Figure 1.
Western blot analysis of fractalkine protein expression in mouse neural retina at P5 until P60 being representative of five independent experiments.
Blots were re-probed with anti-GAPDH antibody. (A). Fractalkine membrane integrated (100-kDa) and soluble (85-kDa) protein forms are present in both control (wt) and rd10 retina lysates during development at P5. (B). Accumulation of presumably intracellular (95-kDa) protein form is seen in both wt (B) and rd10 (C) developing neural retina at P10. Remarkably, no cleaved (85-kDa) protein form was associated with membrane integrated (100-kDa) form in both rd10 and wt retina lysates at P10. Both wt and rd10 retinas show lower to beneath detection level of 100-kDa form and higher level of 95-kDa form at P14 through P60 (D-G). Increased level of 85-kDa form is clearly detectable in rd10 retinas at P45 and P60 (G). Histogram showing relative percent levels of each of the three CX3CL1 protein forms in wt retina (H) and degenerating rd10 retina (I). Data are expressed as percent of densitometric arbitrary units. Values are mean ± SEM, (n = 5). In rd10 retinas, positive correlation between the relative levels of a ∼100-kDa and corresponding 85-kDa bands was found at P45 and P60 (Pearson product-moment correlation coefficient r = 0.683, n = 12, p = 0.014 in RD10 retina at P45 and r = 0.882, n = 9, p = 0.0017 at P60) as well as for the relative levels of a 100-kDa and 95-kDa bands (r = 0.928, n = 11, p<0.0001 at P14, r = 0.861, n = 9, p = 0.0029 at P22, r = 0.892, n = 9, p = 0.0012 at P30, r = 0.966, n = 12, p<0.0001 at P45, and r = 0.628, n = 12, p = 0.0288 at P60 in rd10 retina). The degree of association between the levels of 95 kDa and 85 kDa protein band was r = 0.712, n = 12, p = 0.0094 at P45 and r = 0.907, n = 9, p = 0.0007 at P60. It was not, however, possible, to make similar analysis for the wild type retina samples, as the level of the ∼95-kDa protein form was the highest and the levels of cleaved and full-length forms were far below the limit of detection.
Figure 2.
Comparison of total CX3CL1 protein and mRNA expression levels in rd10 and wt retinas.
(A). Western blot analyses of total CX3CL1 protein levels in rd10 retinas compared to wt retinas determined in five independent experiments. Total protein levels in rd10 retinas are significantly decreased at P10 and remain low at all time points examined. Results (mean ± SEM, n = 5) are expressed as fold change over control in densitometric arbitrary units. *P<0.0001 versus control. (B). Expression of CX3CL1 mRNA in wt and rd10 retinas at P5, P10, P14, P22, P30, P45 and P60, determined by real-time PCR normalized to β-microglobulin levels. Comparable CX3CL1 mRNA expression level in control wt and rd10 retinas at P5, P10 and lower levels at P14 and later time-points in rd10 retinas compare with wt retina. Values are mean ± SEM (n = 5–7; *p<0.05; ns: not significant).
Figure 3.
Localization of CX3CL1 mRNA expressing cells in the retina and brain of wt and rd10 mice.
(A-D). Light micrographs of radial cryosections through mouse central retina probed with a DIG-labeled anti-sense CX3CL1 riboprobe. In both, wt (A) and rd10 (B) mouse retinas, CX3CL1 mRNA signal is present exclusively in the neurons of the ganglion cell layer (GCL) and the inner- and outermost areas of the inner nuclear layer (INL). The outer nuclear (photoreceptor) layer is devoid of CX3CL1 signal at the time points examined (P22 and P30). Enlargements showing juxtanuclear localization of mRNA in the cells of inner nuclear (C) and ganglion (D) cell layers in wt mouse retina. (E-G). Distribution of CX3CL1 mRNA expressing neurons in wt mouse hippocampus. Light micrographs of a paraffin section used as positive control. (E). Numerous CX3CL1 mRNA-positive neurons are located in all hippocampal subfields of the pyramidal (PCL) and in the dentate granule cell layers (GCL). (F). Higher magnification of the dentate gyrus tips and of hilus. (G). Enlargement showing high levels of CX3CL1 mRNA expression within the grey matter of the cerebral cortex. PCL, pyramidal cell layer; GCL, layer of ganglion cells in dentate granule cell layer; H, hilus; T, dentate gyrus tips. Cells expressing CX3CL1 mRNA are visualized as of brown product accumulations in cryosections and red-brown in paraffin sections. The different color of nuclei staining (blue in cryosections and purple in paraffin sections) is due to a hemalaun counterstaining. Scale bars represent: 50 µm (A, B), 35 µm (C, D), 250 µm (E), 125 µm (F) and 25 µm (G).
Figure 4.
Regionally restricted CX3CL1 expression in the retina of adult CX3CL1 cherry: CX3CR1gfp mouse (A-E).
Fluorescent light microscopy analysis of a vertical retina cryosection (A) showing both CX3CL1/Cherry (detected in rhodamine channel, red) (B) and CX3CR1/GFP (detected in FITC channel, green) (C) reporters expression. CX3CR1/GFP signal is assigned to microglial cells with cell bodies and dendritic ramifications in three sublayers (outer plexiform layer, OPL, inner plexiform layer, IPL and ganglion cells layer, GCL). CX3CL1/Cherry fluorescent cells are found exclusively in ganglion cell and inner nuclear layer. No fluorescent cells were observed in outer nuclear layer (ONL). Enlargements show strong Cherry signal in a presumable amacrine cell body in inner nuclear (D, arrow points to dendritic base) and ganglion cell (E) layers, indicating high level of the CX3CL1 chemokine promotor activity in neurons. Cryosections are counterstained with DAPI nucleic acid stain (blue). Distribution of the Cherry reporter positive cells in the retina of CX3CL1cherry: CX3CR1gfp mice coincides mostly with results obtained in in situ hybridization studies. Of note, membrane/lipid inner and outer photoreceptor segment layer exhibit nonspecific fluorescence. Scale bars represent 50 µm (A, B and C), and 10 µm (D, E).