Extended Data Figure 4: Absence of the epiphysis and presence of the ZLI in the develo** brain of the hagfish.

a, b, The adult hagfish has no epiphysis. A rudimentary epiphysis described previously^14,46 was likely to have been an artefact derived from an inappropriate method of fixation. Although von Kupffer (1900) described an epiphysis-like structure in his illustration (a, b, redrawn from ref. 15), he denied its identity as an epiphysis because it is distantly positioned from the posterior commissure (pc). c, The position of the epiphysis (ep) in a shark embryo is shown. d–g, In our present observation of hagfish embryos also, several neuroepithelial cysts were found in the forebrain (arrows in d–f), which were always associated with the development of blood vessel formation in the brain (arrowhead in g). Arrowheads in e indicate the crack between the neuroepithelial cyst and the brain tissue caused during the dehydration process, reminiscent of von Kupffer’s epiphysis-like structure in b. h–k, Hh2 expression in stage-45 (h, i) and stage-53 (j, k) hagfish embryos. Medial sections (h, j) and lateral sections (i, k). Dotted lines in i and k indicate the presumptive ZLI region. See Figs 1, 2, 3 for abbreviations. Scale bars, 500 μm.