Letter | Published:

Inner ear development in cyclostomes and evolution of the vertebrate semicircular canals

Naturevolume 565pages347350 (2019) | Download Citation


Jawed vertebrates have inner ears with three semicircular canals, the presence of which has been used as a key to understanding evolutionary relationships. Ostracoderms, the jawless stem gnathostomes, had only two canals and lacked the lateral canal1,2,3. Lampreys, which are modern cyclostomes, are generally thought to possess two semicircular canals whereas the hagfishes—which are also cyclostomes—have only a single canal, which used to be regarded as a more primitive trait1,4. However, recent molecular and developmental analyses have strongly supported the monophyly of cyclostomes5,6,7, which has left the evolutionary trajectory of the vertebrate inner ear unclear8. Here we show the differentiation of the otic vesicle of the lamprey Lethenteron camtschaticum and inshore hagfish Eptatretus burgeri. This is the first time, to our knowledge, that the development of the hagfish inner ear is reported. We found that canal development in the lamprey starts with two depressions—which is reminiscent of the early developmental pattern of the inner ear in modern gnathostomes. These cyclostome otic vesicles show a pattern of expression of regulatory genes, including OTX genes, that is comparable to that of gnathosomes. Although two depressions appear in the lamprey vesicle, they subsequently fuse to form a single canal that is similar to that of hagfishes. Complete separation of the depressions results in anterior and posterior canals in gnathostomes. The single depression of the vesicle in hagfishes thus appears to be a secondarily derived trait. Furthermore, the lateral canal in crown gnathostomes was acquired secondarily—not by de novo acquisition of an OTX expression domain, but by the evolution of a developmental program downstream of the OTX genes.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding author upon request. The identified cDNA sequences of L. camtschaticum OtxC and OtxD, E. burgeri OtxA, OtxB, OtxC and OtxD, L. camtschaticum PtcB, E. burgeri PtcA and PtcB, L. camtschaticum NeuroD and Ngn, E. burgeri NeuroD and Ngn, and L. camtschaticum Foxg have been deposited in GenBank under accession numbers LC413913LC413921, LC424505LC424508 and LC424955.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


  1. 1.

    Janvier, P. Early Vertebrates (Oxford Univ. Press, Oxford, 1996).

  2. 2.

    Mazan, S., Jaillard, D., Baratte, B. & Janvier, P. Otx1 gene-controlled morphogenesis of the horizontal semicircular canal and the origin of the gnathostome characteristics. Evol. Dev. 2, 186–193 (2000).

  3. 3.

    Gai, Z., Donoghue, P. C., Zhu, M., Janvier, P. & Stampanoni, M. Fossil jawless fish from China foreshadows early jawed vertebrate anatomy. Nature 476, 324–327 (2011).

  4. 4.

    Retzius, G. Das Gehörorgan der Wirbelthiere: I. Das Gehoerorgan der Fische und Amphibien (Samson & Wallin, Stockholm, 1881).

  5. 5.

    Mallatt, J. & Sullivan, J. 28S and 18S rDNA sequences support the monophyly of lampreys and hagfishes. Mol. Biol. Evol. 15, 1706–1718 (1998).

  6. 6.

    Oisi, Y., Ota, K. G., Kuraku, S., Fujimoto, S. & Kuratani, S. Craniofacial development of hagfishes and the evolution of vertebrates. Nature 493, 175–180 (2013)

  7. 7.

    Sugahara, F. et al. Evidence from cyclostomes for complex regionalization of the ancestral vertebrate brain. Nature 531, 97–100 (2016).

  8. 8.

    Janvier, P. Evolutionary biology: born-again hagfishes. Nature 446, 622–623 (2007).

  9. 9.

    Fritzsch, B. & Beisel, K. W. Evolution and development of the vertebrate ear. Brain Res. Bull. 55, 711–721 (2001).

  10. 10.

    Fritzsch, B., Kopecky, B. J. & Duncan, J. S. in Development of Auditory and Vestibular Systems 4th edn (eds Romand, R. & Varela-Nieto, I.), Ch. 12, 339–367 (Academic, Oxford, 2014).

  11. 11.

    Goodrich, E. S. in A Treatise on Zoology, Vol. 9 (ed. Lankster, R.) 1–518 (Adam & Charles Black, London, 1909).

