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Fig. 1. Expression of tctp in the
Xenopus
neural retina. (A) Coronal section of stage 43 retina probed with an anti-Tctp antibody and counterstained with DAPI. Arrowheads indicate the optic fibre layer (OFL). The boxed area is enlarged beneath. The dashed contour delineates the outer plexiform layer. (B) Stage 32 eye explants grown in vitro for 24 h were stained with anti-Tctp antibody (left, phase contrast image; right, Tctp antibody staining). Tctp is detected in the axon shaft, central domain and filopodia. (C) In situ hybridisation (ISH) detection of tctp mRNA expression on coronal sections of stage 43 retinas. Arrowheads indicate the OFL. The boxed area is enlarged in the middle panel. (D,E) Quantitative ISH detection of tctp mRNA expression in the RGC axonal and growth cone compartments was performed using stage 32 eye explants grown in vitro for 24 h. Mean±s.e.m.; ***P<0.0001, one-way ANOVA with Bonferroni correction. (F) RACE amplifications of tctp mRNAs using retinal RNA extracts. FP, forward primer; NUP, nested universal primer; RP, reverse primer; UP, universal primer. (G) Organisation of the tctp gene in X. laevis. cds, coding region; poly(A) signal, polyadenylation signal. (H) Schematic of the laser-capture microdissection procedure used to collect RGC axonal extracts. (I) RACE amplifications of tctp mRNAs using laser-captured axonal extracts. (J) Purity assessment of laser-captured material by RT-PCR. –RT, RNA samples not reverse transcribed. (K) RT-qPCR experimental design. (L,M) Axonal and whole-eye content of tctp mRNAs were analysed by RT-qPCR and normalised to actb expression. In L, data are plotted as ‘tctp-S+tctp-L’ to ‘tctp-L’ expression ratios (*P=0.0175, one-way ANOVA), whereas in M the quantification cycle (Cq) difference relative to actb is shown. Scale bars: 50 μm in A,C; 5 μm in B,D. CMZ, ciliary marginal zone; GCL, ganglion cell layer; IPL/OPL, inner/outer plexiform layer; ONH, optic nerve head; PR, photoreceptor layer. |
