Figure 7

Figure 7

(A) Schematic illustrates Gal4-UAS bipartite expression system, which restricts expression of Flag-tagged Ctsk to either myocardial (driven by myl7:GAL4) or endocardial (driven by fli1a:GAL4) cells. (B) Immunoblotting with anti-Flag antibodies for 1:WT, 2:N216Q, 3: C139S;N216Q Ctsk transgenes in gal4^–or gal4⁺ embryos show each is expressed and reveals increased protein stability and processing of hypersecreted (N216Q) Ctsk variant (red arrows denote cathepsin K) (C) Analyses of Ctsk activity in pools of 15 embryos show that, like MLII embryos, expression of the N216Q-secreted variant is associated with more enzyme activity than induction of the WT Ctsk transgene. Data are presented as mean ± SEM. **P < 0.01. ***P < 0.001 using Dunnett’s test (denoted by red box). n = 4 experiments. (D) Confocal analyses of sections of embryos expressing WT or N216Q-secreted forms of Ctsk stained for the transgene (red) and extracellular matrix (WGA, blue) show WT Ctsk is retained within cells (as it is distinct from WGA stain), while the N216Q variant is secreted. This is illustrated by overlap with extracellular WGA (white arrowheads). Panels 1, 2, and 3 represent higher-power views of regions of interest. (E) Confocal analyses of 4 dpf hearts in progeny of fli1a:gal4 and UAS:ctsk parents expressing either WT, N216Q-, or C139S;N216Q-secreted forms of Ctsk. The genotype with regard to gal4 (+ or –) and ctsk is denoted on each panel. Embryos are cmlc2:EGFP⁺ (green) and stained immunohistochemically for myosin (red). Images show forcing secretion of Ctsk (N216Q) from endocardial cells does not alter heart morphology, although atria are often slightly smaller. (F) Confocal analyses of 4 dpf hearts in progeny of myl7:gal4 and UAS:ctsk parents. The genotype of gal4 (+ or –) and ctsk (WT, N216Q, or C139S;N216Q) is denoted. Images show that forcing secretion of Ctsk (N216Q) from myocardial cells alters heart morphology. n ≥ 30 embryos from 4 experiments. Scale bar: 20 μm. (G and H) In situ analyses of notch1b in animals expressing WT or a cathepsin K variants (N216Q or C139S;N216Q) secreted from endocardia (G, fli1a:gal4) or myocardia (H, myl7:gal4) show secreting active Ctsk from either tissue impairs valve cell differentiation (arrow heads versus expanded arrows). Control animals are gal4^–. Percent values represent the number of animals exhibiting phenotypes. n = 30–35 embryos from 3 experiments. Scale bar: 50 μm. (I) Schematic summarizing outcomes of Gal4-UAS experiments. V, ventricle; A,atrium.