Technical Reference #1618

1618.

In Vitro Guidance of Retinal Axons by a Tectal Lamina-Specific Glycoprotein Nel Yulan Jiang; Hiroya Obama; Soh Leh Juan; Ritsuko Nakamura; Chizu Nakamotot; Zhufeng Ouyang; Masaru Nakamoto, University of Aberdeen, Molecular and Cellular Neuroscience, 41(1618), (2009)

Abstract:
Nel is a glycoprotein containing five chordin-like and six epidermal growth factor-like domains and isstrongly expressed in the nervous system. In this study we have examined expression patterns and in vitrofunctions of Nel in the chicken retinotecta

Materials & Methods:
Chick embryos Fertilized White Leghorn chicken eggs were purchased from Charles River SPAFAS (North Franklin CT) and Henry Stewart (England) and incubated at 38 °C until use. Northern blot analyses Total RNA was prepared from embryonic chicken tecta by using Trizol reagent (Invitrogen Carlsbad CA) separated in 1% formaldehyde agarose gels and transferred to nylon membranes. The membranes were hybridized with a digoxigenin-labeled DNA probe prepared from the full coding region of chicken Nel cDNA. Chemiluminescence detection using alkaline phosphatase-conjugated antidigoxigenin antibody (Roche Applied Science Indianapolis IN) was performed according to the manufacture's instructions. Construction expression and purification of Nel-AP For preparation of a fusion protein of Nel with an alkaline phosphatase tag (Nel-AP) chicken Nel cDNA (from nucleotide 103 to 2565 (Matsuhashi et al. 1996); GenBank accession NM 001030740) was amplified from chicken embryo cDNA by polymerase chain reaction and inserted between the BamHI and MluI sites of the pCMVAP vector. Nel-AP and AP expression constructs were transfected into HEK293T cells using the FuGENE transfection reagent (Roche Applied Science). After 3–4 days the supernatants containing Nel-AP or AP were collected and applied to an anti-AP antibody column. After washing Nel-AP and AP were eluted with 0.1 M glycine–HCl (pH 2.5) neutralized with 0.75 M Tris–HCl (pH 8.8) and dialyzed against PBS. Concentrations of Nel-AP and AP were evaluated by the Bradford assay SDS-PAGE Western blot using anti-AP antibody and by measuring AP activity (Flanagan et al. 2000) and comparable concentrations were used. Heat inactivation of Nel-AP (HI-Nel-AP) was performed at 65 °C for 30 min. Since the human placental alkaline phosphatase used as a tag is heat stable this treatment does not affect the enzyme activity (Flanagan et al. 2000). Affinity probe in situ Affinity probe in situ using Nel-AP was performed essentially as previously described (Flanagan et al. 2000). Briefly unfixed tissues and explants were incubated with Nel-AP AP or HI-Nel-AP for 30 min at room temperature. After washing cells were fixed with 8% formaldehyde for 3 min washed and heated for 60 min at 65 °C to inactivate endogenous APs. Nel-AP binding was detected by incubation with 5-bromo-4-choloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT) or with Cy3-conjugated donkey anti-AP antibody (1:200; Jackson Immuno Research Laboratories West Grove PA). In some experiments cell nuclei were counter-stained with 4′6′- diamidino-2-phenylindole (DAPI). Production and purification of anti-Nel polyclonal antibodies An adult New Zealand white rabbit was immunized with Nel-AP and antisera were collected according to the standard immunization protocol of the Hybridoma Core Facility of Lerner Research Institute. Antisera were first applied to an AP column to remove the antibodies against AP and the flow-through fraction was applied to a Nel-AP column for affinity purification of anti-Nel antibodies. In situ RNA hybridization histology and immunohistochemistry For in situ RNA hybridization two separate anti-sense probes for chicken Nel were used. Both probes gave similar results. One fragment extends from a PstI site at nucleotide 203 to a HindIII site at nucleotide 1028 and the other was from the HindIII site to a KpnI site at nucleotide 1854. In situ hybridization was performed as described previously (Nishida et al. 2002) using digoxigeninlabeled RNA probes. For immunohistochemistry sections were treated with rabbit anti-Nel antibody (0.1 μg/ml) and then with biotin-conjugated donkey anti-rabbit IgG (1:500; Jackson Immuno Research). Detection was performed using the standard ABC method (Vector Laboratories Burlingame CA). 