The present protocol describes designing, preparing, and microinjecting a translational-blocking morpholino against a representative cardiac gene; Heart And Neural Crest Derivatives Expressed2 (hand2) into the yolk of newly fertilized zebrafish embryos to knock down gene function. It also shows a transient rescue of these “morphants” by co-injection of mRNA encoding this gene product.
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.
The utilization of zebrafish as a model for the study of cardiovascular development and disease offers a variety of advantages, including high conservation of gene function, optical transparency, rapid cardiovascular development, and cheaper cost when compared to traditional in vivo models1. Morpholino oligonucleotides (MOs) are the most commonly used antisense gene knockdown tools for the zebrafish model. MOs are frequently used to determine a phenotype or to probe gene function. Dr. James Summerton initially developed the morpholino delivery system for the in vivo inhibition of mRNA translation as an attempt to develop therapeutics for human developmental defects2,3. MOs have been used for in vitro and in vivo model organisms to knockdown genes and investigate the consequence of this knockdown on phenotype. This is done by observing alterations in the development of specific organs, for example, the heart. Knockdown of heart-specific genes in WT zebrafish embryos led to the failure of a proper heartbeat, attesting to the indispensable function of these genes for heart development4,5. These phenotypes were rescued by co-injection of mRNAs for the specific genes. A study involving cardiac troponin T (Tnnt2) showed that the expression of full-length tnnt2 mRNA could rescue sarcomeric phenotypes caused by morpholino knockdown6. Another study revealed that the integrity of A-bands and Z-discs could be restored by overexpression of the regulatory myosin light chain ortholog (cmlc2) mRNA in cmcl2 morphants7.
MOs are commonly used to knock down gene expression by targeting pre-mRNA splicing or by blocking translation. Splice blocking MOs bind and inhibit pre-mRNA by inhibiting the splicesome. Translational blocking occurs when the MO binds to the 5'-untranslated region of complementary mRNA to hinder the ribosome assembly. MOs are the most widely used gene-specific method to knockdown gene expression for in vivo models; they are also the most efficient mRNA blocking agents used in cell cultures. The morpholino itself typically consists of a short-chain (around 25) of morpholino subunit bases. Each MO subunit includes a nucleic acid base, a morpholine ring, and a non-ionic phosphorodiamidate. The different mechanisms of action for the two types of MOs necessitate different tests to verify the efficacy of the knockdown. For translation blocking MOs, a western blot analysis is the most reliable test of efficacy, as the protein of interest should not be produced due to blockage of the ATG translation start site. MOs do not directly degrade their target mRNA; instead, they bind to specific regions and inhibit the expression until naturally degraded. However, the splice blocking MOs modify the pre-mRNA by inducing splice modification, which can be assayed by reverse-transcriptase polymerase chain reaction (RT-PCR) and gel electrophoresis.
Three crucial parts of the MOs screening process must be standardized: (i) The MO dose curve must be tuned for phenotypic recognition. The dose curve also shows the lethal dose 50 (LD50: the dose at which 50% of injected embryos die) for each MO tested to improve the ability to optimize phenotypic 'signal' versus off-target 'noise'3. (ii) The phenotyping nomenclature that was adapted should be well documented; a precise and easily understandable phenotypic description is critical to provide extensive explanations based on existing literature and investigator experience to facilitate information sharing among those who did not directly examine the embryos. (iii) Having well-defined language makes it easy to collect data centrally from Morpholino Database8.
In MO knockdown studies for cardiac genes, animals' heart activity and blood flow dynamics must be monitored in order to determine the impact of MO knockdown experiments on cardiovascular system function. Such analyses require real-time visualizing of the cardiovascular system at high resolution. Zebrafish skin is transparent for the first week of development, enabling visualization of the heart and blood circulation via microscopy. For assessment of heart function, the most calculated physiological parameters are heart rate and cardiac output as well as fractional shortening, fractional area change, and ejection fraction. Blood flow velocities can be measured by tracking moving RBCs, and these measurements are used to determine shear stress levels, a crucial mechanobiological factor on endothelial cells. Such an assessment requires recording time-lapse movies for beating heart and flowing blood via an inverted or a stereomicroscope equipped with a high-speed camera.
This paper shows how to design, prepare, and microinject a translational-blocking morpholino against a gene of interest into the yolk of freshly fertilized zebrafish embryos to knock down gene function. It will also show rescuing these "morphants" by co-injection of mRNA encoding this gene. We will then analyze the efficacy of the morpholino microinjections through phenotypic characterizations as well as cardiac structural and functional analyses. This approach will be demonstrated on a widely studied cardiac gene, hand2.
