Acetylation is one of the key chromatin modifications that control gene transcription during embryonic development and tumorigenesis. The types of genes sensitive to such modifications in vivo are not known to date. We investigated the expression of a number of genes involved in embryonic development after treatment with trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, in the limbs of chicken embryos. Our results show that TSA affects the expression profiles of some genes that play important roles during limb development. The expression of BMP4, SF/HGF and Twist1 increased, whereas the expression of BMP2, FGF8, Shh, Scleraxis, Myf5 and MyoD was decreased or even inhibited. In contrast, the expression of Pax3, Paraxis, Msx1, CREB, and PCNA was not affected. Our results indicate that the chicken embryo can serve as an effective in vivo model for studying the effect of HDAC inhibitors on gene expression and can be helpful for understanding the role of chromatin remodeling and epigenetic control of gene expression.

Keywords:
Deacetylase inhibitor, Gene expression, Limb development, Chicken embryo, Epigenetics
1.
Alappat, S., Z.Y. Zhang, Y.P. Chen (2003) Msx homeobox gene family and craniofacial development. Cell Res 13: 429–442.
2.
Almouzni, G., S. Khochbin, S. Dimitrov, A.P. Wolffe (1994) Histone acetylation influences both gene expression and development of Xenopus laevis. Dev Biol 165: 654–669.
3.
Aoyama, T., T. Okamoto, Y. Kohno, K. Fukiage, S. Otsuka, M. Furu, K. Ito, Y. Jin, S. Nagayama, T. Nakayama, T. Nakamura, J. Toguchida (2008) Cell-specific epigenetic regulation of ChM-I gene expression: crosstalk between DNA methylation and histone acetylation. Biochem Biophys Res Commun 365: 124–130.
4.
Bonafede, A., T. Köhler, M. Rodriguez-Niedenfuhr, B. Brand-Saberi (2006) BMPs restrict the position of premuscle masses in the limb buds by influencing Tcf4 expression. Dev Biol 299: 330–344.
5.
Brand-Saberi, B., T.S. Muller, J. Wilting, B. Christ, C. Birchmeier (1996) Scatter factor/hepatocyte growth factor (SF/HGF) induces emigration of myogenic cells at interlimb level in vivo. Dev Biol 179: 303–308.
6.
Brent, A.E., R. Schweitzer, C.J. Tabin (2003) A somitic compartment of tendon progenitors. Cell 113: 235–248.
7.
Cao, Y., R.M. Kumar, B.H. Penn, C.A. Berkes, C. Kooperberg, L.A. Boyer, R.A. Young, S.J. Tapscott (2006) Global and gene-specific analyses show distinct roles for Myod and Myog at a common set of promoters. EMBO J 25: 502–511.
8.
Capdevila, J., J.C. Izpisua Belmonte (2001) Patterning mechanisms controlling vertebrate limb development. Annu Rev Cell Dev Biol 17: 87–132.
9.
Chang, S., C.S. Pikaard (2005) Transcript profiling in Arabidopsis reveals complex responses to global inhibition of DNA methylation and histone deacetylation. J Biol Chem 280: 796–804.
10.
Chen, A.E., D.D. Ginty, C.M. Fan (2005) Protein kinase A signaling via CREB controls myogenesis induced by Wnt proteins. Nature 433: 317–322.
11.
Chen, B., C.L. Cepko (2007) Requirement of histone deacetylase activity for the expression of critical photoreceptor genes. BMC Dev Biol 7: 78.
12.
Chiba, T., O. Yokosuka, K. Fukai, H. Kojima, M. Tada, M. Arai, F. Imazeki, H. Saisho (2004) Cell growth inhibition and gene expression induced by the histone deacetylase inhibitor, trichostatin A, on human hepatoma cells. Oncology 66: 481–491.
13.
Christ, B., B. Brand-Saberi (2002) Limb muscle development. Int J Dev Biol 46: 905–914.
