Abstract
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.
Similar content being viewed by others
References
Allingham JS, Smith R, Rayment I (2005) The structural basis of blebbistatin inhibition and specificity for myosin II. Nat Struct Mol Biol 12:378–379
Bakkers J (2011) Zebrafish as a model to study cardiac development and human cardiac disease. Cardiovasc Res 91:279–288
Bao J, Ma X, Liu C, Adelstein RS (2007) Replacement of nonmuscle myosin II-B with II-A rescues brain but not cardiac defects in mice. J Biol Chem 282:22102–22111
Betapudi V, Licate LS, Egelhoff TT (2006) Distinct roles of nonmuscle myosin II isoforms in the regulation of MDA-MB-231 breast cancer cell spreading and migration. Cancer Res 66:4725–4733
Bresnick AR (1999) Molecular mechanisms of nonmuscle myosin-II regulation. Curr Opin Cell Biol 11:26–33
Chen JN, Haffter P, Odenthal J, Vogelsang E, Brand M, van Eeden FJ, Furutani-Seiki M, Granato M, Hammerschmidt M, Heisenberg CP, Jiang YJ, Kane DA, Kelsh RN, Mullins MC, Nusslein-Volhard C (1996) Mutations affecting the cardiovascular system and other internal organs in zebrafish. Development 123:293–302
Conti MA, Adelstein RS (2008) Nonmuscle myosin II moves in new directions. J Cell Sci 121:11–18
Conti MA, Even-Ram S, Liu C, Yamada KM, Adelstein RS (2004) Defects in cell adhesion and the visceral endoderm following ablation of nonmuscle myosin heavy chain II-A in mice. J Biol Chem 279:41263–41266
Even-Ram S, Doyle AD, Conti MA, Matsumoto K, Adelstein RS, Yamada KM (2007) Myosin IIA regulates cell motility and actomyosin-microtubule crosstalk. Nat Cell Biol 9:299–309
Golomb E, Ma X, Jana SS, Preston YA, Kawamoto S, Shoham NG, Goldin E, Conti MA, Sellers JR, Adelstein RS (2004) Identification and characterization of nonmuscle myosin II-C, a new member of the myosin II family. J Biol Chem 279:2800–2808
Griffin KJ, Kimelman D (2002) One-eyed pinhead and spadetail are essential for heart and somite formation. Nat Cell Biol 4:821–825
Gritsman K, Zhang J, Cheng S, Heckscher E, Talbot WS, Schier AF (1999) The EGF-CFC protein one-eyed pinhead is essential for nodal signaling. Cell 97:121–132
Huang Y, Wang X, Xu M, Liu M, Liu D (2013) Nonmuscle myosin II-B (myh10) expression analysis during zebrafish embryonic development. Gene Expr Patterns 13:265–270
Kikuchi Y, Agathon A, Alexander J, Thisse C, Waldron S, Yelon D, Thisse B, Stainier DY (2001) Casanova encodes a novel Sox-related protein necessary and sufficient for early endoderm formation in zebrafish. Genes Dev 15:1493–1505
Kikuchi Y, Trinh LA, Reiter JF, Alexander J, Yelon D, Stainier DY (2000) The zebrafish bonnie and clyde gene encodes a mix family homeodomain protein that regulates the generation of endodermal precursors. Genes Dev 14:1279–1289
Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF (1995) Stages of embryonic development of the zebrafish. Dev Dyn 203:253–310
Krendel M, Mooseker MS (2005) Myosins: tails (and heads) of functional diversity. Physiology (Bethesda) 20:239–251
Limouze J, Straight AF, Mitchison T, Sellers JR (2004) Specificity of blebbistatin, an inhibitor of myosin II. J Muscle Res Cell Motil 25:337–341
Ma X, Adelstein RS (2011) In vivo studies on nonmuscle myosin II expression and function in heart development. Front Biosci 17:545–555
Ma X, Jana SS, Conti MA, Kawamoto S, Claycomb WC, Adelstein RS (2010) Ablation of nonmuscle myosin II-B and II-C reveals a role for nonmuscle myosin II in cardiac myocyte karyokinesis. Mol Biol Cell 21:3952–3962
Matsui T, Raya A, Callol-Massot C, Kawakami Y, Oishi I, Rodriguez-Esteban C, Izpisua Belmonte JC (2007) Miles-apart-mediated regulation of cell-fibronectin interaction and myocardial migration in zebrafish. Nature clinical practice. Rev Cardiovasc Med 4(1):S77–S82
