Expression of Brain-Derived Neurotrophic Factor and TrkB in the Lateral Line System of Zebrafish During Development

Fish Maintenance and Materials

In this study, we used normal (Tubingen Long Fin) and transgenic zebrafish from 10 to 180 days post-fertilization (dpf) when they are considered adults. The transgenic strains ET4 and Et(krt4:GFP)sqet4 were kindly provided by Prof. A. J. Hudspeth (Laboratory of Sensory Neuroscience, Rockefeller University, New York, NY, USA) (Nagiel et al. 2008) and bred in the fish facility of the CISS (Centro di Ittiopatologia Sperimentale per la Sicilia), University of Messina in standard conditions. Fish were kept on a 14 h day, 10 h night cycle at a constant temperature of 28.5°C and were fed twice a day. Embryos were raised in system water with the addition of 200 M 1-phenyl-2-thiourea at 1 dpf to reduce pigment formation. All embryos were collected after natural spawning and staged according to Kimmel et al. (1995).

qRT-PCR

Total RNA was extracted from the whole zebrafish using a commercial kit (Trizol Reagent, Invitrogen, Carlsbad, CA, USA), according to manufacturer’s instructions. After precipitation and cold ethanol washing, RNA was dried and dissolved in an appropriate volume of Tris–EDTA buffer (10 mM Tris–HCl pH 8.0 and 1 mM EDTANa₂). Each sample was digested with 1 U of DNase I for 1 h at 37°C to digest genomic DNA. The RNA was precipitated, washed, and dissolved again in the same buffer. RNA solution was quantified at 260 nm (Biomate 3, Thermo Electron Corporation, Waltham, MA, USA) and its purity was assessed by the ratio at 260/280 nm reading. We used the High Capacity cDNA archive kit (Applied Biosystems, Carlsbad, CA, USA) and random hexamers, and 10 μg of total RNA to make cDNA following manufacturer’s instructions. 1 μg of the cDNA was used to detect BDNF and TrkB expression using qPCR. The sequences of the primers used were

• BDNF: F 5′-gttactctttctcttggaggaatacaaaa-3′ R 5′-gctaatactgtcacaaacgctgagtt-3′ Probe 5′–6′FAM-actacctggatgccgcc-MGB-3′

• TkkB: F 5′-ggaaaagcaaaaaccctgtctaga-3′ R 5′-tgtagcatcacttcctgccatt-3′ Probe 5′-6FAM-tgagcatccacgtgtcg-MGB-3′

• Actin: F 5′-ttgccccgaggctctctt-3′ R 5′-agttgaaggtggtctcgtggat-3′ Probe 5′-VIC-ctgggtatggaatctt-MGB-3′

The homemade TaqMan probes were labeled at the 5′ with 6′FAM fluorochromes for the BDNF and TrkB, and VIC fluorochrome for beta-actin, while the 3′ ends were labeled with the Minor Grow Binder (MGB) quencher. The PCR reactions were performed using the TaqMan Universal PCR Master Mix (Applied Biosystems) using 5 pmol of each primers and 9 pmol of both target and beta actin probe. The assays were performed in triplicate using a 7500 PCR real-time System (Applied Biosystems), and quantification was calculated using the 2^−ΔΔCt algorithm. The average value in adult specimens was considered as the standard and the final results were expressed as ±n-fold difference versus standard (relative expression).

