Primer sequences for RNA probes.

Overview and three-dimensional structures of the hagfish brain.

(A) Hagfish brain in dorsal view. (B) Horizontal plane section of the whole-mount lightsheet scanning of the hagfish brain, of which neural somata are stained with fluorescent Nissl dye. (C) Schematic image of the hagfish brain in lateral view, showing the sectioning plane levels for the gene expression analysis. (D) Major brain regions of the hagfish brain in lateral view. (E) Overview of the three-dimensional model of the hagfish brain: all segmented structures (left), deeper structures (middle), and the ventricular system (right). (F–H) Three-dimensional reconstruction of the telencephalic (F), diencephalic (G), and rhombencephalic (G) brain regions. Scale bar: 1 mm in (A) for (A, B).

Phylogenetic trees of the major neurotransmitter markers, based on amino acid sequences.

(A) VGluT, (B) GAD, (C) ChAT, (D) DAT/NET/SerT, (E) TH, (F) Drd1 (D1), (G) Drd2/3/4. The numbers described on some nodes indicate exact bootstrap values. Scale bar represents the number of amino acid substitutions per site.

Gene expression patterns in each brain region.

Histological and gene expression analysis in transversal sections at the anterior olfactory bulb.

(A) Nissl staining, (B) VGluT, (C) GAD1, (D) SerT, (E) AChE staining, (F) ChAT, (G) TH, (H) DATx1, (I) DATx2, (J) Drd1×1, (K) Drd1×2, (L) Drd4. Scale bar: 500 μm in (A) for (A–L).

Histological and gene expression analysis in transversal sections at the posterior olfactory bulb.

(A) Nissl staining, (B) VGluT, (C) GAD1, (D) SerT, (E) AChE staining, (F) ChAT, (G) TH, (H) DATx1, (I) DATx2, (J) Drd1×1, (K) Drd1×2, (L) Drd4. Scale bar: 500 μm in (A) for (A–L).

Histological and gene expression analysis in transversal sections at the anteriormost telencephalon.

(A) Nissl staining, (B) VGluT, (C) GAD1, (D) SerT, (E) AChE staining, (F) ChAT, (G) TH, (H) DATx1, (I) DATx2, (J) Drd1×1, (K) Drd1×2, (L) Drd4. Scale bar: 500 μm in (A) for (A–L), 100 μm in inset (F).

Histological and gene expression analysis in transversal sections at the mid-anterior telencephalon.

(A) Nissl staining, (B) VGluT, (C) GAD1, (D) SerT, (E) AChE staining, (F) ChAT, (G) TH, (H) DATx1, (I) DATx2, (J) Drd1×1, (K) Drd1×2, (L) Drd4. Scale bar: 500 μm in (A) for (A–L).

Histological and gene expression analysis in transversal sections at the mid-posterior telencephalon.

(A) Nissl staining, (B) VGluT, (C) GAD1, (D) SerT, (E) AChE staining, (F) ChAT, (G) TH, (H) DATx1, (I) DATx2, (J) Drd1×1, (K) Drd1×2, (L) Drd4. Scale bar: 500 μm in (A) for (A–L).

Histological and gene expression analysis in transversal sections at the posteriormost telencephalon.

(A) Nissl staining, (B) VGluT, (C) GAD1, (D) SerT, (E) AChE staining, (F) ChAT, (G) TH, (H) DATx1, (I) DATx2, (J) Drd1×1, (K) Drd1×2, (L) Drd4. Scale bar: 500 μm in (A) for (A–L).

Histological and gene expression analysis in transversal sections at the anteriormost diencephalon.

(A) Nissl staining, (B) VGluT, (C) GAD1, (D) SerT, (E) AChE staining, (F) ChAT, (G) TH, (H) DATx1, (I) DATx2, (J) Drd1×1, (K) Drd1×2, (L) Drd4. Scale bar: 500 μm in (A) for (A–L).

Histological and gene expression analysis in transversal sections at the mid-anterior diencephalon.

