Block the function of nonmuscle myosin II by blebbistatin induces zebrafish embryo cardia bifida
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Nonmuscle myosin II (NM II) is the name given to the multi-subunit protein product of three genes encoding different nonmuscle myosin heavy chains including NM II-A, NM II-B, and NM II-C. Blebbistatin is a small molecule that has been shown to be a relatively specific inhibitor of NM II. Blocking the function of NM II by blebbistatin induces zebrafish embryo cardia bifida at a dose-dependent manner. In situ hybridization analysis with ventricular marker ventricular myosin heavy chain (vmhc) and atrial marker atrial myosin heavy chain (amhc) showed each of the heart contained both distinct atria and ventricle. However, the cardia bifida embryos had highly variable distance between two separate ventricles. We also provided evidence that time window from 12 to 20 h post fertilization (hpf) is necessary and sufficient for cardia bifida formation caused by blebbistatin treatment. Expression of spinster homolog 2 (spns2) was decreased in blebbistatin-treated embryos, suggesting the cardia bifida phenotype caused by NM II inhibition was relevant to precardiac mesoderm migration defects. Through in situ hybridization analysis, we showed that foxa1 was expressed in endoderm of blebbistatin-treated embryos at 24-hpf stage, suggesting the endoderm formation is normal in cardia bifida embryos caused by blebbistatin treatment. In addition, we demonstrated that blebbistatin treatment resulted in morphology alteration of zebrafish cardiomyocytes in vivo and neonatal mouse cardiomyocytes in vitro.
KeywordsBlebbistatin Zebrafish Cardia bifida Heart Nonmuscle myosin II
This work is supported by Grants of NSFC (81100116, 31201083, 31400918, 81102524, 31401234), 12KJB180010, SBK201240531, 12Z053, and the initiation funding of the Affiliated Hospital GDMC (XB1226, BK201205).
XQW, MC, XW, HW, JZ, and HX carried out the experiments. XQW and MC did the revision of this work and analyzed the data. DL and JZ were primarily responsible for the experimental design, interpretation of the data, and writing the manuscript. All authors read and approved the final manuscript.
Conflict of interest
The authors declare that they have no competing interests.
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