Abstract
MicroRNAs (miRNAs) regulate gene expression by mediating translational repression or mRNA degradation of their targets, and several miRNAs control developmental decisions through embryogenesis. In the developing heart, miRNA targets comprise key players mediating cardiac lineage determination. However, although several miRNAs have been identified as differentially regulated during cardiac development and disease, their distinct cell-specific localization remains largely undetermined, likely owing to a lack of adequate methods. We therefore report the development of a markedly improved approach combining fluorescence-based miRNA-in situ hybridization (miRNA-ISH) with immunohistochemistry (IHC). We have applied this protocol to differentiating embryoid bodies (EBs) as well as embryonic and adult mouse hearts, to detect miRNAs that were upregulated during EB cardiomyogenesis, as determined by array-based miRNA expression profiling. In this manner, we found specific co-localization of miR-1 to myosin positive cells (cardiomyocytes) of EBs, developing and mature hearts. In contrast, miR-125b and -199a did not localize to cardiomyocytes, as previously suggested for miR-199a, but were rather expressed in connective tissue cells of the heart. More specifically, by co-staining with α-smooth muscle actin (α-SMA) and collagen-I, we found that miR-125b and -199a localize to perivascular α-SMA− stromal cells. Our approach thus proved valid for determining cell-specific localization of miRNAs, and the findings we present highlight the importance of determining exact cell-specific localization of miRNAs by sequential miRNA-ISH and IHC in studies aiming at understanding the role of miRNAs and their targets. This approach will hopefully aid in identifying relevant miRNA targets of both the heart and other organs.
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References
Andersen DC, Andersen P, Schneider M, Jensen HB, Sheikh SP (2009) Murine “cardiospheres” are not a source of stem cells with cardiomyogenic potential. Stem Cells 27(7):1571–1581. doi:10.1002/stem.72
Aplin M, Christensen GL, Schneider M, Heydorn A, Gammeltoft S, Kjolbye AL, Sheikh SP, Hansen JL (2007a) Differential extracellular signal-regulated kinases 1 and 2 activation by the angiotensin type 1 receptor supports distinct phenotypes of cardiac myocytes. Basic Clin Pharmacol Toxicol 100(5):296–301. doi:10.1111/j.1742-7843.2007.00064.x
Aplin M, Christensen GL, Schneider M, Heydorn A, Gammeltoft S, Kjolbye AL, Sheikh SP, Hansen JL (2007b) The angiotensin type 1 receptor activates extracellular signal-regulated kinases 1 and 2 by G protein-dependent and -independent pathways in cardiac myocytes and langendorff-perfused hearts. Basic Clin Pharmacol Toxicol 100(5):289–295. doi:10.1111/j.1742-7843.2007.00063.x
Barroso-del Jesus A, Lucena-Aguilar G, Menendez P (2009) The miR-302–367 cluster as a potential stemness regulator in ESCs. Cell Cycle 8(3):394–398. doi:10.4161/cc.8.3.7554
Cheng Y, Ji R, Yue J, Yang J, Liu X, Chen H, Dean DB, Zhang C (2007) MicroRNAs are aberrantly expressed in hypertrophic heart: do they play a role in cardiac hypertrophy? Am J Pathol 170(6):1831–1840. doi:10.2353/ajpath.2007.061170
Cordes KR, Srivastava D (2009) MicroRNA regulation of cardiovascular development. Circ Res 104(6):724–732. doi:10.1161/CIRCRESAHA.108.192872
