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


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.

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Fig. 1: Inner ears and two phylogenetic hypotheses of vertebrates.
Fig. 2: Development of the inner ear in cyclostomes.
Fig. 3: Gene expressions in the development of the inner ear in cyclostomes.
Fig. 4: Evolutionary scenario of the vertebrate inner ear.

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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.


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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.

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

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Authors and Affiliations



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.

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Correspondence to Shigeru Kuratani.

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Extended data figures and tables

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.

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.

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.

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.

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.

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.

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.

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Higuchi, S., Sugahara, F., Pascual-Anaya, J. et al. Inner ear development in cyclostomes and evolution of the vertebrate semicircular canals. Nature 565, 347–350 (2019).

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