Establishment of Gal4 transgenic zebrafish lines for analysis of development of cerebellar neural circuitry

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Establishment of Gal4 transgenic zebrafish lines for analysis of development of cerebellar neural circuitry Miki Takeuchi a,1, Koji Matsuda b,1, Shingo Yamaguchi b, Kazuhide Asakawa c, Nobuhiko Miyasaka d, Pradeep Lal c, Yoshihiro Yoshihara d, Akihiko Koga e, Koichi Kawakami c, Takashi Shimizu a,b, Masahiko Hibi a,b,n a

Laboratory of Organogenesis and Organ Function, Bioscience and Biotechnology Center, Nagoya University, Nagoya, Aichi 464-8601, Japan Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya, Aichi 464-8602, Japan c Division of Molecular and Developmental Biology, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan d RIKEN Brain Science Institute, Wako, Saitama 351-0198, Japan e Primate Research Institute, Kyoto University, Inuyama 464-8506, Japan b

ar t ic l e i nf o

a b s t r a c t

Article history: Received 20 June 2014 Received in revised form 3 September 2014 Accepted 26 September 2014

The cerebellum is involved in some forms of motor coordination and motor learning. Here we isolated transgenic (Tg) zebrafish lines that express a modified version of Gal4-VP16 (GFF) in the cerebellar neural circuits: granule, Purkinje, or eurydendroid cells, Bergmann glia, or the neurons in the inferior olive nuclei (IO) which send climbing fibers to Purkinje cells, with the transposon Tol2 system. By combining GFF lines with Tg lines carrying a reporter gene located downstream of Gal4 binding sequences (upstream activating sequence: UAS), we investigated the anatomy and developmental processes of the cerebellar neural circuitry. Combining an IO-specific Gal4 line with a UAS reporter line expressing the photoconvertible fluorescent protein Kaede demonstrated the contralateral projections of climbing fibers. Combining a granule cell-specific Gal4 line with a UAS reporter line expressing wheat germ agglutinin (WGA) confirmed direct and/or indirect connections of granule cells with Purkinje cells, eurydendroid cells, and IO neurons in zebrafish. Time-lapse analysis of a granule cell-specific Gal4 line revealed initial random movements and ventral migration of granule cell nuclei. Transgenesis of a reporter gene with another transposon Tol1 system visualized neuronal structure at a single cell resolution. Our findings indicate the usefulness of these zebrafish Gal4 Tg lines for studying the development and function of cerebellar neural circuits. & 2014 Published by Elsevier Inc.

Keywords: Zebrafish Cerebellum Gal4 Neural circuits Cell migration

Introduction The cerebellum is involved in some forms of motor coordination and motor learning (Ito, 2005, 2006, 2008). Since cerebellar neural circuitry is generally conserved among vertebrates (Altman and Bayer, 1997; Butler and Hodos, 1996), studies with zebrafish, which are genetically tractable have been used for studying development and functions of cerebellar neural circuitry (reviewed in (Hashimoto and Hibi, 2012; Hibi and Shimizu, 2012, 2014)). The cerebellum contains several types of neurons. In zebrafish, like mammals, the major excitatory glutamatergic and inhibitory GABAergic neurons in the cerebellum are granule cells and Purkinje cells, respectively (Altman and Bayer, 1997; Bae et al., 2009; Butler

n Corresponding author at: Laboratory of Organogenesis and Organ Function, Bioscience and Biotechnology Center, Nagoya University, Furo, Chikusa, Nagoya, Aichi 464-8601, Japan. Fax: þ 81 52 789 5053. E-mail address: [email protected] (M. Hibi). 1 These authors equally contributed to this work.

and Hodos, 1996) (Fig. 1). Purkinje cells receive inputs from the climbing fibers (CFs), which are neuronal axons emanating from the IO located in the ventro-posterior hindbrain (Fig. 1I). In contrast, granule cells receive inputs from the mossy fibers (MFs), which are neuronal axons projected from precerebellar nuclei, located in various regions of the brain. The signals carried by the MFs are transmitted to the Purkinje cell dendrites by granule cell axons, also known as parallel fibers. Thus, signals carried by both the MFs and the CFs are integrated by the Purkinje cells. In teleosts, neurons known as eurydendroid cells receive signals from both Purkinje cell axons and parallel fibers; these cells further integrate the information and project their axons to targets outside the cerebellum (reviewed in (Ikenaga et al., 2006)). Bergmann glial cells, derived from radial glial cells, are involved in the development and function of the cerebellum (reviewed in (Buffo and Rossi, 2013)). These neurons and glial cells are arranged in three layers, the molecular layer (ML), Purkinje cell layer (PCL), and granule cell layer (GCL), from the superficial to deeper levels in zebrafish (Fig. 1) (Bae et al., 2009; Hashimoto and Hibi, 2012; Hibi and Shimizu, 2012).

http://dx.doi.org/10.1016/j.ydbio.2014.09.030 0012-1606/& 2014 Published by Elsevier Inc.

Please cite this article as: Takeuchi, M., et al., Establishment of Gal4 transgenic zebrafish lines for analysis of development of cerebellar neural circuitry. Dev. Biol. (2014), http://dx.doi.org/10.1016/j.ydbio.2014.09.030i

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Fig. 1. Cerebellar neural circuitry in zebrafish. (A–F) Expression of Vglut1 (A, B), Neurod (C, D), and parvalbumin7 (Pvalb7, E, F) in the cerebellar regions of larvae at 5 dpf (A, C, E, dorsal projection views, with anterior to the left) and adult sagittal sections (B, D, F, optical sections with anterior to the left). Vglut1 and Neurod signals mark the axons and somata of granule cells. Pvalb7 signals mark both neurites (axons and dendrites) and somata of Purkinje cells. (G) Schematic representation of cerebellar neural circuitry. (H, I) Schematic drawing of the cerebellum at 5 dpf (H, dorsal view) and of a sagittal section of the adult midbrain and hindbrain region. Scale bars: 50 μm in A, C, E; 200 μm in B, D, F.

The zebrafish cerebellum is subdivided into three lobes: the valvula cerebelli (Va, anterior lobe), the corpus cerebelli (CCe), and the caudolateral lobe, which consists of the eminentia granularis (EG) and the lobus caudalis cerebelli (LCa) (Fig. 1I) (Bae et al., 2009). This three-lobed cerebellar structure is conserved in other teleost species as well (Altman and Bayer, 1997; Butler and Hodos, 1996). The Va and CCe contain all three cerebellar layers, whereas the caudolateral lobe (EG and LCa) contains only the GCL (Bae et al., 2009). Granule cells in the Va and CCe send their axons to Purkinje cells in the ML, whereas granule cells in the caudolateral lobe (EG and LCa) send their axons to the dendrites of Purkinje cells in the cerebellum and the dendrites of crest cells (Purkinje-like cells) in the crista cerebellaris (CC) of the dorsal hindbrain (Bae et al., 2009; Volkmann et al., 2008; Wullimann and Grothe, 2014) (Table 1). The anatomy and developmental processes of zebrafish cerebellum were analyzed by using various molecular markers and Tg lines (Bae et al., 2009; Kani et al., 2010; Rieger et al., 2009; Tanabe et al., 2010; Volkmann et al., 2010; Volkmann et al., 2008). The cerebellar and IO

Table 1 Abbreviations used in the figures. BG

Bergmann glia

ML

Molecular layer

CC CCe CF EC EG GC GCL IO LCa

Crista cerebellaris Corpus cerebelli Climbing fiber Eurydendroid cells Eminentia granularis Granule cells Granule cell layer Inferior olive nuclei Lobus caudalis cerebelli

MON MF PC PCL PF TeO Va Vam Val

Medial octavolateral nucleus Mossy fiber Purkinje cells Purkinje cell layer Parallel fibers Optic tectum Valvula cerebelli Medial division of valvula cerebelli Lateral division of valvula cerebelli

neuronal development is generally conserved between mammals and zebrafish, and is controlled by orthologous and paralogous genes. The simple neural circuit involving granule cells, Purkinje cells, eurydendroid cells, and the IO neurons is completely formed by 5 days post fertilization (dpf). Although this accumulating information on

Please cite this article as: Takeuchi, M., et al., Establishment of Gal4 transgenic zebrafish lines for analysis of development of cerebellar neural circuitry. Dev. Biol. (2014), http://dx.doi.org/10.1016/j.ydbio.2014.09.030i

