Controlled Rod Cell Ablation in Transgenic Xenopus laevis

Abstract

purpose. Because of their high cone/rod ratio, Xenopus laevis may be a useful system for examining rod-cone interactions during retinal degeneration and mechanisms that underlie secondary cone degeneration. The authors developed an inducible model of retinitis pigmentosa (RP) in X. laevis to investigate these issues.

methods. The authors generated transgenic X. laevis that express a modified caspase-9 (iCasp9) under the control of the X. laevis rod opsin promoter. iCasp9 is activated by the compound AP20187, resulting in an apoptotic cascade. Confocal microscopy, Western blot analysis, and electroretinography (ERG) were used to determine the effects of AP20187 on transgenic retinas.

results. AP20187 induced rod cell apoptosis in transgenic tadpoles and postmetamorphic frogs. Longitudinal results indicate rod cell death led to cone cell dysfunction within 3 months; however, cone function was reinstated after 6 months. Returning cone function may be associated with increased numbers of morphologically normal cone cells and thickening of the inner nuclear layer.

conclusions. These studies indicate that X. laevis may be a useful system for examining cone dysfunction associated with rod death in RP and longer term regeneration of cone responses. This inducible model of RP is unique in that rod death proceeds through a well-understood mechanism, rod death can be carefully controlled to occur at any stage of development, and the stimulus for rod death can be removed at any time.

The response of the vertebrate retina to neuronal loss varies from complete regeneration to severe secondary degeneration, depending on the species and the type of damage. Known mechanisms of regeneration include proliferation of cells in the ciliary marginal zone (CMZ), differentiation of inner nuclear layer (INL) and outer nuclear layer (ONL) progenitors, and transdifferentiation of Müller glia and retinal pigment epithelium (RPE), ^{1 2 3} complementing evidence as early as 1880 ⁴ of retinal regeneration in fish and urodele amphibians (newts). Larval anuran amphibians (frog tadpoles) can regenerate retinal neurons from the CMZ, from RPE transdifferentiation, ^{1 5} and from proliferating INL cells ⁶ after retinal damage. Adult urodele amphibians retain these capacities, whereas anuran regeneration becomes dependent on the CMZ ⁵ and RPE. ⁷ Mammalian retinas have limited regenerative capacity, ⁸ most of which is lost early in life, rendering patients with retinal degeneration (RD) irreversibly vision deprived. The reasons for these differences are the subjects of ongoing research. 

Rod death is an intriguing subset of RD because, in mammals, it is not only irreversible, it is followed by cone death that causes blindness associated with the disorder retinitis pigmentosa (RP). Secondary cone degeneration is an area of intensive research, ^{9 10 11 12 13 14 15 16 17 18 19 20 21 22} but modeling this process is difficult because common laboratory rodents have rod-dominated retinas. Conversely, zebrafish have a cone-dense retina, but their regenerative ability masks secondary cone death. ²³ Xenopus laevis have equal numbers of rods and cones. Some evidence suggests that in X. laevis, cone death occurs in the absence of rods, ²⁴ but the recently reported capacity for postmetamorphic regeneration suggests otherwise. ⁷ Understanding the responses of the X. laevis retina to rod death may improve our understanding of the mechanistic differences that favor regeneration in lower vertebrates compared with progressive RD in higher vertebrates. 

To induce rod ablation through a mechanism consistent with RP, ²⁵ we modified a system described by Spencer et al. ^{26 27} to induce rod apoptosis by activation of procaspase 9, an initiator caspase that cleaves like and effector caspases. Dimerization initiates cleavage of the inactive form at D315, causing separation into large and small subunits. The active subunits set into motion an apoptotic cascade, leading to cleavage of procaspases 3, 7, and 6 (for reviews, see Strasser et al., ²⁸ Kam and Ferch, ²⁹ Hengartner, ³⁰ and Yuan and Horvitz ³¹ ). In this study, we used a transgene encoding a modified, inducible procaspase 9 (iCasp9) incorporating binding domains specific for the compound AP20187 (Ariad Pharmaceuticals, Cambridge, MA), ³² which induces dimerization and autoactivation. We expressed iCasp9 in transgenic X. laevis rods under control of the rod opsin promoter and investigated the effects of AP20187 on the retinas of transgenic and nontransgenic animals using histologic, Western blot, and electrophysiological techniques. 

