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Evidence from cyclostomes for complex regionalization of the ancestral vertebrate brain


The vertebrate brain is highly complex, but its evolutionary origin remains elusive. Because of the absence of certain developmental domains generally marked by the expression of regulatory genes, the embryonic brain of the lamprey, a jawless vertebrate, had been regarded as representing a less complex, ancestral state of the vertebrate brain. Specifically, the absence of a Hedgehog- and Nkx2.1-positive domain in the lamprey subpallium was thought to be similar to mouse mutants in which the suppression of Nkx2-1 leads to a loss of the medial ganglionic eminence1,2. Here we show that the brain of the inshore hagfish (Eptatretus burgeri), another cyclostome group, develops domains equivalent to the medial ganglionic eminence and rhombic lip, resembling the gnathostome brain. Moreover, further investigation of lamprey larvae revealed that these domains are also present, ruling out the possibility of convergent evolution between hagfish and gnathostomes. Thus, brain regionalization as seen in crown gnathostomes is not an evolutionary innovation of this group, but dates back to the latest vertebrate ancestor before the divergence of cyclostomes and gnathostomes more than 500 million years ago.

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Figure 1: Regionalization of the vertebrate brains.
Figure 2: Development of the ventricular system in the hagfish brain.
Figure 3: Gene expression and nervous staining in E. burgeri at stage 53.
Figure 4: Presence of the rhombic lip and MGE in the hagfish and lamprey brains, and an evolutionary model of the vertebrate brain.

Accession codes

Primary accessions

NCBI Reference Sequence

Data deposits

The sequences of the genes described in this study have been deposited in the NCBI GenBank under accessions LC028239LC028243, LC028245 and KT897926KT897927 and KT897930KT897938.


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We thank O. Kakitani for hagfish sampling; K. Shirato for shark sampling; S. Shibuya, K. Yamamoto and Y. Yamamoto for maintenance of aquarium tanks; D. Sipp for critical reading of this manuscript; C. Mitgutsch and M. Kawaguchi for discussions; K. G. Ota and I. Sato for technical support; and H. Nagashima for lamprey sampling and discussions. This research was supported by direct budget supplied by Centre for Developmental Biology, RIKEN, JSPS KAKENHI grant numbers 15H02416 and 25840133, and by Hyogo Science and Technology Association.

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Authors and Affiliations



F.S., J.P.-A., Y.M. and S. Kuratani designed the experiments and wrote the paper. F.S., J.P.-A., Y.O., S.A., N.A. and T.H. performed molecular works. N.S. discussed and interpreted the data. S. Kuraku performed the molecular phylogenetic analysis. W.T. performed the Nkx2.1/2.4 locus synteny conservation analysis.

Corresponding author

Correspondence to Shigeru Kuratani.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Embryonic brain of a shark.

aq, Gene expression patterns and nerve tracts in a stage-27 (ref. 34) embryo of the cloudy catshark (S. torazame), to show the conserved developmental pattern of the jawed vertebrate brain. Whole-mount views (ae), sagittal sections of the brain (gk) and transverse sections at the telencephalon level (mq; the sectioned region is shown by the dotted line in g), stained using probes for FoxG1 (a, g, m), Pax6 (b, h, n), Dlx2 (c, i, o), Nkx2.1 (d, j, p) and Shh (e, k, q). f, l, Confocal images of the central and peripheral nervous systems visualized by immunofluorescence using an anti-acetylated tubulin antibody (green) and DAPI (blue). r, Schematic drawing of the shark telencephalon, showing conserved gene expression patterns among jawed vertebrates. Some of these patterns were described previously7. op, olfactory placode; rp, Rathke’s pouch. See Figs 1 and 2 for other abbreviations. Scale bars, 500 μm.

Extended Data Figure 2 Adult and embryonic brains of E. burgeri, and gene expression and nervous system staining of stage-45 embryo.

a, b, Dorsal view (a) and sagittal section (b) of the adult brain of E. burgeri. ch, E. burgeri embryos at Bashford Dean stages 45 (ce) and 53 (fh)17,18. Dorsal (c, f) and lateral (d, g) views are shown. e, h, Sagittal sections stained with haematoxylin and eosin. Dean stage 53 is very similar to the embryo described in figure 1 in ref. 45. it, A stage 45 E. burgeri embryo observed using histological preparations. Dotted lines indicate the presumptive telencephalic border estimated by EmxB and FoxG expressions21,22. At this stage, Nkx2.1/2.4 and Hh2 have not been expressed yet in the rostral telencephalon (according to the prosomeric model3). cer, cerebrum; met, metencephalon; mes, mesencephalon; ob, olfactory bulb. See Figs 1, 2 and 4 for other abbreviations. Scale bars, 2.0 mm (ac and f), 1.0 mm (e, h) and 500 μm (i, j).

Extended Data Figure 3 Identification of the forebrain–midbrain–hindbrain boundaries in early hagfish embryos.

ac, The mid–hindbrain boundary (MHB) of the hagfish brain is assumed to correspond to the posterior expression boundary of OtxC (b) and focal expression of Fgf8/17 (see Extended Data Fig. 5) (c) that become evident at stages 28–30. ad, On the other hand, the fore–midbrain boundary (FMB) is suggested morphologically (a, b) and by the ventrocaudal portion of Nkx2.1/2.4 expression domain (d, arrowheads). See Figs 1 and 2 for abbreviations. Scale bar, 200 μm.