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zebrafish cerebellar neural circuitry is very informative, it is limited by the fact that very few genetic tools have been available for studying cerebellar neural circuit development and function. For instance, most of zebrafish Tg lines used for the previous studies only express single fluorescent proteins in cerebellar/IO neurons and their progenitors, and they cannot be used to express other reporter or effector proteins in specific neurons. Furthermore, neither Tg line which marks all eurydendroid cells nor all IO neurons in both larvae and adults, nor Tg line which distinguishes granule cells in the caudolateral lobe from those in the Va and CCe was reported. The Gal4-UAS system has been used to facilitate exogenous expression of genes for developmental and CNS studies in invertebrate and vertebrate species (Aigaki et al., 2001; Halpern et al., 2008; Hirsch et al., 2002; Perrimon, 1998; Scott, 2009). The combined use of enhancer and/or gene trap screens using transposon systems (Tol2 and Sleeping Beauty) and the Gal4-UAS system have

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established transgenic zebrafish lines that express genes of interest in specific neurons (Asakawa et al., 2008; Ogura et al., 2009; Scott et al., 2007). The Gal4 lines can also be combined with various reporter or effector systems to elucidate the structure and function of neural circuitry in zebrafish (Asakawa and Kawakami, 2009; Halpern et al., 2008; Scott, 2009). In this report, we isolated Gal4 driver lines that express a modified version of Gal4-VP16, GFF (Asakawa et al., 2008) in subsets of granule cells, Purkinje cells, eurydendroid cells, Bergmann glial cells, and IO neurons, some of which were not previously labeled in Tg lines. The combination of Gal4 lines with UAS reporter lines expressing fluorescent proteins (Asakawa and Kawakami, 2009; Hatta et al., 2006) or a transsynaptic tracer, wheat germ agglutinin (WGA) (Yoshihara, 2002; Yoshihara et al., 1999), or the combination of the Tol1 and Tol2 systems allowed us to study anatomy and developmental processes of cerebellar neural circuitry in zebrafish.

Fig. 2. Granule-specific GFF and GFP transgenic lines. (A, B) gSA2AzGFF152B; UAS:GFP. (C, D) gSAIzGFFM765B; UAS:GFP. (E, F) SAG6A. (G, H) gSAIGFF23C; UAS:GFP. (I, J) SAGFF(LF)128A; UAS:GFP. (K, L) hspGFF57A; UAS:GFP. (M, N) SAGFF(LF)157B; UAS:GFP. (O, P) hspGFFDMC90A; UAS:GFP. To visualize GFF expression, the granule-specific GFF lines (except SAG6A in E, F) were crossed with the UAS:GFP line. The 5-dpf larvae (A, C, E, G, I, K, M, O, dorsal projection views with anterior to the left) and sagittal sections of adult brains (B, D, F, H, J, L, N, P, optical sections with anterior to the left) were stained with anti-GFP antibody. Note that in the gSA2AzGFF152B; UAS:GFP line GFP was specifically expressed in the granule cells of the CCe, while in the gSAIzGFFM765B; UAS:GFP line GFP was expressed in some granule cells in the CCe and most granule cells in the LCa in adult. The SAG6A and gSAIGFF23C; UAS:GFP lines expressed GFP in granule cells primarily in the LCa. In the hspGFFDMC90A; UAS:GFP line GFP was expressed in some granule cells in the GCL and LCa, and in some immature granule cells in the ML. Scale bars: 100 μm in O (applied to A, C, E, G, I, K, M, O); 200 μm in B, D, F, H, J, L, N, P.

Please cite this article as: Takeuchi, M., et al., Establishment of Gal4 transgenic zebrafish lines for analysis of development of cerebellar neural circuitry. Dev. Biol. (2014), http://dx.doi.org/10.1016/j.ydbio.2014.09.030i

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Material and methods Wild-type and transgenic zebrafish lines Wild-type zebrafish (Danio rerio) with the Oregon AB genetic background were used. Some Tg lines were crossed to casper (mitfaw2; roya9) (White et al., 2008). Enhancer and gene trap lines expressing GFF (a fusion protein containing the Gal4 DNA binding domain and a tandem repeat of the VP16 transactivation domain) and GFP were generated using Tol2 transposon-mediated transgenesis, as described previously (Asakawa et al., 2008; Kawakami et al., 2004). hspGFFDMC and hspzGFFgDMC are enhancer trap constructs with GFF and a promoter of hsp70l. gSAG is a gene trap construct with GFP and a splicing acceptor of the gata6 gene. gSAIGFF, gSAIzGFFM, and gSA2AzGFF are gene trap constructs with the gata6 splicing acceptor and GFF, and with the internal ribosomal entry site of encephalomyocarditis virus (gSAIGFF and gSAIzGFFM) or sequence encoding the porcine teschovirus-1 2A peptide (gSA2AzGFF). To visualize GFF expression, the GFF lines were crossed with UAS:GFP, UAS:RFP, or UAS:Kaede lines (nkuasgfp1aTg, nkuasrfp1aTg, and rk8Tg in ZFIN: http://zfin.org/) (Asakawa et al., 2008; Hatta et al., 2006). Tg lines expressing GFP in cerebellar neurons were screened using an MZ16FA Leica fluorescence dissection microscope. The insertion sites of the trapped vectors were determined by inverse PCR as described previously (Urasaki and Kawakami, 2009). Establishment of the aldoca:gap43-Venus line was described previously (Tanabe et al., 2010). To generate the aldoca:GFF and aldoca:gap43-mCherry cassettes, the GFF or gap43-mCherry cDNA, and the SV40 polyadenylation signal (polyA) were inserted downstream of the 5-kb aldoca promoter (Tanabe et al., 2010) in a Gateway vector modified from the Tol2 vector, T2KXIG (Kawakami et al., 2004). To generate the UAS: AcGFP-2A WGA plasmid, in which Aequorea coerulescens GFP (AcGFP) and WGA cDNAs were fused in frame via a 2A sequence, the AcGFP2A WGA cassette was excised from pGEM-T Easy-AcGFP1-P2AtWGA (Ohashi et al., 2011) and inserted between EcoRI and EcoRV site of pT2MUASMCS (Asakawa et al., 2008). To make Tg lines, 25 pg of Tol2 donor plasmid DNA and 25 pg of transposase mRNA were injected into one-cell stage embryos. The olig2:EGFP (vu12) line was reported previously (Shin et al., 2003). The zebrafish were maintained in environmentally controlled rooms at Bioscience and Biotechnology Center, Nagoya University, and Division of Molecular and Developmental Biology, National Institute of Genetics, in accordance with the institutional guidelines for animal experiments. For immunohistochemistry studies, zebrafish embryos/larvae were treated with 0.005% phenylthiourea (PTU) from 12 h post fertilization (hpf). The developmental stages were determined according to ZFIN. The larvae used were between 3 and 29 days old; the adult fish used were at least 90 days old. Requests of the gene and enhancer trap lines described in this report should be addressed to K.K. ([email protected]). Tol1-mediated transgenesis A UAS:GFP-polyA cassette was excised from T2KUASGFP (Asakawa et al., 2008) and was subcloned into a Tol1 donor plasmid pDon122 (Koga et al., 2008; Koga et al., 2007). Tol1 transposase mRNA was generated from pHel105 (Koga et al., 2008; Koga et al., 2007). Immunohistochemistry, imaging, and Kaede photoconversion For immunostaining, anti-GFP (1/1000, rat, Nacalai), anti-parvalbumin 7 (1/1000, mouse monoclonal, ascites) (Bae et al., 2009), anti-Vglut1 (1/1000, rabbit, affinity purified) (Bae et al., 2009), anti-Neurod (1/500, mouse ascites) (Kani et al., 2010), anti-DsRed (1/1000, rabbit, Takara-Clontech), which can recognize mCherry (Kani et al., 2010), anti-Fabp7a (1/1000, mouse monoclonal, ascites)