(A) Nissl staining, (B) VGluT, (C) GAD1, (D) SerT, (E) AChE staining, (F) ChAT, (G) TH, (H) DATx1, (I) DATx2, (J) Drd1×1, (K) Drd1×2, (L) Drd4. Scale bar: 500 μm in (A) for (A–L).

Histological and gene expression analysis in transversal sections at the mid-posterior diencephalon.

(A) Nissl staining, (B) VGluT, (C) GAD1, (D) SerT, (E) AChE staining, (F) ChAT, (G) TH, (H) DATx1, (I) DATx2, (J) Drd1×1, (K) Drd1×2, (L) Drd4. Scale bar: 500 μm in (A) for (A–L).

Histological and gene expression analysis in transversal sections at the posteriormost diencephalon.

(A) Nissl staining, (B) VGluT, (C) GAD1, (D) SerT, (E) AChE staining, (F) ChAT, (G) TH, (H) DATx1, (I) DATx2, (J) Drd1×1, (K) Drd1×2, (L) Drd4. Scale bar: 500 μm in (A) for (A–L).

Histological and gene expression analysis in transversal sections at the anterior mesencephalon.

(A) Nissl staining, (B) VGluT, (C) GAD1, (D) SerT, (E) AChE staining, (F) ChAT, (G) TH, (H) DATx1, (I) DATx2, (J) Drd1×1, (K) Drd1×2, (L) Drd4. Scale bar: 500 μm in (A) for (A–L), 100 μm in inset (F).

Histological and gene expression analysis in transversal sections at the mid-mesencephalon.

(A) Nissl staining, (B) VGluT, (C) GAD1, (D) SerT, (E) AChE staining, (F) ChAT, (G) TH, (H) DATx1, (I) DATx2, (J) Drd1×1, (K) Drd1×2, (L) Drd4. Scale bar: 500 μm in (A) for (A–L).

Histological and gene expression analysis in transversal sections at the posterior mesencephalon/anterior rhombencephalon.

(A) Nissl staining, (B) VGluT, (C) GAD1, (D) SerT, (E) AChE staining, (F) ChAT, (G) TH, (H) DATx1, (I) DATx2, (J) Drd1×1, (K) Drd1×2, (L) Drd4. Scale bar: 500 μm in (A) for (A–L).

Histological and gene expression analysis in transversal sections at the mid-rhombencephalon.

(A) Nissl staining, (B) VGluT, (C) GAD1, (D) SerT, (E) AChE staining, (F) ChAT, (G) TH, (H) DATx1, (I) DATx2, (J) Drd1×1, (K) Drd1×2, (L) Drd4. Scale bar: 500 μm in (A) for (A–L).

Histological and gene expression analysis in transversal sections at the posterior-rhombencephalon.

(A) Nissl staining, (B) VGluT, (C) GAD1, (D) SerT, (E) AChE staining, (F) ChAT, (G) TH, (H) DATx1, (I) DATx2, (J) Drd1×1, (K) Drd1×2, (L) Drd4. Scale bar: 500 μm in (A) for (A–L).

Molecular profiling of the cerebellum-like area.

(A–C, E–G) Histological and gene expression analysis in transversal sections: Nissl staining (A), VGluT (B), GAD1 (C), AChE staining (E), ChAT (F), Pax6 (G). (D) Schematic summary of the molecular profiling. (H) Schematic image of the cerebellum-like neural circuit. Scale bar: 100 μm in (A) for (A–C, E–G).

Molecular profiling of the NCvl.

(A) Three-dimensional reconstruction of the NCvl with vlt and vld in ventrolateral view. (B–F) Histological and gene expression analysis in transversal sections: Nissl staining (B), Tbr1 (C), GAD1 (D), AChE staining (E), mAchR1×1 (G). Scale bar: 200 μm in (B) for (B–F).

Schematic image of the forebrain structure development.

The cross sections of the mouse (top), lamprey (middle), and hagfish (bottom) forebrains are shown from early (left) to later (right) stages.