  12. 12.

    Haddon, C. M. & Lewis, J. H. Hyaluronan as a propellant for epithelial movement: the development of semicircular canals in the inner ear of Xenopus. Development 112, 541–550 (1991).

  13. 13.

    Lowenstein, O., Osborne, M. P. & Thornhill, R. A. The anatomy and ultrastructure of the labyrinth of the lamprey (Lampetra fluviatilis L.). Proc. R. Soc. Lond. B 170, 113–134 (1968).

  14. 14.

    Thornhill, R. A. The development of the labyrinth of the lamprey (Lampetra fluviatilis Linn. 1758). Proc. R. Soc. Lond. B 181, 175–198 (1972).

  15. 15.

    Hagelin, L.-O. Studies on the development of the membranous labyrinth in lampreys, Lampetra fluviatilis Linne, Lampetra planneri (Bloch) and Petromyzon marinus Linne. Acta Zool. Suppl., 1–218 (1974).

  16. 16.

    Avallone, B. et al. The membranous labyrinth during larval development in lamprey (Lampetra planeri, Bloch, 1784). Hear. Res. 201, 37–43 (2005).

  17. 17.

    Ota, K. G. & Kuratani, S. The history of scientific endeavors towards understanding hagfish embryology. Zool. Sci. 23, 403–418 (2006).

  18. 18.

    Tahara, Y. Normal stages of development in the lamprey, Lampetra reissneri (Dybowski). Zool. Sci. 5, 109–118 (1988).

  19. 19.

    Maklad, A., Reed, C., Johnson, N. S. & Fritzsch, B. Anatomy of the lamprey ear: morphological evidence for occurrence of horizontal semicircular ducts in the labyrinth of Petromyzon marinus. J. Anat. 224, 432–446 (2014).

  20. 20.

    Waterman, R. E. & Bell, D. H. Epithelial fusion during early semicircular canal formation in the embryonic zebrafish, Brachydanio rerio. Anat. Rec. 210, 101–114 (1984).

  21. 21.

    Tiecke, E. et al. Identification and developmental expression of two Tbx1/10-related genes in the agnathan Lethenteron japonicum. Dev. Genes Evol. 217, 691–697 (2007).

  22. 22.

    Raft, S., Nowotschin, S., Liao, J. & Morrow, B. E. Suppression of neural fate and control of inner ear morphogenesis by Tbx1. Development 131, 1801–1812 (2004).

  23. 23.

    Hammond, K. L. & Whitfield, T. T. The developing lamprey ear closely resembles the zebrafish otic vesicle: otx1 expression can account for all major patterning differences. Development 133, 1347–1357 (2006).

  24. 24.

    Zarei, S., Zarei, K., Fritzsch, B. & Elliott, K. L. Sonic hedgehog antagonists reduce size and alter patterning of the frog inner ear. Dev. Neurobiol. 77, 1385–1400 (2017).

  25. 25.

    Lowenstein, O. The electrophysiological study of the responses of the isolated labyrinth of the lamprey (Lampetra fluviatilis) to angular acceleration, tilting and mechanical vibration. Proc. R. Soc. Lond. B 174, 419–434 (1970).

  26. 26.

    Chang, W., Brigande, J. V., Fekete, D. M. & Wu, D. K. The development of semicircular canals in the inner ear: role of FGFs in sensory cristae. Development 131, 4201–4211 (2004).

  27. 27.

    Stensiö, E. A. The Downtonian and Devonian vertebrates of Spitsbergen. I, Family Cephalaspidae. Skr. Svalbard Nordishavet 12, 1–391 (1927).

  28. 28.

    Acampora, D. et al. Murine Otx1 and Drosophila otd genes share conserved genetic functions required in invertebrate and vertebrate brain development. Development 125, 1691–1702 (1998).

  29. 29.

    Morsli, H. et al. Otx1 and Otx2 activities are required for the normal development of the mouse inner ear. Development 126, 2335–2343 (1999).

  30. 30.

    Fritzsch, B., Signore, M. & Simeone, A. Otx1 null mutant mice show partial segregation of sensory epithelia comparable to lamprey ears. Dev. Genes Evol. 211, 388–396 (2001).

  31. 31.