Methods

Molecular Biology

The iCasp9 cDNA from pSH1/S-Fvls-p30casp9-E,²⁷ provided by David Spencer, was subcloned into pXOP0.8-eGFP-N1, and the plasmid (XOP0.8-iCasp9-N1) was prepared for transgenesis, as previously described. ³³  

Generation, Rearing, and AP20187 Treatment of Transgenic X. laevis

We generated transgenic X. laevis as previously detailed. ^{34 35} We coinjected XOP0.8-iCasp9-N1 and XOP0.8-eGFP-N1 plasmids (2:1 ratio) and identified transgenic animals by screening for enhanced green fluorescent protein (GFP) expression 5 days postfertilization (dpf). Tadpoles and frogs were reared as described. ³³ AP20187 (Ariad Pharmaceuticals, Cambridge, MA) was added to tadpole medium at a final concentration of 10 nM. Postmetamorphic frogs received subcutaneous injections of 10 μL of 1 mM AP20187/g body weight. Procedures adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 

Western Blot Analysis

Solubilized eye extracts and Western blots were prepared and imaged as described. ³³ Antibody dilutions were 1:1000 for anti-HA (Cedarlane Laboratories Limited, Hornby, ON, Canada), 1:10 for B630N (Paul Hargrave), and 1:10,000 for IRDye800-CW conjugated anti-mouse secondary antibody (Rockland, Gilbertsville, PA). 

Microscopy

Eyes were fixed and processed for microscopy as previously described. ³³ In addition to the antibodies listed, we used anti-calbindin (EMD, San Diego, CA) diluted 1:250 and Cy3- or Cy5-conjugated anti-mouse and anti-rabbit secondary antibodies (Jackson ImmunoResearch, West Grove, PA) diluted 1:750. TUNEL staining was performed (ApoTag Red Kit; Millipore, Billerica, MA), and sections were imaged with laser scanning confocal microscope (LSM510; Zeiss, Thornwood, NY). We counted cells manually and measured distances using Zeiss software. Quantification was performed on consecutive sections containing the optic nerve, excluding the most peripheral region. TUNEL-positive nuclei were tallied for entire sections, whereas cone counts were averaged from five consecutive 150-μm samples of each section, and measurements of INL width were averaged from 10 measurements per section. 

Electroretinography

Postmetamorphic animals were dark adapted for 12 hours and anesthetized in 0.05% tricaine. Initial experiments used a Ganzfeld stimulator and recording unit (UTAS-E 2000 LKC; LKC Technologies, Gaithersburg, MD). The corneal electrode was a ringer-filled glass micropipette, and needle electrodes were used for reference and ground. Electrodes were connected to a model 1800 AC amplifier and headstage (AM Systems, Sequim, WA). Stimuli included scotopic flashes at five intensities, 0-dB flicker flashes at five frequencies, and 0-dB flashes superimposed on a lit background (photopic stimuli). Four or five responses were averaged for intensity series, and 10 responses were averaged for photopic waveforms. Later experiments (see 1 2 3 4 5 6 Fig. 7 ) were similarly conducted using a stimulator (Colordome; Diagnosys LLC, Lowell, MA) and recording unit (Espion; Diagnosys LLC) and a gold wire corneal electrode. Equations used to fit a-wave and intensity data were the Shady et al. ³⁶ modification of the Lamb and Pugh ³⁷ equation  

\[R(I,\ t){=}{\{}1{-}\mathrm{exp}{[}{-}0.5IS(t{-}t_{\mathrm{d}})^{2}{]}{\}}R_{\mathrm{mp3}}\]

and the Naka-Rushton ³⁸ equation  

\[V(I){=}V_{\mathrm{max}}\mathrm{{[}}I^{n}/(I^{n}{+}K^{n}){]}.\]

 