(Bae et al., 2009), and anti-WGA (1/10000, rabbit, Sigma) antibodies were used. Immunostaining of larvae and cryostat sections was performed as described previously (Bae et al., 2009; Kani et al., 2010). Alexa Fluor 405 goat anti-rabbit, Alexa Fluor 488 goat antirat, and Alexa Fluor 568 goat anti-mouse or anti-rabbit IgG (HþL, Molecular Probes, Life Technologies) were used as secondary antibodies. Optical clearing of some fixed samples was carried out with SeeDB reagent according to the previously reported protocol (Ke et al., 2013; Ke and Imai, 2014). The fluorescent images were captured with an LSM700 confocal laser-scanning microscope or an AxioPlan-2 microscope with AxioCam CCD camera (Zeiss). The images were constructed from Z-stack sections by the 3D projection program associated with the microscope (Zen, Zeiss) or by Imaris (Bitplane). The figures were constructed using Adobe Photoshop and Adobe Illustrator. The Alexa Fluor 405, 488, and 568 signals appear cyan, green, and magenta, respectively, in the figures. For photoconversion of the Kaede protein, local irradiation with a laser (405 nm) was performed using the confocal microscope and a bleaching protocol. In situ hybridization The vglut2a probes were generated as previously described (Higashijima et al., 2004a; Higashijima et al., 2004b). To make an antisense riboprobe for GFP, EGFP cDNA was subcloned into pBluescript II SKþ (Agilent). Whole-mount in situ hybridization and in situ hybridization of sections were carried out as previously (Bae et al., 2009; Kani et al., 2010). For the single-color observation, BM purple was used as a substrate for alkaline phosphatase (Suppl. Fig. 3). When the samples were stained with a riboprobe and antibody, in situ hybridization was performed first, followed by immunohistochemistry. tyramide signal amplification (TSA) kit with Alexa Fluor 555 (Molecular Probe, Life Technology) was used for in situ hybridization (Suppl. Fig. 4).

Results Gene/enhancer trap line screening To investigate the cerebellar neural circuitry in zebrafish, Tg lines expressing GFF or GFP in cerebellar neurons and input neurons were generated using the Tol2 transposon system. We performed a large-scale gene and enhancer trap screens, crossed GFF trap lines with the UAS:GFP line, and observed the resulting progeny under a fluorescence microscope at 5 dpf (Asakawa et al., 2008; Kawakami et al., 2010). Tg lines that had GFP expression in the cerebellar neural circuitry were further analyzed by co-staining with antibodies to the cerebellar neuronal markers Vglut1, Neurod, parvalbumin 7 (Pvalb7), and fatty acid binding protein7a (Fabp7a), as previously reported (Bae et al., 2009). Vglut1 marks the axons of granule cells in the ML and CC (Fig. 1A, B). Neurod is a neurogenic factor that plays an important role in generation of granule cells (Mueller and Wullimann, 2002, 2003; Wullimann et al., 2011) and it marks the nuclei of immature granule cells located in the ML and of mature granule cells in the GCL, EG, and LCa (Fig. 1C, D). Pvalb7 is a marker for Purkinje cells and is expressed in both the neurites (axons and dendrites) and somata of Purkinje cells (Fig. 1E, F). Fabp7a (also known as brain lipid binding protein Blbp) marks Bergmann glial cells (Fig. 8). Using these markers, we identified eight granule cell-specific trap lines (seven GFF and one GFP line), one eurydendroid cell-specific GFF line, one IO neuron-specific GFF line, and two Bergmann glial cell-specific GFF lines, from approximately one thousand trap lines. The trap lines are listed in Table 2. Expression patterns of GFP in the trap lines at 5 dpf are shown in Suppl. Fig. 1 (entire larva) and Suppl. Fig. 2 (head region).

Please cite this article as: Takeuchi, M., et al., Establishment of Gal4 transgenic zebrafish lines for analysis of development of cerebellar neural circuitry. Dev. Biol. (2014), http://dx.doi.org/10.1016/j.ydbio.2014.09.030i

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Fig. 3. Granule cell-specific GFF lines, co-staining with granule cell markers. (A–H, Q–T) gSA2AzGFF152B; UAS:GFP. (I–P, U–X) gSAIGFF23C; UAS:GFP. The 5-dpf larvae (A–P, dorsal views with anterior to the left) and sagittal sections of adult brains (Q–X, anterior to the left) were stained with anti-GFP (green) and Vglut1 or Neurod (magenta) antibodies. High magnification images of green (B, F, J, R, V), magenta (C, G, K, O, S, W), merged (D, H, L, P, T, X) of the box in A, E, I, M, Q, U. Projection views (A–D, E, I–L, M, Q–U) and optical sections (F–H, N–P, V–X). Note that GFP þ axons and somata were co-stained with anti-Vglut1 (arrowheads in D, L) and Neurod antibodies, respectively. There are Neurod þ and GFP- cells observed in the lobus caudalis of 5-dpf gSAIGFF23C; UAS:GFP cerebellum (Fig. 3N P), indicating that gSAIGFF23C line marks most but not all granule cells in the lobus caudalis cerebelli. Scale bars: 100 μm in A, E, I, M; 100 μm in Q, U; 20 μm in D, H, L, P (applied to B–D, F–H, J–L, N–P, respectively); 20 μm in T, X (applied to R–T, V–X, respectively).

Please cite this article as: Takeuchi, M., et al., Establishment of Gal4 transgenic zebrafish lines for analysis of development of cerebellar neural circuitry. Dev. Biol. (2014), http://dx.doi.org/10.1016/j.ydbio.2014.09.030i

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Fig. 4. Purkinje-specific GFF and membrane tagged Venus/mCherry lines. (A–H) aldoca:gap43-Venus larvae (A–D, dorsal views with anterior to the left) and adult sagittal sections (E–H, with anterior to the left) were co-stained with anti-GFP (Venus, green) and anti-Pvalb7 (magenta) antibodies. Venus signals colocalized with Pvalb7 signals in all the Purkinje cells of 5-dpf larvae (D) and the adult cerebellum (H). Cerebellovestibular tracts are indicated by arrowheads (A, B). (I–L) aldoca:gap43-mCherry larvae (dorsal views with anterior to the left) were co-stained with anti-DsRed (mCherry, magenta) and anti-Pvalb7 (green) antibodies. (M–S) aldoca:GFF; UAS:GFP larvae (M–P, dorsal views with anterior to the left) and adult sagittal sections (Q–V, anterior to the left) were co-stained with anti-GFP (Venus, green) and anti-Pvalb7 (magenta) antibodies. (T–V) High magnification views of the box in S. Only a portion of the Pvalb7 þ Purkinje cells stained positive for GFP (L, P). Scale bars: 100 μm in A; 50 μm in D (applied to B–D); 200 μm in E; 50 μm in H (applied to F–H); 100 μm in I; 50 μm in L (applied to J–L); 100 μm in M; 50 μm in P (applied to N–P); 200 μm in S (applied to Q–S); 100 μm in V (applied to T–V).

Please cite this article as: Takeuchi, M., et al., Establishment of Gal4 transgenic zebrafish lines for analysis of development of cerebellar neural circuitry. Dev. Biol. (2014), http://dx.doi.org/10.1016/j.ydbio.2014.09.030i

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Table 2 Transgenic lines. A: Gene/enhancer trap lines Name of Tg lines GFF/ GFP

Cell types

Cerebellar circuitry expression

Expression outside cerebellar circuitry

2 dpf-adult

Telencephalon, tectum, tegmentum

2 dpf-adult

Integration site

gSA2AzGFF152B

GFF

gSAIzGFFM765B

GFF

Granule cells (Va, CCe 4 LCa, EG) Granule cells

gSAG6A

GFP

Granule cells (LCa, EG)

3 dpf-adult

Retina, telencephalon, habenula nuclei, hindbrain, spinal cord Retina, tectum, tegmentum, dorsal hindbrain

gSAIGFF23C

GFF

Granule cells (LCa, EG)

4 dpf-adult

Olfactory bulb, telencephalon, ventral hindbrain

SAGFF(LF)128A

GFF

Granule cells

3 dpf-adult

Weakly expressed in various tissues

hspGFF57A SAGFF(LF)157B

GFF GFF

Granule cells Granule cells

3 dpf-adult 2 dpf-adult

hspGFFDMC90A hspzGFFgDMC156A hspGFFDMC28C

GFF GFF GFF

Granule cells Eurydendroid cells Climbing fibers

2 dpf-adult 3 dpf-adult 3 dpf-adult

SAGFF(LF)251A SAGFF(LF)226B

GFF GFF

Bergmann glia Bergmann glia

4 dpf-adult 2 dpf-ND

Tectum, skeletal muscle, heart Olfactory epithelium, lateral line, various neurons in the brain Retina, telencephalon, tectum Tegmentum, skeletal muscle Telencephalon, small number of neurons in various brain regions Midline of spinal cord cnot4b, intron (Chr4)e5 Retina, telencephalon, midline of hindbrain and spinal Chr1f6 cord

