The loss of tamalin and Arf6 synergistically induces the upregulation of glutamate receptor expression. (a) The isolation of tubb:EGFP+ neurons from the spinal cord of Tg(tubb:gal4;uas:egfp), Tg(tubb:gal4;uas:egfp)/tamalin-/-, and Tg(tubb:gal4;uas:egfp)/tamalin-/-/arf6 MO-injected embryos by fluorescent activated cell sorting (FACS). (b) qRT-PCR analysis to compare mglur5a/5b expression level in the neurons isolated from the spinal cord of Tg(tubb:gal4;uas:egfp) and Tg(tubb:gal4;uas:egfp)/tamalin-/- embryos. (c) qRT-PCR analysis to compare mglur5a/5b expression levels in the spinal cord of adult Tg(tubb:gal4;uas:egfp) and Tg(tubb:gal4;uas:egfp)/tamalin-/- zebrafish (*** p < 0.001 **** p < 0.0001). (d) qRT-PCR analysis to compare neuronal mglur5a/5b expression level isolated from the spinal cord of Tg(tubb:gal4;uas:egfp)/tamalin-/- and Tg(tubb:gal4;uas:egfp)/tamalin-/-/arf6 MO-injected larvae (**** p < 0.0001). (e,f) Transverse sections of the spinal cord of tamalin KO/Arf6 MO-injected embryos not treated € and treated with MPEP (f). Immunolabeling with anti-Hu antibody (green) and TUNEL staining (red) has been performed to detect neuronal apoptotic cell death. (g) Quantification of the number of Tunel+ Hu+ cells. (** p = 0.01). (e,f): n = 10 sections from five zebrafish. Scale bars: (e,f), 10 μm. EGFP, enhanced green fluorescent protein; MO, morpholino oligonucleotides; Qrt-PCR, quantitative real time polymerase chain reaction; Arf6, ADP-ribosylation factor 6; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; and MPEP, 2-Methyl-6-(phenylethynyl)-pyridine.
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