Dgcr8 deletion in the primitive heart uncovered novel microRNA regulating the balance of cardiac-vascular gene program

Abstract

Primitive mammalian heart transforms from a single tube to a four-chambered muscular organ during a short developmental window. We found that knocking out global microRNA by deleting Dgcr8 microprocessor in Mesp1 cardiovascular progenitor cells lead to the formation of extremely dilated and enlarged heart due to defective cardiomyocyte (CM) differentiation. Transcriptome analysis revealed unusual upregulation of vascular gene expression in Dgcr8 cKO hearts. Single cell RNA sequencing study further confirmed the increase of angiogenesis genes in single Dgcr8 cKO CM. We also performed global microRNA profiling of E9.5 heart for the first time, and identified that miR-541 was transiently highly expressed in E9.5 hearts. Interestingly, introducing miR-541 back into microRNA-free CMs partially rescued their defects, downregulated angiogenesis genes and significantly upregulated cardiac genes. Moreover, miR-541 can target Ctgf and inhibit endothelial function. Our results suggest that micro-RNAs are required to suppress abnormal angiogenesis gene program to maintain CM differentiation.

Keywords

microRNA / Dgcr8 / Cardiovascular progenitor cells / miRNA-541 / Single cell RNA sequencing

References

[1] Ambros V (2004) The functions of animal microRNAs. Nature 431:350–355

[2] Babic AM, Chen C-C, Lau LF (1999) Fisp12/mouse connective tissue growth factor mediates endothelial cell adhesion and migration through Integrin αv β3, promotes endothelial cell survival, and induces angiogenesis in vivo. Mol Cell Biol 19:2958–2966

[3] Bondue A, Tännler S,Chiapparo G, Chabab S, Ramialison M, Paulissen C, Beck B, Harvey R, Blanpain C (2011) Defining the earliest step of cardiovascular progenitor specification during embryonic stem cell differentiation. J Cell Biol 192:751–765

[4] Buckingham M, Meilhac S, Zaffran S (2005) Building the mammalian heart from two sources of myocardial cells. Nat Rev Genet 6:826–835

[5] Cassidy JJ, Straughan AJ, Carthew RW (2016) Differential masking of natural genetic variation by miR-9a in Drosophila. Genetics 202:675–687

[6] Chen J-F, Murchison EP, Tang R, Callis TE, Tatsuguchi M, Deng Z, Rojas M, Hammond SM, Schneider MD, Selzman CH (2008) Targeted deletion of Dicer in the heart leads to dilated cardiomyopathy and heart failure. Proc Natl Acad Sci USA 105:2111–2116

[7] DeLaughter DM, Bick AG, Wakimoto H, McKean D, Gorham JM, Kathiriya IS, Hinson JT, Homsy J, Gray J,Pu W (2016) Single-cell resolution of temporal gene expression during heart development. Dev Cell 39:480–490

[8] Dornhöfer N, Spong S, Bennewith K, Salim A, Klaus S, Kambham N, Wong C, Kaper F, Sutphin P, Nacalumi R (2006) Connective tissue growth factor-specific monoclonal antibody therapy inhibits pancreatic tumor growth and metastasis. Cancer Res 66:5816–5827

[9] Duisters RF, Tijsen AJ, Schroen B, Leenders JJ, Lentink V, Van Der Made I,Herias V, Leeuwen RE Van, Schellings MW, Barenbrug P (2009) miR-133 and miR-30 regulate connective tissue implications for a role of microRNAs in myocardial matrix remodeling. Circ Res 104:170–178

[10] Ebert MS, Sharp PA (2012) Roles for microRNAs in conferring robustness to biological processes. Cell 149:505–524

[11] Fish JE, Santoro MM, Morton SU, Yu S, Yeh RF, Wythe JD, Ivey KN, Bruneau BG, Stainier DYR, Srivastava D (2008) miR-126 regulates angiogenic signaling and vascular integrity. Dev Cell 15:272–284

[12] Friedländer MR, Chen W, Adamidi C, Maaskola J, Einspanier R, Knespel S, Rajewsky N (2008) Discovering microRNAs from deep sequencing data using miRDeep. Nat Biotechnol 26:407–415

[13] Han J, Lee Y, Yeom KH, Nam JW, Heo I, Rhee JK, Sohn SY, Cho Y, Zhang BT, Kim VN (2006) Molecular basis for the recognition of primary microRNAs by the Drosha-DGCR8 complex. Cell 125:887–901

[14] He Y, Chang G, Zhan S, Song X,Wang X, Luo Y (2008) Soluble tissue factor has unique angiogenic activities that selectively promote migration and differentiation but not proliferation of endothelial cells. Biochem Biophys Res Commun 370:489–494

[15] Heidersbach A, Saxby C, Carver-moore K, Huang Y (2013) microRNA-1 regulates sarcomere formation and suppresses smooth muscle gene expression in the mammalian heart. Elife 2:1–22

[16] Ivey KN, Muth A, Arnold J, King FW, Yeh RF, Fish JE, Hsiao EC, Schwartz RJ, Conklin BR, Bernstein HS (2008) microRNA regulation of cell lineages in mouse and human embryonic stem cells. Cell Stem Cell 2:219–229

[17] Kasper DM, Moro A, Ristori E, Narayanan A, Hill-Teran G, Fleming E, Moreno-Mateos M, Vejnar CE, Zhang J, Lee D (2017) microRNAs establish uniform traits during the architecture of vertebrate embryos. Dev Cell 40(552–565):e5

[18] Kita-Matsuo H, Barcova M, Prigozhina N, Salomonis N, Wei K, Jacot JG, Nelson B, Spiering S, Haverslag R, Kim C (2009) Lentiviral vectors and protocols for creation of stable hESC lines for fluorescent tracking and drug resistance selection of cardiomyocytes. PLoS ONE 4:e5046

