Abstract
purpose. To determine the relationship between the presence of kinesin-2 and photoreceptor cell viability and opsin transport, by generating RHO-Cre transgenic mice and breeding them to mice with a floxed kinesin-2 motor gene.
methods. Different lines of RHO-Cre transgenic mice were generated and characterized by transgene expression, histology, and electrophysiology. Mice from one line, showing uniform transgene expression, were crossed with Kif3a flox /Kif3a flox mice. The time courses of photoreceptor Cre expression, KIF3A loss, ectopic opsin accumulation, and photoreceptor cell death were determined by Western blot analysis and microscopy.
results. One of the RHO-Cre lines effected synchronous expression of Cre and thus uniform excision of Kif3a flox in rod photoreceptors across the retina. After the neonatal production of CRE and the initiation of KIF3A loss, ectopic accumulation of opsin was detected by postnatal day (P)7, and ensuing photoreceptor cell death was evident after P10 and almost complete by P28. Of importance, the photoreceptor cilium formed normally, and the disc membranes of the nascent outer segment remained normal until P10.
conclusions. The RHO-Cre-8 mice provide an improved tool for studying gene ablation in rod photoreceptor cells. Regarding kinesin-2 function in photoreceptor cells, the relatively precise timing of events after CRE excision of Kif3a flox allows us to conclude that ectopic opsin is a primary cellular lesion of KIF3A loss, consistent with the hypothesis that opsin is a cargo of kinesin-2. Moreover, it demonstrates that KIF3A loss results in very rapid photoreceptor cell degeneration.
Many genes that are important in ocular function and disease have been studied with traditional gene-targeting strategies in the mouse. However, some genes that function in the eye are also critical for normal development and homeostasis of the animal and are thus not amenable to simple knockout techniques.
1 2 The development of conditional knockout strategies has enabled the study of these other genes. The use of CRE and other recombinases for conditional targeting permits the controlled removal or activation of genes in specific tissues and at specific times of development.
3 4
In a previous study, we used
Cre-loxP mutagenesis to test for motor transport by kinesin-2 in photoreceptor cells.
5 Vertebrate photoreceptor cells include two distal compartments: an inner segment, which contains much of the cellular machinery, and an outer segment, which is a specialized sensory cilium dedicated to phototransduction. The outer segment is linked to the inner segment by a connecting cilium, which is analogous to the transition zone of a primary cilium.
6 Trafficking between the inner and outer segments occurs along the connecting cilium and the axoneme of the outer segment and is essential for the function and viability of the cells. Large amounts of phototransductive proteins, including the visual receptor, opsin, are transported in an anterograde direction as part of the continuous renewal of the outer segment.
7 Moreover, at least three proteins, arrestin, transducin, and recoverin, redistribute between the inner and outer segments according to ambient lighting.
8 9 10 11 12 13 14 Kinesin-2 is a likely candidate to provide motor transport along the connecting cilium and axoneme of photoreceptor cells, based on its role in the movement of proteins along cilia and flagella (“intraflagellar transport”) in a variety of organisms, from single cell flagellates to mammals.
15 16 Moreover, the motor subunits of kinesin-2, KIF3A, and KIF3B, have been detected in the photoreceptor connecting cilium.
17 18 19 20
In the previous study, mice were generated in which a region of the
Kif3a gene was flanked by loxP sites and thus could be excised in the presence of CRE. CRE was introduced into the photoreceptor cells by way of an
IRBP-Cre transgene, whose expression was restricted primarily to the photoreceptor cells.
5 With this strategy, excision of the
Kif3a gene occurred in photoreceptor cells, beginning after the second postnatal week. The consequential removal of KIF3A from the photoreceptor cells not only perturbed the flow of protein to the outer segment, but also killed some of the photoreceptor cells. Although this study demonstrated a requirement for kinesin-2 in photoreceptor cell protein transport and viability, gene excision was incomplete and asynchronous across each retina, and its extent varied among different animals, thus limiting the usefulness of this approach. In particular, these animals were not suitable for any type of biochemical study.