14.
Davidson, D.R., A. Crawley, R.E. Hill, C. Tickle (1991) Position-dependent expression of two related homeobox genes in developing vertebrate limbs. Nature 352: 429–431.
15.
Dillon, R., C. Gadgil, H.G. Othmer (2003) Short- and long-range effects of Sonic hedgehog in limb development. Proc Natl Acad Sci USA 100: 10152–10157.
16.
Eberharter, A., R. Ferreira, P. Becker (2005) Dynamic chromatin: concerted nucleosome remodelling and acetylation. Biol Chem 386: 745–751.
17.
Edom-Vovard, F., B. Schuler, M.A. Bonnin, M.A. Teillet, D. Duprez (2002) Fgf4 positively regulates scleraxis and tenascin expression in chick limb tendons. Dev Biol 247: 351–366.
18.
Fasolini, M., X. Wu, M. Flocco, J.Y. Trosset, U. Oppermann, S. Knapp (2003) Hot spots in Tcf4 for the interaction with beta-catenin. J Biol Chem 278: 21092–21098.
19.
Firulli, B.A., D. Krawchuk, V.E. Centonze, N. Vargesson, D.M. Virshup, S.J. Conway, P. Cserjesi, E. Laufer, A.B. Firulli (2005) Altered Twist1 and Hand2 dimerization is associated with Saethre-Chotzen syndrome and limb abnormalities. Nat Genet 37: 373–381.
20.
Füchtbauer, E.M. (2002) Inhibition of skeletal muscle development: less differentiation gives more muscle. Results Probl Cell Differ 38: 143–161.
21.
Gibbons, R.J. (2005) Histone modifying and chromatin remodelling enzymes in cancer and dysplastic syndromes. Hum Mol Genet 14(spec No 1): R85–R92.
22.
Gurvich, N., M.G. Berman, B.S. Wittner, R.C. Gentleman, P.S. Klein, J.B. Green (2005) Association of valproate-induced teratogenesis with histone deacetylase inhibition in vivo. FASEB J 19: 1166–1168.
23.
Hamburger, V., H.L. Hamilton (1992) A series of normal stages in the development of the chick embryo. 1951. Dev Dyn 195: 231–272.
24.
Han, M., X. Yang, J.E. Farrington, K. Muneoka (2003) Digit regeneration is regulated by Msx1 and BMP4 in fetal mice. Development 130: 5123–5132.
25.
Hasan, S., P.O. Hassa, R. Imhof, M.O. Hottiger (2001) Transcription coactivator p300 binds PCNA and may have a role in DNA repair synthesis. Nature 410: 387–391.
26.
Hornik, C., B. Brand-Saberi, S. Rudloff, B. Christ, E.M. Füchtbauer (2004) Twist is an integrator of SHH, FGF, and BMP signaling. Anat Embryol (Berl) 209: 31–39.
27.
Hu, E., E. Dul, C.M. Sung, Z. Chen, R. Kirkpatrick, G.F. Zhang, K. Johanson, R. Liu, A. Lago, G. Hofmann, R. Macarron, M. de los Frailes, P. Perez, J. Krawiec, J. Winkler, M. Jaye (2003) Identification of novel isoform-selective inhibitors within class I histone deacetylases. J Pharmacol Exp Ther 307: 720–728.
28.
Hurlstone, A., H. Clevers (2002) T-cell factors: turn-ons and turn-offs. EMBO J 21: 2303–2311.
29.
Iezzi, S., G. Cossu, C. Nervi, V. Sartorelli, P.L. Puri (2002) Stage-specific modulation of skeletal myogenesis by inhibitors of nuclear deacetylases. Proc Natl Acad Sci USA 99: 7757–7762.
30.
Jenuwein, T., C.D. Allis (2001) Translating the histone code. Science 293: 1074–1080.
31.
