Fig. 4

Microinjection of MMP21 mRNA c.731G > A and c.1459A > G failed to rescue the zebrafish heart looping disorder. The WT and mutant human MMP21 mRNAs synthesized using in vitro transcription were separately microinjected or co-microinjected (WT/mutant = 1:1) into 1–2 cell stage zebrafish embryos accompanied by SB-MO to compensate for the mmp21 knockdown. The zebrafish heart looping disorder phenotype at 48 hfp was observed and analyzed. Heart tube looping abnormalities include L-loop, ambiguous looping, or no looping. Left panels show all phenotypes after separate microinjection or co-microinjection of WT and mutant MMP21 mRNAs to zebrafish embryos. The right panels show normal and abnormal heart looping phenotypes. A, B Compared to the SB-MO group, MMP21 WT mRNA effectively reduced the number of malformed phenotypes. Mut1 and Mut3 could not effectively compensate for the effects of mmp21 knockdown. The effect of Mut2 was similar to that of the WT mRNA. C, D When co-microinjected with the WT and mutant mRNAs at a 1:1 ratio, no statistical difference in the malformation rate between the WT&Mut2 group and the WT mRNA group was noted. The malformation rate of the WT&Mu1 and WT&Mu3 groups was significantly reduced compared to that of the SB-MO group; however, it remained significantly higher than that of the WT mRNA group (chi-square test was used for statistical calculation, $ p < 0.0001 versus control MO group, #### p < 0.0001 versus SB-MO group, ## p < 0.01 versus SB-MO group, **** p < 0.0001 versus WT mRNA group, *** p < 0.001 versus WT mRNA group, ns: nonsignificant)