Primary culture of avian embryonic heart forming region cells to study the regulation of vertebrate early heart morphogenesis by vitamin A

BMC Developmental Biology

Indrikis Muiznieks¹  Maija H Zile²  Janis Ancans¹  Ilva Nakurte⁴  Martins Boroduskis¹  Una Riekstina³  Inese Cakstina¹ 

[1] Division of Microbiology and Biotechnology, Department of Biology, University of Latvia, Riga, Latvia;Department Food Science & Human Nutrition, Michigan State University, East Lansing, MI, USA;Division of Pharmacy, Department of Medicine, University of Latvia, Riga, Latvia;Laboratory of Biodosimetry and Bioanalytical Methods, Department of Biology, University of Latvia, Riga, Latvia

关键词: Early cardiovascular development;    TGFβ2;    Retinoic acid;    in vitro culture;    Chicken heart forming region cells;   

Others  :  1085157
DOI  :  10.1186/1471-213X-14-10

 received in 2013-11-25, accepted in 2014-02-10,  发布年份 2014

【 摘 要 】

Background

Important knowledge about the role of vitamin A in vertebrate heart development has been obtained using the vitamin A-deficient avian in ovo model which enables the in vivo examination of very early stages of vertebrate heart morphogenesis. These studies have revealed the critical role of the vitamin A-active form, retinoic acid (RA) in the regulation of several developmental genes, including the important growth regulatory factor, transforming growth factor-beta2 (TGFβ2), involved in early events of heart morphogenesis. However, this in ovo model is not readily available for elucidating details of molecular mechanisms determining RA activity, thus limiting further examination of RA-regulated early heart morphogenesis. In order to obtain insights into RA-regulated gene expression during these early events, a reliable in vitro model is needed. Here we describe a cell culture that closely reproduces the in ovo observed regulatory effects of RA on TGFβ2 and on several developmental genes linked to TGFβ signaling during heart morphogenesis.

Results

We have developed an avian heart forming region (HFR) cell based in vitro model that displays the characteristics associated with vertebrate early heart morphogenesis, i.e. the expression of Nkx2.5 and GATA4, the cardiogenesis genes, of vascular endothelial growth factor (VEGF-A), the vasculogenesis gene and of fibronectin (FN1), an essential component in building the heart, and the expression of the multifunctional genes TGFβ2 and neogenin (NEO). Importantly, we established that the HFR cell culture is a valid model to study RA-regulated molecular events during heart morphogenesis and that the expression of TGFβ2 as well as the expression of several TGFβ2-linked developmental genes is regulated by RA.

Conclusions

Our findings reported here offer a biologically relevant experimental in vitro system for the elucidation of RA-regulated expression of TGFβ2 and other genes involved in vertebrate early cardiovascular morphogenesis.

