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. 2010 Nov 2;10:110.
doi: 10.1186/1471-213X-10-110.

Optogenetic in Vivo Cell Manipulation in KillerRed-expressing Zebrafish Transgenics

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Free PMC article

Optogenetic in Vivo Cell Manipulation in KillerRed-expressing Zebrafish Transgenics

Cathleen Teh et al. BMC Dev Biol. .
Free PMC article

Abstract

Background: KillerRed (KR) is a novel photosensitizer that efficiently generates reactive oxygen species (ROS) in KR-expressing cells upon intense green or white light illumination in vitro, resulting in damage to their plasma membrane and cell death.

Results: We report an in vivo modification of this technique using a fluorescent microscope and membrane-tagged KR (mem-KR)-expressing transgenic zebrafish. We generated several stable zebrafish Tol2 transposon-mediated enhancer-trap (ET) transgenic lines expressing mem-KR (SqKR series), and mapped the transposon insertion sites. As mem-KR accumulates on the cell membrane and/or Golgi, it highlights cell bodies and extensions, and reveals details of cellular morphology. The photodynamic property of KR made it possible to damage cells expressing this protein in a dose-dependent manner. As a proof-of-principle, two zebrafish transgenic lines were used to affect cell viability and function: SqKR2 expresses mem-KR in the hindbrain rhombomeres 3 and 5, and elsewhere; SqKR15 expresses mem-KR in the heart and elsewhere. Photobleaching of KR by intense light in the heart of SqKR15 embryos at lower levels caused a reduction in pumping efficiency of the heart and pericardial edema and at higher levels - in cell death in the hindbrain of SqKR2 and in the heart of SqKR15 embryos.

Conclusions: An intense illumination of tissues expressing mem-KR affects cell viability and function in living zebrafish embryos. Hence, the zebrafish transgenics expressing mem-KR in a tissue-specific manner are useful tools for studying the biological effects of ROS.