Haghikia A, Missol-Kolka E, Tsikas D, Venturini L, Brundiers S, Castoldi M, Muckenthaler MU, Eder M, Stapel B, Thum T, Petrasch-Parwez E, Drexler H, Hilfiker-Kleiner D, Scherr M (2010) Signal transducer and activator of transcription 3-mediated regulation of miR-199a-5p links cardiomyocyte and endothelial cell function in the heart: a key role for ubiquitin-conjugating enzymes. Eur Heart J. doi:10.1093/eurheartj/ehq369
Hellemans J, Mortier G, De Paepe A, Speleman F, Vandesompele J (2007) qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol 8(2):R19. doi:10.1186/gb-2007-8-2-r19
Ivey KN, Muth A, Arnold J, King FW, Yeh RF, Fish JE, Hsiao EC, Schwartz RJ, Conklin BR, Bernstein HS, Srivastava D (2008) MicroRNA regulation of cell lineages in mouse and human embryonic stem cells. Cell Stem Cell 2(3):219–229. doi:10.1016/j.stem.2008.01.016
Keller G (2005) Embryonic stem cell differentiation: emergence of a new era in biology and medicine. Genes Dev 19(10):1129–1155. doi:10.1101/gad.1303605
Latronico MV, Condorelli G (2009) MicroRNAs and cardiac pathology. Nat Rev Cardiol 6(6):419–429. doi:10.1038/nrcardio.2009.56
Le MT, Teh C, Shyh-Chang N, Xie H, Zhou B, Korzh V, Lodish HF, Lim B (2009) MicroRNA-125b is a novel negative regulator of p53. Genes Dev 23(7):862–876. doi:10.1101/gad.1767609
Liu N, Olson EN (2010) MicroRNA regulatory networks in cardiovascular development. Dev Cell 18(4):510–525. doi:10.1016/j.devcel.2010.03.010
Liu N, Williams AH, Kim Y, McAnally J, Bezprozvannaya S, Sutherland LB, Richardson JA, Bassel-Duby R, Olson EN (2007) An intragenic MEF2-dependent enhancer directs muscle-specific expression of microRNAs 1 and 133. Proc Natl Acad Sci USA 104(52):20844–20849. doi:10.1073/pnas.0710558105
Nuovo GJ, Elton TS, Nana-Sinkam P, Volinia S, Croce CM, Schmittgen TD (2009) A methodology for the combined in situ analyses of the precursor and mature forms of microRNAs and correlation with their putative targets. Nat Protoc 4(1):107–115. doi:10.1038/nprot.2008.215
Obernosterer G, Martinez J, Alenius M (2007) Locked nucleic acid-based in situ detection of microRNAs in mouse tissue sections. Nat Protoc 2(6):1508–1514. doi:10.1038/nprot.2007.153
Rane S, He M, Sayed D, Vashistha H, Malhotra A, Sadoshima J, Vatner DE, Vatner SF, Abdellatif M (2009) Downregulation of miR-199a derepresses hypoxia-inducible factor-1alpha and Sirtuin 1 and recapitulates hypoxia preconditioning in cardiac myocytes. Circ Res 104(7):879–886. doi:10.1161/CIRCRESAHA.108.193102
Sayed D, Hong C, Chen IY, Lypowy J, Abdellatif M (2007) MicroRNAs play an essential role in the development of cardiac hypertrophy. Circ Res 100(3):416–424. doi:10.1161/01.RES.0000257913.42552.23
Sempere LF, Preis M, Yezefski T, Ouyang H, Suriawinata AA, Silahtaroglu A, Conejo-Garcia JR, Kauppinen S, Wells W, Korc M (2010) Fluorescence-based codetection with protein markers reveals distinct cellular compartments for altered MicroRNA expression in solid tumors. Clin Cancer Res 16(16):4246–4255. doi:10.1158/1078-0432.CCR-10-1152
Silahtaroglu AN, Nolting D, Dyrskjot L, Berezikov E, Moller M, Tommerup N, Kauppinen S (2007) Detection of microRNAs in frozen tissue sections by fluorescence in situ hybridization using locked nucleic acid probes and tyramide signal amplification. Nat Protoc 2(10):2520–2528. doi:10.1038/nprot.2007.313
