Fig. 2

Fig. 2

Characterization of the transgenic zebrafish line that expresses the apoaequorin and EGFP genes, which are targeted to the skeletal muscle via a muscle specific α-actin promoter. (A) Expression of EGFP (see arrowhead) in the trunk musculature of a representative α-actin-aeq transgenic (F₁) embryo at 26 hpf. This embryo was bred by crossing the single pα-KS-Aeq-IRES-EGFP plasmid-injected founder fish (F₀) generated, with a wild-type fish. Ant. and Pos. are anterior and posterior, respectively. (B) Profile of apoaequorin expression in the F₁ transgenic embryos from 12 hpf to 24 hpf. (Bi) Representative example (n=3) of a Western blot and (Bii) a line graph (mean ± SEM; n≥3) to illustrate the relative level and temporal expression profile of apoaequorin in extract prepared from the transgenic embryos. Aeq, aequorin control; WT, wild-type embryos. Extract prepared from 3 embryos was loaded into each lane. (C) Cross section of the myotome of a representative (n=5) transgenic zebrafish embryo at 24 hpf illustrating (Ci) the expression of EGFP, (Cii) the slow muscles (labeled via immunohistochemistry with the F59 myosin heavy chain antibody), and (Ciii) the EGFP and F59-labeled images when merged, such that the yellow color indicates the region of overlap. (Di) A representative example (n=4) of the spatial pattern of Ca²⁺ transients generated in the trunk of an apoaequorin expressing transgenic embryo at ~18.5 hpf. This image represents 10 sec of accumulated light superimposed on to the appropriate bright-field image. The region of the embryo bounded by the black dashed square is shown at higher magnification in Dii. S1, S12 and S14 are somites 1, 12 and 14, respectively. Color scale indicates luminescent flux in photons/pixel. Scale bars are (A) 250 μm, (C) 10 μm, and (D) 200 μm.