In the present study, we first set out to establish a more robust expression of Cre—one that would effect widespread and synchronous recombination across the retina and thus would be more useful for the study of Kif3a and other genes in photoreceptor cells. We settled on a line of RHO-Cre transgenic mice that fulfills these criteria and have characterized the expression and effects of this transgene. We have also used this line to study further the requirement of KIF3A in photoreceptor cells, and especially the time course of the change in gene expression in relation to the ensuing effects on the photoreceptor cells. Of note, we found that an abnormal accumulation of opsin is the primary cellular defect, occurring when all other aspects of cellular organization appear normal.
Each retina was homogenized in 100 μL of PBS buffer with protease inhibitors (Sigma-Aldrich, St. Louis, MO) and 25 μL of Laemmli sample buffer. Equal proportions of the retinal homogenate were loaded on a 10% highly porous sodium dodecyl sulfate polyacrylamide gel for electrophoresis (SDS-PAGE). The running gel was transblotted on to nitrocellulose membranes (Immobilon-P; Millipore, Bedford, MA) and immunolabeled with KIF3A antibodies (BD Transduction Laboratories, Lexington, KY) and alkaline phosphatase-conjugated secondary antibody (Sigma-Aldrich) for staining with nitro blue tetrazolium chloride/5-bromo-4-chloro-3′-inodylphosphate p-toluidine salt (NBT/BCIP; Roche). Quantification of the KIF3A labeling was performed with ImageJ software (available by ftp at zippy.nimh.nih.gov/ or at http://rsb.info.nih.gov/nih-imageJ; developed by Wayne Rasband, National Institutes of Health, Bethesda, MD).
After ERG analyses, mice were deeply anesthetized and killed by cardiac perfusion with 4% paraformaldehyde in phosphate-buffered saline (PBS). The eyes were isolated and postfixed in 4% paraformaldehyde in PBS overnight. After they were rinsed with PBS, eye cups were made, and one eye cup was processed for plastic sectioning. The eye cup for plastic sectioning was first dehydrated by placing the tissue for 1 hour in each of 70%, 95%, and 100% ethanol solutions. The dehydrated tissue was then infiltrated overnight in infiltration solution (JB-4 Plus; Polysciences Inc., Warrington, PA) and embedded in resin (JB-4 Plus). Histologic sections were cut at 3-μm thickness and stained with Richardson’s stain for 30 seconds. The slides were washed under running water for 2 minutes and mounted (Permount; Fisher Scientific, Pittsburgh, PA. Bright-field digital images were captured (model TE300; Nikon, Tokyo, Japan) with a microscope equipped with a digital camera (Spot RT; Diagnostic Instruments, Sterling Heights, MI).
For use in X-gal staining or immunofluorescence analyses, the other eye cup was infiltrated in 30% sucrose, frozen in OCT freezing medium, and cryosectioned at 10 μm. For immunostaining, retinal sections were blocked in PBS containing 1% or 2% normal goat serum, 1% bovine serum albumin, and 0.1% or 0.5% Triton X-100 for 1 hour and then incubated overnight with primary antibodies at 4°C. After they were rinsed with PBS, the sections were treated with fluorochrome-conjugated secondary antibodies for 1 hour (sometimes including 4′,6′-diamino-2-phenylindole (DAPI), diluted 1:10,000), washed in PBS, and mounted (Fluoromount-G; Southern Biotechnology Associates, Birmingham, AL).
26 The primary antibodies used were monoclonal anti-Cre-recombinase (BabCO-CRP, Inc., Vienna, VA) and anti-red/-green cone opsin (JH492).
27 Cy2- and Cy3-conjugated secondary antibodies were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). Alexa 468– and Alexa 584–conjugated secondary antibodies were from Invitrogen-Molecular Probes (Eugene, OR). Control sections were treated with preimmune anti-C′-Rp1 or without primary antibodies. Stained sections were viewed with a confocal microscope (model LSM510; Carl Zeiss Meditec, Inc., Dublin, CA), and images were processed with the accompanying software (Meta 510; Carl Zeiss Meditec, Inc.).