Results

AP20187-Induced Rod Apoptosis in Primary Transgenic Tadpoles

Fan et al. ²⁷ developed several death switches based on caspases. For these experiments, we selected a caspase 9-based switch because apoptosis was more dramatic in preliminary cell transfection and transgenesis experiments (not shown). We generated double-transgenic X. laevis expressing iCasp9 and GFP under the control of the X. laevis rod opsin promoter. ³⁹ This dual transgene method allowed noninvasive identification of iCasp9-positive animals because of the high frequency of transgene cointegration. ^{34 40} Given that the iCasp9 construct incorporates an HA epitope tag degraded on procaspase cleavage, visualization and quantification of the inactive procaspase form is possible. At 14 dpf, we separated 14 GFP-positive transgenic animals into treatment and control groups (n = 7 for each group). The treatment group received 10 nM AP20187 for 48 hours, after which all tadpoles were killed. One eye from each animal was solubilized for Western blot analysis, and the contralateral eye was fixed for immunohistochemistry. 

Western blot analysis of the untreated GFP-positive transgenic tadpoles showed that detectable levels of intact iCasp9 were expressed in 6 of 7 GFP-positive tadpoles, though expression levels varied (Fig. 1A) . Therefore, GFP expression was a reliable indicator of iCasp9 expression. Immunofluorescence microscopy of contralateral eyes confirmed rod-specific expression of intact iCasp9 and GFP, coexpressed in most cells, and normal rod morphology (Fig. 1C) . 

In GFP-positive AP20187 treated tadpoles, HA signal was undetectable in Western blot analysis (P = 0.039, compared with untreated, Mann-Whitney U test; n = 7; Fig. 1B ), indicating no intact caspase, as anticipated for iCasp9 activation. Rhodopsin level (a measure of rod viability because it is expressed exclusively and abundantly in rods) was not significantly reduced at this time point (P = 0.528; Mann-Whitney U test; n = 7; not shown). Microscopy of contralateral eyes showed ongoing rod death in treated retinas despite high rhodopsin levels, indicated by classic apoptotic morphology, including blebbing and pyknotic nuclei (Fig. 1D) . In contrast, identical AP20187 treatments did not initiate apoptosis in wild-type retinas (not shown). Together these results demonstrate that iCasp9 was expressed in the rods of GFP-positive primary transgenic X. laevis tadpoles and that AP20187 was absorbed from the medium, transported to the retina, and interacted with iCasp9 to initiate apoptosis. 

ICasp9 Did Not Cause Rod Death in the Absence of AP20187

Although we did not observe evidence of abnormal apoptosis in the absence of AP20187 in 14 dpf transgenic tadpoles, it is possible that high-level iCasp9 expression eventually caused cell death through spontaneous autoactivation. To examine the long-term effects of iCasp9 expression, three GFP-positive postmetamorphic primary transgenic frogs were killed 3 months after fertilization, and their retinas were examined through immunohistochemistry. These retinas appeared healthy and were anti-HA reactive, indicating consistent iCasp9 expression with minimal autoactivation (not shown). 

To confirm these results, we used electroretinography to measure retinal function in two GFP-positive postmetamorphic frogs, two GFP-negative frogs, and 10 age-matched wild-type frogs. We fit trough-to-peak ERG amplitudes obtained at increasing stimulus intensities to the Naka-Rushton equation. Functional data from the two GFP-positive frogs (1 and 3) and two GFP-negative frogs (2 and 4) were not distinguishable from each other, or from wild-type responses, because none of the responses from either experimental group fell outside 1 SD of the averaged wild-type data (Fig. 2) . In conjunction with histology, the strong ERG response in the GFP-positive postmetamorphic frogs confirmed negligible drug-independent activation of iCasp9. 

