Optogenetic Control of Apoptosis in Targeted Tissues of Xenopus laevis Embryos

Abstract

KillerRed (KR) is a recently discovered fluorescent protein that, when activated with green light, releases reactive oxygen species (ROS) into the cytoplasm, triggering apoptosis in a KR-expressing cell. This property allows for the use of KR as a means of killing cells in an organism with great temporal and spatial specificity, while minimizing the nonspecific effects that can result from mechanical or chemical exposure damage techniques. Such optogenetic control of cell death, and the resulting ability to induce the targeted death of specific tissues, is invaluable for regeneration/repair studies-particularly in Xenopus laevis, where apoptosis plays a key role in regeneration and repair. We here describe a method by which membrane-bound KR, introduced to Xenopus embryos by mRNA microinjection, can be activated with green light to induce apoptosis in specific organs and tissues, with a focus on the developing eye and pronephric kidney.

Keywords: KillerRed; Xenopus; apoptosis; optogenetics; regeneration.

Figure 1

KillerRed (KR)-mediated light-induced apoptosis results in ablated eye phenotype. Xenopus laevis embryos were injected with KR mRNA at Nieuwkoop–Faber (NF) stage 2–3 and selected for KR expression in the left eye at NF stage 26–28. Notes: (A) Five minute activation of KR by exposure to green light induced photobleaching. (B, C) 1.5–3.5 hours after light treatment in eye (or at matching stages for unlit tadpoles), immunohistochemistry for active Caspase-3 showed that light activation of KR resulted in significantly higher levels of apoptosis over tadpoles not exposed to light, or expressing KR. (Fisher’s exact; *p < 0.01; **p < 0.001). Apoptosis was spatially restricted to the illuminated region. (D) After 24 hours, tadpoles were visually inspected for the presence of an ablated phenotype in the left eye. (E) Light activation of KR resulted in significantly higher incidence of ablated eye phenotype. (Fisher’s exact; *p < 0.001). All scale bars are 500 μm. n indicates the number of tadpoles across all replicates, and N indicates the number of replicates shown. (A, B: TRITC; D: Brightfield).

Figure 2

Photoactivation of KillerRed (KR) increases reactive oxygen species only within the illuminated region. Dihydroethidium (DHE) is a fluorescent indicator of superoxides. Following five minute activation of KR by exposure to green light, DHE fluorescence greatly increased within the illuminated region (dashed circle). However, it did not similarly increase in nearby, non-illuminated KR-expressing regions, like the forebrain and cement gland (arrowheads). All scale bars are 200 μm. (Top: TRITC; middle, bottom: Mermaid.)

Figure 3

KillerRed (KR)-mediated light-induced apoptosis results in ablated pronephros. Xenopus laevis embryos were injected with KR mRNA at Nieuwkoop–Faber (NF) stage 2–3 and selected for KR expression in the left pronephros at NF stage 37. Notes: (A) Five minute activation of KR by exposure to green light induced photobleaching in pronephros (circled). (B, C) 1.5–3.5 hours after light treatment in pronephros (or at matching stages for unlit tadpoles), immunohistochemistry for active Caspase-3 showed that light activation of KR resulted in a significant increase in the number of Caspase-positive loci over tadpoles not exposed to light, or expressing KR (Tukey–Kramer; *p < 0.001). Mean number of Caspase-positive loci per tadpole pronephros is shown, with error bars indicating the standard error of the mean. Apoptosis was spatially restricted to the illuminated region. (D) After 24 hours, in situ hybridization for senescence marker protein 30 (SMP30) was performed to visualize proximal tubules, and areas of SMP30 expression were measured on each side of the tadpole using SPOT Advanced imaging software. (E) Light activation of KR resulted in ablation of proximal tubules on the left side, compared to tadpoles with KR expression alone and tadpoles with no treatment at all. For (B) (inset) scale bars are 100 μm; all other scale bars are 500 μm. n indicates the number of tadpoles across all replicates, and N indicates the number of replicates shown. (A, B: TRITC; D: Brightfield.)