Kang, J.H., M.J. Kim, S.Y. Chang, S.S. Sim, M.S. Kim, Y.H. Jo (2008) CCAAT box is required for the induction of human thrombospondin-1 gene by trichostatin A. J Cell Biochem 104: 1192–1203.
32.
Kardon, G., B.D. Harfe, C.J. Tabin (2003) A Tcf4-positive mesodermal population provides a prepattern for vertebrate limb muscle patterning. Dev Cell 5: 937–944.
33.
Kimura, S., K. Shiota (1996) Sequential changes of programmed cell death in developing fetal mouse limbs and its possible roles in limb morphogenesis. J Morphol 229: 337–346.
34.
Knudsen, T.B., D.M. Kochar (1981) The role of morphogenetic cell death during abnormal limb-bud outgrowth in mice heterozygous for the dominant mutation Hemimelia-extra toe (Hmx). J Embryol Exp Morphol 65 (suppl): 289–307.
35.
Köhler, T., F. Pröls, B. Brand-Saberi (2005) PCNA in situ hybridization: a novel and reliable tool for detection of dynamic changes in proliferative activity. Histochem Cell Biol 123: 315–327.
36.
Kouraklis, G., S. Theocharis (2006) Histone deacetylase inhibitors: a novel target of anticancer therapy (review). Oncol Rep 15: 489–494.
37.
Koussoulakos, S. (2004) Vertebrate limb development: from Harrison’s limb disk transplantations to targeted disruption of Hox genes. Anat Embryol (Berl) 209: 93–105.
38.
Kramer, J., C. Hegert, K. Guan, A.M. Wobus, P.K. Muller, J. Rohwedel (2000) Embryonic stem cell-derived chondrogenic differentiation in vitro: activation by BMP-2 and BMP-4. Mech Dev 92: 193–205.
39.
Lagger, G., D. O’Carroll, M. Rembold, H. Khier, J. Tischler, G. Weitzer, B. Schuettengruber, C. Hauser, R. Brunmeir, T. Jenuwein, C. Seiser (2002) Essential function of histone deacetylase 1 in proliferation control and CDK inhibitor repression. EMBO J 21: 2672–2681.
40.
Lallemand, Y., M.A. Nicola, C. Ramos, A. Bach, C.S. Cloment, B. Robert (2005) Analysis of Msx1; Msx2 double mutants reveals multiple roles for Msx genes in limb development. Development 132: 3003–3014.
41.
Lee, H., R. Habas, C. Abate-Shen (2004) MSX1 cooperates with histone H1b for inhibition of transcription and myogenesis. Science 304: 1675–1678.
42.
Lewandoski, M., X. Sun, G.R. Martin (2000) Fgf8 signalling from the AER is essential for normal limb development. Nat Genet 26: 460–463.
43.
Li, Y., B. Lal, S. Kwon, X. Fan, U. Saldanha, T.E. Reznik, E.B. Kuchner, C. Eberhart, J. Laterra, R. Abounader (2005) The scatter factor/hepatocyte growth factor: c-met pathway in human embryonal central nervous system tumor malignancy. Cancer Res 65: 9355–9362.
44.
Maestro, R., A.P. Dei Tos, Y. Hamamori, S. Krasnokutsky, V. Sartorelli, L. Kedes, C. Doglioni, D.H. Beach, G.J. Hannon (1999) Twist is a potential oncogene that inhibits apoptosis. Genes Dev 13: 2207–2217.
45.
McGlinn, E., C.J. Tabin (2006) Mechanistic insight into how Shh patterns the vertebrate limb. Curr Opin Genet Dev 16: 426–432.
46.
McKinsey, T.A., C.L. Zhang, E.N. Olson (2001) Control of muscle development by dueling HATs and HDACs. Curr Opin Genet Dev 11: 497–504.
47.
Mehra-Chaudhary, R., H. Matsui, R. Raghow (2001) Msx3 protein recruits histone deacetylase to down-regulate the Msx1 promoter. Biochem J 353: 13–22.