    Oisi, Y., Kakitani, O., Kuratani, S. & Ota, K. G. in In Situ Hybridization Methods (ed. Hauptmann, G.), Ch. 12, 249–262 (Springer, New York, 2015).

  32. 32.

    Dean, B. On the Embryology of Bdellostoma stouti: a General Account of Myxinoid Development from the Egg and Segmentation to Hatching (Harvard Univ. Press, Harvard, 1899).

  33. 33.

    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, 234–256 (2012).

  34. 34.

    Ballard, W. W., Mellinger, J. & Lechenault, H. A series of normal stages for development of Scyliorhinus canicula, the lesser spotted dogfish (Chondrichthyes, Scyliorhinidae). J. Exp. Zool. 267, 318–336 (1993).

  35. 35.

    Song, C., Murata, K. & Suzaki, T. Intracellular symbiosis of algae with possible involvement of mitochondrial dynamics. Sci. Rep. 7, 1221 (2017).

  36. 36.

    Kopecky, B., Johnson, S., Schmitz, H., Santi, P. & Fritzsch, B. Scanning thin-sheet laser imaging microscopy elucidates details on mouse ear development. Dev. Dyn. 241, 465–480 (2012).

  37. 37.

    Ke, M. T. et al. Super-resolution mapping of neuronal circuitry with an index-optimized clearing agent. Cell Rep. 14, 2718–2732 (2016).

  38. 38.

    Pascual-Anaya, J. et al. Hagfish and lamprey Hox genes reveal conservation of temporal colinearity in vertebrates. Nat. Ecol. Evol. 2, 859–866 (2018).

  39. 39.

    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, 1217–1226 (2011).

  40. 40.

    Ueki, T., Kuratani, S., Hirano, S. & Aizawa, S. Otx cognates in a lamprey, Lampetra japonica. Dev. Genes Evol. 208, 223–228 (1998).

  41. 41.

    Suda, Y. et al. Evolution of Otx paralogue usages in early patterning of the vertebrate head. Dev. Biol. 325, 282–295 (2009).

  42. 42.

    Mehta, T. K. et al. Evidence for at least six Hox clusters in the Japanese lamprey (Lethenteron japonicum). Proc. Natl Acad. Sci. USA 110, 16044–16049 (2013).

  43. 43.

    Adachi, N., Pascual-Anaya, J., Hirai, T., Higuchi, S. & Kuratani, S. Development of hypobranchial muscles with special reference to the evolution of the vertebrate neck. Zoological Lett. 4, 5 (2018).

  44. 44.

    Edgar, R. C. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797 (2004).

  45. 45.

    Kumar, S., Stecher, G. & Tamura, K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33, 1870–1874 (2016).

  46. 46.

    Capella-Gutiérrez, S., Silla-Martínez, J. M. & Gabaldón, T. trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics 25, 1972–1973 (2009).

  47. 47.

    Ronquist, F. et al. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst. Biol. 61, 539–542 (2012).

  48. 48.

    Sugahara, F., Murakami, Y. & Kuratani, S. in In Situ Hybridization Methods (ed. Hauptmann, G.), Ch. 13, 263–278 (Springer, New York, 2015).

  49. 49.

    Germot, A. et al. Structural evolution of Otx genes in craniates. Mol. Biol. Evol. 18, 1668–1678 (2001).

  50. 50.

    Pauley, S., Lai, E. & Fritzsch, B. Foxg1 is required for morphogenesis and histogenesis of the mammalian inner ear. Dev. Dyn. 235, 2470–2482 (2006).

Download references


We thank T. Hirasawa, R. Kusakabe, T. Kohsokabe, M. Uesaka and D. Sipp for critical reading of the manuscript; O. Kakitani and K. G. Ota for hagfish sampling; K. Shirato for catshark sampling; T. Suzaki for providing Chlorogonium capillatum; S. Shibuya, K. Yamamoto and E. Momota for maintenance of aquarium tanks; S. Kuraku, K. Itomi, C. Tanegashima, K. Tatsumi and O. Nishimura from the Phyloinformatics Team, RIKEN BDR, for gene-sequence analyses; M. Tanaka and K. Onimaru for providing the LcTbx1/10A plasmid. The RIKEN Kobe Light Microscopy Facility supported the imaging experiments. This work was supported by Grant-in-Aid for Scientific Research (A) 15H02416, Grant-in-Aid for Scientific Research on Innovative Areas (Research in a Proposed Research Area) 17H06385 and a Naito Grant for the Promotion of Focused Research (The Naito Foundation) to S.K.; Grant-in-Aid for JSPS Fellows 16J00069 to S.H.; Grant-in-Aid for Young Scientists (B) 16K18594 and Grant-in-Aid for Researchers from Hyogo College of Medicine, 2017 to F.S.

