Characterization of yulink-MO KD zebrafish morphants. a Expression of yulink in zebrafish using whole-mount in situ hybridization. Embryos were fixed overnight at 4 °C in 4% paraformaldehyde buffered with 1 × phosphate-buffered saline (PFA/PBS). After hybridization with Dig-labeled antisense or control sense RNA probes of yulink, embryos were incubated with anti-Dig antibody conjugated to AP and developed with NBT-BCIP reagents. The yulink was expressed in whole zebrafish embryo ubiquitously from 0.5 hpf (zygote stage) to 3 dpf (larval stage) with lateral overview. The black arrows point to heart regions at 24–30 hpf and 3 dpf with ventral overview. The black arrowheads point to yolk of embryos, hour post-fertilization (hpf); day post-fertilization (dpf). b After injection with yulink-MO to knockdown yulink expression, the yulink KD morphants presented with small eyes, a small head, abnormal blood circulation, and pericardial edema at 3 dpf. Increasing the yulink-MO dosage from 2.3 ng (n = 119) to 4.6 ng (n = 119 or 9.2 ng (n = 156) caused the proportion of severely affected embryos with pericardial edema to increase from 6 to 54% (purple). c Hemodynamic changes. Yulink KD morphants had a slower heart rate and reduced cardiac output, as compared to WT embryos at 2 dpf (** p < 0.01, Student’s t test). d Diagram of the EYFP-fusion construct with the yulink 5′-UTR region and partial coding region (amino acids 1–79). The position of the corresponding MO binding site is indicated by a bar. To demonstrate the specificity of the yulink-MO, embryos were injected with pYulink-EYFP plasmid (100 pg/embryo) alone, or together with yulink-MO or the mismatch control, yulink-5mmMO (4.6 ng/embryo). About 58% of embryos injected with pYulink-EYFP plasmid alone exhibited fluorescence at 1 dpf, while fluorescence was absent in embryos co-injected with yulink-MO. In contrast, co-injection with yulink-5mmMO did not affect EYFP expression
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