All experiments were carried out in accordance with the accepted standards of humane animal care under the regulation of the IACUC at QU; animals were held in the zebrafish facility under Qatar University Biomedical Research Center (QU-BRC). All animals used in these experimental studies were under 3 days post-fertilization (dpf).
NOTE: For each experimental group, it is advisable to use at least 30 embryos for statistical rigor. The experimental groups are as follows:
Control group: This group includes embryos cultured in egg water without any injections. Results here will form the control baseline.
Negative Control group: This group includes embryos cultured in egg water injected with scrambled MOs.
Injected group: This group includes embryos injected with hand2 MO alone and hand2 MO with hand2 mRNA to rescue the phenotype. Results here will confirm that observed phenotypes appeared due to injected MOs. Comparison of experimental groups will enable assessment of the influence of inhibiting and rescue of hand2 on heart function precisely.
1. Morpholino designs for hand2.
NOTE: MO sequences can be adapted from the literature9,10,11. Alternatively, these oligos can be designed online by Gene-tools. Gene-tools offers a free and fast online design service, which can be accessed through their website12. A custom MO can readily be designed by providing information about the genes of interest, such as sequence information or accession numbers. The following specific steps summarize how to design MOs against hand2 in zebrafish:
2. Preparation of morpholino injection
3. Injection of MO and mRNA solution into the yolk
4. Western blot to verify the success of morpholino knockdown
5. Cardiac structure and function assessment:
The graph in Figure 6 illustrates the average percent of embryos surviving at 24, 48, and 72 hpf for both HAND2- specific MO and control scrambled MO-injected embryos. The 1 mM (8 ng/µL) and 0.8 mM (6.4 ng/µL) MO-injected embryos showed a significant reduction in survival percentage compared to control scrambled MO-injected embryos. This was observed across each measured time point where lethality or malformation was observed. The results indicated that a high concentration of HAND2MO had a significantly lower survival percentage due to off-target effects and toxicity of MO. While 0.4 mM (3.2 ng/nL) HAND2 MO-injected embryos did not show a significant reduction in survival percentage; however, some similar phenotypes in the experimental group were noted, such as delayed in heart development, elongated tub-like structure heart, and pericardial edema in comparison to control scrambled MO-injected embryos (Figure 6).
HAND2 knockdown embryos exhibited distinct and specific phenotypes. Phenotypes of the morphants were examined between 24 and 72 hpf under a Light Microscope. HAND2 loss of function gave rise to embryos with a delay in heart development and an elongated heart, shaped like a tube with pericardial edema (Figure 7). As a result, embryos with this defect in heart formation behaved differently in terms of cardiac performance in comparison to scrambled MO (Figure 8).
In our investigation, we have characterized the changes in the zebrafish heart shape, cardiac output, and blood flow in control and HAND2 knockdown animals. Blood flow velocity was measured by tracking down moving RBCs, which were used to determine shear stress levels, an important mechanobiological factor on endothelial cells.
Cardiac output and blood flow analyses showed that HAND2 knockdown triggered a delay in heart development in comparison to wild-type embryos. This alteration resulted in a decrease in DA blood velocity, a drop in heart pulse, and a reduction in cardiac output (Figure 8A). In agreement with the negative impact of HAND2 knockdown on cardiac function, shear stress was also reduced significantly (Figure 8A) in comparison to the control scrambled MO-injected embryos. Most of the heart function failure was rescued when HAND2 MO was co-injected with human HAND2 mRNA, which resulted in an increase in DA blood velocity, increase in heart pulse, and restoration in cardiac output (Figure 8A). These results are in agreement with cardiac output and blood flow analysis results for PCV (Figure 8B). Collectively, the data suggested that HAND2 might play a critical role in cardiomyogenesis during zebrafish development.
