Craniofacial development of hagfishes and the evolution of vertebrates

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Cyclostomes, the living jawless vertebrates including hagfishes and lampreys, represent the most basal lineage of vertebrates. Although the monophyly of cyclostomes has been supported by recent molecular analyses, the phenotypic traits of hagfishes, especially the lack of some vertebrate-defining features and the reported endodermal origin of the adenohypophysis, have been interpreted as hagfishes exhibiting a more ancestral state than those of all other vertebrates. Furthermore, the adult anatomy of hagfishes cannot be compared easily with that of lampreys. Here we describe the craniofacial development of a series of staged hagfish embryos, which shows that their adenohypophysis arises ectodermally, consistent with the molecular phylogenetic data. This finding also allowed us to identify a pan-cyclostome pattern, one not shared by jawed vertebrates. Comparative analyses indicated that many of the hagfish-specific traits can be explained by changes secondarily introduced into the hagfish lineage. We also propose a possibility that the pan-cyclostome pattern may reflect the ancestral programme for the craniofacial development of all living vertebrates.

At a glance


  1. Cyclostomes and gnathostomes.
    Figure 1: Cyclostomes and gnathostomes.

    a–c, Eptatretus burgeri (hagfish). a, Dorsal view. b, Ventral view of a hagfish head showing a single nostril (en) and jawless mouth (mo). c, A diagram of the head anatomy. dg, Lethenteron japonicum (lamprey). d, Lateral view. e, Ventral view to show the oral funnel surrounding the mouth. f, Dorsal view to show the dorsal nostril. g, A diagram of the head anatomy. h, i, Scyliorhinus torazame (shark). h, Lateral view. i, Frontal view to show upper and lower jaws (uj and lj), and paired nostrils (en). j, Diagram of the generalized gnathostome head. ah, adenohypophysis; et, ethmoidal region; llp, lower lip; ne, nasal epithelium; ons, oronasohypophyseal septum; T1–4, tentacles 1–4; ulp, upper lip.

  2. Craniofacial development of E. burgeri.
    Figure 2: Craniofacial development of E. burgeri.

    af, Frontal (a, c, e) and lateral (b, d, f) views. The ectoderm is coloured light blue, the endoderm is coloured yellow, and the mesoendoderm is coloured orange. Arrows indicate the SOM. g, h, Sagittal sections at the plane represented in e. ip, Lateral views (i, k, m, o) and sagittal sections (j, l, n, p). Inset in p shows the differentiated adenohypophysis (ah). e, eye; ht, hypothalamus; nhd, nasohypophyseal duct; nhp, nasohypophyseal plate; nt, notochord; oc, oral cavity; onc, oronasohypophyseal cavity; opm, oropharyngeal membrane; pcp, prechordal plate; p1, pharyngeal pouch 1; st, stomodeum. See Fig. 1 for other abbreviations. Scale bars, 100μm. Numbers indicate developmental stages.

  3. Embryonic gene expression in Eptatretus burgeri.
    Figure 3: Embryonic gene expression in Eptatretus burgeri.

    af, Expression of genes involved in the NHP development from stages 26 to 40. g, Dorsal view of three-dimensional-reconstructed image. The blue area shows pan-placodal domain. h, Schematic diagrams summarizing the gene expression in the embryos. In the graph, the hypothalamic domain is characterized by EbHh1, EbNkx2.1 and EbSix3/6 expression. Adenohypophysis-like expression (blue bars) is restricted in the ectodermal domain rostral to and including the oropharyngeal membrane (opm). ch, chiasma; fb, forebrain; hb, hindbrain; mb, midbrain; ot, otic placode. See Figs 1 and 2 for other abbreviations. Scale bars, 100 μm.

