Microinjector

The morpholino (MO) antisense oligonucleotide MO-Tbx5a (5′-
GAAAGGTGTCTTCACTGTCCGCCAT-3′) was purchased from Gene Tools (Philomatch, OR, USA). Human miR-30a, mir-9 and a negative control were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Zebrafish were raised under standard conditions at 28.5 °C. Each 1-2-cell stage embryo was injected with a constant injection of 5 ng MO, 100 pg TBX5 mRNA and 100 pg miRNAs using a microinjector (Narishige, Japan). Twelve hours post-injection, the dead embryos were removed, leaving only viable embryos that were used for further analysis. Consistent with the previously published studies [18 (link), 55 (link)], all live embryos were divided into the four categories according to their heart morphologies. At 48-h post fertilization (hpf), images were acquired with an Olympus stereomicroscope microscope or Leica TCS-SP5 LSM confocal microscope. For confocal imaging analysis of zebrafish embryos, they were anesthetized with egg water/0.16 mgml⁻¹ tricaine/1% 1- phenyl-2-thiourea (Sigma-Aldrich, St Louis, MO, USA) and embedded in 0.6% low melting agarose. Confocal imaging analysis was performed using Imaris software. Two transgenic zebrafish lines: Tg(vmhc:eGFP) and Tg(vmhc:mCherry-NTR) were used as described in previous work [55 (link)]. Whole-embryo microRNA sensor assay in zebrafish was carried out as described previously [56 (link)].

We used a Narishige microinjector to inject one-cell-stage zebrafish embryos with 2 ng of translation blocker cdh23_morpholino (Söllner et al., 2004 (link)) or 4 ng of the translation blocker cdk5_morpholino (5′-CCAGCTTCTCAT -ACTTTTGCATGGT-3′) (Easley-Neal et al., 2013 (link)). Efficiency of the cdk5_morpholino was evaluated as previously described (Tanaka et al., 2012 (link)). Injection volumes were estimated using a micrometer.

PADI1-targeting morpholino (5′-GTCGAGCTTCCAGTCTCCTGGTC-3′) was purchased from Gene Tools LLC (Philomath, OR, USA), and then diluted with water to give a working concentration of 1 mM. About 5 pl morpholino solution was microinjected into the zygotes obtained as described above using a Narishige microinjector (Tokyo, Japan). A non-targeting MO was injected as a control. After microinjection, zygotes cultured in fresh KSOM medium under mineral oil in a 5% CO2 incubator at 37 °C for subsequent development.

Oocytes were microinjected as previously described [77 (link)] using an Narishige microinjector (Narishige Inc., Sea Cliff, NY, USA) with an Olympus IX70 microscope (Olympus, Center Valley, PA, USA) equipped with an Eppendorf TM FemtoJet TM 4i microinjector (Eppendorf, Hauppauge, NY, USA) and completed within 30 min. For the overexpression experiments, 10 pL Myc-Tfap2a mRNA solution (1 μg/μL) was injected into the cytoplasm of denuded GV oocytes. The same amount of Myc mRNA (1 μg/μL) was injected as a control group. Following microinjection, the oocytes were arrested at the GV stage in α-MEM containing 10% (v/v) fetal bovine serum (Gibco-Invitrogen, Karlsruhe, Germany) and 200 nM IBMX for 12 h. Then, the oocytes were washed and cultured in an IBMX-free α-MEM medium (containing 10% (v/v) fetal bovine serum) under a humidified atmosphere of 5% CO₂ at 37 °C for 14 h.