B: Promoter-driven transgenic lines Name of Tg lines Reporter Cells types aldoca:GFF aldoca:gap43Venus aldoca:gap43mCherry

ctxn1, intron (Chr2)a1 kcnip3b, intron (Chr10)b2 wu:fj39g12, exon (Chr16)c3 cuedc1b, intron (Chr15)d4

Stage

GFF/GFP Venus

Purkinje cells Purkinje cells

3 dpf-adult 3 dpf-adult

mCherry

Purkinje cells

3 dpf-adult

dpf: days post fertilization, ND: Not determined, Va: valvula cerebelli, CCe: corpus cerebelli, LCa: lobus caudalis cerebelli, EG: eminentia granularis, Chr: chromosome. GFF expression was determined by crossing to the UAS:GFP line. Cell types and the stages of reporter gene expression in the cerebellar neural circuitry are described in the table. GFF expression was determined by crossing to the UAS:GFP line. Although gap43-Venus was detected in all Purkinje cells in the aldoca:gap43-Venus line, GFP was observed in only a portion of Purkinje cells in the aldoca:GFF; UAS:GFP line, in both larvae and adults (Fig. 3). dpf: days post fertilization. a

1 ctxn1: cortexin 1. 2 kcnip3b: Kv channel interacting protein 3b, calsenilin. c 3 wu:fj39g12: gene predicted to encode natriuretic peptide precursor C-like protein (nppcl). d 4 cuedc1b: CUE domain containing 1b. e 5 cnot4b: CCR4-NOT transcription complex, subunit 4b. f 6 There is no known gene found to be located adjacently to the integration site. b

Analysis of the granule cell-specific Tg lines In the zebrafish cerebellum, mature granule cells are located in two regions, the GCL of the Va/CCe and the EG/LCa (Fig. 1H, I). Granule cells in the two domains exhibit distinct neural circuit connections and developmental processes (Kani et al., 2010; Volkmann et al., 2008). In the current study some of the granule cell-specific trap lines expressed GFP in the granule cells of both the Va/CCe and EG/LCa in larval and adult cerebella (gSAIzGFFM765B, SAGFF(LF)128A, hspGFF57A, hspGFF157B, and hspGFFDMC90A, Fig. 2C, D, I–P). The Tg line gSA2AzGFF152B expressed GFP in most of the granule cells of the Va/CCe and in fewer granule cells of the EG/LCa (Fig. 2A, B), whereas the Tg lines gSAG6A and gSAIGFF23C expressed GFP only in the EG and LCa (Fig. 2E–H). There were additional variations in GFP expression levels and in the proportion of GFP-positive cells within the domains. For example, the Tg lines gSA2AzGFF152B, hspGFF57A, and hspGFF157B expressed GFP strongly in most of the granule cells in the GFP-expressing domains (Fig. 2A, B, K–N), whereas the Tg lines gSAIzGFFM765B and hspGFFDMC90A expressed GFP in only a subset of granule cells in these domains (Fig. 2C, D, O, P). In most of these Tg lines, GFP was also detected in cells outside the cerebellum; however, the GFP signals within the cerebellum were restricted to granule cells. In some lines, such as gSA2AzGFF152B, the GFP expression was relatively specific to cerebellar granule cells (Table 2A). Most of these lines started to express the GFP reporter from 2 or 3 dpf when

differentiation of granule cells initiate (except gSAIGFF23C, the expression started at 4 dpf in gSAIGFF23C), and the expression continued in adult cerebellum. In all of the granule cell-expressing Tg lines, the GFP-positive somata colocalized with Neurod signals in the granule cell nuclei (gSA2AzGFF152B and gSAIGFF23C, Fig. 3E–H, M–X, Fig. 10, data not shown for the other lines) and with Vglut1 expression in the granule cell axons of larval and adult cerebella (Fig. 3A–D, I–L). We further confirmed that the GFP transcripts were localized specifically in the granule cell somata in 5-dpf and adult gSA2AzGFF152B; UAS:GFP fish (Suppl. Fig. 3). Analysis of the Purkinje cell-specific lines We previously reported that an approximately 5-kb genomic fragment extending upstream from the translation initiation site of the aldolase Ca (aldoca) gene, which encodes zebrin II (Ahn et al., 1994), can drive Purkinje cell-specific expression, and we established an aldoca:gap43-Venus Tg line (Tanabe et al., 2010). We confirmed that gap43-Venus was expressed in the axons, dendrites, and somata of all the Pvalb7-positive Purkinje cells from 3 dpf in the larval cerebellum after several generations (Table 2B, Fig. 4A–D), and that its expression continued in the adult cerebellum (Fig. 4E–H). Gap43Venus was additionally detected in the cerebellovestibular tracts of Purkinje cells in this line (Fig. 4A). Using the aldoca promoter, we also established aldoca:gap43-mCherry and aldoca:GFF Tg lines. Similar to the aldoca:gap43-Venus line, gap43-mCherry was detected in

Please cite this article as: Takeuchi, M., et al., Establishment of Gal4 transgenic zebrafish lines for analysis of development of cerebellar neural circuitry. Dev. Biol. (2014), http://dx.doi.org/10.1016/j.ydbio.2014.09.030i

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most Purkinje cells in the aldoca:gap43-mCherry line (Fig. 4I–L). However, after crossing aldoca:GFF with the UAS:GFP reporter line, the GFP expression exhibited a mosaic pattern and was only detected in a portion of the Purkinje cells in the medial and lateral parts of the larval cerebellum from 3 dpf (Fig. 4M–V). The mosaic expression continued in adult cerebellum (Fig. 4Q–V). These data suggested that, although the aldoca promoter has the ability to drive gene expression in all Purkinje cells, all reporters may not be equivalently expressed. Analysis of the eurydendroid cell-specific line The eurydendroid cell somata are located in the vicinity of the PCL and receive signals from Purkinje and granule cell axons, and project their axons to targets outside the cerebellum (Ikenaga et al., 2005, 2006; Murakami and Morita, 1987). In hspzGFFDMC156A; UAS:GFP larvae and adults, GFP signals were detected in somata in close proximity to Pvalb7 þ Purkinje cell somata, in dendrites in the ML (Fig. 5D, L–P), and in axons that projected rostrally from the cerebellum (Fig. 5A, C, E–G, adult stage data not shown) from 5 dpf to adult. More than 75% of the GFP þ somata received Pvalb7 þ Purkinje cell axons in the CCe of the adult cerebellum (Fig. 5N–P, Table 3). The localization of GFP signals in this line indicated that they represent eurydendroid cells. Further analysis showed that the majority of nonPurkinje neurons that received Purkinje axons expressed GFP in this line (data not shown), suggesting that hspzGFFDMC156A marks major population of eurydendroid cells. Although there were some GFP þ cells that did not receive Purkinje cell axons (Table 3), it is not clear whether these cells represent a eurydendroid population that receive granule cell but not Purkinje cell axons. We and others previously reported that a subset of eurydendroid cells express the proneural gene olig2 and are GFP þ in the olig2:EGFP line (Bae et al., 2009; Kani et al., 2010; McFarland et al., 2008). We therefore crossed the olig2:EGFP and hspzGFFDMC156A; UAS:RFP lines, and examined the localization of signals in the larval and adult cerebella. We found that both olig2:EGFP þ and olig2:EGFP- cells expressed RFP in the larval (Fig. 5H–K) and adult cerebella (Fig. 5Q–S; 7/22 olig2:EGFP þ ; hspzGFFDMC156A þ , 15/22 olig2:EGFP  ; hspzGFFDMC156A þ , n¼ 2), consistent with the previous finding that there are olig2 þ and olig2  eurydendroid cells in zebrafish (Bae et al., 2009). Furthermore, we found that the GFP þ axons crossed at the rostro-ventral side of the cerebellum, and projected to the contralateral side of the tegmental region (Fig. 5E–G), indicating that at least a portion of the eurydendroid axons are directed to this region of the brain. Analysis of the IO-specific line In the hspGFFDMC28C; UAS:GFP line, GFP signals were detected in somata located in the ventro-posterior hindbrain of embryos, starting at 3 dpf and extending through the adult stage (Fig. 6, 3 dpf data not shown). The GFP þ axons of these neurons reached the Purkinje cells by 5 dpf (Fig. 6A–H), indicating that the GFP þ cells in this line were IO neurons that extend CFs to Purkinje cells in the cerebellum. At least a portion of the GFP þ axons crossed at the midline between left and right IO and then turned rostrally at 5 dpf (Fig. 6D). During adulthood, GFP expression was more restricted to the IO neurons, and the crossing of the GFP þ CFs at the midline was more obvious (Fig. 6K). The GFP þ CFs took different routes to the cerebellum; some CFs turned vertically toward the rostral side above the IO, whereas others curve gently to the rostral direction (Fig. 6I, Jb). The GFP þ axonal termini were found to project onto the somata or proximal dendrites of Purkinje cells (Fig. 6H, M). We confirmed that most of the GFP þ cells in the ventro-posterior hindbrain are vglut2a-positive (Suppl. Fig. 4). This is consistent with the notion that the IO neurons are vglut2positive glutamatergic neurons (Bae et al., 2009; Hisano et al., 2002; Miyazaki et al., 2003).