  4. Comparison of vertebrate heads.
    Figure 4: Comparison of vertebrate heads.

    ah, Mid-sagittal sections (a, e) and ventral views (bd, fh) of Eptatretus burgeri (a, stage 45; b, stage 40; c, stage 50; d, stage 53; SOM has been removed from bd) and Lethenteron japonicum (e, stage 26; f, stage 23; g, stage 26; h, stage 27). ik, Ventral views of Scyliorhinus torazame (i, stage 20; j, stage 25; k, stage 28). The premandibular domain (asterisk) is comparable to the post-hypophyseal process (php) of cyclostomes. The blue domain (lateral and medial nasal prominences (fnp+lnp)) resembles the anterior nasal process (anp) of cyclostomes (ref. 22). l, Generalized patterns of vertebrate heads. ma, mandibular arch; np, nasal placodes. See Figs 1 and 2 for other abbreviations. Scale bars, 100μm.

  5. Evolution of the vertebrate head.
    Figure 5: Evolution of the vertebrate head.

    A model based on the assumption that the pan-cyclostome pattern (in box) represents a plesiomorphic pattern for the entire vertebrates. The hagfish oronasohypophyseal septum (ons) and the lamprey upper lip (ulp) are both derivatives of the post-hypophyseal process (php). Separation of the NHP into the nasal epithelium (ne) and adenohypophysis (ah) may have led to the pattern of galeaspids42. Further bilateral separation of the nasal epithelia is regarded as a prerequisite for acquisition of the trabecula (tr) and upper and lower jaws (uj and lj) in crown gnathostomes. ph, pharynx. See Fig. 1, 2 and 4 for other abbreviations.