Matsumura F (2005) Regulation of myosin II during cytokinesis in higher eukaryotes. Trends Cell Biol 15:371–377
Miura GI, Yelon D (2011) A guide to analysis of cardiac phenotypes in the zebrafish embryo. Methods Cell Biol 101:161–180
Osborne N, Brand-Arzamendi K, Ober EA, Jin SW, Verkade H, Holtzman NG, Yelon D, Stainier DY (2008) The spinster homolog, two of hearts, is required for sphingosine 1-phosphate signaling in zebrafish. Curr Biol 18:1882–1888
Robinson DN, Spudich JA (2004) Mechanics and regulation of cytokinesis. Curr Opin Cell Biol 16:182–188
Sellers JR (2000) Myosins: a diverse superfamily. Biochim Biophys Acta 1496:3–22
Shu S, Liu X, Korn ED (2005) Blebbistatin and blebbistatin-inactivated myosin II inhibit myosin II-independent processes in Dictyostelium. Proc Natl Acad Sci U S A 102:1472–1477
Stainier DY (2001) Zebrafish genetics and vertebrate heart formation. Nat Rev Genet 2:39–48
Stainier DY, Fouquet B, Chen JN, Warren KS, Weinstein BM, Meiler SE, Mohideen MA, Neuhauss SC, Solnica-Krezel L, Schier AF, Zwartkruis F, Stemple DL, Malicki J, Driever W, Fishman MC (1996) Mutations affecting the formation and function of the cardiovascular system in the zebrafish embryo. Development 123:285–292
Stainier DY, Lee RK, Fishman MC (1993) Cardiovascular development in the zebrafish. I. myocardial fate map and heart tube formation. Development 119:31–40
Straight AF, Cheung A, Limouze J, Chen I, Westwood NJ, Sellers JR, Mitchison TJ (2003) Dissecting temporal and spatial control of cytokinesis with a myosin II Inhibitor. Science 299:1743–1747
Swailes NT, Colegrave M, Knight PJ, Peckham M (2006) Non-muscle myosins 2A and 2B drive changes in cell morphology that occur as myoblasts align and fuse. J Cell Sci 119:3561–3570
Takeda K, Kishi H, Ma X, Yu ZX, Adelstein RS (2003) Ablation and mutation of nonmuscle myosin heavy chain II-B results in a defect in cardiac myocyte cytokinesis. Circ Res 93:330–337
Trinh LA, Stainier DY (2004) Cardiac development. Methods Cell Biol 76:455–473
Tu S, Chi NC (2012) Zebrafish models in cardiac development and congenital heart birth defects. Differentiation 84:4–16
Tullio AN, Accili D, Ferrans VJ, Yu ZX, Takeda K, Grinberg A, Westphal H, Preston YA, Adelstein RS (1997) Nonmuscle myosin II-B is required for normal development of the mouse heart. Proc Natl Acad Sci U S A 94:12407–12412
Urven LE, Yabe T, Pelegri F (2006) A role for non-muscle myosin II function in furrow maturation in the early zebrafish embryo. J Cell Sci 119:4342–4352
Vicente-Manzanares M, Ma X, Adelstein RS, Horwitz AR (2009) Non-muscle myosin II takes centre stage in cell adhesion and migration. Nat Rev Mol Cell Biol 10:778–790
Wylie SR, Chantler PD (2001) Separate but linked functions of conventional myosins modulate adhesion and neurite outgrowth. Nat Cell Biol 3:88–92
Yelon D (2001) Cardiac patterning and morphogenesis in zebrafish. Dev Dyn 222:552–563
Zhang J, Talbot WS, Schier AF (1998) Positional cloning identifies zebrafish one-eyed pinhead as a permissive EGF-related ligand required during gastrulation. Cell 92:241–251
Acknowledgments
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).
Author’s contributions
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.
Author information
Authors and Affiliations
Corresponding authors
Additional information
Editor: T. Okamoto
Xueqian Wang and Mei Chong contributed equally to this work.
Electronic supplementary material
Below is the link to the electronic supplementary material.
(AVI 1439 kb)
(AVI 2293 kb)
Rights and permissions
About this article
Cite this article
Wang, X., Chong, M., Wang, X. et al. Block the function of nonmuscle myosin II by blebbistatin induces zebrafish embryo cardia bifida. In Vitro Cell.Dev.Biol.-Animal 51, 211–217 (2015). https://doi.org/10.1007/s11626-014-9836-0
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11626-014-9836-0