AP20187 Severely Compromised Retinal Function in Primary Transgenic Postmetamorphic Frogs

The untreated GFP-positive (1 and 3) and GFP-negative (2 and 4) frogs described in the previous section and four wild-type frogs received subcutaneous injections of AP20187 and were retested by electroretinography 5 days after drug administration. From Naka-Rushton curve fits, we derived the projected maximal b-wave amplitude and K _m (intensity required for a half-maximal response). As a measure of normal function, we used the 66% confidence interval for the 10 untreated wild-type animals (1 SD). GFP-positive animals 1 and 3 demonstrated dramatic reductions in scotopic ERG response on AP20187 injection (Fig. 2A) , with Rmp3 and b-wave amplitude measures dramatically reduced (Fig. 2B) . b-Wave amplitude dropped substantially in both frogs, rendering K _m an insignificant parameter (Fig. 2C) , suggesting almost complete destruction of rods and of INL abnormalities. Based on the dramatic changes in a- and b-waves in these GFP-positive frogs, we are confident that AP20187 induced rod death, as demonstrated in our initial experiments on tadpoles. 

In contrast, the four wild-type animals were unaffected by AP20187 injections (not shown). Similarly, a- and b-wave amplitude measures were virtually unchanged in GFP-negative animals 2 and 4. However, in animal 4, K _m increased considerably, suggesting mild rod dysfunction on AP20187 injection. This unexpected result will be discussed in a later section. 

Consistent iCasp9 Expression and Induction of Apoptosis in F1 Tadpoles

We analyzed the progeny of the first animal to reach sexual maturity and found consistent iCasp9 expression in GFP-positive tadpoles, which was depleted after 7 days of AP20187 exposure (P = 0.0088; Mann-Whitney U test; n = 7; Fig. 3A ). This was associated with a significant reduction of rhodopsin levels (P = 0.0004, Mann-Whitney U test; n = 7; Fig. 3B ) and of rod clearance from the ONL (not shown). Since expression was more consistent in the F1 generation, more advanced analyses were possible. 

To generate a timeline of events from drug administration to rod clearance, we analyzed F1 tadpoles exposed to AP20187 at times ranging from 1 hour to 4 days. iCasp9 and rhodopsin expression were quantified from Western blots, and the number of apoptotic nuclei were counted from TUNEL-labeled sections; results are summarized in Figure 4A . iCasp9 and rhodopsin expression increased for 12 hours after drug exposure because of tadpole growth. By 12 hours, TUNEL-positive nuclei were apparent in the ONL, and iCasp9 and rhodopsin levels began to decline. Two days after drug exposure, HA signal was undetectable, and the number of TUNEL-positive nuclei in the ONL peaked. Rhodopsin levels continued to decrease as the number of TUNEL-positive nuclei declined. In contrast to treated transgenic animals, no TUNEL-positive nuclei were found in the ONL of untreated transgenic animals (Fig. 4B)or treated control animals (data not shown). 

Small numbers of TUNEL-positive nuclei were also present in other retinal layers of treated and untreated animals. TUNEL-positive nuclei were more abundant in the RPE of treated transgenic animals, closely following trends in the ONL and likely representing phagocytosed rods. However, this trend was not statistically significant. 

Longitudinal Studies on Postmetamorphic Frogs Confirm Rod Death and Suggest Cone Abnormalities

Cone Function in Primary Transgenic Frogs after Prolonged Treatment with AP20187.

For 8 months, we monitored the AP20187-treated primary transgenic postmetamorphic and four treated wild-type X. laevis frogs previously described. To counteract the addition of healthy rods at the CMZ as a result of eye growth or of possible rod regeneration, we injected each animal monthly with AP20187. We repeatedly recorded the ERG response to 10-Hz flicker and photopic stimuli as a measure of cone function. Figure 5summarizes the photopic waveforms generated by transgenic animals compared with averaged nontransgenic AP20187-treated frogs. Control animals had consistent responses, whereas GFP-positive transgenic animals had compromised and irregular cone function within 3 months of the initial AP20187 injection, which subsequently improved despite chronic AP20187 administration. 

Rod Morphology in Primary Transgenic Frogs after 5 Months of AP20187 Treatment.