Accession codes

Primary accessions


  1. Mallatt, J. & Sullivan, J. 28S and 18S rDNA sequences support the monophyly of lampreys and hagfishes. Mol. Biol. Evol. 15, 17061718 (1998)
  2. Kuraku, S., Hoshiyama, D., Katoh, K., Suga, H. & Miyata, T. Monophyly of lampreys and hagfishes supported by nuclear DNA-coded genes. J. Mol. Evol. 49, 729735 (1999)
  3. Takezaki, N., Figueroa, F., Zaleska-Rutczynska, Z. & Klein, J. Molecular phylogeny of early vertebrates: monophyly of the agnathans as revealed by sequences of 35 genes. Mol. Biol. Evol. 20, 287292 (2003)
  4. Kuraku, S. Insights into cyclostome phylogenomics: pre-2R or post-2R. Zoolog. Sci. 25, 960968 (2008)
  5. Heimberg, A. M., Cowper-Sal-lari, R., Semon, M., Donoghue, P. C. & Peterson, K. J. microRNAs reveal the interrelationships of hagfish, lampreys, and gnathostomes and the nature of the ancestral vertebrate. Proc. Natl Acad. Sci. USA 107, 1937919383 (2010)
  6. Janvier, P. Early Vertebrates (Oxford Univ. Press, 1996)
  7. Gorbman, A. Early development of the hagfish pituitary gland: evidence for the endodermal origin of the adenohypophysis. Am. Zool. 23, 639654 (1983)
  8. Gorbman, A. & Tamarin, A. In Evolutionary Biology of Primitive Fishes (eds Foreman, R. E., Gorbman, A., Dodd, J. M. & Olsson, R.) 165185 (Plenum, 1985)
  9. Wicht, H. & Tusch, U. in The Biology of Hagfish (eds Jørgensen, J. M., Lomholt, J. R., Weber, R. E. & Molte, H.) 431451 (Chapman & Hall, 1998)
  10. Soukup, V., Horácek, I. & Cerny, R. Development and evolution of the vertebrate primary mouth. J. Anat.. (16 July 2012)
  11. Forey, P. & Janvier, P. Agnathans and the origin of jawed vertebrates. Nature 361, 129134 (1993)
  12. Gess, R. W., Coates, M. I. & Rubidge, B. S. A lamprey from the Devonian period of South Africa. Nature 443, 981984 (2006)
  13. Marinelli, W. & Strenger, A. Vergleichende Anatomie und Morphologie der Wirbeltiere 2. Myxine Glutinosa (Franz. Deuticke, 1956)
  14. Candiani, S., Holland, N. D., Oliveri, D., Parodi, M. & Pestarino, M. Expression of the amphioxus Pit-1 gene (A mphiPOU1F1/Pit-1) exclusively in the developing preoral organ, a putative homolog of the vertebrate adenohypophysis. Brain Res. Bull. 75, 324330 (2008)
  15. Schlosser, G. Evolutionary origins of vertebrate placodes: insights from developmental studies and from comparisons with other deuterostomes. J. Exp. Zool. B 304, 347399 (2005)
  16. von Kupffer, C. Studien zur Vergleichenden Entwicklungsgeschichte des Kopfes der Kranioten. Heft 4: Zur Kopfentwicklung von Bdellostoma 1–86 (Lehmann, 1900)
  17. Adachi, N. & Kuratani, S. Development of head and trunk mesoderm in the dogfish, Scyliorhinus torazame. I. Embryology and morphology of the head cavities and related structures. Evol. Dev. 14, 234256 (2012)
  18. Jean, D., Bernier, G. & Gruss, P. Six6 (Optx2) is a novel murine Six3-related homeobox gene that demarcates the presumptive pituitary/hypothalamic axis and the ventral optic stalk. Mech. Dev. 84, 3140 (1999)
  19. Sugahara, F. et al. Involvement of Hedgehog and FGF signalling in the lamprey telencephalon: evolution of regionalization and dorsoventral patterning of the vertebrate forebrain. Development 138, 12171226 (2011)
  20. Uchida, K., Murakami, Y., Kuraku, S., Hirano, S. & Kuratani, S. Development of the adenohypophysis in the lamprey: evolution of epigenetic patterning programs in organogenesis. J. Exp. Zool. B 300, 3247 (2003)
  21. Charles, M. A. et al. PITX genes are required for cell survival and Lhx3 activation. Mol. Endocrinol. 19, 18931903 (2005)
  22. Kuratani, S. Evolution of the vertebrate jaw from developmental perspectives. Evol. Dev. 14, 7692 (2012)
  23. Kuratani, S. & Ota, K. G. Primitive versus derived traits in the developmental program of the vertebrate head: views from cyclostome developmental studies. J. Exp. Zool. B 310, 294314 (2008)
  24. Maruoka, Y. et al. Comparison of the expression of three highly related genes, Fgf8, Fgf17 and Fgf18, in the mouse embryo. Mech. Dev. 74, 175177 (1998)
  25. Koyama, H., Kishida, R., Goris, R. C. & Kusunoki, T. Organization of sensory and motor nuclei of the trigeminal nerve in lampreys. J. Comp. Neurol. 264, 437448 (1987)