[19] Kubota S, Takigawa M (2007) CCN family proteins and angiogenesis: from embryo to adulthood. Angiogenesis 10:1–11

[20] Lescroart F, Wang X, Lin X, Swedlund B, Gargouri S, Sànchezdànes A, Dubois C, Paulissen C, Kinston S, Göttgens B (2018) Defining the earliest step of cardiovascular lineage segregation by single-cell RNA-seq. Science 4174:1–9

[21] Lewis BP, Shih I, Jones-Rhoades MW, Bartel DP, Burge CB (2003) 33-prediction of mammalian microRNA targets. Cell 115:787–798

[22] Lewis BP, Burge CB, Bartel DP (2005) Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120:15–20

[23] Li G, Xu A, Sim S, Priest JR, Tian X, Khan T, Quertermous T, Zhou B, Tsao PS, Quake SR (2016) Transcriptomic profiling maps anatomically patterned subpopulations among single embryonic cardiac cells. Dev Cell 39:491–507

[24] Liu N, Olson EN (2010) microRNA regulatory networks in cardiovascular development. Dev Cell 18:510–525

[25] Liu F, Li N, Long B, Fan Y-Y, Liu C-Y, Zhou Q-Y, Murtaza I,Wang K, Li P (2014) Cardiac hypertrophy is negatively regulated by miR-541. Cell Death Dis 5:e1171

[26] Liu Z,Wang L, Welch DJ, Ma H, Zhou Y, Vaseghi RH, Yu S, Wall BJ, Alimohamadi S, Zheng M (2017) Single cell transcriptomics reconstructs fate conversion from fibroblast to cardiomyocyte. Nature 551:100

[27] Milgrom-Hoffman M, Harrelson Z, Ferrara N, Zelzer E, Evans SM, Tzahor E (2011) The heart endocardium is derived from vascular endothelial progenitors. Development 138:4777–4787

[28] Patro R, Duggal G, Love MI, Irizarry RA, Kingsford C (2017) Salmon provides fast and bias-aware quantification of transcript expression. Nat Methods 14:417–419

[29] Picelli S, Faridani OR, Björklund AK, Winberg G, Sagasser S, Sandberg R (2014) Full-length RNA-seq from single cells using Smart-seq2. Nat Protoc 9:171–181

[30] Rao PK, Toyama Y, Chiang HR, Gupta S, Bauer M, Medvid R, Reinhardt F, Liao R, Krieger M, Jaenisch R (2009) Loss of cardiac microRNA-mediated regulation leads to dilated cardiomyopathy and heart failure. Circ Res 105:585–594

[31] Saga Y, Miyagawa-Tomita S, Takagi A, Kitajima S, Miyazaki JI, Inoue T (1999) MesP1 is expressed in the heart precursor cells and required for the formation of a single heart tube. Development 126:3437–3447

[32] Shi R, Chiang VL (2005) Facile means for quantifying microRNA expression by real-time PCR. Biotechniques 39:519–524

[33] Shimo T,Nakanishi T, Kimura Y, Nishida T, Ishizeki K, Matsumura T, Takigawa M (1998) Inhibition of endogenous expression of connective tissue growth factor by its antisense oligonucleotide and antisense RNA suppresses proliferation and migration of vascular endothelial cells. J Biochem 124:130–140

[34] Shimo T, Nakanishi T,Nishida T, Asano M, Kanyama M, Kuboki T, Tamatani T, Tezuka K, Takemura M, Matsumura T (1999) Connective tissue growth factor induces the proliferation, migration, and tube formation of vascular endothelial cells in vitro, and angiogenesis in vivo. J Biochem 126:137–145

[35] Showell C, Conlon FL (2007) Decoding development in Xenopus tropicalis. Genesis 45:418–426

[36] Srivastava D (2006) Making or breaking the heart: from lineage determination to morphogenesis. Cell 126:1037–1048

[37] von Mering C, Jensen LJ, Snel B, Hooper SD, Krupp M, Foglierini M, Jouffre N, Huynen MA, Bork P (2005) STRING: known and predicted protein-protein associations, integrated and transferred across organisms. Nucleic Acids Res 33:433–437

[38] Wang Y, Medvid R, Melton C, Jaenisch R, Blelloch R (2007) DGCR8 is essential for microRNA biogenesis and silencing of embryonic stem cell self-renewal. Nat Genet 39:380–385

[39] Wang S, Aurora AB, Johnson BA, Qi X, McAnally J, Hill JA, Richardson JA, Bassel-Duby R, Olson EN (2008a) The endothelial-specific microRNA miR-126 governs vascular integrity and angiogenesis. Dev Cell 15:261–271

[40] Wang Y, Baskerville S, Shenoy A, Babiarz JE, Baehner L, Blelloch R (2008b) Embryonic stem cell-specific microRNAs regulate the G1-S transition and promote rapid proliferation. Nat Genet 40:1478–1483

[41] Xin M, Olson EN, Bassel-Duby R (2013) Mending broken hearts: cardiac development as a basis for adult heart regeneration and repair. Nat Publ Gr 14:529–541

[42] Zhang F, Wang L, Li Y, Liu W, Duan F, Huang R, Chen X, Chang SCN, Du Y, Na J (2017) Optimizing mesoderm progenitor selection and three-dimensional microniche culture allows highly efficient endothelial differentiation and ischemic tissue repair from human pluripotent stem cells. Stem Cell Res. Ther 8:6

[43] 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 129:303–317

[44] Zhou F, Li X,Wang W, Zhu P, Zhou J, He W, Ding M,Xiong F, Zheng X, Li Z (2016) Tracing haematopoietic stem cell formation at single-cell resolution. Nature 533:1–17

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