ZFIN Image: Flanagan-Steet et al., 2018, Fig. 7

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Figure Caption

Fig. 7

chst11 Inhibition Inhibits Ctsk Processing

(A) Immunoblot of Chst11 in MO-inhibited WT embryos 2–4 d. MLII data and quantitation are shown in Figure S6. n = 3 experiments. d, dpf.

(B) Immunohistochemistry for C4-S (red) in chst11 MO-inhibited embryos. n = 15–20 embryos per condition from 3 experiments. Percent values are the number of embryos that resemble the picture. Scale bar, 10 μm.

(C) Gels of BMV109-labeled chst11-inhibited embryos. n = 4 experiments.

(D) Quantitation of the Ctsk band. Error bars, SD; ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.

(E) Live confocal analyses of the BMV109 label (red) in chst11-inhibited EGFP-positive chondrocytes. n = 10–15 embryos per condition. Percent values are the number of embryos that resemble the picture. Scale bar, 10 μm.

(F) Alcian blue-stained WT, MLII, and chst11-inhibited embryos. n = 100–150 embryos per sample from 3 experiments. Percent values are the number of embryos resembling the picture. Scale bar, 10 μm.

(G and H) Immunoblot of Ctsk 24–80 hpf in chst11-inhibited WT (G) and MLII (H) embryos. n = 3 experiments.

(I) Table listing the abundance (relative to all Ctsk) of the pro and mature form.

(J) Live confocal analyses of cartilage-localized (green) TGF-ß signaling using the SBE:nucCherry reporter. n = 10–15 embryos per condition from 2–3 independent experiments. Percent values are the number of embryos resembling the picture. Scale bar, 10 μm.

(K) Schematic of the current model suggesting that TGF-ß-mediated increases in C4-S promote increased activation of extracellular Ctsk in the lysosomal disease MLII. Although not currently known, it is also possible that reduced expression of inhibitory GAGs may contribute.