Loss of muscleblind-like 1 promotes invasive mesenchyme formation in endocardial cushions by stimulating autocrine TGFβ3

BMC Developmental Biology

Andrea N Ladd¹  Natalie A Vajda¹  Samantha J Stillwagon¹  Yotam Blech-Hermoni¹  Kathryn E LeMasters¹ 

[1] Department of Cell Biology, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Ave., Mail code NC10, Cleveland, OH, 44195, USA

关键词: Cell invasion;    Epithelial-mesenchymal transition;    Endocardial cushions;    TGFβ;    Alternative splicing;    Muscleblind-like 1;   

Others  :  1086556
DOI  :  10.1186/1471-213X-12-22

 received in 2012-05-09, accepted in 2012-08-01,  发布年份 2012

【 摘 要 】

Background

Valvulogenesis and septation in the developing heart depend on the formation and remodeling of endocardial cushions in the atrioventricular canal (AVC) and outflow tract (OFT). These cushions are invaded by a subpopulation of endocardial cells that undergo an epithelial-mesenchymal transition in response to paracrine and autocrine transforming growth factor β (TGFβ) signals. We previously demonstrated that the RNA binding protein muscleblind-like 1 (MBNL1) is expressed specifically in the cushion endocardium, and knockdown of MBNL1 in stage 14 embryonic chicken AVC explants enhances TGFβ-dependent endocardial cell invasion.

Results

In this study, we demonstrate that the effect of MBNL1 knockdown on invasion remains dependent on TGFβ3 after it is no longer required to induce basal levels of invasion. TGFβ3, but not TGFβ2, levels are elevated in medium conditioned by MBNL1-depleted AVC explants. TGFβ3 is elevated even when the myocardium is removed, indicating that MBNL1 modulates autocrine TGFβ3 production in the endocardium. More TGFβ3-positive cells are observed in the endocardial monolayer following MBNL1 knockdown. Addition of exogenous TGFβ3 to AVC explants recapitulates the effects of MBNL1 knockdown. Time course experiments demonstrate that knockdown of MBNL1 induces precocious TGFβ3 secretion, and early exposure to excess TGFβ3 induces precocious invasion. MBNL1 expression precedes TGFβ3 in the AVC endocardium, consistent with a role in preventing precocious autocrine TGFβ3 signaling. The stimulatory effects of MBNL1 knockdown on invasion are lost in stage 16 AVC explants. Knockdown of MBNL1 in OFT explants similarly enhances cell invasion, but not activation. TGFβ is necessary and sufficient to mediate this effect.

Conclusions

Taken together, these data support a model in which MBNL1 negatively regulates cell invasion in the endocardial cushions by restricting the magnitude and timing of endocardial-derived TGFβ3 production.