Two days after the 5-month AP20187 injection, we sectioned one eye from GFP-negative animal 4 (Fig. 6A ; see also Figs. 2 5 ) and GFP-positive animal 3 (Fig. 6B ; see also Figs. 2 5 ). We observed predominantly healthy rod outer segments in animal 4 (Fig. 6A) . However, we also noticed faint GFP expression in some rods of animal 4, undetected by in vivo screening, and small patches of rod ablation (Fig. 6F) . These findings were consistent with the decreased sensitivity (increased K _m) in animal 4 presented in Figure 2C , suggesting limited and mosaic expression of iCasp9. In subsequent analyses, we separated the rod-rich (Fig. 6C , labeled 4) and rodless (Fig. 6F , labeled 4*) portions of the retina for further analysis. 

In contrast, in GFP-positive animal 3, we found almost complete rod ablation (Fig. 6B) . The only exceptions were occasional dysmorphic rods in the central retina (Fig. 6B , arrowheads). This suggested the few rods present in the central retina were undergoing apoptosis in response to the most recent AP20187 injection (given 2 days before cryosectioning), similar to the dysmorphic cells apparent 2 days after drug administration in our initial tadpole experiments (Fig. 1D) . 

Retinal Morphology in Primary Transgenic Frogs after 5 Months of AP20187 Treatment.

Two days after the 5-month time point, cones were abundant in the rod-deprived retina of animal 3 and in the rod-rich retina of animal 4 (Figs. 6C 6D) ; in fact, the density of cones was higher in transgenic animal 3 than in nontransgenic controls (Fig. 6E)despite the functional depression measured in the photopic ERG (Fig. 5) . 

The widths of the INL and of the total retina differed between the rodless animals and controls. Figures 6A and 6Bdemonstrate that the INL was 30% thicker in animal 3 than in animal 4. Interestingly, in the rodless regions of the retina in animal 4, the INL was also thicker than in rod-rich areas (Fig. 6F)to an extent similar to that of the INL of rod-ablated animal 3 (Fig. 6G) . These rodless regions in the retina of animal 4 did not have increased cone counts, as occurred in rod-ablated animal 3 (Fig. 6E) , and suggest retinal remodeling after rod death. 

Cone Dysfunction in F1 Frogs after Prolonged AP20187 Treatment.

The electrophysiological effects described in Figures 2 and 5were confirmed in a similar analysis of F1 animals. We identified a group of F1 animals, the offspring of a single transgenic male, that carried the iCasp9 transgene and responded to AP20187 with a decrease in ERG sensitivity. On drug treatment, there was a 50% decrease in V _max within this group compared with predrug levels. By 1 month after treatment (with treatment readministered monthly), photopic and flicker ERGs were repressed by 1 order of magnitude (Fig. 7A) . At 3 months, two animals were killed and their eyes were cryosectioned. Again, though rods were almost completely absent, these retinas contained numerous cones (Fig. 7B) . Cone density was not significantly different from that observed in control animals. We did observe tearing of the cone outer segment and ellipsoid region away from the cell body, but this was likely an artifact associated with fixation in the absence of rods because the detached fragments would have been rapidly cleared had this occurred in vivo. Thus, the negative effects of rod ablation on cone responses that we observed in X. laevis retina appear to have resulted from a functional deficit rather than from cone death. At this 3-month point, INL thickness was not different from that of control retinas (as it was at 5 months), suggesting that proliferation in the INL may be linked to the return of the photopic ERG. 

Discussion

We have successfully coexpressed iCasp9 and GFP in X. laevis rod photoreceptors and identified animals expressing iCasp9 by GFP expression. Furthermore, we were able to induce rapid, controlled apoptosis of rods by administering AP20187 to these animals. In the absence of AP20187, the transgene products were nontoxic and had no significant effect on retinal physiology or morphology. 

AP20187 was successfully administered by epithelial absorption in tadpoles, and subcutaneous injection in postmetamorphic frogs. The drug crossed the blood-retinal barrier and reached photoreceptors. This was not unexpected given that the structurally similar compound FK506 crosses the blood-brain barrier. ⁴¹ AP20187 did not cause apoptosis in the absence of iCasp9. 

