates that the presence of cartilaginous axial skeletal elements can be considered as a characteristic common to gnathostomes, lampreys and hagfishes. Hence, we can no longer exclude the hagfish from the Vertebrata simply due to the absence of a cartilaginous axial skeleton, which has now been disproved (Fig. 5).

Figure 5: Hypothetical scenario of vertebral evolution. This phylogenetic tree is based mainly on molecular data and hagfishes are clustered with lampreys as members of monophyletic cyclostomes25,26,27,28. The origins of the cartilaginous vertebrae, consisting of dorsal and ventral elements, as well as associated developmental mechanisms are assumed to have been obtained before the divergence of gnathostomes, lampreys, and hagfish. The vertebral elements of the extant vertebrates are coloured blue, and those of the common ancestor of entire extant vertebrates are indicated by dotted lines. bd, basi-dorsal; bv, basi-ventral; id, inter-dorsal; iv, inter-dorsal; n, notochord; nt, neural tube. Full size image

Although our developmental study suggested a sclerotomal origin for the ventral elements, the origin of the dorsal element, reminiscent of the neural arches and spine of gnathostomes, remains unclear (Fig. 1b). Nevertheless, given the anatomical similarity of this element to the gnathostome counterpart (Figs 1e and 2e,f), it is highly possible that they share the same developmental patterns, suggesting that the paraxial mesoderm of the entire common ancestor of gnathostomes, lampreys and hagfishes could form cartilaginous elements on both the dorsal and ventral sides of the notochord. Taking into account the recently reconstructed phylogenetic trees based on molecular data, which support a monophyly of hagfishes and lampreys25,26,27,28, it seems reasonable to hypothesize that the dorsal and ventral vertebral elements were obtained from a common ancestor of the entire vertebrates, and that subsequent secondary losses would have led to loss of the dorsal vertebral elements in the hagfishes on one hand, and to a ventral vertebral element in the lamprey on the other (Fig. 5). To test this hypothesis, further comparative developmental studies will be required to exclude the possibility of independent evolutionary occurrence of vertebral elements in the extant agnathan lineages, because it is still unclear whether the entire part of the vertebral elements of extant agnathans is derived from sclerotomal cells, as occurs in gnathostomes12. We still cannot rule out completely the possibility that some parts of the vertebral elements of lamprey and hagfish are derived from non-sclerotomal cells (for example, neural crest cells).

Finally, it remains to be determined whether the morphological pattern of hagfish axial skeletal elements represents a secondary loss of segmentation or an ancestral state before acquisition of the segmental pattern. However, the former scenario is more plausible under the assumption of the monophyly of the extant agnathans (Fig. 5). This scenario implies that the common ancestor of the extant gnathostomes and agnathans had the developmental mechanism to form a segmental axial skeleton consisting of two topographically different cartilaginous nodules, one located at the level of the myoseptum and the other at the intermediate level between the two myosepta, along the anterior–posterior axis, as seen in the vertebral elements of lampreys and gnathostomes (Figs 2 and 5). The origin of this developmental mechanism could date back to around 500 million years ago based on the divergence time between agnathans and gnathostomes26,27. Also problematic is the absence of cartilaginous tissue from the pre-cloacal trunk, as well as the absence of dorsal skeletal elements in most of the post-cloacal region (Fig. 1). Further study is required to elucidate whether such a loss can be simply explained by adaptation to a hagfish lifestyle or by the long evolutionary period2. The origin of the axial skeleton remains an issue in evolutionary developmental biology, and will require consideration of paleontological, embryological and molecular data12,13,25,26,27,28,29,30. We are hopeful that further advances in hagfish embryology will provide more insights into the evolution of the axial skeletal element in the early vertebrates, especially into the origin of its segmental pattern.