  26. Nishizawa, H., Kishida, R., Kadota, T. & Goris, R. C. Somatotopic organization of the primary sensory trigeminal neurons in the hagfish, Eptatretus burgeri. J. Comp. Neurol. 267, 281295 (1988)
  27. Song, J. & Boord, R. L. Motor components of the trigeminal nerve and organization of the mandibular arch muscles in vertebrates: phylogenetically conservative patterns and their ontogenetic basis. Acta Anat. 148, 139149 (1993)
  28. Ogasawara, M., Shigetani, Y., Hirano, S., Satoh, N. & Kuratani, S. Pax1/Pax9-related genes in an agnathan vertebrate, Lampetra japonica: expression pattern of LjPax9 implies sequential evolutionary events toward the gnathostome body plan. Dev. Biol. 223, 399410 (2000)
  29. Tiecke, E. et al. Identification and developmental expression of two Tbx1/10-related genes in the agnathan Lethenteron japonicum. Dev. Genes Evol. 217, 691697 (2007)
  30. Strahan, R. The velum and the respiratory current of Myxine. Acta Zool. 39, 227240 (1958)
  31. Stockard, C. R. The development of the mouth and gills in Bdellostoma stouti. Am. J. Anat. 5, 481517 (1906)
  32. Sower, S. A., Freamat, M. & Kavanaugh, S. I. The origins of the vertebrate hypothalamic–pituitary–gonadal (HPG) and hypothalamic–pituitary–thyroid (HPT) endocrine systems: new insights from lampreys. Gen. Comp. Endocrinol. 161, 2029 (2009)
  33. Uchida, K. et al. Evolutionary origin of a functional gonadotropin in the pituitary of the most primitive vertebrate, hagfish. Proc. Natl Acad. Sci. USA 107, 1583215837 (2010)
  34. Stockard, C. R. The embryonic history of the lens in Bdellostoma stouti in relation to recent experiments. Am. J. Anat. 6, 511515 (1906)
  35. Ota, K. G., Fujimoto, S., Oisi, Y. & Kuratani, S. Identification of vertebra-like elements and their possible differentiation from sclerotomes in the hagfish. Nature Commun. 2, 373 (2011)
  36. Wicht, H. & Northcutt, R. G. Ontogeny of the head of the Pacific hagfish (Eptatretus stouti, Myxinoidea): development of the lateral line system. Phil. Trans. R. Soc. Lond. B 349, 119134 (1995)
  37. Yalden, D. W. Feeding mechanisms as evidence for cyclostome monophyly. Zool. J. Linn. Soc. 84, 291300 (1985)
  38. Janvier, P. Early jawless vertebrates and cyclostome origins. Zoolog. Sci. 25, 10451056 (2008)
  39. Bardack, D. First fossil hagfish (Myxinoidea): a record from the Pennsylvanian of Illinois. Science 254, 701703 (1991)
  40. Davis, S. P., Finarelli, J. A. & Coates, M. I. Acanthodes and shark-like conditions in the last common ancestor of modern gnathostomes. Nature 486, 247250 (2012)
  41. Stensiö, E. A. The Downtonian and Devonian Vertebrates of Spitsbergen. Part 1: Family Cephalaspidae (Arno, 1927)
  42. Gai, Z., Donoghue, P. C., Zhu, M., Janvier, P. & Stampanoni, M. Fossil jawless fish from China foreshadows early jawed vertebrate anatomy. Nature 476, 324327 (2011)
  43. Ota, K. G., Kuraku, S. & Kuratani, S. Hagfish embryology with reference to the evolution of the neural crest. Nature 446, 672675 (2007)
  44. Dean, B. On the embryology of Bdellostoma stouti. A genera account of myxinoid development from the egg and segmentation to hatching. Festschrift zum 70ten Geburststag Carl von Kupffer 220–276 (Gustav Fischer, 1899)
  45. Katoh, K., Kuma, K., Toh, H. & Miyata, T. MAFFT version 5: improvement in accuracy of multiple sequence alignment. Nucleic Acids Res. 33, 511518 (2005)
  46. Steinberg, M. A nonnutrient culture medium for amphibian embryonic tissues. Year B. Carnegie Inst. Wash. 56, 347348 (1957)
  47. Tahara, Y. Normal stages of development in the lamprey, Lampetra reissneri (Dybowski). Zoolog. Sci. 5, 109118 (1988)
  48. Horigome, N. et al. Development of cephalic neural crest cells in embryos of Lampetra japonica, with special reference to the evolution of the jaw. Dev. Biol. 207, 287308 (1999)
  49. Takio, Y. et al. Hox gene expression patterns in Lethenteron japonicum embryos: insights into the evolution of the vertebrate Hox code. Dev. Biol. 308, 606620 (2007)
  50. Ballard, W. W., Mellinger, J. & Lechenaut, H. A series of normal stages for development of Scyliorhinus canicula, the lesser spotted dogfish (Chondrichthyes: Scyliorhinidae). J. Exp. Zool. 267, 318336 (1993)