Semithin sections were obtained from eye cups that were fixed in 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3), processed for embedding in resin (LR White; EMS, Fort Washington, PA), and immunolabeled using the PAP (peroxidase anti-peroxidase) technique. Sections (0.7 μm) were washed in PBS and incubated overnight at room temperature with opsin pAb 01 (generated against bovine rod opsin; 1:500) in PBS plus 1% BSA and 2% goat serum, 2 hours at 37°C with a goat anti-rabbit secondary antibody (1:20; Jackson ImmunoResearch), and 2 hours at 37°C with a rabbit-PAP complex (1:10; Jackson ImmunoResearch). The sections were washed in 0.1 M Tris-HCl (pH 7.6) for 10 minutes, and the peroxidase was detected by incubating the sections in 3,3′-diaminobenzidine (DAB) for 20 to 30 minutes. The sections were counterstained with toluidine blue, and then dehydrated and mounted with a rapid mounting medium (Entellan; EMS). For quantification of photoreceptor cell nuclei, three dorsoventral semithin sections from each retina were used. Photoreceptor nuclei were counted in the areas, located 500 μm each side of the optic nerve head.
We have generated mice that express Cre recombinase in rod photoreceptor cells under control of the RHO promoter. In one of the lines, RHO-Cre-8, the expression of Cre was synchronous and robust throughout the retina. When used to excise a Kif3a flox gene, we were able to define the time course of events, from the neonatal expression of Cre, to the decline in photoreceptor KIF3A, the mislocalization of opsin, and, finally, the complete loss of the photoreceptor cells in the ensuing weeks.
Several photoreceptor-specific,
Cre-expressing transgenic mouse lines have now been reported. After the study of the
Kif3a flox ;
IRBP-Cre mice,
5 cone opsin
Cre lines were generated,
29 30 and a mouse rod opsin
Cre line was reported recently.
31 However, these new lines have yet to be tested in a genetic study of a photoreceptor gene. To study the
Otx2 homeobox gene in photoreceptor cell fate during development, Nishida et al.
32 produced a
Crx-Cre mouse line, in which CRE is present embryonically. With our
RHO-Cre-8 line, retinal degeneration was initiated between 4 and 7 weeks of age. It is likely that this loss of photoreceptor cells is due to overexpression of the CRE protein, as mice that express a lower level of CRE in rod photoreceptors do not demonstrate degeneration up to 8 months of age.
31 It is unclear whether the observed toxicity is due to a specific effect of the CRE recombinase or to general protein overexpression. Because overexpression of other proteins in photoreceptor cells has been observed to lead to cell death, simple overexpression of protein in these sensitive cells appears to be the more likely cause of degeneration.
33 It is noteworthy that the level of retinal CRE continued to increase up to 6 weeks of age in the
RHO-Cre-8 mice
(Fig. 3B) . CRE-mediated genomic toxicity in cultured cells has been reported, although similar effects of CRE expression in transgenic mice have not been described.
34 35
CRE was detected in the
RHO-Cre-8 mice just after birth. The finding that no photoreceptor cell death was observed until 7 weeks of age demonstrates that rod cells tolerate transient overexpression of
Cre. Thus, the
RHO-Cre-8 mice will be useful for conditional gene targeting experiments during the first postnatal month. This finding also suggests that mice that express
Cre for a limited time might be ideal for conditional gene targeting in photoreceptor cells. Limiting
Cre expression can be effected by incorporating loxP sites into the transgene, so that it self-excises.
35 36
A relatively high level of expression of
Cre is likely to be needed to achieve the synchronous excision observed in the present study. The effect of
RHO-Cre-8 on
Kif3a flox contrasts with that of
IRBP-Cre, which had variable effects, spatially and temporally, across the retina and among different animals.
5 Although the study with
Kif3a flox ;
IRBP-Cre mice
5 showed that the knockout of
Kif3a resulted in opsin mislocalization in some cells and the death of some photoreceptor cells, the extent of this effect was not determined. It was not clear whether a given normal looking photoreceptor cell in a
Kif3a flox ;
IRBP-Cre retina was unaffected because there had been no gene excision or because the gene excision had no effect in that cell. From the present study, with gene excision and opsin redistribution evident in every rod photoreceptor cell and occurring during well-defined and sequential intervals, we can conclude that KIF3A and the delivery of opsin to the outer segment are inextricably linked.
The clearance of opsin from the inner segment was found to be very sensitive to the presence of KIF3A. A decline in KIF3A, rather than its complete loss, was sufficient to cause the accumulation of opsin in the inner segment. Perhaps, however, the relatively abrupt change in concentration of KIF3A contributed to the defect, as well as the lower concentration itself.
Kif3a heterozygotes, which have retinal KIF3A levels that are only 50% of wild-type levels, do not undergo retinal degeneration.