Western Blot

Frozen larvae 20 dpf were processed for western blot. Experiments were performed in triplicate as follows: the whole larvae were rinsed in cold saline, then pooled, and homogenized (1:2, w/v) with a Potter homogenizer in Tris–HCl-buffered saline (TBS; 0.1 M, pH 7.5) containing 1 μM leupeptin, 10 μM pepstatin, and 2 mM phenylmethylsulfonyl fluoride. The homogenates were then centrifuged at 25000×g for 15 min at 4°C, and the resulting pellet dissolved in 10 mM Tris–HCl, pH 6.8, 2% SDS, 100 mM DTT, and 10% glycerol at 4°C. The pellets were thawed and analyzed by electrophoresis in 10% (for Trks) and 15% (for NTs) polyacrylamide SDS gels. After electrophoresis, proteins were transferred onto a nitrocellulose membrane and unspecific binding was blocked by incubation for 3 h in PBS containing 5% dry milk, and 0.1% Tween 20. The membranes were then incubated at 4°C for 2 h with primary antibodies against BDNF and TrkB proteins. We used rabbit polyclonal antibodies against a sequence of amino-terminal mouse BDNF (sequence H₂N-HSDPARRGEL-COOH; dilution 1:500; Chemicon International Inc., Temecula, CA, USA; catalog no. AB1534SP) and TrkB (dilution 1:500, directed against the residues 794–808 of the introcytoplasmatic domain of human TrkB; Santa Cruz Biotechnology, Santa Cruz, CA, USA; catalog no. sc-12). After incubation, the membranes were washed with TBS (pH 7.6) containing 20% Tween 20, and incubated again for 1 h with the goat anti-rabbit IgG diluted 1:100 at room temperature. Membranes were washed again and incubated with the PAP complex diluted 1:100 for 1 h at room temperature and the reaction was visualized using the ECL kit (Amersham Pharmaceuticals, Buckinghamshire, UK). Marker proteins were visualized by staining with Brilliant Blue.

Localization of TrkB and BDNF Immunohistochemistry

Embryos of 10 and 20 dpf were processed for whole mount immunofluorescence. The animals were fixed overnight in PBS containing 1% Tween 20 and 4% paraformaldehyde, then rinsed three times for 15 min in PBS and transferred to blocking solution for 30 min. Thereafter, they were incubated overnight at 4°C with antibodies raised against pro-BDNF (mouse monoclonal antibody, Santa Cruz Biotechnology; catalog no. sc-65513) and TrkB (described above; Santa Cruz Biotechnology) both used diluted 1:100. The samples were then rinsed three times in PBS for 1 h, and incubated overnight at 4°C with Alexa Fluor 488 (Invitrogen, diluted 1:200), and with Alexa Fluor 568 (Invitrogen, diluted 1:20). Samples were washed in PBS, mounted in Vectashield (Vector Laboratories, Burlingame, CA, USA), and visualized on an Olympus FV1000 laser-scanning confocal microscope with a 60×, 1.42 NA PlanApochromat objective lens at a scan rate of 8 μs per pixel with Kalman averaging.

The immunohistochemical study in zebrafish older than 30 dpf was carried out on histological sections. The animals were anesthetized with MS222 (ethyl-m-amino benzoate; 0.4 g/l), killed, fixed in toto in Bouin’s fixative for 24 h at 4°C, and then routinely processed for paraffin embedding. The pieces were cut in serial frontal, horizontal, and sagittal sections 10-μm thick, and collected on gelatin-coated microscope slides. The sections were then processed for indirect peroxidase immunohistochemistry as previously described (Germanà et al. 2008). In brief, deparaffinized and rehydrated sections were rinsed in Tris–HCl buffer (0.05 M, pH 7.5) containing 0.1% bovine serum albumin and 0.2% Triton-X-100. The endogenous peroxidase activity and non-specific binding were blocked with 3% H₂O₂ and 25% fetal calf serum, respectively, and sections were incubated overnight with the rabbit antibodies raised against BDNF and TrkB (described in the “Western blot” section), both used diluted 1:100. Then sections were washed in the same buffer and incubated for 1.5 h at room temperature with peroxidase-labeled goat anti-rabbit IgG (Amersham), diluted 1:100 or with Texas red-labeled sheep anti-rabbit IgG (Amersham) diluted 1:50. The immunoreaction was visualized using 3-3′-diaminobenzidine as a chromogen.

For control purposes, sections were processed in the same way substituting the primary antibodies by non-immune rabbit serum; or omitting the primary or the secondary antibodies in the incubation. For TrkB, additional controls were performed incubating the sections with a specifically preabsorbed TrkB antibody (20 μg/ml of the homologous antigen; blocking peptide sc-12 P from Santa Cruz Biotechnology). Moreover, to avoid cross reactivity of TrkB antisera to other Trks, aliquots of each Trk antiserum were absorbed with an excessive amount of heterologous antigen (see for details Hannestad et al. 2000; Catania et al. 2007). Under these conditions no specific immunostaining was detected.