【 授权许可】

   
2014 Cakstina et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

【 参考文献 】

• [1]Li P, Pashmforoush M, Sucov H: Retinoic acid regulates differentiation of the secondary heart field and TGFβ-mediated outflow tract septation. Dev Cell 2010, 18:480-485.
• [2]Niederreither K, Vermont J, Messaddeg N, Schuhbaur B, Chambon P, Dolle P: Embryonic retinoic acid synthesis is essential for heart morphogenesis in the mouse. Development 2011, 128:1019-1031.
• [3]Zile MH: Vitamin A requirement for early cardiovascular morphogenesis specification in the vertebrate embryo: insights from the avian embryo. J Exptl Biol Med 2004, 229:598-606.
• [4]Germain P, Chambon P, Eichele G, Evans RM, Lazar MA, Leid M, de Lera AR, Lotan R, Mangelsdorf DJ, Gronemeyer H: International union of pharmacology. LX. Retinoic acid receptors. Pharmacol Rev 2006, 58:712-725.
• [5]Germain P, Chambon P, Eichele G, Evans RM, Lazar MA, Leid M, de Lera AR, Lotan R, Mangelsdorf DJ, Gronemeyer H: International union of pharmacology. LXIII. Retinoid X receptors. Pharmacol Rev 2006, 58:760-772.
• [6]Zile MH: Vitamin A - not for your eyes only: requirement for heart formation begins early in embryogenesis. Nutrients 2010, 2:532-550.
• [7]Dersch H, Zile MH: Induction of normal cardiovascular development in the vitamin A- deprived quail embryo by natural retinoids. Dev Biol 1993, 160:424-433.
• [8]Roberts AB, Sporn MB: Mechanistic interrelationships between two superfamilies: the steroid/retinoid receptors and transforming growth factor-beta. Cancer Rev 1992, 14:205-220.
• [9]Hoover LL, Burton EG, O’Neil ML, Brooks BA, Sreedharan S, Dawson NA, Kubalak SW: Retinoids regulate TGFbeta signaling at the level of Smad2 phosphorylation and nuclear accumulation. Biochim Biophys Acta 2008, 1783:2279-2286.
• [10]Chang H, Brown CW, Matzuk MM: Genetic analysis of the mammalian transforming growth factor-beta superfamily. Endocr Rev 2002, 23:787-823.
• [11]Wu MY, Hill CS: TGF-β superfamily signaling in embryonic development and homeostasis. Dev Cell 2009, 16:329-343.
• [12]Lee MK, Pardoux C, Hall MC, Lee PS, Warburton D, Quing J, Smith SM, Derynck R: TGF-beta activates Erk MAP kinase signaling through direct phosphorylation of ShcA. EMBO J 2007, 26:3957-3967.
• [13]Mahmood R, Flanders KC, Morriss-Kay GM: Interactions between retinoids and TGFβs in mouse morphogenesis. Development 1992, 115:67-74.
• [14]Ghatpande SK, Zhou HR, Cakstina I, Carlson C, Rondini EA, Romeih M, Zile MH: Transforming growth factor beta2 is negatively regulated by endogenous retinoic acid during early heart morphogenesis. Dev Growth Differ 2010, 52:433-455.
• [15]Goumans MJ, Mummery C: Functional analysis of the TGFβ receptor/Smad pathway through gene ablation in mice. Int J Dev Biol 2000, 44:253-265.
• [16]Jakowlew SB, Ciment G, Tuan RS, Sporn MB, Roberts AB: Expression of transforming growth factor-β2 and -β3 mRNAs and proteins in the developing chicken embryo. Differentiation 1994, 55:105-118.
• [17]Mercado-Pimentel ME, Runyan RB: Multiple transforming growth factor-beta isoforms and receptors function during epithelial-mesenchymal cell transformation in the embryonic heart. Cells Tissues Organs 2007, 185:146-156.
• [18]Glick AB, McCune BK, Abdulkareem N, Flanders KC, Lumadue JA, Smith JM, Sporn MB: Complex regulation of TGFβ expression by retinoic acid in the vitamin A deficient rat. Development 1991, 111:1081-1086.
• [19]Kubalak SW, Hutson DR, Scott KK, Shannon RA: Elevated transforming growth factor beta2 enhances apoptosis and contributes to abnormal outflow tract and aortic sac development in retinoic X receptor alpha knockout embryos. Development 2002, 129:733-746.
• [20]LaRue AC, Argraves WS, Zile MH, Drake CJ: Critical role for retinol in the generation/differentiation of angioblasts required for embryonic blood vessel formation. Dev Dyn 2004, 230:666-674.
• [21]Bohnsack BL, Lai L, Dolle P, Hirschi KL: Signaling hierarchy downstream of retinoic acid that independently regulates vascular remodeling and endothelial cell proliferation. Genes Dev 2004, 18:1345-1358.
• [22]Wendler CC, Schmoldt A, Flentke GR, Case LC, Quadro L, Blaner WS, Lough J, Smith SM: Increased fibronectin deposition in embryonic hearts of retinol-binding protein-null mice. Circ Res 2003, 92:920-928.
• [23]Verrecchia F, Chu ML, Mauviel A: Identification of novel TGF-β/Smad gene target in dermal fibroblasts using a combined cDNA microarray/promoter transactivation approach. J Biol Chem 2001, 276:17058-17062.
• [24]Cole SJ, Bradford D, Cooper HM: Neogenin: a multi-functional receptor regulating diverse developmental processes. Int J Biochem Cell Biol 2007, 39:1569-1575.
• [25]Hagihara M, Endo M, Hata K, Higuchi C, Takaoka K, Yoshikawa H, Yamashita T: Neogenin, a receptor for bone morphogenetic proteins. J Biol Chem 2011, 286:5157-5165.
• [26]Ghatpande S, Brand T, Zile M, Evans T: Bmp2 and GATA4 function additively to rescue heart tube development in the absence of retinoids. Dev Dyn 2006, 235:2030-2039.
