Figure 2

Enhanced accumulation of autophagosomes and phagolysosomes is observed in the injured cardiac tissue upon amputation. The transgenic line Tg(b-actin2:mRFP-GFP-Lc3) allows the observation of autophagosome formation and processing in the regenerating zebrafish heart ventricle at different time points after apex amputation. Autophagosomes are depicted both as GFP- and RFP-puncta, while the lower pH-sensitivity of RFP additionally allows the visualization of phagolysosomes. Images were taken immediately after heart excision, and upon overnight incubation of the zebrafish in 2 mM CQ, which acts as a lysosomal inhibitor. Quantitative analysis revealed a significant increase in the number of both vesicles, as well as in the average autophagosome/phagolysosome size (red puncta) in the regenerating tissue, which gradually decreased over time. A schematic view of the ventricle orientation and the region of interest considered for image acquisition is shown, while higher magnifications exemplifying the accumulation of bigger autophagic vesicles are presented next to the mosaic images. Scale bars represent 200 μm. *p ≤ 0.05. N ≥ 3.