Fig. 1

A Mortality, B hatching, and C malformations in zebrafish larvae 72 h after intra-yolk microinjections of MiR92b-3p mimic at 0.5 ng/embryo (MIM 0.5 ng; cyan diamonds) or 5 ng/embryo (MIM 5 ng; violet triangles), or intra-yolk microinjections with C. elegans MiR39-3p mimic at 5 ng/embryo (mismatch; green squares) compared to untreated zebrafish larvae (control; red circles). Individual points show rate of occurrence in individual Petri dishes used for incubation (i.e., replicates; n = 5 per group). Black horizontal line indicates mean rate of occurrence. p values for the difference in the rate of occurrence between the control and exposed groups were calculated using logistic regression
Fig. 2

Development of zebrafish embryos and larvae throughout the experiment. A Normal development of control larvae and B developmental malformations in embryos and larvae after intra-yolk microinjections with MiR92b-3p at 5 ng/embryo (MIM 5 ng). Embryos from the exposed group exhibited a yolk void at 24 hpf that developed into pericardial edema (black arrows)
Fig. 3

Representative phenotypes of zebrafish larvae 72 h after intra-yolk microinjections with A C. elegans MiR39-3p at 5 ng/embryo (mismatch) or B MiR92b-3p mimic at 5 ng/embryo (MIM 5 ng). In the mismatch group, zebrafish larvae had normal phenotypes, whereas the MIM-exposed larvae developed pericardial edema (black arrows) and had smaller eyes with abnormal morphology (white arrowheads)
Fig. 4

Morphology of eyes of zebrafish larvae at 72 hpf. A Normal eye structure of larvae from the control group and B–E morphological abnormalities of eyes in larvae microinjected with MiR92b-3p mimic at 5 ng/embryo (MIM 5 ng). The morphological changes in the exposed zebrafish usually manifested as smaller eye diameter, and as a lack of a lens and typical retina stratification (black arrow). In some zebrafish individuals, a vacuolized lens (or its remnants) was located in the delaminated cornea that was expanding outward (white arrowhead)
Fig. 5

Locomotor activity of zebrafish embryos at 24 hpf. A Burst count and B mean burst duration of zebrafish that received intra-yolk microinjections of MiR92b-3p mimic at 0.5 ng/embryo (MIM 0.5 ng; cyan diamonds) or 5 ng/embryo (MIM 5 ng; violet triangles), those that received C. elegans MiR39-3p mimic at 5 ng/embryo (mismatch; green squares), or those that were not treated with microinjections (control; red circles). Individual points represent values from individual Petri dishes used for incubation (i.e., replicates; n = 5 per group). Black horizontal lines indicate group means, and different letters indicate statistically significant differences between group means (one-way independent ANOVA followed by Tukey’s HSD test; p < 0.05)
Fig. 6

Successful delivery of MiR92b-3p into fertilized zebrafish eggs via intra-yolk microinjections. A Distribution of the MiR92b-3p mimic 3′-labeled with Alexa Fluor 555 in the yolks of the fertilized eggs up to 180 min after mimic injection at a dose of 1 ng/embryo (AF-MIM). B Localization of the fluorescent material in the zebrafish at 24, 48, and 72 hpf. Strong fluorescence was observed inside the eggs of the developing zebrafish embryos, but it was not observed in the hatched larvae at 72 hpf
Fig. 7

Relative expression of A MiR92b-3p and B MiR92a-3p in zebrafish larvae (at 72 hpf) that received intra-yolk microinjections of MiR92b-3p mimic at 0.5 ng/embryo (MIM 0.5 ng; cyan diamonds) or 5 ng/embryo (MIM 5 ng; violet triangles), those that received C. elegans MiR39-3p mimic at 5 ng/embryo (mismatch; green squares), or those that were not treated with microinjections (control; red circles). Individual points represent values from individual Petri dishes used for incubation (i.e., replicates; n = 5 per each group). Black horizontal lines indicate means, and different letters indicate statistically significant differences between group means (one-way independent ANOVA followed by Tukey’s HSD test; p < 0.05)
Fig. 8

Relative expression of A gata5, B sox17, C pax6a, and D pax6b mRNA in zebrafish larvae (at 72 hpf) that received intra-yolk microinjections of MiR92b-3p mimic at 0.5 ng/embryo (MIM 0.5 ng; cyan diamonds) or 5 ng/embryo (MIM 5 ng; violet triangles), those that received C. elegans MiR39-3p mimic at 5 ng/embryo (mismatch; green squares), or those that were not treated with microinjections (control; red circles). Individual points represent values collected from individual Petri dishes used for incubation (i.e., replicates; n = 5 per each group). Black horizontal lines indicate means
Acknowledgments
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ J. Appl. Genet.
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