Extended Data Figure 4 Absence of the epiphysis and presence of the ZLI in the developing brain of the hagfish.

a, b, The adult hagfish has no epiphysis. A rudimentary epiphysis described previously14,46 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. dg, In our present observation of hagfish embryos also, several neuroepithelial cysts were found in the forebrain (arrows in df), 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. hk, 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.

Extended Data Figure 5 Molecular phylogenetic trees of genes identified in this study.

The trees were inferred with the maximum-likelihood method using PhyML3.0 with the JTT+G4 model. Members of individual gene families were collected from public databases, and groupings of the sequences identified in this study into the subfamilies shown in this figure were confirmed by preliminary phylogenetic inferences including other subfamilies. a, Hedgehog homologues (shape parameter for the gamma distribution alpha = 0.80). A total of 249 amino acid sites unambiguously aligned without any gap were used in the inference. b, Nkx2.1/2.4 homologues (86 sites; alpha = 0.60). c, Wnt1 homologues (196 sites; alpha = 0.60). d, Ptf1a homologues (67 sites; alpha = 0.92). e, Atoh1 homologues (60 sites; alpha = 0.74). f, Fgf8/17/18/24 homologues including the hagfish homologue identified previously19 (74 amino acid sites; alpha = 0.87). g, FoxG1 homologues (115 amino acid sites; alpha = 0.16). Hagfish, lamprey and catshark sequences are shown in red, green and blue, respectively. Support values at nodes are bootstrap probabilities in the maximum-likelihood method and those in the neighbour-joining method (under the above-mentioned substitution model), in order.

Extended Data Figure 6 Expression patterns of hagfish Hedgehog genes.

ai, In situ hybridization staining of four Hedgehog genes (Hh14) of E. burgeri at stage-45 (af) and stage-53 (gi) embryos. The Hedgehog expressions were found in notochord (n), floor plate (fp), pharyngeal endoderm (pe), oral ectoderm (oe) and subregions of the forebrain (hypothalamus, ZLI and MGE; see Fig. 3). The overall expressions of hagfish Hh1–4 exhibited patterns similar to those of Hedgehog genes in jawed vertebrates, although each Hh expression showed slight differences in some domains. j, Summary of the expression patterns of Hedgehog genes (Hh1–4) of E. burgeri in stage 45 and stage 53 embryos. Scale bars, 500 μm.

Extended Data Figure 7 Rhombic lip gene expression patterns in shark, hagfish and lamprey embryos.

Gene expression patterns in stage-27 (whole-mount) and stage-31 (transverse sections) embryos of a catshark (S. torazame) (al), stage-53 embryo of a hagfish (E. burgeri) (m, n), and stage-26 embryo of lamprey (L. japonicum) (ot), stained using probes for the Pax6, Wnt1, Atoh1 and Ptf1a gene homologues. u, Schematic transverse section of the vertebrate rhombic lip showing crucial gene expression patterns based on ref. 27 and the present study. Lines in a indicate the levels of the transverse sections shown in eh (rhombomere 1: the cerebellar primordia) and il (posterior rhombomeres). The line in o indicates the level of the transverse sections shown in (o′s′) (around rhombomere 4). Arrowheads indicate rhombic lip gene expressions. Asterisks indicate non-rhombic lip expressions of Pax6 through the neural tube. See Figs 1 and 2 for abbreviations. Scale bars, 500 μm (a, e, i, m, o) and 100 μm (o′).

Extended Data Figure 8 Synteny conservation between the genomic regions containing Nkx2.1/2.4 among gnathostomes and L. japonicum.

Numbered boxes represent single protein-coding genes; all the genes used for synteny analysis and the given numbers are listed in Extended Data Table 1. Colours of boxes are as follows: red, Nkx2-1/2-4 orthologue; orange, Nkx2-2/2-8 orthologue; grey, a gene showing high sequence similarity to two gnathostome ohnologues as a result of the TBLASTN search. The green line represents orthology between the neighbouring vertebrate genomes, whereas the purple line indicates a paralogous relationship of each gene among the lamprey genome scaffolds. At present, there are no descriptions of chicken NKX2-4 (nor in birds) and we have not been able to find it. This putative lack is marked by a question mark.

Extended Data Figure 9 Telencephalic gene expression patterns in lamprey embryos.

ap, Gene expression patterns in L. japonicum stage-26 and -27 (ref. 33) embryos, stained using probes for Nkx2.1/2.4A (a, b), Nkx2.1/2.4B (c, d), Nkx2.1/2.4C (e, f), Pax6 (g), DlxA (h), HhA (i, j), HhB (k, l), HhC (m, n) and HhD (o, p). Dotted lines indicate the telencephalic border distinguished by the anterior intraencephalic sulcus25. The expression domains of Pax6 (g) and DlxA (h) in the telencephalon pallial and subpallial subdivisions, respectively. Although the previously reported Nkx2.1/2.4A is not expressed in the telencephalon36, Nkx2.1/2.4B and Nkx2.1/2.4C are expressed in the rostral telencephalon (arrowheads), suggesting the presence of the MGE in the lamprey. We did not detect expression of any Hedgehog genes in the rostral telencephalon25 (ip). q, Summary of the expression patterns of Nkx2.1/2.4 and Hedgehog genes of L. japonicum at embryonic stages 17–30 examined by whole-mount in situ hybridization. en, endostyle; ptu, posterior tuberculum47. See Figs 1 and 2 for other abbreviations. Scale bar, 200 μm.

Extended Data Table 1 List of genes used in the synteny conservation analysis

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

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