48.
Menegola, E., F. Di Renzo, M.L. Broccia, M. Prudenziati, S. Minucci, V. Massa, E. Giavini (2005) Inhibition of histone deacetylase activity on specific embryonic tissues as a new mechanism for teratogenicity. Birth Defects Res B Dev Reprod Toxicol 74: 392–398.
49.
Milaire, J., M. Rooze (1983) Hereditary and induced modifications of the normal necrotic patterns in the developing limb buds of the rat and mouse; facts and hypotheses. Arch Biol 94: 459–490.
50.
Minucci, S., P.G. Pelicci (2006) Histone deacetylase inhibitors and the promise of epigenetic (and more) treatments for cancer. Nat Rev Cancer 6: 38–51.
51.
Moon, A.M., M.R. Capecchi (2000) Fgf8 is required for outgrowth and patterning of the limbs. Nat Genet 26: 455–459.
52.
Morgan, H.D., F. Santos, K. Green, W. Dean, W. Reik (2005) Epigenetic reprogramming in mammals. Hum Mol Genet 14(spec No 1): R47–R58.
53.
Nervi, C., U. Borello, F. Fazi, V. Buffa, P.G. Pelicci, G. Cossu (2001) Inhibition of histone deacetylase activity by trichostatin A modulates gene expression during mouse embryogenesis without apparent toxicity. Cancer Res 61: 1247–1249.
54.
Newman, S.A., G.B. Muller (2005) Origination and innovation in the vertebrate limb skeleton: an epigenetic perspective. J Exp Zoolog B Mol Dev Evol 304: 593–609.
55.
Niswander, L., S. Jeffrey, G.R. Martin, C. Tickle (1994) A positive feedback loop coordinates growth and patterning in the vertebrate limb. Nature 371: 609–612.
56.
O’Rourke, M.P., K. Soo, R.R. Behringer, C.C. Hui, P.P. Tam (2002) Twist plays an essential role in FGF and SHH signal transduction during mouse limb development. Dev Biol 248: 143–156.
57.
Puri, P.L., V. Sartorelli, X.J. Yang, Y. Hamamori, V.V. Ogryzko, B.H. Howard, L. Kedes, J.Y. Wang, A. Graessmann, Y. Nakatani, M. Levrero (1997) Differential roles of p300 and PCAF acetyltransferases in muscle differentiation. Mol Cell 1: 35–45.
58.
Qian, D.Z., Y. Kato, S. Shabbeer, Y. Wei, H.M. Verheul, B. Salumbides, T. Sanni, P. Atadja, R. Pili (2006) Targeting tumor angiogenesis with histone deacetylase inhibitors: the hydroxamic acid derivative LBH589. Clin Cancer Res 12: 634–642.
59.
Reinhold, M.I., R.M. Kapadia, Z. Liao, M.C. Naski (2006) The Wnt-inducible transcription factor Twist1 inhibits chondrogenesis. J Biol Chem 281: 1381–1388.
60.
Riddle, R.D., R.L. Johnson, E. Laufer, C. Tabin (1993) Sonic hedgehog mediates the polarizing activity of the ZPA. Cell 75: 1401–1416.
61.
Robert, B. (2007) Bone morphogenetic protein signaling in limb outgrowth and patterning. Dev Growth Differ 49: 455–468.
62.
Scaal, M., A. Bonafede, V. Dathe, M. Sachs, G. Cann, B. Christ, B. Brand-Saberi (1999) SF/HGF is a mediator between limb patterning and muscle development. Development 126: 4885–4893.
63.
Scherz, P.J., B.D. Harfe, A.P. McMahon, C.J. Tabin (2004) The limb bud Shh-Fgf feedback loop is terminated by expansion of former ZPA cells. Science 305: 396–399.
64.
