Fig. 2

Lysosome accumulation during cardiac valve development (A) Experimental set up for in vivo lysosome imaging in the developing zebrafish heart. Transgenic zebrafish larvae were imaged at 48, 56, 72 and 96 h post fertilization (hpf) using light-sheet microscopy and following a 3D+t acquisition mode. (B) Schematic overview of the process of autophagy-lysosomal degradation involving phagosome and autophagosome formation, fusion with lysosome, and lysosomal degradation. Lysosomes were tracked using LysoTracker or a transgenic reporter line expressing lamp2 fused with red fluorescent protein mRFP. (C) Graphic representation of the cardiac region imaged, corresponding to the atrioventricular canal (AVC). EnCs, endocardial cells; VIC, valve interstitial cells; EnVCs, endocardial valve cells. (D–F) Reconstructed live image acquisitions by 3D+t light-sheet microscopy. Shown are optical sections through hearts at the indicated developmental stages. Anterior is to the top, the ventricle to the left and atria to the right. Upper panels show merged channels (color), lower panels single channels (inverted greyscale). Arrows mark mRFP+-puncta in the atrioventricular valves. Graphs show quantification of fluorescent puncta in the ventricle and AVC regions at different developmental stages. Shown are numbers in individual animals as well as median and quartile values. Each dot represents one larva. Statistical test: two-way ANOVA, ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001). Scale bars represent 10 μm. (D) Tg(cmv:EGFP-Lc3) embryos were stained with LysoTracker to observe GFP+ autophagosome and LysoTracker+ acidic lysosomal vesicles in the developing heart. Note that EGFP signal is also strongly detected in circulating erythrocytes between 48 and 56 hpf, but later localizes mainly to the in AVC. LysoTracker-labelled lysosomes are scarcely observed before 56 hpf in cardiac tissues, but accumulate in the AVC at 72 hpf and are prominent in the developing valves at 96 hpf. (E and F) The lysosome reporter Tg(lamp2:RFP) was crossed into Tg(fli1:GFP) (E) or Tg(myl7:GFP) (F) to assess lysosomes colocalization in endocardium and myocardium, respectively. (G) Cardiac sections of Tg(cmv:GFP-LC3); (lamp2:RFP) immunostained for GFP (green), RFP (magenta) and ALCAM (yellow). Arrows point to RFP/GFP double positive puncta. ALCAM staining allows to demarcate the myocardium at the AVC. Scale bars represent 10 μm. See also Figure S3.