【 授权许可】

   
2012 LeMasters et al.; licensee BioMed Central Ltd.

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【 参考文献 】

• [1]Krug EL, Mjaatvedt CH, Markwald RR: Extracellular matrix from embryonic myocardium elicits an early morphogenetic event in cardiac endothelial differentiation. Dev Biol 1987, 120(2):348-355.
• [2]Krug EL, Runyan RB, Markwald RR: Protein extracts from early embryonic hearts initiate cardiac endothelial cytodifferentiation. Dev Biol 1985, 112(2):414-426.
• [3]Mjaatvedt CH, Markwald RR: Induction of an epithelial-mesenchymal transition by an in vivo adheron-like complex. Dev Biol 1989, 136(1):118-128.
• [4]Nakajima Y, Krug EL, Markwald RR: Myocardial regulation of transforming growth factor-beta expression by outflow tract endothelium in the early embryonic chick heart. Dev Biol 1994, 165(2):615-626.
• [5]Person AD, Klewer SE, Runyan RB: Cell biology of cardiac cushion development. Int Rev Cytol 2005, 243:287-335.
• [6]Boyer AS, Ayerinskas II, Vincent EB, McKinney LA, Weeks DL, Runyan RB: TGFbeta2 and TGFbeta3 have separate and sequential activities during epithelial-mesenchymal cell transformation in the embryonic heart. Dev Biol 1999, 208(2):530-545.
• [7]Potts JD, Dagle JM, Walder JA, Weeks DL, Runyan RB: Epithelial-mesenchymal transformation of embryonic cardiac endothelial cells is inhibited by a modified antisense oligodeoxynucleotide to transforming growth factor beta 3. Proc Natl Acad Sci U S A 1991, 88(4):1516-1520.
• [8]Ghosh S, Brauer PR: Latent transforming growth factor-beta is present in the extracellular matrix of embryonic hearts in situ. Dev Dyn 1996, 205(2):126-134.
• [9]Nakajima Y, Yamagishi T, Nakamura H, Markwald RR, Krug EL: An autocrine function for transforming growth factor (TGF)-beta3 in the transformation of atrioventricular canal endocardium into mesenchyme during chick heart development. Dev Biol 1998, 194(1):99-113.
• [10]Ramsdell AF, Markwald RR: Induction of endocardial cushion tissue in the avian heart is regulated, in part, by TGFbeta-3-mediated autocrine signaling. Dev Biol 1997, 188(1):64-74.
• [11]Kalsotra A, Xiao X, Ward AJ, Castle JC, Johnson JM, Burge CB, Cooper TA: A postnatal switch of CELF and MBNL proteins reprograms alternative splicing in the developing heart. Proc Natl Acad Sci U S A 2008, 105(51):20333-20338.
• [12]Ladd A, Stenberg M, Swanson M, Cooper T: A dynamic balance between activation and repression regulates pre-mRNA alternative splicing during heart development. Dev Dyn 2005, 233(3):783-793.
• [13]Masuda A, Andersen HS, Doktor TK, Okamoto T, Ito M, Andresen BS, Ohno K: CUGBP1 and MBNL1 preferentially bind to 3' UTRs and facilitate mRNA decay. Sci Rep 2012, 2:209.
• [14]Vajda NA, Brimacombe KR, LeMasters KE, Ladd AN: Muscleblind-like 1 is a negative regulator of TGF-beta-dependent epithelial-mesenchymal transition of atrioventricular canal endocardial cells. Dev Dyn 2009, 238(12):3266-3272.
• [15]Nakajima Y, Mironov V, Yamagishi T, Nakamura H, Markwald RR: Expression of smooth muscle alpha-actin in mesenchymal cells during formation of avian endocardial cushion tissue: a role for transforming growth factor beta3. Dev Dyn 1997, 209(3):296-309.
• [16]Koli K, Saharinen J, Hyytiainen M, Penttinen C, Keski-Oja J: Latency, activation, and binding proteins of TGF-beta. Microsc Res Tech 2001, 52(4):354-362.
• [17]Nakajima Y, Miyazono K, Kato M, Takase M, Yamagishi T, Nakamura H: Extracellular fibrillar structure of latent TGF beta binding protein-1: role in TGF beta-dependent endothelial-mesenchymal transformation during endocardial cushion tissue formation in mouse embryonic heart. J Cell Biol 1997, 136(1):193-204.
• [18]Krishnan S, Deora AB, Annes JP, Osoria J, Rifkin DB, Hajjar KA: Annexin II-mediated plasmin generation activates TGF-beta3 during epithelial-mesenchymal transformation in the developing avian heart. Dev Biol 2004, 265(1):140-154.
• [19]Sun D, Vanderburg CR, Odierna GS, Hay ED: TGFbeta3 promotes transformation of chicken palate medial edge epithelium to mesenchyme in vitro. Development 1998, 125(1):95-105.
• [20]Lorda-Diez CI, Montero JA, Garcia-Porrero JA, Hurle JM: Tgfbeta2 and 3 are coexpressed with their extracellular regulator Ltbp1 in the early limb bud and modulate mesodermal outgrowth and BMP signaling in chicken embryos. BMC Dev Biol 2010, 10:69. BioMed Central Full Text
• [21]Kanadia RN, Urbinati CR, Crusselle VJ, Luo D, Lee YJ, Harrison JK, Oh SP, Swanson MS: Developmental expression of mouse muscleblind genes Mbnl1, Mbnl2 and Mbnl3. Gene Expr Patterns 2003, 3(4):459-462.
• [22]Hamburger V, Hamilton HL: A series of normal stages in the development of the chick embryo. 1951. Dev Dyn 1992, 195(4):231-272.
• [23]Mjaatvedt CH, Lepera RC, Markwald RR: Myocardial specificity for initiating endothelial-mesenchymal cell transition in embryonic chick heart correlates with a particulate distribution of fibronectin. Dev Biol 1987, 119(1):59-67.
• [24]Runyan RB, Markwald RR: Invasion of mesenchyme into three-dimensional collagen gels: a regional and temporal analysis of interaction in embryonic heart tissue. Dev Biol 1983, 95(1):108-114.
• [25]Willems E, Leyns L, Vandesompele J: Standardization of real-time PCR gene expression data from independent biological replicates. Anal Biochem 2008, 379(1):127-129.