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  1. Department of Biology, Graduate School of Science, Kobe University, Kobe 657-8501, Japan

    • Yasuhiro Oisi
  2. Laboratory for Evolutionary Morphology, RIKEN Center for Developmental Biology, Kobe 650-0047, Japan

    • Yasuhiro Oisi,
    • Satoko Fujimoto &
    • Shigeru Kuratani
  3. Marine Research Station, Institute of Cellular and Organismic Biology, Academia Sinica, Yilan 26242, Taiwan

    • Kinya G. Ota
  4. Genome Resource and Analysis Unit, RIKEN Center for Developmental Biology, Kobe 650-0047, Japan

    • Shigehiro Kuraku


Y.O. performed sample collection, maintenance of aquarium tanks, histological preparation and three-dimensional reconstructions. Y.O. and S.F. performed the molecular cloning of EbPitxA, EbSix3/6A, EbFgf8/17A, EbHh1, EbTbx1/10A, EbSoxB1, EbLhx3/4A and EbNkx2.1 genes and in situ hybridization. S. Kuraku performed the molecular evolutionary analysis. Y.O. and S. Kuratani wrote the first draft of the manuscript. K.G.O., S. Kuraku and S. Kuratani wrote the final version of the manuscript. All of the authors discussed the results and commented on the manuscript.

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The authors declare no competing financial interests.

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Sequences for EbPitxA, EbSix3/6A, EbFgf8/17, EbHh1, EbTbx1/10A, EbSoxB1, EbLhx3/4A and EbNkx2.1 from E. burgeri are deposited in DDBJ/GenBank/EMBL under accession numbers AB703678AB703682, AB729075AB729076, and AB747372.

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  1. Supplementary Information (12.5M)

    This file contains Supplementary Figures 1-12, which show embryonic morphological data, Supplementary Table 1 and additional references.


  1. Report this comment #54730

    Peter Gibson said:

    Oisi et al have given an anatomical explanation for the single nasal opening in hagfish and lamprey. This does not appear to take into account the function of this orifice and that of the month. The two are perhaps generally confused. The present nasal opening was possibly the mouth in the early chordates. That is why it is a single opening. Filter feeders such as Amphioxus only needs one opening (the pharynx). Possibly the species evolved from an entirely planktonic stage (e.g. A. palagicus). This was trapped above a thermocline and only later, after destruction of the thermocline, become benthic. The emphasis was then on eating solids or for which a mouth and teeth were necessary. This has remained so to the present. The original opening was possibly used for this purpose in the Ostracodrems which were benthic. Clearly this is unsatisfactory so the present day mouth with teeth was separated by septum (Fig 5, ONS). This probably grew in from the edge of the pharynx leaving a passage to the gut. The original mouth was used for respiration as it still is and for sensing nature of the water (close proximity to the central nerve chord) (Fig 5, ne, ah). As food became larger so did the mouth. In the clycostomes the new mouth was clamped onto prey and could not be used for respiration. The same is still true vertebrates when eating.
    Amphioxus has a single anterior orifice ? the pharynx and atrium. In evolution the gill slits moved caudally (or the preoral region moved rostrally). The atrium becomes the mouth with teeth (adapted scales moving into the new mouth). In the detailed analysis of head of hagfish and lamprey Oisi et al suggest that the nasal tube become separated (shut off) from the evolving mouth and buccal cavity (Fig 3, OMP). To show this convincingly must be difficult. I suggest that the nasal passage has always remained open. Since the function of the duct was for respiration and not feeding there was no need for it to remain as a single opening. Two opening, as with eyes, gives a degree of directional location.
    The larval-like form of Amphioxus and other chordates (as well as invertebrates in general) have a single feeding opening in the side of the body. This is the pharynx. The ciliated band on the body surface and pharynx may have given rise to the gills (ciliated structures) through overgrowth of the sides of the body to give the atrium and its atriopore. The gills in vertebrate evolution gave rise to jaws and ears. The Eurasian tube may be a remnant of the atrium (divided atriopore). This is a demonstration that nothing is lost during evolution: structures are shuffled about the body to be used for other purposes.

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