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 Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
Morpholino (MO) technology has been extensively used in zebrafish, xenopus, sea urchins, and more recently in cell culture model systems. With most methods, along with the benefits, there are also pitfalls that the experimenter should be aware of. One of the major pitfalls with MO technology includes the concern that phenotypic effects observed by the MO-mediated gene knockdown approach are not due to the loss of function associated with the primary gene product but that some other genes along with the primary gene or independent of the primary gene that has been targeted, and thus this so-called "off-target" effect is the reason for the "observed" morphant phenotype. This point came to the forefront when poor correlation was observed between morphants and mutants for the same gene in zebrafish32. Thus, several recommendations and guidelines for morpholino use in zebrafish field2,3 have been established, and it is recommended that experimenters follow these guidelines. In our view, when possible, a second independent complementary approach (CRISPR) confirming the morphant or mutant phenotype is desirable. A second limitation includes the ability to selectively knockdown a gene in space and time using MOs. Yes, photoactivatable MOs33 are available, but genes with complex expression patterns and those that have distinct functions at different times in development (early vs. late) will be difficult to target using this method. Further, the use of high light intensities to uncage/activate the MOs may damage the embryonic tissue, which precludes its widespread use. A third limitation of MO is a general one for knockdown technology wherein only transient blocking of gene transcription is possible, and from a model system perspective, MOs will only work effectively in organisms like zebrafish, Xenopus, sea urchins where organogenesis occurs within a short time (2-3 days) period. This time frame limits the use of the technology in higher order organisms, and in adults. Some reports have emerged for in vivo MO use in mice34 although, such approaches have not gained widespread adoption. Finally, non-coding RNAs that comprise the bulk of our genome is a challenge to target with MOs. MOs have successfully targeted short non-coding RNAs (miRNAs)35 but long (>200 bp) non-coding RNAs without translational start site or definitive exon-intron splicing sequence information remains a challenge.
In this study, we investigate HAND2, a transcription factor that regulates cardiac development. Its expression domain determines the heart-forming region in the anterior lateral plate mesoderm (ALPM)36,37,38. HAND2 has an early impact on cardiac progenitor cells formation; consequently, HAND2 knockdown embryos were used as a positive control to assess defective heart development. To examine the involvement of HAND2 in zebrafish embryonic heart development, its translation was interrupted using antisense MO that was designed to target the 5' untranslated region (UTR). The lack of evidence describing the optimal concentration of MO required for HAND2 silencing in zebrafish necessitated the optimization of the MO dosage.
Zebrafish injected with scrambled MO had a similar survival percentage as compared to un-injected embryos. This finding rules out any embryo lethality attributed to damage from the morpholino injection solution. However, the low survival percentage before 12 hpf could be attributed to the injection of unfertilized embryos. Several concentrations were tested to ascertain the optimal dosage in which knockdown would be achieved while maintaining the normal development of the fish. Fish should appear healthy and not developmentally stunted, which would indicate a delicate balance between phenotypic 'signal' versus off-target 'noise has been obtained3. For clarity, only the optimum amount used is shown in Figure 6.
After successfully optimizing the condition for MO injection, we further examined whether HAND2 knockdown affected heart function. Cardiac defects in HAND2 knockdown zebrafish embryos generally manifested as pericardial edema and faulty circulation at an early stage39. For functional analysis, we began with studying heart rate, as it is an important factor for the assessment of pathological heart function in zebrafish27,40. HAND2 MO-injected embryos exhibited a significant decrease in heart rate, confirming that HAND2 is important for heart function. Successful rescue of HAND2 expression was confirmed at both structural and functional levels by assessing the protein level as well as the embryo's phenotype, as shown in cardiac function analyses. The results in Figure 8 show that HAND2 expression was significantly diminished and that expression was significantly rescued when the HAND2 MO was co-injected with human HAND2mRNA. This result indicated that HAND2 knockdown is the cause of the induced phenotype.
Our investigations confirmed that in comparison to wild-type, HAND2 knockdown embryos exhibited a delay in heart development accompanied by a reduction in the number of red blood cells and an overall decrease in blood flow and cardiac output.
The MO-based knockdown system has been used to identify the roles played by various gene products through a lack of functional testing. It has the advantage in biological stability over DNA oligos since MOs are not susceptible to enzymatic degradation. For optimal effectiveness, they are injected into 1-4 cell stage embryos. The temporal efficacy of knockdown is variable, and the oligo is believed to eventually lose its effect due to dilution. This protocol details the biological analysis and structural/functional assessment steps to determine if a specific phenotypic abnormality is produced when a cardiac-specific gene is targeted via MOs in zebrafish.
The authors have nothing to disclose.
The publication of this article was covered with a generous support from BARZAN HOLDINGS. RR is partly supported by R61HL154254 and funds from Department of Pediatrics and Children’s Hospital.
