Transient expression of RNA transcripts in zebrafish embryos. The injection dosage of each RNA transcript was optimised for comparison (RAF1: 50 pg/embryo; RIT1: 400 pg/embryo; A2ML1: 200 pg/embryo). A morphometric analysis was performed to compare the effect of the VUSs compared to the wild-type transcript at three dpf. Statistical significance was derived using a two-sided Fisher’s exact test to compare mutants with the corresponding WT. **p < 0.01; ***p < 0.001; ns: not significant. NIC: No injection control; (a) Proportion of zebrafish embryos with normal or diseased phenotypes. (b) Representative zebrafish embryos with a normal phenotype (c) craniofacial dysmorphism (d) gross malformations and (e) cardiac oedema.

Craniofacial assessment of zebrafish embryos. The zebrafish embryos were treated with Alcian blue and washed with acidic alcohol 3 dpf. The ratio of the width of the ceratohyal (x) to the tip of Meckel’s cartilage (y) was used as a measure of craniofacial defects. (a) Comparison of the x-to-y ratio between mutant and wild-type zebrafish embryos. The data are the mean ± SD. Statistical significance was derived using an unpaired t-test comparing the mutants with the corresponding WT. **p < 0.01; ****p < 0.0001; ns: not significant. NIC: No injection control. The number in bold represents the total count of zebrafish embryos in the experimental group. (b) Alcian-blue stained cartilage in the zebrafish. The x-axis shows the width of the ceratohyal, and the y-axis shows the length of the tip of Meckel’s cartilage. (c) Representative embryos with different x-to-y ratios.

Structural assessment of cardiac tubes by in situ hybridization of cmlc1. Statistical significance was derived using a two-sided Fisher’s exact test to compare mutants with the corresponding WT. **p < 0.01; ns: not significant. (a) The proportion of zebrafish embryos with a normal or deformed heart tube structure. (b) Representative embryos with a normal and (c) deformed heart tube.