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Fig. 1. Expression pattern of rab8WT, rab867L, and rab822N determined by fluorescence microscopy. Frozen sections of eyes from 6-week-old transgenic tadpoles were imaged by confocal microscopy. Selected sections were labeled with the nuclear stain Hoechst 33342 and either antirhodopsin mAb mabE followed by CY3-conjugated secondary antibody (A, B, and G) or TR-WGA (CâF, H, and I). In tri-color pictures, the GFP signal is represented in green, Hoechst stain in blue, and antibody or TR-WGA labeling in red. Grayscale images show only the GFP signal. For all three transgene constructs, green fluorescence was restricted to the major rod photoreceptors, whose OS bound mabE (A, B, and G). GFP fusion proteins were primarily observed in the IS of these rods. GFP-rab8WT did not cause retinal degeneration (A). However, both GFP-rab8Q67L (B) and GFP-rab8T22N (G) caused central retinal rod degeneration as evidenced by the lack of green and red fluorescence in the central retinas in these tadpoles. Retinal degeneration caused by GFP-rab8T22N was more extensive, sparing only a few peripheral rod photoreceptors. At higher magnification, the transgene products GFP-rab8WT and GFP-rab8Q67L appeared to be primarily associated with internal membranes of the IS (C and E), although fluorescence was also observed in the cytoplasm of the IS, and to a lesser extent in the OS. Membrane-associated GFP-rab8WT and GFP-rab8Q67L colocalized with TR-WGA label (D and F), indicating that these fusion proteins were associated with Golgi and post-Golgi membranes. A bright spot of green fluorescence was often observed just below the IS/OS junction near the predicted location of the PRC (white arrows, C and J). In contrast, GFP-rab8T22N appeared to be primarily cytoplasmic (H and I). Imaging of GFP-rab8T22N was more limited because the only surviving rods were the small arched rods confined to the peripheral retina. GFP-rab8WT was also observed along axially aligned structures in the IS (J, yellow arrowheads) and in the calycal processes that surround the OS (J, white arrowheads), but its relative abundance in calyces was low. Bars, A, B, and G, 250 μm; C, D, E, F, H, and I, 5 μm; J, 2.5 μm.
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Fig. 2. Expression of GFP-rab8T22N results in a dramatic increase in tubulo-vesicular membranes adjacent to the PRC. Rod photoreceptors throughout the retina were examined by electron microscopy, except in the case of GFP-rab8T22N-expressing retinas, in which only peripheral rods survived. (A) The IS/OS junction of a rod from a nontransgenic retina, showing the PRC region and CC (white arrows). (B and C) Similar regions of photoreceptors from retinas expressing high levels of GFP-rab8WT (B) and GFP-rab8Q67L (C). The few vesicles found adjacent to the PRC regions resembled those seen in nontransgenic rods. (D and E) Junctional regions of photoreceptors from retinas expressing GFP-rab8T22N. Abundant tubulo-vesicular membranes accumulated in the region beneath the CC (indicated by brackets). (F) Similar region of a photoreceptor from a retina expressing GFP-rab8T22N processed for immuno-electron microscopy and labeled with antirhodopsin mAb 11D5, followed by a secondary antibody conjugated to 10-nm gold particles. The tubulo-vesicular membranes and OS membranes are heavily labeled by antirhodopsin. Note that the adjacent IS plasma membrane (arrowhead) is relatively unlabeled. IS, inner segment; OS, outer segment; m, mitochondria. Bar, 500 nm
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Fig. 3. F1 offspring of transgenic frogs expressing either GFP-rab8T22N or GFP-rab8Q67L have an inherited retinal degeneration; relative toxic effects of the various fusion proteins. F1 offspring expressing the various transgene constructs were obtained from several primary transgenic adults. Eyes of individual F1 tadpoles were imaged at 6, 10, and 14 dpf with the use of a standard fluorescence microscope (left panels). The animals were sacrificed at d 14, and frozen sections of the eyes were stained with TR-WGA (red) and Hoechst nuclear stain (blue). Stained sections were imaged by confocal microscopy with the use of identical laser amplitude and detector gain settings for each image (center panels), avoiding saturation of the GFP signal (green). The GFP fluorescence intensities are therefore comparable between the various images. To the right of the confocal image, the GFP fluorescence is represented as a three-dimensional profile, where the height of the peaks corresponds to the signal intensity in arbitrary fluorescence units. The GFP fluorescence intensity is also represented by a color scale (blue, low intensity; red, high intensity). Retinas of offspring of founder A, which expressed relatively high levels of the GFP-rab8WT fusion protein (A), had little or no degeneration, similar to nontransgenic control animals (E). Significant depletion of rods was seen in the F1 offspring of founder B, which expressed high levels of the GFP-rab8Q67L fusion protein (B), although some central rods survived. The surviving (younger) peripheral rods expressed the fusion protein at levels similar to or lower than the GFP-rab8WT–expressing retinas (A). F1 offspring of founder C expressed GFP-rab8Q67L fusion protein at a lower level (C) and did not have retinal degeneration, demonstrating that lower levels of this fusion protein are less toxic. F1 offspring of founder E expressed GFP-rab8T22N and had severe retinal degeneration with only peripheral rods remaining, despite expressing very low relative levels of fusion protein that were almost undetectable at this level of sensitivity (D). Numerous closely apposed cones were still present, and the layers of the inner retina were of normal thickness. Fusion protein could be easily detected with more sensitive microscope settings (see Figure 1). Interestingly, it was possible to diagnose retinal degeneration in these tadpole eyes by observing the decrease in fluorescence with time (B and D, left panels) in contrast to eyes without degeneration where fluorescence levels increased or remained constant (A and C, left panels). Bar, 100 μm.
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Fig. 4. Expression of GFP-rab8WT and GFP-rab8Q67L downregulates the expression of endogenous rab8 in transgenic tadpoles. Equivalents of 1/8 of the retinas isolated from stage 51 tadpoles were subjected to SDS-PAGE and immunoblotted with anti-rab8 antibody (left panel) and anti-GFP antibody (right panel). In addition to a 23-kDa protein recognized by anti-rab8 antibody, transgenic retinas contain a 50-kDa protein that is also recognized by anti-GFP. The expression of GFP-rab8WT fusion protein under the control of theX. laevis rhodopsin promoter does not exceed that of endogenous rab8 as measured in the nontransgenic retina. However, the amount of the endogenous rab8 in these tadpoles is <50% of the nontransgenic animal. Therefore, the total content of rab8 (endogenous plus the fusion protein) is â¼1.5- to 2-fold higher than in the nontransgenic animals. In GFP-rab8Q67L tadpoles endogenous rab8 is also reduced to similar levels (<50% of control) as in GFP-rab8WT animals, and the fusion protein is in a threefold excess over the endogenous rab8. Because of the progressive retinal degeneration in GFP-rab8Q67L animals, the contribution of rod photoreceptors is lower than that in the retinas expressing GFP-rab8WT protein.
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