Acrylamide 40% | Sigma | Sigma, cat. no. C977M88 | |
Agarose | Sigma-Aldrich | Sigma-Aldrich cat. no A9539-250G | |
All Prep DNA/RNA Mini Kit | Qiagen | Qiagen cat. no. 80204. | |
alpha Tubulin | Abcam | Abcam- ab4074 | Rabbit polyclonal to alpha Tubulin lot GR3 180877-1 (50 kDa) |
Ammonium persulfate molecular grade | Sigma | Sigma, cat. no C991U65 | |
BV10 capillary beveller | Sutter Instruments Product | Sutter Instruments Product Catalog # BV10 | |
Chemiluminescence Imaging Gene Gnome | SYNGENE | SYNGENE | |
Cleaver Scientific Blotting | CVS10D_OmniPAGEMini | CVS10D_OmniPAGEMini | |
Coomassie | Thermo Fisher | Thermo Fisher cat. no C861C44 | |
Electrochemiluminescence (ECL) kit | Abcam Biochemicals | Abcam Biochemicals cat. no ab65623 | |
Glycine | Sigma | Sigma, cat. no C988U91 | |
Goat anti Rabbit | Abcam | Abcam- ab6721 | Goat Anti-Rabbit IgG H&L (HRP) 2nd antibodies lot GR3179871-1 |
HAND2 | Gene tools | Custom made for HAND2 (NM_021973) | 5'-CCTCCAACTAAACTCATGGCGAC AG-3' |
Hand2 | Abcam | Abcam- ab10131 | Rabbit polyclonal Anti-HAND2 antibody lot GR143200-9 (24- 26 kDa) |
HAND2 (NM_021973) Human Tagged ORF Clone | OriGene Technologies, Inc | RC224436L3 | Vector: pLenti-C-Myc-DDK-P2A-Puro (PS100092) |
IBI DNA/RNA/Protein Extraction Kit | IBI Scientific | IBI Scientific cat. no -r IB47702 | |
Imaging System | iBright | iBright CL1000 Imaging System | |
Isopropanol | Sigma-Aldrich | Sigma-Aldrich cat. no 278475-2L | |
Laemmli sample loading buffer (4x) | Sigma-Aldrich | Sigma-Aldrich cat. no 70607 | |
Mercaptoethanol | Sigma | Sigma, cat. no M6250-1L | |
Microplate Spectrophotometer with the Gen5 Data Analysis software interface | Epoch | Epoch | |
Microscope | Ziess SteREO Lumar V12 Flourescence Microscope | Ziess SteREO Lumar V12 Flourescence Microscope | |
Mineral oil | Fisher Scientific | Fisher Scientific cat. no 0121-1 | |
mMESSAGE mMACHINE T7/T3/SP6 Transcription Kit | Thermo Fisher | Thermo Fisher cat. no.AM1340 | for mRNA generation |
Nuclease-free water | New England Biolabs | New England Biolabs cat. no B1500L | |
PC-100 Micropipette Puller | NARISHIGE GROUP Product | NARISHIGE GROUP Product Catalog # PC-100 | |
Phenol red | Sigma | Sigma, cat. no. P-0290 | |
Picolitre Injector | Harvard Apparatus | Harvard Apparatus cataloge # PLI-90A | |
Pierce Bicinchoninic acid assay (BCA) Protein Assay kit | Thermo Fisher | Thermo Fisher cat. no 23227 | |
PMSF, Protease inhibitor as protease inhibitors | Thermo Fisher | Thermo Fisher cat. no 36978 | |
Ponceau S | Sigma-Aldrich | Sigma-Aldrich cat. no 10165921001 | |
Protease Inhibitor Cocktail | Thermo Fisher | Thermo Fisher cat. no 88668 | |
Protein ladder | SMOBiO | SMOBiO cat. no PM2500 | |
Radioimmunoprecipitation Assay (RIPA) | Thermo Fisher | Thermo Fisher cat. no 89900 | |
Ringer’s solution | Thermofisher | Catalog No.S25513 | |
SDS | Sigma | Sigma, cat. no 436143 | |
Standard Control | Gene tools | SKU: PCO-StandardControl-100 | 5'-CCTCTTACCTCAGTTACAATTTAT A-3'- that targets a human beta-globin intron mutation |
Stripping buffer | Sigma-Aldrich | Sigma-Aldrich cat. 21059 | |
Temed | IBI scientific | IBI scientific cat. no C000A52 | |
Tris Base | Thermo Fisher | Thermo Fisher cat. no BP-152-500 | |
Tween | sigma life science | sigma life science cat. no P2287 | |
Zebra Box Revolution-Danio Track system chamber with the EthoVision XT 11.5 software | Noldus Information Technology, NL | Noldus Information Technology, NL | |
Zeiss Axiocam ERc 5s | Zeiss | Stemi 508 Zeiss | |
Zeiss Stemi 2000-C | Zeiss | Stemi 2000-C |
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