In the presence of sufficient AP20187, autoactivation of iCasp9 leading to apoptosis was robust, as previously demonstrated in studies carried out by Spencer et al. ^{26 27 42} On AP20187 administration, we observed the depletion of intact iCasp9 and rhodopsin and fragmenting anti-rhodopsin reactive cell bodies, as well as pyknotic and TUNEL-positive nuclei in the ONL, all hallmarks of rod apoptosis. This was corroborated functionally by compromised scotopic ERGs after AP20187 treatment in postmetamorphic frogs. iCasp9 activation occurred in less than 1 day, and rod apoptosis was apparent from 2 to 4 days after drug interaction. This was consistent in primary transgenic, F1 generation, and postmetamorphic X. laevis. Therefore, we are confident that it is possible to induce controlled rod apoptosis at any point in X. laevis development. 

After inducing rod ablation in postmetamorphic animals, we noticed a decrease in amplitude of photopic and flicker ERGs compared with controls, indicating rod death was affecting cone function, viability, or both, as commonly observed in human RP. Understanding this interaction may help us understand the mechanisms underlying the secondary cone death that causes blindness in RP. Several theories have been proposed to explain this phenomenon, including direct interaction, ⁴³ indirect toxicity, ^{17 44} lack of protection from secreted factors, ^{16 19} and functional loss through ectopic synaptogenesis and retinal reorganization. ^{12 21} Our data are consistent with the last hypothesis because INL changes were the only overt abnormalities 2 months after reduction in cone function. It is intriguing to speculate that a decline in cone function may precede cone death and that there may be the potential for rescue of cones even in retinas with markedly decreased cone function. 

We also noted a reinstatement of photopic responses 5 months after rod ablation, and increased numbers of cones, indicating functional restoration or regeneration. In addition to normal growth from the periphery, we noticed blebbing cell bodies immunopositive for anti-rhodopsin in the central retina 2 days after AP20187 administration, after 5 months of continued rod cell ablation, suggesting that without continued AP20187 administration, central rods may regenerate. Proliferation from the CMZ and transdifferentiation of the RPE are generally accepted as the only mechanisms of regeneration in postmetamorphic X. laevis. ^{5 7} Given the INL thickening in conjunction with the presence of new photoreceptors, it seems plausible that regeneration could initiate from progenitor cells residing in this region, just as in adult newts ⁴⁵ and in X. laevis larvae ⁶ after retinal damage. 

Thickening of the INL (as a sign of reorganization and/or regeneration in response to rod death) and loss of b-wave amplitude in scotopic and photopic ERGs could reflect changes in Müller cell glia. Intriguingly, similar thickening of the retina has recently been observed by optical coherence tomography in RP patients. ⁴⁶ These cells are associated with the ERG b-wave, have somas located in the INL, and can transdifferentiate into progenitor cells capable of replacing retinal cells. ⁴⁷ It will be interesting to further probe the involvement of Müller cell glia in events after rod death. 

For most of the experiments described, we used animals expressing uniform high levels of transgene products and measured robust effects; however, mosaic expression patterns commonly occurred in our system and could be used to examine the subtleties of regional versus complete ablation, as demonstrated for animal 4 in which rod-rich and rodless regions were compared. In future studies involving primary transgenic animals, this property will be used to examine the implications of partial rod ablation. 

RP is the most common cause of inherited blindness, ¹¹ and the common path of rod death, despite more than 160 different initiating mutations (http://www.sph.uth.tmc.edu/Retnet/), is apoptosis. ²⁵ We have developed a transgenic X. laevis model of RP in which rod apoptosis is initiated by the administration of a normally innocuous compound. This experimental model is distinguished from other inducible models (such as light damage) in that the cell death pathway is well characterized, and rod photoreceptors are the only cells affected by the primary insult. Thus, these animals provide an excellent system for examining the effects of rod apoptosis on other retinal cell types at any stage of development. Rod-cone interactions are of particular interest because of the high rod- cone ratio (1:1) in the X. laevis retina and the involvement of secondary cone degeneration in human RP. Furthermore, because AP20187 can be administered for the short term and long term, it will be possible to examine the regenerative capacity of the developing and mature X. laevis retina. 

