Fig. 5.

Development of the axial skeleton of A. burtoni. a Neural and haemal arches develop between 102 hpf and 126 hpf, as shown by Alcian blue staining. In teleost fish, vertebral centra do not form through a cartilaginous intermediate, and ossified centra become first visible by Alizarin red staining (fluorescent microscopy image) at 138 hpf. Blue asterisk in 126 hpf indicates absence of mineralized centra. b Visualization of somites, using the muscle sarcomere specific antibody MF20 together with Alcian blue staining, shows that the basioccipital (yellow arrowhead) has formed by 100 hpf within somites 1 and 2, and extends up to the somite 2–3 boundary. The first developing neural arch is present in somite 4. This shows that, as in other anamniotes, the basioccipital develops from the first three somites. The horizontal dotted line indicates the somite 2–3 boundary. c Ribs develop around 138 hpf (red arrowhead). Interestingly, we observe what appear to be rib Anlagen associated with pre-caudal vertebrae 1 and 2 (indicated with a red asterisk), which in the adult skeleton are not rib-bearing. At 156 hpf, these “cryptic” ribs are however no longer present (indicated with a red asterisk). Epicentrals develop much later and only start developing around 9 dpf as membrane bones (black arrowhead indicates epicentral on pre-caudal vertebra 1; the black asterisk indicates the forming epicentral on pre-caudal vertebra 2; the blue arrowhead indicates a transversal process on pre-caudal vertebra 4, which is not further discussed here). d The axial skeleton forms in the following sequence: the first element to appear is the basioccipital (stage I, before 100 hpf), followed by the formation of neural and haemal arches dorsal and ventral of the notochord (stage II and III). Further elements to appear are the vertebral centra and the ribs (stage IV), followed by the epicentrals (stage V). Abbreviations: dpf: days post fertilization; hpf: hours post fertilization; AZR: Alizarin red