37 Yet deleterious effects are evident in
Kif3a flox /
Kif3a flox ;
RHO-Cre-8 mice after the decrease in photoreceptor KIF3A to 40% of wild-type levels that occurs in the first postnatal week.
Some 3 days after the start of opsin accumulation within the inner segment, the photoreceptor connecting cilium and the disc membranes of the nascent outer segment still appeared unperturbed
(Fig. 5H) . At no stage, even in the photoreceptors remaining in advanced degenerate retinas, were ultrastructural abnormalities evident in the photoreceptor connecting cilium. These observations, in addition to the relatively precise timing of the events, indicate that KIF3A loss disrupts motor traffic without immediately affecting the supporting infrastructure. They thus support the hypothesis that kinesin-2 transports opsin, rather than having a less direct role in opsin delivery to the outer segment, such as by maintenance of the structural integrity of the connecting cilium. They also support the notion that the critical element leading to apoptosis is the abnormal accumulation of opsin outside of the outer segment, rather than any structural perturbation of the axoneme or outer segment.
The presence of opsin throughout the photoreceptor cell has been reported during early photoreceptor development
38 39 40 and before cell death in some other inherited retinal degenerations that appear to be unrelated to opsin transport (e.g., those in the RCS rat and
rd1 and
rds mice
41 ). The accumulation of opsin outside the outer segments of
Kif3a flox /
Kif3a flox ;
RHO-Cre-8 mouse photoreceptors, as observed herein, differs from the first case, in that it occurred after this early developmental stage, when the opsin distribution was fully polarized in the control photoreceptor cells
(Figs. 5A 5C) . It may differ from both cases, in that the initial accumulation is primarily
within the inner segments (at P7,
Fig. 5F ). The ectopic opsin distribution during development and in other photoreceptor degenerations has been demonstrated only in the plasma membrane.
38 39 40 41 42 In
Kif3a flox /
Kif3a flox ;
RHO-Cre-8 mouse photoreceptors, significant ectopic distribution in the plasma membrane was not evident until a slightly later stage (at P10,
Fig. 5H ). Opsin in the plasma membrane of the inner segment, nuclear region, and synapse may indicate leakage from the outer segment in ailing cells. By contrast, an accumulation of opsin within the inner segment is consistent with a backlog of trafficking to the outer segment. An accumulation along the anabolic pathway (from a defect in targeting rather than retention) may be a more important trigger for cell death and may be responsible for the surprisingly rapid degeneration that follows the loss of kinesin-2.
In conclusion, the RHO-Cre-8 mice are useful for studying gene ablation in rod photoreceptor cells and clearly provide a new and improved tool for such studies. The high and widespread expression of Cre results in relatively synchronous excision that is necessary for many experiments, especially biochemical ones. In the present study, it has enabled us to determine the time course of events ensuing from Kif3a excision and to provide a clearer depiction of the role of kinesin-2 in opsin transport and photoreceptor cell viability.
Contributed equally to the work and therefore should be considered equivalent authors.
Present affiliation: Department of Ophthalmology and Visual Sciences, Washington University, St. Louis, Missouri.
Supported in part by Grants EY12910 (EAP) and EY13408 from the National Eye Institute (DSW, LSBG) and by funding from Research to Prevent Blindness, The Foundation Fighting Blindness, the Rosanne Silbermann Foundation, and the Mackall Foundation Trust.
Submitted for publication January 11, 2006; revised May 26, June 3, 2006; accepted September 12, 2006.
Disclosure:
D. Jimeno, None;
L. Feiner, None;
C. Lillo, None;
K. Teofilo, None;
L.S.B. Goldstein, None;
E.A. Pierce, None;
D.S. Williams, None
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “
advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Each of the following is a corresponding author: Eric A. Pierce, F. M. Kirby Center for Molecular Ophthalmology, University of Pennsylvania School of Medicine, 305 Stellar-Chance Labs, 422 Curie Boulevard, Philadelphia, PA 19104;
[email protected]. David S. Williams, Department of Pharmacology, UCSD School of Medicine, Mail code 0912, 9500 Gilman Drive, La Jolla, CA 92093-0912;
[email protected].
The authors thank Helen Khalafbeigi, Elizabeth Roberts, Erin Legacki, and Maithili Navaratnarajah for help with parts of the project and Martin Friedlander’s laboratory (TSRI) for rd1 mouse eyes.
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