To clarify the CF projections further, we crossed hspGFFDMC28C with a UAS reporter line expressing the photoconvertible protein, Kaede (Hatta et al., 2006) (Fig. 7). Photoconverted red Kaede expressed in an axon can be retrogradely transported to the soma (Kimura et al., 2013). We labeled axon termini of the CF in one side of the larval cerebellum with laser irradiation (Fig. 7 D, E). After 7 h, red Kaede was detected primarily in the contralateral IO somata (Fig. 7, L, M). Similar contralateral projections were observed from independent experiments (n¼20, 100%). These data confirm that CFs primarily project contralaterally in zebrafish, as in mammals. Analysis of Bergmann glia-specific lines We identified two GFF trap lines, SAGFF(LF)251A; UAS:GFP and SAGFF(LF)226B; UAS:GFP in which GFP expression was colocalized with Fabp7a in the larval cerebellum (Fig. 8A–C, J–L). Although, GFP signals were not detected in the Bergmann glia of adult cerebella in the SAGFF(LF)226B; UAS:GFP line, GFP signals in adults of the SAGFF(LF)251A; UAS:GFP line colocalized with Fabp7a signals in the cerebellum, but were not detected in Fabp7a þ radial glia outside the cerebellum (Fig. 8D–I). Thus, these lines are Bergmann glia-specific GFF lines, and the SAGFF(LF)251A line can be used for studies of Bergman glial cells in both larval and adult cerebella. Gal4-UAS-mediated WGA reporter detects multi-synaptic connections To demonstrate that GFF lines can be used for the anatomical analysis of neural circuitry, we crossed the granule cell-specific GFF line gSA2AzGFF152B; UAS:GFP and the UAS:AcGFP-P2A-WGA line, and determined the localization of WGA in the adult cerebellum by immunohistochemistry with an anti-WGA antibody. In adults, the GFP signals were only detected in granule cells within the cerebellar neural circuitry and were not observed in Purkinje cells, eurydendroid cells, or the IO neurons (Figs. 2B, 9A, E, I, J, N, R, S, U). WGA was detected in GFP þ granule cell somata in the GCL and granule cell axons in the ML (Fig. 9C, L). WGA was also observed in the Pvalb7 þ Purkinje cells (Fig. 9, B–D, F–H), and the eurydendroid cells, which received the Pvalb7 þ axons and did not express Pvalb7 (Fig. 9K–M, O–Q), indicating that WGA was transferred to Purkinje and eurydendroid cells. Moreover, IO somata were also positive for WGA (Fig. 9T, V), suggesting that WGA was transferred to the IO neurons from either Purkinje cells or eurydendroid cells. These findings demonstrate that the Gal4UAS-mediated WGA reporter system detected direct and/or indirect connections of granule cells with Purkinje cells, eurydendroid cells, and IO neurons in zebrafish. Gal4-UAS system can be used for studying anatomy and developmental processes of granule cells Granule cells are differentiated from atoh1-expressing neuronal progenitors in the rhombic lip and migrate ventrally from 3 to 5 dpf (Kani et al., 2010; Volkmann et al., 2008). To examine migration processes of granule cells, we carried out time-lapse analysis with gSA2AzGFF152B; UAS:GFP lines. In this line, GFP þ differentiating granule cells were first detected in the dorsal superficial domain of the cerebellum at 2 dpf (Fig. 10A, E). The GFP þ somata moved randomly at 3 dpf near the dorsal surface (Fig. 10A–D, Supplemental Movie 1). At 2–3 dpf, some of the GFP þ somata started to migrate ventrally (Fig. 10E–G, Supplemental Movie 2). To visualize granule cell structure at a single cell resolution, we employed two methods: Tol1-mediated transgenesis of a reporter gene (Fig. 10H–K) and photoconversion with a mosaic UAS:Kaede line (Fig. 10L–P). Tol1 and Tol2 are different transposons isolated from albino mutants of medaka (Koga et al., 2008; Koga

Please cite this article as: Takeuchi, M., et al., Establishment of Gal4 transgenic zebrafish lines for analysis of development of cerebellar neural circuitry. Dev. Biol. (2014), http://dx.doi.org/10.1016/j.ydbio.2014.09.030i

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Fig. 5. Analysis of the eurydendroid-specific GFF line, hspzGFFgDMC156A. (A–G, L–P) The hspzGFFgDMC156A line was crossed with UAS:GFP. Larvae at 5 dpf (A–G) and adult brain sections (L–P) were stained with anti-GFP (green) and anti-Pvalb7 (magenta) antibodies. (A–C) Dorsal views with anterior to the left. (D) Cross-section image of the dotted box in (C). (E) Dorso-anterior view. (F, G) Dorsal (F) and lateral (G) views of eurydendroid cell axons, with anterior to the left. The eurydendroid axons are marked by a dotted line with an arrowhead in F and G. The midlines are indicated by dotted lines in D and E. (L–P) GFP and Pvalb7 expression in the cerebellum of hspzGFFgDMC156A; UAS:GFP adults. (N–P) High magnification views of the box in M. GFP-expressing cells received the axons of Pvalb7 þ Purkinje cells (marked by asterisks in N–P). (H–K, Q–S) Co-expression of GFP and RFP in hspzGFFgDMC156A; UAS:RFP; olig2:EGFP eurydendroid cells. The 5-dpf larvae (H–K) and adult brain sections (Q–S) were stained with antiGFP (green) and anti-RFP (magenta) antibodies. (I–K) High magnification views of the box in H. (Q–S) Sagittal sections. Some of the RFP þ cells were also GFP þ (marked by arrowheads in I–K, Q–S). Projection views (A–H), optical sections (I–K), and histological sections (L–S). Scale bars: 100 μm in A (applied to A–C); 20 μm in D; 50 μm in F; 50 μm in G; 20 μm in H; 20 μm in I (applied to I–K); 50 μm in L (applied to L, M); 50 μm in P (applied to N–P); 50 μm in S (applied to Q–S).

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Table 3 Features of hspzGFFgDMC156A; UAS:GFP þ eurydendroid cells. Region Sample 1 Va CCe Sample 2 Va CCe Sample 3 Va CCe

GFP þ cells

GFP þ cells receiving Purkinje axons

Ratio (%)

23 90

8 79

34.8 87.8

30 41

5 33

16.7 80.5

32 118

2 92

6.3 78.0

Sagittal sections (14 μm) of three adult hspzGFFgDMC156A; UAS:GFP brains were generated, and seven typical sections from each brain were used for the analysis. The numbers of GFP þ cells and GFP þ somata surrounded by Pvalb7 þ Purkinje cell axons in the Va and CCe (Fig. 4K) were counted. The ratio of GFP þ cells receiving Purkinje cell axons to the total number of GFP þ cells is indicated. Va: valvula cerebelli, CCe: corpus cerebelli.

et al., 1995; Koga et al., 2007). Since all the Tg lines described in this study were generated by the Tol2 system and expression of Tol2 but not Tol1 transposase in the Tg lines could mobilize the integrated GFF cassette (Urasaki et al., 2008), we thought that another transgene can be introduced into these trap lines by Tol1mediated transgenesis without affecting existing Tol2-transgenes. We constructed a UAS:GFP reporter gene in a Tol1 donor vector and injected it with Tol1 transposase mRNA into gSA2AzGFF152B embryos. When 5 pg of the reporter DNA (5 pg) and Tol1 transposase mRNA (50 pg) were injected into the one-cell stage embryos, most granule cells in the corpus cerebelli expressed GFP at 6 dpf like those in gSA2AzGFF152B; UAS:GFP larvae (Fig. 10H, I). However, injection of the reporter DNA (6.6 pg) and transposase mRNA (16.6 pg) into the 4-to-8-cell stage embryos led to labeling of a few granule cells (Fig. 10J, K). In those embryos, axon and dendrite structures were observed at a single cell resolution (Fig. 10K). It is reported that UAS reporter genes are often silenced by DNA methylation (Akitake et al., 2011). Nevertheless mosaic UAS reporters can be used to label fewer cells. We crossed gSA2AzGFF152B and a mosaic UAS:Kaede line, and labeled granule cell somata one by one with the laser irradiation. Photoconverted red Kaede was anterogradely transported to the axon and visualized the axonal structure of each granule cell (Fig. 10L–P). Confocal imaging revealed that granule cell axons showed typical T-shaped structure of parallel fibers in the corpus cerebelli, like in mammals (Fig. 10P). Supplementary material related to this article can be found online at http://dx.doi.org/10.1016/j.ydbio.2014.09.030.