Figures

Figure 1
Figure 1
Expression of the membrane-tethered KillerRed in some of enhancer trap transgenic lines. (A-B) The head of SqKR1, lateral view. (B) The magnified view of the eye. (C-D) The head of SqKR2, lateral view. Mem-KR is expressed in rhombomeres 3 and 5 (r3 and r5). (D) A magnified view of the box in C. (E-F) The head of SqKR4, dorsal view. mem-KR is expressed in the optic tectum (ot) and hypothalamus (hyp). (F) A magnified view of the box in E. (G-H) The head of SqKR11, dorsal view. The habenula (ha), optic tectum (ot), hindbrain (hb) is highlighted in this projection. (H) A magnified view of the box in G enclosing the habenula. (I-J) The head of SqKR15, lateral view. (J) A magnified view of the heart, ventral view. (K-L) The head of SqKR19, lateral view. (L) Expression of mem-KR in the choroid plexus (chp) of SqKR19, dorsal view. All scale bars correspond to 100 μm, unless otherwise stated.
Figure 2
Figure 2
Illumination of the hindbrain of SqKR2 caused bleaching of KR followed by increase in apoptosis. (A-I) Efficient photobleaching of KR was achieved by intense green light using the UV lamp of the compound microscope in widefield mode. (A-C) Fluorescent merged images of the SqKR2 embryo at various time points, before and after illumination with white light. (E-I) Fluorescent merged images of the SqKR2 embryo at various time points, before and after illumination with green light. F and H are the bright field and fluorescent merged images of E and G, respectively. (J-L) Merged fluorescent/DIC images of KR-expressing cells (green) and TUNEL-positive cells (red) in SqKR2 (J), SqKR15B (K) and wild type zebrafish embryos (L). (J, L) Embryos were double stained for TUNEL (red) and (J, K) in addition by anti-KR antibody (green). (M) When compared to illuminated controls apoptosis increased more than two-fold in the hindbrain of illuminated SqKR2. A bar chart documenting the average number of TUNEL-positive cells per embryo in 10 embryos of three illuminated groups (SqKR2, SqKR15B and WT control). Values presented as mean ± SEM. Paired T test between the illuminated SqKR2 embryos and controls showed that the difference in the average number of TUNEL-positive cells is significant (P < 0.05). P values between groups are highlighted by the enclosing brackets.
Figure 3
Figure 3
KillerRed expression in SqKR15 is present in all layers of the heart. (A) Co-localization of GFP and KR in the endocardium of the ET33-mi84A:SqKR15 double transgenic embryo (arrow - endocardium). (B) Co-localization of GFP and KR in the endocardium and myocardium of the ET33-mi103:SqKR15 double transgenic embryo. (C) Co-localization of GFP and KR in the myocardium of the ET33-mi3A:SqKR15 double transgenic embryo (arrow - myocardium). (A, B, C) merged images; (A', B', C') - KR expression; (A'', B'', C'') - GFP expression.
Figure 4
Figure 4
Illumination of KR-expressing heart bleaches KR and causes cardiac edema.(A-B) KR expression in the 3dpf beating heart of SqKR15 was reduced to 21% after 5 min of exposure to intense green light. (A) Expression of GFP and KR in the ET33-mi3A:SqKR15 double transgenic embryo prior to illumination and (B) after illumination. (C-D) Larvae at 4 dpf, one day after illumination: C - ET33-mi3A (control), D - ET33-mi3A:SqKR15 (experimental sample). Cardiac edema developed in ET33-mi3A:SqKR15 larvae one day after illumination (D). Apoptosis in the heart of SqKR15 is similar to that in control.
Figure 5
Figure 5
Increased illumination increased apoptosis in the KR-expressing heart. (A-B) 8 min illumination of the 3dpf heart of SqKR15 embryo with intense green light reduced fluorescence intensity to 17%. (A) GFP and SqKR15 fluorescence in the ET33-mi3A:SqKR15 double transgenic embryo before and (B) after illumination. (C-D) same larvae one day after illumination: C - ET33-mi3A (control), D - SqKR15/ET33-mi3A (sample). TUNEL (+) cells in the heart of illuminated sample (D') and control (C'), a day after illumination. (E-F) TUNEL staining of transverse sections of SqET33-mi3A larva (control) and (G-H) SqKR15/ET33-mi3A larva (sample), at different magnification one day after illumination. Examples of TUNEL (+) cells in the myocardial layer are boxed in white. All scale bars are 50 μm in length unless otherwise stated.
Figure 6
Figure 6
A reduction in cardiac output was observed in KR-expressing larvae immediately after illumination. (A-C) Heartbeat and contractility in SqKR15/ET33-mi3A (A), SqKR15B/ET33-mi3A (B) and KR-negative Sq ET33-mi3A (C) 3dpf larvae before illumination and 20 min after illumination. Panel (I) in (A-B) shows confocal images of corresponding double transgenic larvae, taken at the same gain setting, before and after illumination. M-modes depicting a heartbeat for 10 sec before and after illumination are in panels A-B, II-III and C, I-II. Images of five consecutive ventricular systole and diastole are shown in A-B, IV-V and C, III-IV. Cardiac output was specifically reduced after illumination of SqKR15/ET33-mi3A larva as the reduction in heartbeat is accompanied by a decrease in contractility indicated by a decrease in value of %FS. (D) Larvae at 4dpf, one day after illumination: (I) - SqKR15/ET33-mi3A (sample), (II) - SqKR15B/ET33-mi3A with skin mem-KR expression as a positive control and (III) - SqET33-mi3A as a negative control. Only SqKR15/ET33-mi3 developed prominent cardiac edema next day after illumination [D (I-I')].
Figure 7
Figure 7
Cardiac output is consistently reduced in all illuminated SqKR15 larvae. (A-B) A bar chart comparing the percentage change in heartbeat (A) and contractility (B) after illumination of SqKR15/ET33-mi3A (sample) and controls (SqKR15B/ET33-mi3A; SqET33-mi3A) across three illuminated groups of five embryos each. Values presented are mean % change in heartbeat ± SEM (A) or mean % change in contractility ± SEM (B), where 100% indicates no change in heart beat or contractility after illumination. Paired T test between illuminated SqKR15 larvae and controls showed that the difference in value is significant (P < 0.05). P values between groups are highlighted by the enclosing brackets.

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References

    1. Asakawa K, Kawakami K. Targeted gene expression by the Gal4-UAS system in zebrafish. Dev Growth Differ. 2008;50(6):391–399. doi: 10.1111/j.1440-169X.2008.01044.x. - DOI - PubMed
    1. Palmiter RD, Behringer RR, Quaife CJ, Maxwell F, Maxwell IH, Brinster RL. Cell lineage ablation in transgenic mice by cell-specific expression of a toxin gene. Cell. 1987;50(3):435–443. doi: 10.1016/0092-8674(87)90497-1. - DOI - PubMed
    1. Kurita R, Sagara H, Aoki Y, Link BA, Arai K, Watanabe S. Suppression of lens growth by alphaA-crystallin promoter-driven expression of diphtheria toxin results in disruption of retinal cell organization in zebrafish. Dev Biol. 2003;255(1):113–127. doi: 10.1016/S0012-1606(02)00079-9. - DOI - PubMed
    1. Wan H, Korzh S, Li Z, Mudumana SP, Korzh V, Jiang YJ, Lin S, Gong Z. Analyses of pancreas development by generation of gfp transgenic zebrafish using an exocrine pancreas-specific elastaseA gene promoter. Exp Cell Res. 2006;312(9):1526–1539. doi: 10.1016/j.yexcr.2006.01.016. - DOI - PubMed
    1. Remington SJ. Fluorescent proteins: maturation, photochemistry and photophysics. Curr Opin Struct Biol. 2006;16(6):714–721. doi: 10.1016/j.sbi.2006.10.001. - DOI - PubMed

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