 Supported by Foundation Fighting Blindness (Canada); the Michael Smith Foundation for Health Research; and the Canadian Institutes of Health Research (CIHR). OLM is a Michael Smith Scholar, a CIHR New Investigator, and a W.K. Stell Scholar. LMH was funded by a fellowship from the FFB (Canada).

 Submitted for publication May 26, 2008; revised August 22, 2008; accepted December 16, 2008.

 Disclosure: L.M. Hamm, None; B.M. Tam, None; O.L. Moritz, 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.

 

Figure 7.

 

Confirmation of decrease in rod function, and decrease and subsequent return of cone function, in F1 offspring. (A) Plot of measures of rod and cone function over time in animals treated with AP20187. Rod function is represented by the V _max value obtained from Naka-Rushton plots of the a- to b-wave amplitudes of the scotopic ERG. Cone function is represented by the amplitudes of the photopic ERG and 10-Hz flicker ERGs. Data are based on five F1 animals to the time point marked with an asterisk and on three animals thereafter. Error bars are ± SEM. The data reveal a decline and protracted deficit in rod function and a temporary decline and return of cone function. (B) Confocal microscopy analysis of the retina of an animal killed at time point marked with the asterisk. Cryosections were labeled with anti-calbindin (white), Hoechst dye (blue), and wheat germ agglutinin (red). All cells of the ONL were stained with calbindin, indicating that they are cone photoreceptors, though some irregular membrane fragments may be derived from small numbers of dysmorphic/degenerating rods (arrows). There was no evidence of cone death, despite greatly decreased cone function. The frequently observed separation of the cone OS and ellipsoid region from the cell body was likely an artifact of fixation.

Figure 1.

 

Induced rod cell ablation in primary transgenic tadpoles. Western blot and immunohistochemical assays of primary transgenic tadpoles untreated or treated with AP20187 for 2 days. (A) Western blot analysis shows variable expression of intact iCasp9 (anti-HA signal from epitope on caspase prodomain) in 6 of 7 GFP-positive untreated animals. (B) In tadpoles exposed to AP20187 for 2 days, the anti-HA signal was absent, consistent with degradation of the epitope after autoactivation. (C) Microscopy confirms that anti-HA labeling (red) colocalized with GFP (green) exclusively in rod cells and that these cells retained structural health in the absence of AP20187. (D) Treated retinas were depleted of intact iCasp9 and displayed morphologic hallmarks of apoptosis, including loss of structural integrity, cell blebbing (arrows), and pyknotic nuclei (arrowheads), indicating that activation of iCasp9 was associated with rod cell death. Scale bar, 20 μm.

Figure 2.

 

AP20187 injection caused compromised retinal function in primary transgenic postmetamorphic frogs. Scotopic ERG recordings from before (black) and 5 days after (gray) AP20187 injection in two GFP-positive (1 and 3) and two GFP-negative (2 and 4) frogs. (A) Raw waveforms at increasing stimulus intensity superimposed for each animal show that scotopic retinal function was normal in untreated animals but was severely compromised in GFP-positive frogs after drug injection. (B) Projected a-wave asymptote (log R _{m p3}) and b-wave amplitude for the maximal flash intensities confirm photoreceptor and resultant INL functional abnormalities (error bars = SE). (C) The amplitude of response to each of five stimulus intensities was fit to the Naka-Rushton equation. The fit from 10 wild-type frogs was averaged, and the 66% confidence intervals (±1 SD) derived from these animals were superimposed on the graph (bars). All untreated animals fell within the 66% confidence interval, but when treated with AP20187 animals 1, 3, and 4 did not. (Animals 1 and 3 were also outside the 96% confidence interval at the three highest flash intensities.)

Figure 3.

 

Inducible apoptosis was more consistent in the F1 generation. Western blot assays of F1 transgenic tadpoles, untreated or treated with AP20187 for 7 days. (A) Untreated GFP-positive tadpoles showed consistent iCasp9 expression (upper) and elimination of HA signal 7 days after drug administration (lower). (B) Rhodopsin levels (B630N signal) were high and consistent in untreated animals (upper) and severely reduced in treated tadpoles (lower). A control sample (untreated primary transgenic retina) was included on each blot (asterisks).