Song XW, Li Q, Lin L, Wang XC, Li DF, Wang GK, Ren AJ, Wang YR, Qin YW, Yuan WJ, Jing Q (2010) MicroRNAs are dynamically regulated in hypertrophic hearts, and miR-199a is essential for the maintenance of cell size in cardiomyocytes. J Cell Physiol 225(2):437–443. doi:10.1002/jcp.22217
Stary M, Schneider M, Sheikh SP, Weitzer G (2006) Parietal endoderm secreted S100A4 promotes early cardiomyogenesis in embryoid bodies. Biochem Biophys Res Commun 343(2):555–563. doi:10.1016/j.bbrc.2006.02.161
Tatsuguchi M, Seok HY, Callis TE, Thomson JM, Chen JF, Newman M, Rojas M, Hammond SM, Wang DZ (2007) Expression of microRNAs is dynamically regulated during cardiomyocyte hypertrophy. J Mol Cell Cardiol 42(6):1137–1141. doi:10.1016/j.yjmcc.2007.04.004
Thum T, Galuppo P, Wolf C, Fiedler J, Kneitz S, van Laake LW, Doevendans PA, Mummery CL, Borlak J, Haverich A, Gross C, Engelhardt S, Ertl G, Bauersachs J (2007) MicroRNAs in the human heart: a clue to fetal gene reprogramming in heart failure. Circulation 116(3):258–267. doi:10.1161/CIRCULATIONAHA.107.687947
van Rooij E (2011) The art of microRNA Research. Circ Res 108(2):219–234. doi:10.1161/CIRCRESAHA.110.227496
van Rooij E, Sutherland LB, Liu N, Williams AH, McAnally J, Gerard RD, Richardson JA, Olson EN (2006) A signature pattern of stress-responsive microRNAs that can evoke cardiac hypertrophy and heart failure. Proc Natl Acad Sci USA 103(48):18255–18260. doi:10.1073/pnas.0608791103
Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3(7): RESEARCH0034. doi:10.1186/gb-2002-3-7-research0034
Villeneuve LM, Kato M, Reddy MA, Wang M, Lanting L, Natarajan R (2010) Enhanced levels of microRNA-125b in vascular smooth muscle cells of diabetic db/db mice lead to increased inflammatory gene expression by targeting the histone methyltransferase Suv39h1. Diabetes 59(11):2904–2915. doi:10.2337/db10-0208
Yang B, Lin H, Xiao J, Lu Y, Luo X, Li B, Zhang Y, Xu C, Bai Y, Wang H, Chen G, Wang Z (2007) The muscle-specific microRNA miR-1 regulates cardiac arrhythmogenic potential by targeting GJA1 and KCNJ2. Nat Med 13(4):486–491. doi:10.1038/nm1569
Zhao Y, Samal E, Srivastava D (2005) Serum response factor regulates a muscle-specific microRNA that targets Hand2 during cardiogenesis. Nature 436(7048):214–220. doi:10.1038/nature03817
Zhao Y, Ransom JF, Li A, Vedantham V, von Drehle M, Muth AN, Tsuchihashi T, McManus MT, Schwartz RJ, Srivastava D (2007) Dysregulation of cardiogenesis, cardiac conduction, and cell cycle in mice lacking miRNA-1–2. Cell 129(2):303–317. doi:10.1016/j.cell.2007.03.030
Acknowledgments
We thank Mette Christensen for performing the array analysis, and Tonja Jørgensen, Sussi Mortensen and Bettina Mentz for technical help. This work was supported by The John and Birthe Meyer Foundation and The Danish Heart Foundation. Wilhelm Johannsen Centre for Functional Genome Research is established by The Danish National Research Foundation.
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Schneider, M., Andersen, D.C., Silahtaroglu, A. et al. Cell-specific detection of microRNA expression during cardiomyogenesis by combined in situ hybridization and immunohistochemistry. J Mol Hist 42, 289–299 (2011). https://doi.org/10.1007/s10735-011-9332-8
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DOI: https://doi.org/10.1007/s10735-011-9332-8