Discussion Establishment of Gal4 driver lines that can mark cells in cerebellar neural circuits Several Tg lines were used for studying development of cerebellar neural circuits. These include gata1a:GFP (Koster and Fraser, 2006; Volkmann et al., 2010) and neurod:EGFP (Obholzer et al., 2008), which express GFP in granule cells; olig2:EGFP (Kani et al., 2010; McFarland et al., 2008) and a Gal4 trap line s1168t (Heap et al., 2013), which express a reporter in eurydendroid cells; pou4f1-hsp70l:EGFP (Kani et al., 2010), hoxb4a:YFP, and hoxd4a: YFP (Punnamoottil et al., 2008) express a reporter in IO neurons; the gfap:GFP line expresses GFP in glial cells (Bernardos and Raymond, 2006). Most of these lines express only single fluorescent proteins and cannot express other reporter/effector proteins, except s1168t. The Gal4 drivers that we established in this study

can be used to express different types of reporter/effector proteins by crossing UAS lines. In addition, the Gal4 Tg lines can mark cell populations which are different from those marked by the published Tg lines. The most granule cell specific lines such as gata1a: GFP and neurod:EGFP mark all granule cells (Koster and Fraser, 2006; Volkmann et al., 2010) (data not shown for neurod:EGFP), whereas some of the granule cell specific Tg lines established in this study could distinguish granule cells in between the Va/CCe and the EG/LCa (Figs. 2, 3). The olig2:EGFP and s1168t lines express a reporter only in a subset of eurydendroid cells (Heap et al., 2013; Kani et al., 2010), whereas hspzGFFgDMC156A expressed a reporter gene in the major population of eurydendroid cells (Fig. 5, Table 3). The pou4f1-hsp70l:EGFP and hoxb4a/hoxd4a:YFP lines express a reporter in not only IO neurons but also many other neurons (Kani et al., 2010; Punnamoottil et al., 2008), whereas hspGFFDMC28C express a reporter gene mainly in IO neurons in both larvae and adults (Fig. 6). Unlike the gfap:GFP line, the SAGFF (LF)251A and SAGFF(LF)226B lines expressed a reporter only in Bergmann glial cells. These data suggest that the Gal4 driver Tg lines display unique properties that can be used for studying detailed structure and development of the cerebellar neural circuits. Distinct compartments of granule cells Granule cells are localized to two major domains in the zebrafish cerebellum: the GCL in the Va/CCe and the EG/LCa. The granule cells in these two domains are involved in the formation of two distinct types of circuits: rostral non-vestibulocerebellar and caudal vestibulocerebellar circuits (Bae et al., 2009; Volkmann et al., 2008) (Fig. 1). Granule cells in the two domains are derived from spatially separated neuronal progenitors, located in the upper rhombic lip (Kani et al., 2010; Volkmann et al., 2008). The developmental processes of the two different granule cell populations are also distinct; granule cells in the Va/CCe exhibit tangential and radial migration, while those in the EG/LCa exhibit limited tangential and no radial migration (Kani et al., 2010; Volkmann et al., 2008). We also previously reported the identification of genes that are differentially expressed in the two domains (Bae et al., 2009). In this report, we identified GFF/GFP lines in which the reporter was preferentially expressed in the Va/CCe (gSA2AzGFF152B) and lines in which the reporter was expressed only in the EG/LCa (gSAG6A and gSAIGFF23C) (Figs. 2, 3). These lines can be used to investigate anatomical, developmental, and functional differences between the granule cells in the two domains. We identified the trapped locus of the EG/LCa-specific lines; the gene encoding Kv channel-interacting protein 3b (kcnip3b) was trapped in gSAG6A, and the gene encoding natriuretic peptide precursor C-like protein (wu:fj39g12, nppcl) was trapped in gSAIGFF23C (Table 2). It was previously reported that nppcl is expressed in the GCL of both the CCe and LCa in the adult cerebellum (Toro et al., 2009). Thus, the integrated GFF cassette may have captured an enhancer/promoter specific for expressing nppcl in the GCL of the CCe, or trapped a CCespecific variant of the nppcl transcript, as the trap vector contains a splicing acceptor. A detailed analysis of the integration site and transcripts generated in the line should clarify this issue. Purkinje cells Analysis of the aldoca:gap43-Venus and aldoca:gap43-mCherry lines confirmed that Purkinje cells do not only project their axons to eurydendroid cells, and showed that Purkinje cells in the lateral regions directly project to vestibular nuclei in the hindbrain (cerebellovestibular tracts) (Bae et al., 2009), as seen in other vertebrates (Altman and Bayer, 1997; Han and Bell, 2003; Meek, 1992). These two types of axonal projections could function differently. The isolation of Tg lines that distinguish Purkinje cells in the two domains will be

Please cite this article as: Takeuchi, M., et al., Establishment of Gal4 transgenic zebrafish lines for analysis of development of cerebellar neural circuitry. Dev. Biol. (2014), http://dx.doi.org/10.1016/j.ydbio.2014.09.030i

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Fig. 6. Climbing fibers visualized with the hspGFFDMC28C line. Immunostaining of hspGFFDMC28C; UAS:GFP larvae (A–H) and adult brains (I–-M) with anti-GFP (green) and anti-Pvalb7 (magenta) antibodies. CFs are indicated by arrowheads (A, E) or dotted lines (F, Jb, K). (A–C) Dorsal views of the 5-dpf larval hindbrain, with anterior to the left. (D) High magnification view of the box in A. CFs crossing the midline are indicated by arrowheads. (E) Lateral view of the 5-dpf larval hindbrain, with anterior to the left. (F–H) High magnification views of the box in E. (I) Lateral view (with anterior to the left) of the adult brain, subjected to whole mount staining and optical clearing. (Ja, Jb) High magnification views of the boxes in I. (K) Dorsal view of the IO region. (L) Sagittal section, with anterior to the left. (M) High magnification view of the box in L. The localization of GFP þ axonal termini on the Pvalb7 þ somata of Purkinje cells is indicated by arrowheads. Scale bars: 100 μm in A (applied to A–C); 20 μm in D; 100 μm in E; 20 μm in F (applied to F–H); 400 μm in I; 200 μm in Ja; 100 μm in Jb; 200 μm in K; 400 μm in L; 20 μm in M.

necessary to dissect their developmental processes and functions. Zebrin II and Aldoc exhibit compartment-specific expression in Purkinje cells (Ahn et al., 1994; Brochu et al., 1990; Lannoo et al., 1991a; Lannoo et al., 1991b; Meek et al., 1992), and Aldoc:Venus knock-in mice recapitulate the striped expression pattern of Aldoc observed in the Purkinje cells of native mice (Fujita et al., 2014). However, neither the aldoca:gap43-Venus nor aldoca:gap43mCherry cerebellum exhibited this expression pattern (Fig. 5), consistent with a previous finding that all Purkinje cells express zebrin II in zebrafish (Bae et al., 2009), and the difference from mice may be related to the simpler cerebellar neural circuitry in zebrafish.

