The cbln12 promoter drives transgene expression in granule cells (GCs). (A) Genomic structure of the cbln12 gene. cbln12 has four exons and a total length of about 4.2 kbp. The start codon is in exon 2, and the stop codon is in exon 4. We isolated a 2-kbp fragment extending upstream from the start codon (cbln12 promoter). (B) Schematic drawing of the Tol2 plasmid used to express Venus in the GCs. We fused the cbln12 promoter to Venus cDNA and inserted it into a Tol2 vector. This construct was used to establish Tg(cbln12:Venus) lines. (C–E) Immunostaining of 5-days post fertilization (dpf) Tg(cbln12:Venus) larvae with anti-GFP (green) and anti-Neurod1 antibodies (magenta). Venus (Ca,Da,Ea), Neurod1 (Cb,Db,Eb), and merged images (C,D,E) are shown. Dorsal views with rostral to the left (C,Ca,Cb,D,Da,Db) or to the top (E,Ea,Eb). (D,E) High-magnification images of the boxes in (C,D). Venus was expressed in the Tel, the TL, and the Cb. In the medial region and the caudal edge of the Cb, some Neurod1⁺ cells did not express Venus (D). These were probably immature GCs. In the ventral region of the Cb (E), most of the Neurod1⁺ neurons co-expressed Venus (E). (F) Sagittal section of an adult Tg(cbln12:Venus) brain was stained with an anti-GFP antibody. Scale bars: (C) 100 μm, (D) 20 μm, (E) 10 μm, and (F) 200 μm.

Transgenic lines for tracing Purkinje cell (PC) afferents. (A) Schematic drawing of the Tol2 plasmids used to express TVA-mCherry or rabies virus glycoprotein (G). The approximately 5-kbp aldoca promoter was used to express transgenes specifically in PCs. To identify Tg fish harboring the G transcription unit, the Tol2 vector pBH (Bleeding Heart vector), which expresses mCherry in the heart was used. (B,C) Immunostaining of sagittal sections of the adult Tg(aldoca:TVA-mCherry) fish cerebellum with anti-mCherry (anti-DsRed, magenta) and anti-parvalbumin7 (anti-Pvalb7, green) antibodies. (C) High-magnification images of the box in (B). TVA-mCherry (Ba,Ca), Pvalb7 (Bb,Cb), and merged images (B,C) are shown. PCs that expressed TVA-mCherry also expressed Pvalb7. Scale bars: (B) 100 μm, and (C) 20 μm.

Transgenic lines for tracing GC afferents. (A) Schematic drawing of the Tol2 plasmids used to express TVA-mCherry or RVG. The cbln12 promoter was used to express transgenes in GCs. pBH was used to identify G-expressing fish. (B,C) Immunostaining of sagittal sections of the adult Tg(cbln12:TVA-mCherry) cerebellum with anti-mCherry (magenta) and anti-Neurod1 (green) antibodies. (C) High-magnification images of the box in (B). TVA-mCherry (Ba,Ca), Neurod1 (Bb,Cb), and merged images (B,C) are shown. Note that TVA-mCherry was located on the cell membrane of Neurod1⁺ GCs (C). Scale bars: (B) 50 μm, and (C) 5 μm.

Tracing of the presynaptic neurons of PCs using the RV method. A solution of pseudotyped RV was injected into the left side of the cerebellum of adult Tg(aldoca:TVA-mCherry); Tg(aldoca:G) fish. The injected fish were reared at 34–35.5°C for 5 days. The brains were then harvested, fixed, and subjected to immunostaining. (A–E) Staining of sagittal sections with anti-mCherry (magenta) and anti-GFP (green) antibodies, and the nuclear marker Hoechst (cyan). (B,C,Ea,Eb) Higher-magnification images of the boxes in (A,D). GFP (Ba,Ca,Ea), TVA-mCherry (Bb,Cb,Eb), and merged images (B,C) are shown. GFP was observed in the molecular layer (ML) and PC layer (PCL; A). GFP was detected in the TVA-mCherry⁺ PCs (B). GFP but not TVA-mCherry was also detected in neurons in the granular layer (GL; C) and the inferior olivary (IOs; E). (F) Schematic summary of the PC afferent tracing results. Scale bar: (A) 100 μm, (B) 20 μm, (C) 20 μm, (D) 200 μm, and (E) 20 μm.

Tracing of mossy fibers (MFs) using the RV method. The RV solution was injected into the left side of the cerebellum of adult Tg(cbln12:TVA-mCherry); Tg(cbln12:G) fish. The injected fish were then reared at 34–35.5°C for 10 days. The brains were harvested, fixed, and subjected to immunostaining. (A–K) Staining of brain cross sections with anti-mCherry (magenta) and anti-GFP (green) antibodies, and Hoechst (cyan). In the injected regions, GFP and TVA-mCherry were detected in neurons and neurites in the cerebellum (A,Aa). GFP but not TVA-mCherry was detected in neurons in the CPN (B), the paracommissural nucleus (PCN; C,D), the nucleus lateralis valvulae (NLV; E–G), the GC (H), the medial octavolateralis nucleus (MON; I), and the descending octaval nucleus (DON; J,K). Scale bar: (A) 200 μm, (B’) 50 μm. The magnification in (B–K) and (C’–K’) was the same as in (A) and (B’), respectively.