Figure 4.

 

Rod cell apoptosis was complete within 4 days of AP20187 administration. Signal intensity from Western blots probed for intact iCasp9 (anti-HA signal) and rhodopsin (B630N signal) and counts of TUNEL-positive nuclei from retinal sections graphed for eight time points after AP20187 administration in F1 tadpoles. Each time point represents an averaged, normalized value for five animals. (A) ICasp9 and rhodopsin levels increased up to 12 hours after drug exposure. At 12 hours, the first TUNEL-positive nuclei were apparent in the photoreceptor layer. Subsequently, the HA epitope of iCasp9 was progressively degraded, and rhodopsin levels were progressively decreased. The number of TUNEL-positive cells peaked at day 2 (six nuclei per section) and subsequently declined. (B) Numerous TUNEL-positive cells (red) were observed in the ONL of treated transgenic animals 36 hours after AP20187 administration but were absent from the ONL of untreated transgenic animals (blue: Hoechst 33342). Scale bar, 20 μm.

Figure 5.

 

Photopic responses were compromised after AP20187 injection but showed subsequent improvement. The graphs summarize the responses to photopic stimuli over an 8-month period. Animals 1 and 3 are the same as described in Figure 2 , and control waveforms (two GFP-negative and five wild-type frogs) were averaged together for each month. (A) Responses to photopic stimuli were irregular 3 and 4 months after the initial injection but subsequently improved. (B) Calculated a- to b-wave amplitudes for each waveform showed a decrease between 1 and 3 months after initial injection and also subsequently improved. (Animal 3 was killed after the 5-month ERG analysis; morphologic data from this animal are shown in Fig. 6 ).

Figure 6.

 

Five months after initial AP20187 injection, rod cells were ablated, the INL thickened, and cone cells were intact and more abundant in GFP-positive animal 3 than in animal 4. Animals 3 and 4 were killed 2 days after the 5-month AP20187 injection. (A) Animal 4 had healthy ROSs, visualized by B630N labeling (red), and a 22-μm thick INL (±3.1), measured from boundaries of Hoechst 3392-labeled nuclei (blue). (B) Animal 3 had ablated rod cells (absence of B630N labeled outer segments), except for centrally located dysmorphic rods (arrowheads) and a thickened INL (34.9 ± 4.2 μm). (C) Retinal sections from animal 4 labeled with anti-calbindin displayed normal cone cells. (D) Retinal sections from animal 3 labeled with anti-calbindin also demonstrate healthy cones despite functional irregularity, shown in Figure 5 , and lack of intact rod cells displayed in (B). (E) Quantification of cone cells in untreated and AP20187-treated controls (D−, D+), animal 3, and the rod-rich (4) and rodless (4*) portions of animal 4 show that the density of cone cells was greater in rodless animal 3 but not in the small rodless portions of otherwise normal animal 4. (F) Area of retina from animal 4 showing two regions of rod-deprived retina (rod −) flanking a relatively normal rod-rich region (rod +). This panel shows the Hoechst dye channel only, with rod outer segments visible because of autofluorescence. In the rod-deprived regions can be seen a local increase in INL thickness, as in the retina of animal 3, but the INL is of normal thickness in the intervening region. (G) Relative to treated and untreated controls, the INL was thicker in both the rod-ablated retina of animal 3 and the small rod-ablated portions of animal 4. INL thickening appeared to be a local, possibly direct, effect of rod ablation. Scale bars: (A, B, F) 50 μm; (C, D) 20 μm. Error bars = SE.

The authors thank David Spencer for providing the pSH1/S-Fvls-p30casp9-E plasmid, Paul Hargrave for providing the B6–30N antibody, Joseph Bilotta for helpful comments on electroretinography, Gerald Li for developing initial ERG procedures, and ARIAD Pharmaceuticals Inc. (www.ariad.com/regulationkits) for providing AP20187.