Schweitzer, R., J.H. Chyung, L.C. Murtaugh, A.E. Brent, V. Rosen, E.N. Olson, A. Lassar, C.J. Tabin (2001) Analysis of the tendon cell fate using Scleraxis, a specific marker for tendons and ligaments. Development 128: 3855–3866.
65.
Selever, J., W. Liu, M.F. Lu, R.R. Behringer, J.F. Martin (2004) Bmp4 in limb bud mesoderm regulates digit pattern by controlling AER development. Dev Biol 276: 268–279.
66.
Shetty, S., T. Takahashi, H. Matsui, R. Ayengar, R. Raghow (1999) Transcriptional autorepression of Msx1 gene is mediated by interactions of Msx1 protein with a multi-protein transcriptional complex containing TATA-binding protein, Sp1 and cAMP-response-element-binding protein-binding protein (CBP/p300). Biochem J 339(Pt 3): 751–758.
67.
Steinbach, O.C., A.P. Wolffe, R.A. Rupp (2000) Histone deacetylase activity is required for the induction of the MyoD muscle cell lineage in Xenopus. Biol Chem 381: 1013–1016.
68.
Sundaram, N., Q. Tao, C. Wylie, J. Heasman (2003) The role of maternal CREB in early embryogenesis of Xenopus laevis. Dev Biol 261: 337–352.
69.
Tabin, C., L. Wolpert (2007) Rethinking the proximodistal axis of the vertebrate limb in the molecular era. Genes Dev 21: 1433–1442.
70.
Tajbakhsh, S., D. Rocancourt, G. Cossu, M. Buckingham (1997) Redefining the genetic hierarchies controlling skeletal myogenesis: Pax-3 and Myf-5 act upstream of MyoD. Cell 89: 127–138.
71.
te Welscher, P., A. Zuniga, S. Kuijper, T. Drenth, H.J. Goedemans, F. Meijlink, R. Zeller (2002) Progression of vertebrate limb development through SHH-mediated counteraction of GLI3. Science 298: 827–830.
72.
Tickle, C. (2004) The contribution of chicken embryology to the understanding of vertebrate limb development. Mech Dev 121: 1019–1029.
73.
Tickle, C. (2006) Making digit patterns in the vertebrate limb. Nat Rev Mol Cell Biol 7: 45–53.
74.
Turner, B.M. (2002) Cellular memory and the histone code. Cell 111: 285–291.
75.
van der Hoeven, F., T. Schimmang, A. Volkmann, M.G. Mattei, B. Kyewski, U. Ruther (1994) Programmed cell death is affected in the novel mouse mutant Fused toes (Ft). Development 120: 2601–2607.
76.
Wan, M., X. Cao (2005) BMP signaling in skeletal development. Biochem Biophys Res Commun 328: 651–657.
77.
Wang, Y., D. Sassoon (1995) Ectoderm-mesenchyme and mesenchyme-mesenchyme interactions regulate Msx-1 expression and cellular differentiation in the murine limb bud. Dev Biol 168: 374–382.
78.
Yoo, C.B., P.A. Jones (2006) Epigenetic therapy of cancer: past, present and future. Nat Rev Drug Discov 5: 37–50.
79.
Yoshida, M., M. Kijima, M. Akita, T. Beppu (1990) Potent and specific inhibition of mammalian histone deacetylase both in vivo and in vitro by trichostatin A. J Biol Chem 265: 17174–17179.
80.
Zhang, Z., Y. Song, X. Zhao, X. Zhang, C. Fermin, Y. Chen (2002) Rescue of cleft palate in Msx1-deficient mice by transgenic Bmp4 reveals a network of BMP and Shh signaling in the regulation of mammalian palatogenesis. Development 129: 4135–4146.
81.
Zuniga, A., R. Quillet, F. Perrin-Schmitt, R. Zeller (2002) Mouse Twist is required for fibroblast growth factor-mediated epithelial-mesenchymal signalling and cell survival during limb morphogenesis. Mech Dev 114: 51–59.
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