Zebrafish larval cardiogenesis and response to rescuing agents.
(A) The serial cryo-sections of whole larvae at 3 dpf were HE-stained and examined for the integrity of heart chambers. (B-E) Larval heart beats were measured at 3 dpf. The cardiac ejection fraction and cardiac output were calculated from the dimension of cardiac chamber (D; circled area) in the video still images (lateral view with anterior to the right) as described in Materials and Methods. Data were collected from at least 3 independent experiments with the total sample number of 14–23 for each group. (F) The cardiac development in both control and FD larvae was characterized by WISH with the riboprobe specific to cmlc2, a cardiac primordium marker. Representative WISH images of cmlc2 showed decreased or spatially altered (arrowheads) signal in FD larvae at 2 dpf. Images were taken from ventral view (upper panel) and lateral view with the head to the left (lower panel). (G) The severity of cardiac developmental anomalies observed in embryos with/without rescuing agents supplementation were categorized into normal, mild and severe and quantified based on the distribution pattern and intensity of the cmlc2 WISH signal, as shown in (F). (H) The distribution of bmp4 transcripts in embryos at 21 hpf was characterized by WISH. Bmp4 expressed predominantly on the left side of embryonic cardiac disc in both control and FD embryos. Embryos were shown in dorsal view with head to the top and the boxed region magnified in inset at the lower panel. (I-K) The larval responses to rescuing agents were also evaluated based on cardiac function, including heart beats, cardiac ejection fraction and cardiac output on 3-dpf larvae exposed to the indicated compounds. Data were collected from at least 3 independent experiments with a total embryo number of 15–50 for each group. C or CTL, heat-shocked non-fluorescent transgenic control; FD, folate deficiency; M or MFD, mild folate deficiency; S or SFD, strong folate deficiency; A, atrium; AVC, atrium-ventricle canal; V, ventricle; EDA, end-diastolic area; ESA, end-systolic area; 5-CHO, 5-formyltetrahydrofolate; NAC, N-acetyl-L-cysteine; FA, folic acid. *, p<0.05; **, p<0.01; ***, p<0.001.