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Fig. 1 Autophagosome formation accompanies valve development (A) Experimental plan followed for in vivo imaging of the developing zebrafish heart. Transgenic zebrafish larvae were imaged at 48, 56, 72 and 96 hpf upon 3 h treatment with chloroquine (2 mM) to observe autophagosomes and lysosomes in the beating heart 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. The transgenic autophagosome tagged reporter proteins used in this study are illustrated. (C) Graphic representation of the cardiac region imaged. AVC, atrioventricular canal; EnCs, endocardial cells; VIC, valve interstitial cells; EnVCs, endocardial valve cells. (D–F) Reconstructed image acquisitions by 3D+t light-sheet microscopy. Shown are always merged (color) and single channel views (inverted greyscale). Anterior is to the top, ventricle to the left, atria to the right. Middle part constriction is the AVC. Developmental stages are indicated within the panels as hours postfertilization (hpf). Graphs show number of mRFP-GFP + puncta within ventricle and AVC regions at the different developmental stages. Shown are numbers in individual animals as well as median and quartile values. Each point represents one larva. Statistical test: two-way ANOVA, ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001). Scale bars represent 10 μm. (D) Tg(β-actin2:mRFP-GFP-Lc3) larvae show mRFP-GFP+ autophagosomes ubiquitously distributed in the developing heart and accumulation of mRFP+-only autolysosomes in the atrioventricular canal (AVC) during development. Arrows point to double-positive puncta in atrioventricular valves. TgKI(mRFP-Lc3) expresses an mRFP-tagged version of the endogenous autophagosome backbone protein Lc3. This reporter line was crossed into the endocardial Tg(fli1a:GFP) (E) and myocardial Tg(myl7:GFP) (F) lines. Arrows mark mRFP+-puncta in endocardium and myocardium, respectively. See also Figure S2.