Reviewer information

Nature thanks B. Fritzsch, P. Janvier and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information


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

    • Shinnosuke Higuchi
  2. Laboratory for Evolutionary Morphology, RIKEN Center for Biosystems Dynamics Research (BDR), Kobe, Japan

    • Shinnosuke Higuchi
    •  & Shigeru Kuratani
  3. Division of Biology, Hyogo College of Medicine, Nishinomiya, Japan

    • Fumiaki Sugahara
  4. Evolutionary Morphology Laboratory, RIKEN Cluster for Pioneering Research (CPR), Kobe, Japan

    • Fumiaki Sugahara
    • , Juan Pascual-Anaya
    •  & Shigeru Kuratani
  5. Physiology Laboratory, Atmosphere Ocean Research Institute, The University of Tokyo, Kashiwa, Japan

    • Wataru Takagi
  6. Laboratory for Haptic Perception and Cognitive Physiology, RIKEN Center for Brain Science, Wako, Japan

    • Yasuhiro Oisi


  1. Search for Shinnosuke Higuchi in:

  2. Search for Fumiaki Sugahara in:

  3. Search for Juan Pascual-Anaya in:

  4. Search for Wataru Takagi in:

  5. Search for Yasuhiro Oisi in:

  6. Search for Shigeru Kuratani in:


S.H., F.S. and S.K. conceived the project, designed the experiments and wrote the paper. S.H. and W.T. obtained the hagfish embryos. S.H., F.S., J.P.-A. and Y.O. performed experiments. All authors analysed and discussed the data. All authors approved the final version of the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Shigeru Kuratani.

Extended data figures and tables

  1. Extended Data Fig. 1 Comparison of the development of the inner ear in the hagfish, lamprey and zebrafish.

    af, Developing left inner ears are compared at morphogenetic stages between E. burgeri (a, b, stage 53), L. camtschaticum (c, d, 12-mm stage larva), and D. rerio (e, f, 57 hours post fertilization). a, c, e are seen from lateral views, and b, d, f are seen from anterior views. Red arrows show positions of vesicular depressions before the pillar formation. See main-text figures for abbreviations.

  2. Extended Data Fig. 2 The inner ear of L. camtschaticum.

    a, The lateral view of the left membranous labyrinth was excised from the adult lamprey, and is shown in dorsolateral view (n = 3 biological replicates). b, A 3D model of the adult lamprey reconstructed from histological serial sections, shown in a lateral view. ch, Transverse histological sections of the inner ear at levels indicated by dashed lines in b. Note that the anterior and posterior semicircular canals and the ciliated chamber are not completely separated from each other, and that the canals are only defined by the presence of membranous septations. The common crus in the inner ear of the lamprey has previously been described10. Our sections, however, indicate an interstitial region between the anterior and posterior canal that does not show any tubular structure. By contrast, the typical common crus in jawed vertebrates is a vertical tubular structure shared by both the anterior and posterior canals (Fig. 2f, g). In the sea lamprey Petromyzon marinus, two ‘medial horizontal ducts’ have recently been reported19. We found similar canal bifurcations in L. camtschaticum, which were not as conspicuous as in P. marinus (data not shown). See Fig. 1 for abbreviations. Scale bar, 500 μm.

  3. Extended Data Fig. 3 The developing right inner ear of the hagfish.

    a, A 3D model of the stage-51–53 embryo, reconstructed from histological serial sections (n = 1 biological replication), shown in a dorsal view, with anterior to the left and lateral to the top. bf, Sagittal histological sections of the inner ear at levels indicated by dashed lines in a. There can be variations in inner ear development among hagfish species. For example, the central hole of the Myxine glutinosa inner ear is penetrated by cartilage4, whereas the equivalent space is filled by mesenchyme in E. burgeri (c). Image shown with anterior to the left, and dorsal to the top. oc, otic capsule. See Fig. 2 for other abbreviations. Scale bar, 100 μm.