30 μm cryosections of embryonic chicken tectum were used for both RNA in situ hybridization and immunohistochemistry. Immunoblot analyses Embryonic chicken tecta were homogenized in cold lysis buffer (50mMHEPES (pH 7.4) 50mMNaCl1% Triton X-100 5mMEDTA1× protease inhibitor mixture (Sigma-Aldrich St. Louis MO) 2 mM sodium vanadate 25mMsodium fluoride) and centrifuged for 10 min at 15000 ×g to remove insoluble material. The lysates were separated by SDS-PAGE (7.5% polyacrylamide gel) and the proteins were transferred onto Immobilon-P PVDF membrane (Millipore Billerica MA). The filters were blocked with 5% skim milk in PBS containing 0.05% Triton X-100 and treated with rabbit anti-Nel antibody (0.1 μg/ ml) and then with HRP-conjugated donkey anti-rabbit IgG (1:40000; Jackson Immuno Research Laboratories). As a control anti-β-actin antibody (1:10000; Sigma) and HRP-conjugated donkey anti-mouse IgG (1:40000; Jackson Immuno Research Laboratories) were used. The ECL plus system (Amersham Piscataway NJ) was used for detection. Tissue culture and axon outgrowth assays Retinal explantswere prepared as described previously (Nakamoto et al.1996). Briefly retinaewere dissected from E6 chick and flattened onto cellulose nitrate filters (0.45 μm pore size Sartorius). The filters with retinae were cut into 300 μmwide strips using a McIllwain tissue chopper. Olfactory bulbs were dissected out from E10 chick embryos and cut into small pieces. Retinal strips and olfactory bulb tissues were put on a Nucleopore membrane (Whatman Clifton NJ) that was first coated with 50 μg/ml of laminin and then with 25 μg/ml of Nel-AP AP or HI-Nel-AP. Explants were cultured in the retinal culture medium (DMEM:F12=1:1 10% fetal bovine serum 5% calf serum 1% penicillin/streptomycin 0.6% glucose). After 48 h axons were labeled by incubating the cultures in 33 μM carboxylfluorescein diacetate succinimidyl ester (Molecular Probes Eugene OR) for 5 min. Axon outgrowth for each explant was scored on a graded 0 to 3 scale in which 0 was no or very sparse outgrowth from a living explant and 3 was very robust outgrowth. Axon outgrowth assays of dissociated retinal ganglion cells were performed according to the protocol of Stepanek et al. (2001) except that we analyzed only neurofilament and Islet-1 double positive cells. 35 mm dishes (MatTek Corporation Ashland MA) were coated with 10 μg/ml of laminin for 1 h and then with 25 μg/ml of Nel-AP AP or HI-Nel-AP for 30 min. The dishes were rinsed and blocked with the retinal culture medium. Dissociated retinal cells were plated at 1×105 cells/dish and allowed to growovernight before fixation. After antigen retrieval the cells were incubated with rabbit anti-neurofilament antibody (1:200 Millipore Billerica MA) and anti-Islet 1 antibody (4D51:100 Developmental Studies Hybridoma Bank at the University of Iowa) and then with Alexa Fluor 594-conjugated donkey antirabbit IgG (2 μg/ml Invitrogen Paisley Scotland) and Alexa Fluor 488-conjugated donkey anti-mouse IgG (5 μg/ml Invitrogen). In each dish the lengths of at least 15 axons from double stained cells were measured. The assay was repeated three times. Growth cone collapse assays Growth cone collapse assays were performed essentially as described previously (Goshima et al. 1995). Retinal explants were prepared from E6 chick and cultured for 18 h on glass coverslips coated with 100 μg/ml of laminin in 24-well plates in the retinal culture medium. Then Nel-AP or control AP or HI-Nel-APwas added to the culture. The cells were incubated at 37 °C for up to 30 min fixed and stained with Alexa Fluor 488 Phalloidin (Invitrogen). In each experiment at least 30 growth cones were scored for each treatment as collapsed or not collapsed and three independent experiments were performed. In a separate set of experiments growth cone behavior was directly observed under phase contrast microscopy during treatment with AP and Nel-AP. Statistical analyses Statistical significance of the data was determined using Student's t-test.

Microscopic Technique
Phase Contrast Microscopy

Cell Type(s)
HEK-293