Transmission Electron Microscopy

Samples of neuromasts identified in semi-thin sections were processed for Durcupan^® ACM (Fluka, Buchs, Switzerland) resin-embedding as follows: pieces fixed in 2.5% glutaraldehyde in 0.2 M PBS (pH 7.4) for 2 h at 4°C were washed repeatedly in the same buffer and postfixed in 1% osmium tetroxide in 0.2 M PBS for 1 h at 4°C. The tissues were then dehydrated with increasing alcohol concentrations: 50, 70, 90, and 100%. The dehydrated pieces were embedded in Araldite. Finally, sections were obtained with a Reichert Jung Ultracut E ultramicrotome. Semi-thin sections (1 μm) obtained with a diamond knife were stained with toluidine blue. Then, ultra-thin sections (70 nm) were obtained from selected areas of the semi-thin sections, collected on copper grids, stained with uranyl acetate and lead citrate and examined, and photographed with a JEOL-JEM-T8 electron microscope.

BDNF and TrkB mRNA Expressions in Zebrafish

At all ages studied detectable levels of both BDNF and TrkB mRNAs were found (Fig. 1). BDNF and TrkB expressions were found to peak at 20 days, reaching values ranging from 12- to 2-fold those found in adults. Later on, the levels of expression of BDNF decreased in parallel to the levels of expression of TrkB to the low, but detectable, expression levels found in adult animals.

BDNF and TrkB Expressions in Zebrafish

The antibodies used to identify BDNF and TrkB at the protein level, detected by western blot a single protein with estimated molecular masses of approximately 13 and 145 kDa, respectively (Fig. 2). These molecular weights were consistent with those expected for the mature BDNF in mammals and mammalian full-length TrkB. The TrkB detected is a full-length catalytic TrkB isoform.

BDNF and TrkB Localizations in Hair Cells of Developing Zebrafish

Using confocal-laser microscopy, the localization of BDNF and TrkB was investigated in the neuromasts of the transgenic ET4 strain of zebrafish during the larval period (until 20 dpf). In these animals, the hairy sensory cells were identified because they express green fluorescent protein (Fig. 3a). Expression of BDNF (Fig. 3b) and TrkB (Fig. 3c) was restricted to the hair cells and that for TrkB as well. Therefore, at these larval stages, BDNF and TrkB co-localize in the same cells. Moreover, because the precise co-localization of BDNF and TrkB with GFP, expression of BDNF or TrkB in non-hairy cells of the neuromast, i.e. supporting and mantle cells, can be ruled out.

In fish older than 30 dpf, the localization of BDNF and TrkB was investigated in sections from fixed and paraffin-embedded material. TrkB immunoreactivity was regularly found in most of the neuronal somata of the anterior and posterior lateral line ganglia, which contains the neurons that innervate the neuromast of the body canal, at all the ages examined. Interestingly, expression of TrkB was not detected neither in the axons of these neurons nor in the nerve fibers supplying neuromasts (Fig. 4a). Nevertheless, in the neuromasts themselves there was a clear age-dependent change in the levels of expression of TrkB. From 30 up to 50 dpf, all sensory cells were TrkB positive (Fig. 4b), whereas in adult animals (180 dpf) TrkB immunoreactivity is restricted to a few cells located at the periphery of the neuromast with faint intensity but specific immunolabeling (Fig. 4c).

Regarding the cellular localization of BDNF, between 30 and 50 dpf, there was a remarkable decrease in the density of sensory cells expression BDNF (data not shown). This decrease was more evident in adult animals in which BDNF expression was found to be restricted to a few cells located in the sensory part of the neuromast; they showed particular “bottle with large neck” morphology (Fig. 5a, b). To identify the BDNF-positive cells, we used transmission electron microscopy carried out in neuromasts of adult animals. Based on their morphological features, the cells that express BDNF were identified as maturing hair sensory cells (Fig. 5c).