Characterization of zebrafish Atx. (A) Expression patterns of zebrafish Atx during early development was observed by WISH. (B) Embryos injected with 5 ng Std MO, or 2.5 or 5 ng atx MO were cultured to shield stage and subjected to western blotting analysis. Expression of Atx was dose-dependently inhibited by atx MO. Molecular weight is shown in KD on the left-hand side. (C) Uninjected embryos and embryos injected with 2.5 ng of an atx splicing blocking MO (sMO) were incubated to 26-28 hfp and subjected to cardiac jogging analysis by WISH against cmlc2. Total number of embryos used in each treatment is shown on top of each column hereafter. (D) Human ATX (hATX) and zebrafish Atx (drATX) were expressed in HEK293T cells. LysoPLD activity of secreted Atx in the medium was measured by hydrolysis of FS3 (2.5 mM) as described in Materials and methods. (E) Representative patterns of cardiac jogging phenotypes in Atx- LPAR3 loss-of-function conditions. (F) Embryos were injected with or without HA51 at the one-cell stage, incubated and fixed at 48 hpf. Fixed embryos were subjected to o-dianosidine staining and WISH against cmlc2. Representative images are shown. (G) Cardiac jogging of embryos incubated with various concentrations of HA130 from 50% epiboly to tail bud stage (marked by ‘E’ for early) or embryos incubated with various concentrations of HA130 from 60% epiboly to tail bud stage (marked by ‘L’ for late) was examined and quantified (n=3). (H) Cardiac jogging of embryos incubated with various concentrations of Ki16425 from 50% epiboly to tail bud stage was examined and quantified (n=3).
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