Eurydendroid cells Eurydendroid cells in the teleost cerebellum display several characteristic features: (1) their dendrites form extensive arbors in the ML, (2) their somata are primarily located in the PCL, with some in the GCL, (3) they send their axons to targets outside of the cerebellum (Finger, 1978; Ikenaga et al., 2005; Ito and Yoshimoto, 1990; Murakami and Morita, 1987; Nieuwenhuys et al., 1974; Pouwels, 1978), and (4) a subset of eurydendroid cells express olig2 (Bae et al., 2009; Kani et al., 2010; McFarland et al., 2008). We found that many hspzGFFgDMC156A þ neurons exhibited these

Please cite this article as: Takeuchi, M., et al., Establishment of Gal4 transgenic zebrafish lines for analysis of development of cerebellar neural circuitry. Dev. Biol. (2014), http://dx.doi.org/10.1016/j.ydbio.2014.09.030i

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Fig. 7. Tracing with Kaede shows contralateral projections of CFs in zebrafish. A CF terminal region of the left side of 5-dpf hspGFFDMC28C; UAS:Kaede larvae was laser irradiated. Expression of Kaede and photoconverted Kaede were examined. Cerebellum (CCe, A–I) and IO (J–M) regions. Dorsal views with anterior to the left. The midlines are indicated by dotted lines in A, J–M. (B–E) High magnification views of the box a in A (right side of the cerebellum). (F–I) High magnification views of the box b in A (left side). The CF axon termini on the right side were irradiated. The irradiated area is marked by a dotted box in B. Expression of green (A, B, D, F, H) and photoconverted Kaede (white, C, E, G, I) was detected in the cerebellum (CCe) before (B, C, F, G) and 7 h after laser-mediated photoconversion (D, E, H, I). Expression of green (J, L) and photoconverted Kaede (K, M) was also detected in the IO region before (J, K) and 7 h after the photoconverstion (L, M). Similar contralateral projections were detected from twenty independent experiments. Scale bars: 50 μm in A; 50 μm in B (applied to B, C, G, H); 50 μm in D (applied to D, E, H, I); 40 μm in J (applied to J–M).

features (Fig. 4), resulting in their identification as eurydendroid cells. Molecular markers specific for eurydendroid cells have not yet been reported. Thus, eurydendroid cells were visualized by either retrograde or anterograde labeling of cerebellar efferent fibers, or identified as cells receiving Purkinje cell axons (Ikenaga et al., 2006). Thus, the hspzGFFgDMC156A line provides a genetic tool for studying the anatomy and function of eurydendroid cells. Although we found that the majority of hspzGFFgDMC156A þ cells received Purkinje cell axons in the CCe, many of the hspzGFFgDMC156A þ cells in the Va did not receive Purkinje cell axons (Table 3). This may be partly due to the difficulty in visualizing all Purkinje axons by Pvalb7 staining, or there may be eurydendroid cell subsets that only receive granule cell axon inputs. Future electrophysiological and histological studies of the hspzGFFgDMC156A þ cells will be required to further characterize the eurydendroid cells. Anterograde labeling of the cerebellum with neural tracers in various teleost species has identified potential eurydendroid cellprojection targets, which are distributed from the diencephalon to the medulla (Finger, 1978; Folgueira et al., 2006; Ikenaga et al., 2005,

2006; Meek et al., 1986a, b; Murakami and Morita, 1987; Wullimann and Northcutt, 1988). Similarly, retrograde labeling with tracer injections showed that the nucleus ventromedial thalami, nucleus ruber, oculomotor nucleus, reticular formation, and nucleus lateralis valvulae are also possible targets of eurydendroid cells (Torres et al., 1992; Yang et al., 2004). Tracer experiments reveal the axonal projections extending from or to the cerebellum, but do not provide direct evidence that these areas are targets of eurydendroid cell projections. Our findings using the hspzGFFgDMC156A line showed that the major eurydendroid cell axons crossed at the midline under the cerebellum and projected contralaterally to a wide area in the tegmentum and rostral hindbrain, where the nucleus ruber, oculomotor nucleus, and reticular formation are located, during the larval stages (Fig. 5). Although eurydendroid cells are quite different from projection neurons in the mammalian deep cerebellar nuclei, contralateral projection is the common feature of efferent tracts from the cerebellum (Altman and Bayer, 1997). Eurydendroid cell projections to the optic tectum and thalamus were reported previously (Heap et al., 2013; Torres et al., 1992; Yang et al., 2004); however, we did not

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Fig. 8. Bergmann glia-specific GFF lines. The SAGFF(LF)251A and SAGFF(LF)226B lines were crossed with UAS:GFP. (A–I) SAGFF(LF)251A; UAS:GFP. (J–L) SAGFF(LF)226B; UAS: GFP. The 5-dpf larvae (A–C, J–L, dorsal projection views with anterior to the left) and sagittal sections of adult brains (D–F, anterior to the left) were stained with anti-GFP (green, A, C, D, F, G, I, J, L) and anti-Fabp7 (magenta, B, C, E, F, H, I, K, L) antibodies. (G–I) High magnification views of the box in F. Note that GFP and Fabp7a staining colocalized only in the cerebella of larvae and adults (marked by arrowheads, B, C, F), indicating that SAGFF(LF)251A; UAS:GFP was expressed only in Bergmann glial cells, and not in the radial glia of other brain regions. SAGFF(LF)226B; UAS:GFP-positive signals were detected in the Bergmann glia of larvae, but not adult cerebella. Scale bars: 100 μm in C (applied to A-C); 200 μm in F (applied to D–F); 20 μm in I (applied to G–I); 100 μm in L (applied to J-L).

detect these projections from hspzGFFgDMC156A þ neurons (Fig. 4). It is possible that although hspzGFFgDMC156A þ cells are the major population of eurydendroid cells, there are other types of eurydendroid cells that project to these regions. Climbing fibers and neural connections Previous studies with hoxb4a/hoxd4a:YFP lines and the pou4f1-hsp70l:GFP line revealed that the CFs form by 5 dpf (Bae et al., 2009; Punnamoottil et al., 2008). Retrograde labeling and IO ablation experiments in early-stage larvae (6 or 7 dpf) showed that CFs function in motor adaptation during these stages (Ahrens et al., 2012). These results are consistent with our findings that the hspGFFDMC28C; UAS:GFP þ axons, projecting from the IO neurons, reached the Purkinje cells by 5 dpf (Fig. 6). Tracer experiments with teleost species revealed the presence of CF contralateral projections during adult stages (Folgueira et al., 2006; Meek et al., 1986a, b; Wullimann and Northcutt, 1988, 1989); however, the precise

connections between IO neurons and Purkinje cells were unknown. Here, the midline crossing of the CFs was observed from the larval through adult stages; photoconversion of Kaede in the CF termini in the cerebellum resulted in retrograde labeling of the contralateral IO (Figs. 6 and 7), revealing that contralateral projection is a conserved feature of the CFs from zebrafish to mammals (Altman and Bayer, 1997). A clear topographic map of the mammalian CFs has been developed, which shows that neurons in a particular region of the IO project to Purkinje cells in a specific area of the cerebellum; the CF projection pattern is correlated with Purkinje cell compartmentalization and the pattern of zebrin II expression (Apps and Hawkes, 2009; Sasamura et al., 2013; Sugihara, 2006; Sugihara et al., 2007; Sugihara and Quy, 2007; Sugihara and Shinoda, 2004). In zebrafish, we found several distinct routes for cerebellar CFs; a few major CF axon bundles reached the cerebellum during the larval stage, while several different CF bundles were projected from the IO during the adult stage (Fig. 6). As hspGFFDMC28C expressed reporter genes in most of the IO neurons, it was difficult to identify the individual axons of the CFs.

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Fig. 9. Detection of cerebellar neural circuitry with WGA tracing. The gSA2AzGFF152B; UAS:GFP line was crossed with UAS:AcGFP-P2A-WGA. Sagittal sections of the adult cerebellum were stained with anti-GFP (green), anti-Pvalb7 (magenta), and anti-WGA (cyan) antibodies. (A–D) and (J–M) represent two different areas of the ML/GCL boundary of the cerebellum. Expression of GFP (A, J), Pvalb7 (B, K), and WGA (C, L), and merged images (D, M) are shown. Projection views of histological sections. (E–I) High magnification views of the box in D. (N-R) High magnification views of the box in M. Optical sections (E–I, N–R). Pvalb7 þ Purkinje cells that incorporated WGA are indicated by arrowheads (B–D, F–I). Eurydendroid cell with a Pvalb7  soma, that received Pvalb7 þ axon(s) and incorporated WGA, is indicated by arrowheads (K–M, O–R). Note both Purkinje and eurydendroid cells are GFP-negative. (S, T) Low magnification views of the hindbrain. Expression of GFP (S–V) and WGA (T, V). (U, V) High magnification views of the box in T (IO region). The ventral bottoms of the hindbrain are indicated by dotted lines (U, V). Note that IO neurons incorporated WGA but GFP-negative. Scale bars: 20 μm in A (applied to A–D); 20 μm in E (applied to E–I); 20 μm in J (applied to J–M); 25 μm in N (applied to N-R); 200 μm in S (applied to S, T); 40 μm in U (applied to U, V).