  4. Extended Data Fig. 4 Development of the hagfish vestibular ganglion, in dorsal view.

    Right inner ears reconstructed from histological sections of E. burgeri embryos from stages 28 to 60 (n = 1 biological replication). The ganglionic anlage is coloured in yellow; anterior is to the left, and lateral to the top. Similar to the other vertebrate embryos, the vestibular ganglionic anlage (vg) of the hagfish first appears as a single cell mass at stage 30, which begins to be separated secondarily into paired anlagen (anterior and posterior vg) at stage 50. See Fig. 2 for other abbreviations.

  5. Extended Data Fig. 5 Phylogenetic trees of vertebrate OTX and PTC genes.

    a, A 5-million-generation tree (3.7-million post-burn trees) of OTX genes. Orthology between cyclostome and gnathostome OTX genes is not supported. We named E. burgeri genes according to their orthology relationship to the L. camtschaticum counterparts, which were described before the Myxine counterparts49. We suggest that the names of OtxC and OtxD genes for Myxine (indicated by asterisks) should be updated according to our phylogenetic analysis, which shows very robust orthology relationships among four different OTX clades in cyclostomes (OtxA, OtxB, OtxC and OtxD) to avoid confusion. b, A 3-million-generation tree (2.25-million post-burn trees) of PTC genes. As for OTX genes, orthology is not supported between cyclostome and gnathostome counterparts.

  6. Extended Data Fig. 6 Shared pattern of vestibular nerve branching between the cyclostomes and jawed vertebrates.

    The nervous system is coloured in yellow in all panels. a, 12-mm stage larva of the lamprey (n = 4 biological replicates). The lateral view of the branches that innervate the dorsal macula, anterior crista and posterior crista. b, The same inner ear as shown in a from the ventrolateral view. From the main trunk of the vestibular nerve (CnVIII), a branch first arises that innervates the posterior crista; this branch then bifurcates into two branches, one of which innervates the posterior crista and the other the dorsal macula. c, Inner ear of a stage-27 embryo of the shark S. torazame, from the lateral view (n = 2 biological replicates). d, A mouse embryo at embryonic day 11.5, from the lateral view (n = 2 biological replicates). Both in shark and in mouse, CnVIII bifurcates into several branches, of which the posterior one innervates the posterior crista and the rest the anterior and lateral cristae. e, Schematic of the shared pattern of innervation and nerve branching of the vertebrate CnVIII. The dorsal macula refers to a dorsally located sensory patch described only in the lamprey4, the function of which remains unknown30. In terms of the above-described innervation pattern, the dorsal macula has been suggested to be homologous with the lateral crista of jawed vertebrates30; the dorsal macula in the 11-mm stage lamprey larva is located in the anterolateral part of the otic vesicle, and is innervated by an equivalent nerve branch for the lateral crista. f, Reconstruction of the right inner ear primordium (blue) and the vestibular nerve fibres (yellow) in a stage-53 embryo of E. burgeri (n = 1 biological replicate). The dorsal macula is apparently missing; however, its homologous part may be present, as is suggested by the innervation pattern. The anterior and posterior branches of the vestibular nerve innervate the anterior crista and posterior crista, respectively. There is a small branch that issues from the anterior branch of the vestibular nerve specifically to innervate the middle part of the common macula (red arrowheads). The morphological innervation pattern possibly reflects selective innervation of common macula subdivisions, reminiscent of the dorsal macula innervation in the lamprey (a, b), as well as that of the lateral crista of jawed vertebrates (c, d). The hagfish dorsal macula may be fused with the common macula, but its position is probably indicated by the above noted innervation area (f). ma, maculae. See main-text figures for other abbreviations.

  7. Extended Data Fig. 7 Foxg expression in the otic vesicle of lamprey.

    a, Expression of Foxg in L. camtschaticum stage-25 embryo (n = 3 biological and technical replicates). b, Cleared and enlarged otic vesicle region in a. The lamprey Foxg expression in the whole otic vesicle is comparable with that of gnathostome Foxg1, which is responsible for the development of lateral crista50. The lamprey expression is consistent with the potential homology of the dorsal macula and lateral crista. See main-text figures for abbreviations. Scale bars, 100 μm.

Supplementary information

About this article

Publication history




Issue Date




By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.