• [27]Ogura T, Evans RM: Evidence for two distinct retinoic acid response pathways for HOXB1 gene regulation. Proc Natl Acad Sci USA 1995, 92:392-396.
• [28]Maden M, Gale E, Kostetskii I, Zile MH: Vitamin A-deficient quail embryos have half a hindbrain and other neural defects. Curr Biol 1996, 6:417-426.
• [29]Dong D, Zile MH: Endogenous retinoids in the early avian embryo. Biochem Biophys Res Commun 1995, 217:1026-1103.
• [30]Niederreither K, Dolle P: Retinoic acid in development: towards an integrated view. Nat Rev Genetics 2008, 9:541-553.
• [31]Kostetskii I, Yuan SY, Kostetskaia E, Linask K, Blanchet S, Seleiro E, Michaille JJ, Brickell P, Zile MH: Initial retinoid requirement for early avian development coincides with retinoid receptor coexpression in the precardiac fields and induction of normal cardiovascular development. Dev Dyn 1998, 213:188-198.
• [32]Zile MH, Kostetskii I, Yuan S, Kostetskaia E, St. Amand TR, Chen Y, Jaing W: Retinoid signaling is required to complete the vertebrate cardiac left/right asymmetry pathway. Dev Biol 2000, 223:323-338.
• [33]Noma T, Glick AB, Geiser AG, O’Reilly MA, Miller J, Roberts AB, Sporn MB: Molecular cloning and structure of the human transforming growth factor-β2 gene promoter. Growth Factors 1991, 4:247-255.
• [34]Schule R, Rangarjan P, Yang N, Kliewer S, Ransone L, Bolado J, Verma IM, Evans RM: Retinoic acid is a negative regulator of AP-1-responsive genes. Proc Natl Acad Sci USA 1991, 88:6092-6096.
• [35]Borel V, Marceau G, Gallot D, Blanchon L, Sapin V: Retinoids regulate human amniotic tissue-type plasminogen activator gene by a two-step mechanism. J Cell Mol Med 2010, 14:1793-1805.
• [36]Comb M, Goodman HM: CpG methylation inhibits proenkephalin gene expression and binding of the transcription factor AP-2. Nucleic Acids Res 1990, 18:3975-3982.
• [37]Tulchinsky EM, Georgiev GP, Lukanidin EM: Novel AP-1 binding site created by DNA-methylation. Oncogene 1996, 12:1737-1745.
• [38]Rishi V, Bhattacharya P, Chatterjee R, Rozenberg J, Zhao J, Glass K, Fitzgerald P, Vinson C: CpG methylation of half-CRE sequences creates C/EBPalpha binding sites that activate some tissue-specific genes. Proc Natl Acad Sci USA 2010, 107:20311-20316.
• [39]Dean DC, Bowlus CL, Bourgeois S: Cloning and analysis of the promoter region of the human fibronectin gene. Proc Natl Acad Sci USA 1987, 84:1876-1880.
• [40]Scita G, Darwiche N, Greenwald E, Rosenberg M, Politi K, De Luca LM: Retinoic acid down-regulation of fibronectin and retinoic acid receptor alpha proteins in NIH-3 T3 cells. Blocks of this response by ras transformation. J Biol Chem 1996, 271:6502-6508.
• [41]Feng XH, Derynck R: Specificity and versatility in TGFβ2 signaling through Smads. Annu Rev Cell Dev Buil 2005, 21:659-693.
• [42]Ferrara N, Houck KA, Jakeman LB, Winer J, Leung DW: The vascular endothelial growth factor family of polypeptides. J Cell Biochem 1991, 47:211-218.
• [43]Sidell N, Feng Y, Hao L, Wu J, Yu J, Kane MA, Napoli JL, Taylor RN: Retinoic acid is a cofactor for translational regulation of vascular endothelial growth factor in human endometrial stromal cells. Mol Endocrinol 2010, 24:148-160.
• [44]Tanabe K, Hirade K, Ishisaki A, Shu E, Suga H, Kitajima Y, Katagiri Y, Dohi S, Kozawa O: Possible involvement of p44/p42 MAP kinase in retinoic acid-stimulated vascular endothelial growth factor release in aortic smooth muscle cells. Artherosclerosis 2004, 175:245-251.
• [45]Kotake D, Hirasawa N: Activation of a retinoic acid pathway by thiazolidinediones induces production of vascular endothelial growth factor/vascular permeability factor in OP9 adipocytes. Eur J Pharm 2013, 707:95-103.
• [46]Balmer JE, Blomhoff R: Gene expression regulation by retinoic acid. J Lipid Res 2002, 43:1773-1808.
• [47]Diaz BV, Lenoir MC, Ladoux A, Frelin C, Demarchez M, Michel S: Regulation of vascular endothelial growth factor expression in human keratinocytes by retinoids. J Biol Chem 2000, 275:642-650.
• [48]Weninger W, Rendl M, Mildner M, Tschachler E: Retinoids downregulate vascular endothelial growth factor/vascular permeability factor production by normal human keratinocytes. J Invest Dermatol 1998, 111:907-911.
• [49]Pal S, Iruela-Arispe ML, Harvey VS, Zeng H, Nagy JA, Dvorak HF, Mukhopadhyay D: Retinoic acid selectively inhibits the vascular permeabilizing effect of VPF/VEGF, an early step in the angiogenic cascade. Microvasc Res 2000, 60:112-120.
• [50]Winer J, Jung CK, Shackel I, Williams PM: Development and validation of real-time quantitative reverse transcriptase-polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro. Anal Biochem 1999, 270:41-49.
• [51]Miyagi M, Yokoyama H, Shiraishi H, Matsumoto M, Ishii H: Simultaneous quantification of retinol, retinal, and retinoic acid isomers by high-performance liquid chromatography with a simple gradiation. J Chromatogr B Biomed Sci Appl 2001, 757:365-368.