Analysis of the hspGFFDMC28C line with single-cell labeling techniques, such as Brainbow/Zebrabow (Livet et al., 2007; Pan et al., 2013) or mosaic reporter gene expression (Fig. 10) (Asakawa et al., 2013; Miyasaka et al., 2014; Miyasaka et al., 2009), will enable the establishment of a topographic map of the CFs in zebrafish. Use of cerebellar neural circuit GFF lines in anatomical, developmental, and functional studies of the cerebellar neural circuitry in zebrafish The GFF lines reported in this study can be used for various types of analyses. Using a granule cell-specific GFF line, we demonstrated that WGA tracing revealed connections between granule cells and Purkinje/eurydendroid cells (Fig. 9). Since eurydendroid cells receive inputs from both granule cells and Purkinje cells, eurydendroid cells

may receive WGA directly from granule cells and/or indirectly through Purkinje cells. In this experiment, WGA was also detected in the IO neurons (Fig. 9). It was also reported that WGA can be incorporated in the IO neurons when WGA is expressed in Purkinje cells in mouse and zebrafish (Matsui et al., 2014; Yoshihara et al., 1999). Since previous studies suggested that Purkinje cells do not send outputs to the IO neurons (Bae et al., 2009) and eurydendroid cells do not receive inputs from the CFs (Han and Bell, 2003; Ikenaga et al., 2006), our data suggest two possibilities. The WGA might be anterogradely transported in the axons of the eurydendroid cells and then transferred to the somata of the IO neurons. Alternatively the WGA might be retrogradely transferred from the Purkinje cells to the CFs and then transported to the somata of the IO neurons. Although WGA is known to be transported in an anterograde manner, its retrograde transport is also reported (Braz et al., 2002; Yoshihara

Please cite this article as: Takeuchi, M., et al., Establishment of Gal4 transgenic zebrafish lines for analysis of development of cerebellar neural circuitry. Dev. Biol. (2014), http://dx.doi.org/10.1016/j.ydbio.2014.09.030i

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Fig. 10. Development and anatomy of granule cells revealed by gSA2AzGFF152B line. (A–G) Dorsal projection views (A–D) and cross section images (E-G) of gSA2AzGFF152B; UAS:GFP larval cerebella. Single cells on the left and right sides (arrowheads and arrows in A–D, asterisks in E–G) are traced during the indicated periods. Trajectories of the marked somata are shown in D (the positions at 70, 70.5, and 71.5 hpf are indicated by white circles and squares). (H–K) Tol1-medated transgenesis of UAS:GFP reporter in gSA2AzGFF152B line. 50 pg of UAS:GFP reporter DNA in a Tol1 donor vector (T1-UAS:GFP) and 50 pg of Tol1 transposase mRNA were injected into an one-cell stage gSA2AzGFF152B embryo (H, I). 6.6 pg of T1-UAS:GFP DNA and 16.6 pg of Tol1 transposase mRNA (J, K) were injected into one blastomere of a 4-to-8-cell stage gSA2AzGFF152B embryos. GFP expression was detected at 6 dpf. Axon and dendrite structure are indicated by arrowhead and arrow, respectively, in K. (L–O) Combination of mosaic UAS:Kaede and laser irradiation shows the structure of granule cells at a single cell resolution. gSA2AzGFF152B line was crossed with mosaic (partially silenced) UAS: Kaede line. Single somata of granule cells were marked by laser-irradiation one by one at 3 dpf (one in L, three in M, and four in N, marked by arrowheads). Expression of original (green) and photoconverted Kaede (magenta) is shown in L–N. (O, P) Dorsal view and cross section image for photoconverted Kaede in N. Note granule cells in the corpus cerebelli shows typical T-shaped parallel fibers (marked by dotted lines with arrowheads in P). The midlines are indicated by dotted lines. Scale bars: 25 μm in A (applied to A–D); 20 μm in E (applied to E–G); 50 μm in H, J; 20 μm in I, K; 20 μm in L (applied to L–O); 20 μm in P.

et al., 1999). Thus we cannot exclude both possibilities. Future studies using anterograde or retrograde-specific tracers will clarify this issue. In any case our data indicate the WGA tracing can be used to detect mono- and multi-transsynaptic connections. The combination of WGA tracing and other techniques, such as electrophysiology and virus-mediated monosynaptic tracing, will reveal the precise connections in zebrafish cerebellar neural circuitry. We further demonstrate that a granule cell-specific GFF line can be used for studying development and structure of granule cells in detail. Time lapse imaging of the gSA2AzGFF152B; UAS:GFP line revealed random movements of granule cell somata near the

dorsal surface and their ventral migration (Fig. 10, Movie 1, 2). Although the ventral migration of granule cells was previously reported (Kani et al., 2010; Volkmann et al., 2008), relationship between the random movements, ventral migration, and axogenesis of granule cells is not clear. At 2–3 hpf, parallel fibers and Bergmann glial processes were scarcely detected (data not shown), suggesting that the initial random movements and ventral migration of granule cells are not linked with their axongenesis and do not depend on glial fibers. Furthermore the combinations of the gSA2AzGFF152B line with the Tol1 reporter expression or mosaic UAS:Kaede could visualize granule cell structure at a single cell

Please cite this article as: Takeuchi, M., et al., Establishment of Gal4 transgenic zebrafish lines for analysis of development of cerebellar neural circuitry. Dev. Biol. (2014), http://dx.doi.org/10.1016/j.ydbio.2014.09.030i

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resolution (Fig. 10). Future studies with reporters marking organelles, cytoskeleton, or cell polarities will reveal cellular mechanisms controlling the cell movements of granule cells during neural circuit formation. Many UAS reporter/effector zebrafish Tg lines are available to monitor or manipulate the cerebellar neural circuitry. The reporters include fluorescent Ca2 þ indicators (Muto and Kawakami, 2013; Muto et al., 2011), fluorescent proteins that monitor cell polarity and cytoskeletons (Distel et al., 2010; Distel et al., 2011), multicolor fluorescent proteins (Zebrabow) that label the axons/dendrites of individual neurons (Pan et al., 2013), nitroreductase for cell ablation (Pisharath et al., 2007), tetanus toxin for neuronal synaptic transmission blockade (Asakawa et al., 2008; Koide et al., 2009), and optogenetic tools, such as channelrhodopsin and halorhodopsin, for neuronal activation and suppression (Arrenberg et al., 2009; Douglass et al., 2008; Kimura et al., 2013; Umeda et al., 2013). Combining the GFF lines with these reporter/effector lines should enable further characterization of development and functions of the cerebellar neural circuitry in zebrafish. Together with cerebellar mutants (Bae et al., 2009), the Tg lines provide valuable tools for elucidating the cerebellar neural circuitry in zebrafish.

Acknowledgments We thank B. Appel for the olig2:EGFP line, K. Hatta for UAS:Kaede line, Y. Ohashi and Y. Miyashita for AcGFP-P2A-WGA plasmid, K. Kondoh and S. Tsukazaki for fish care, the National BioResource Project (NBRP) for zebrafish maintenance, G. Abe and H. Kikuta for gene and enhancer trap constructs, and the members of the Hibi laboratory for helpful discussions. This work was supported by Grants-in-Aid for Scientific Research (A, B, and C) from the Ministry of Education, Science, Sports and Technology (MEXT), Japan (23241063 to K.K., 24370088 to M.H., 25440104 to T.S.); Grants-inAid for Scientific Research on Innovative Areas from MEXT (25111709, 26115512 to M.H.); the Uehara Memorial Foundation (to M.H.); Takeda Science Foundation (to M.H.); the Naito Foundation Natural Science Scholarship (to M.H.).

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Please cite this article as: Takeuchi, M., et al., Establishment of Gal4 transgenic zebrafish lines for analysis of development of cerebellar neural circuitry. Dev. Biol. (2014), http://dx.doi.org/10.1016/j.ydbio.2014.09.030i

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