Design and Microinjection of Morpholino Antisense Oligonucleotides and mRNA into Zebrafish Embryos to Elucidate Specific Gene Function in Heart Development

Abstract

The morpholino oligomer-based knockdown system has been used to identify the function of various gene products through loss or reduced expression. Morpholinos (MOs) have the advantage in biological stability over DNA oligos because they are not susceptible to enzymatic degradation. For optimal effectiveness, MOs are injected into 1-4 cell stage embryos. The temporal efficacy of knockdown is variable, but MOs are believed to lose their effects due to dilution eventually. Morpholino dilution and injection amount should be closely controlled to minimize the occurrence of off-target effects while maintaining on-target efficacy. Additional complementary tools, such as CRISPR/Cas9 should be performed against the target gene of interest to generate mutant lines and to confirm the morphant phenotype with these lines. This article will demonstrate how to design, prepare, and microinject a translation-blocking morpholino against hand2 into the yolk of 1-4 cell stage zebrafish embryos to knockdown hand2 function and rescue these "morphants" by co-injection of mRNA encoding the corresponding cDNA. Subsequently, the efficacy of the morpholino microinjections is assessed by first verifying the presence of morpholino in the yolk (co-injected with phenol red) and then by phenotypic analysis. Moreover, cardiac functional analysis to test for knockdown efficacy will be discussed. Finally, assessing the effect of morpholino-induced blockage of gene translation via western blotting will be explained.

Figure 1:. Steps for injection of MO solution into the yolk.

(A) Injection chamber. (B) Using a transfer pipette, transfer the collected embryos to the furrows. (C) Insert the needle through the chorion, inject near the boundary of cell/yolk to the embryo.

Figure 2:. Mounting and imaging Zebrafish embryos.

(A) The concave well imaging slide with zebrafish embryo under the microscope. (B) Magnified view of the well. Fill the wells ¾ of volume with E3 medium. (C) Positioning of the embryo should be on its left side. (D) Ventricle can be seen clearly in this configuration (zoomed image on left, ventricle borders are highlighted). Anterior is to the left. (E) Moving red blood cells (RBC) in major vessels, such as the dorsal aorta or the Posterior Cardinal Vein. Anterior is to the left.

Figure 3:. Analysis of the whole heart to detect the presence of cardiac edema and/or any structural defects.

(A) A normal 3 dpf embryo heart. (B) Cardiac edema and looping defect in 3 dpf zebrafish embryo. Anterior is to the left.

Figure 4:. Measurement of ventricle size.

(A) Ventricular cavity and myocardial wall are highlighted. Long axis and short axis diameters are seen for (B) end-diastole and (C) end-systole.

Figure 5:. Automatic detection of vessels using ZebraLab.

(A,B) The two major blood vessels in zebrafish: the dorsal aorta (DA) and the posterior cardinal vein (PCV). (C) Viewpoint program used to quantify blood flow and vessel diameter in DA.

Figure 6:. Hand2 MO titration.

Embryonic survival for different Hand2 MO concentrations was converted to a percentage, averaged, and plotted. The percent survival of MO-injected embryos was calculated at 24, 48, and 72 hpf. All data points represent mean ± SEM (100 embryos were used in each group; experiment was performed in triplicate). The analysis was done by two-way-ANOVA with Dunnett test. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Figure 7:. Hand2 morphants exhibit distinct and specific phenotypes.

Hearts and tails of 72 hpf embryos were examined under a light microscope (100x magnification). (A,B) Control scrambled MO shows normal heart development without pericardial edema. (C,D) 0.4 mM HAND2 MO-injected embryos as positive control shows tube-like heart structure.

Figure 8:. Assessment of cardiac function

(A) Dorsal aorta blood flow analysis, (B) Posterior Cardinal Vein (PCV) blood flow analysis. 1–4 cell stage zebrafish embryos were injected as groups with scrambled MO, Hand2 MO, Hand2 MO + Hand2 mRNA rescue, and scrambled MO + Hand2 mRNA rescue. Un-injected embryos were used as control. All data are presented as mean ± SEM (6 embryos were used in each group; experiment was performed in triplicate). The analysis was done by one-way ANOVA with Sidak post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.