Fig. 5

apol1 interacts with myh9 in an anemic context.

To test for epistatic effects of apol1 and myh9 in zebrafish, we first co-injected both apol1-MO (1.0ng/nl dose) and myh9-MO (6.0ng/nl dose) into zebrafish larvae and scored for edema formation at 5 dpf. (n = 39–89 embryos/injection; repeated three times). However, under this co-suppression model (A, B), we observed no significantly increased edema formation compared to each MO alone. We next tested for an interaction between apol1 and myh9 in the context of atpif1a suppression, predicting that the added stress of anemia would mimic our initial observations in sickle cell disease patients. 70kDa dextran-FITC conjugate was injected into the cardiac venous sinus of 48 hpf zebrafish larvae and fluorescence intensity in the eye vasculature was measured at 24 and 48 hours later. (C) Representative eye image series of zebrafish embryos for each injection group show relatively stable or decreased fluorescence intensity over time. (E) Bar graphs summarize the changes observed for each injection group. Zebrafish embryos injected with all three MOs show a significant increase in dextran clearance from the vasculature compared to co-suppression of apol1 and myh9. (D, F) These data are reproduced using butafenacil induced anemia (0.195 µM in embryo media, treated at 48 hpf). Dextran values are in relative fluorescence intensity, mean ± SE. Control, sham-injected control (n = 19); atpif1a MO injected (n = 14); apol1-MO+myh9-MO (n = 12); apol1-MO+myh9-MO+atpif1a-MO (n = 11); Butafenacil (n = 48); But+myh9-MO+apol1-MO (n = 18). hpf, hours post-fertilization; hpi, hours post-injection. *p<0.001.